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1.  Pervasive Genetic Hitchhiking and Clonal Interference in 40 Evolving Yeast Populations 
Nature  2013;500(7464):571-574.
The dynamics of adaptation determines which mutations fix in a population, and hence how reproducible evolution will be. This is central to understanding the spectra of mutations recovered in evolution of antibiotic resistance1, the response of pathogens to immune selection2,3, and the dynamics of cancer progression4,5. In laboratory evolution experiments, demonstrably beneficial mutations are found repeatedly6–8, but are often accompanied by other mutations with no obvious benefit. Here we use whole-genome whole-population sequencing to examine the dynamics of genome sequence evolution at high temporal resolution in 40 replicate Saccharomyces cerevisiae populations growing in rich medium for 1,000 generations. We find pervasive genetic hitchhiking: multiple mutations arise and move synchronously through the population as mutational “cohorts.” Multiple clonal cohorts are often present simultaneously, competing with each other in the same population. Our results show that patterns of sequence evolution are driven by a balance between these chance effects of hitchhiking and interference, which increase stochastic variation in evolutionary outcomes, and the deterministic action of selection on individual mutations, which favors parallel evolutionary solutions in replicate populations.
doi:10.1038/nature12344
PMCID: PMC3758440  PMID: 23873039
2.  Synthetic biology tools for programming gene expression without nutritional perturbations in Saccharomyces cerevisiae 
Nucleic Acids Research  2014;42(6):e48.
A conditional gene expression system that is fast-acting, is tunable and achieves single-gene specificity was recently developed for yeast. A gene placed directly downstream of a modified GAL1 promoter containing six Zif268 binding sequences (with single nucleotide spacing) was shown to be selectively inducible in the presence of β-estradiol, so long as cells express the artificial transcription factor, Z3EV (a fusion of the Zif268 DNA binding domain, the ligand binding domain of the human estrogen receptor and viral protein 16). We show the strength of Z3EV-responsive promoters can be modified using straightforward design principles. By moving Zif268 binding sites toward the transcription start site, expression output can be nearly doubled. Despite the reported requirement of estrogen receptor dimerization for hormone-dependent activation, a single binding site suffices for target gene activation. Target gene expression levels correlate with promoter binding site copy number and we engineer a set of inducible promoter chassis with different input–output characteristics. Finally, the coupling between inducer identity and gene activation is flexible: the ligand specificity of Z3EV can be re-programmed to respond to a non-hormone small molecule with only five amino acid substitutions in the human estrogen receptor domain, which may prove useful for industrial applications.
doi:10.1093/nar/gkt1402
PMCID: PMC3973312  PMID: 24445804
3.  A molecular barcoded yeast ORF library enables mode-of-action analysis of bioactive compounds 
Nature biotechnology  2009;27(4):10.1038/nbt.1534.
We present a yeast chemical-genomics approach designed to identify genes that when mutated confer drug resistance, thereby providing insight about the modes of action of compounds. We developed a molecular barcoded yeast open reading frame (MoBY-ORF) library in which each gene, controlled by its native promoter and terminator, is cloned into a centromere-based vector along with two unique oligonucleotide barcodes. The MoBY-ORF resource has numerous genetic and chemical-genetic applications, but here we focus on cloning wild-type versions of mutant drug-resistance genes using a complementation strategy and on simultaneously assaying the fitness of all transformants with barcode microarrays. The complementation cloning was validated by mutation detection using whole-genome yeast tiling microarrays, which identified unique polymorphisms associated with a drug-resistant mutant. We used the MoBY-ORF library to identify the genetic basis of several drug-resistant mutants and in this analysis discovered a new class of sterol-binding compounds.
doi:10.1038/nbt.1534
PMCID: PMC3856559  PMID: 19349972
4.  Rapid Synthesis and Screening of Chemically Activated Transcription Factors with GFP-based Reporters 
Synthetic biology aims to rationally design and build synthetic circuits with desired quantitative properties, as well as provide tools to interrogate the structure of native control circuits. In both cases, the ability to program gene expression in a rapid and tunable fashion, with no off-target effects, can be useful. We have constructed yeast strains containing the ACT1 promoter upstream of a URA3 cassette followed by the ligand-binding domain of the human estrogen receptor and VP16. By transforming this strain with a linear PCR product containing a DNA binding domain and selecting against the presence of URA3, a constitutively expressed artificial transcription factor (ATF) can be generated by homologous recombination. ATFs engineered in this fashion can activate a unique target gene in the presence of inducer, thereby eliminating both the off-target activation and nonphysiological growth conditions found with commonly used conditional gene expression systems. A simple method for the rapid construction of GFP reporter plasmids that respond specifically to a native or artificial transcription factor of interest is also provided.
doi:10.3791/51153
PMCID: PMC3992113  PMID: 24300440
Genetics; Issue 81; transcription; transcription factors; artificial transcription factors; zinc fingers; Zif268; synthetic biology
5.  Phylogenetic Portrait of the Saccharomyces cerevisiae Functional Genome 
G3: Genes|Genomes|Genetics  2013;3(8):1335-1340.
The genome of budding yeast (Saccharomyces cerevisiae) contains approximately 5800 protein-encoding genes, the majority of which are associated with some known biological function. Yet the extent of amino acid sequence conservation of these genes over all phyla has only been partially examined. Here we provide a more comprehensive overview and visualization of the conservation of yeast genes and a means for browsing and exploring the data in detail, down to the individual yeast gene, at http://yeast-phylogroups.princeton.edu. We used data from the OrthoMCL database, which has defined orthologs from approximately 150 completely sequenced genomes, including diverse representatives of the archeal, bacterial, and eukaryotic domains. By clustering genes based on similar patterns of conservation, we organized and visualized all the protein-encoding genes in yeast as a single heat map. Most genes fall into one of eight major clusters, called “phylogroups.” Gene ontology analysis of the phylogroups revealed that they were associated with specific, distinct trends in gene function, generalizations likely to be of interest to a wide range of biologists.
doi:10.1534/g3.113.006585
PMCID: PMC3737173  PMID: 23749449
yeast; evolution; phylogeny; orthology; genome
6.  Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae 
The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.
doi:10.3791/50382
PMCID: PMC3727456  PMID: 23793137
Genetics; Issue 76; Molecular Biology; Cellular Biology; Microbiology; Biochemistry; Genomics; Life Sciences (General); FISH; single cells; mRNA; transcripts;  Saccharomyces cerevisiae; yeast cells; single-molecule; yeast
7.  Genetic Basis of Ammonium Toxicity Resistance in a Sake Strain of Yeast: A Mendelian Case 
G3: Genes|Genomes|Genetics  2013;3(4):733-740.
High concentrations of ammonium at physiological concentrations of potassium are toxic for the standard laboratory strain of Saccharomyces cerevisiae. In the original description of this metabolic phenotype, we focused on the standard laboratory strains of Saccharomyces. In this study, we screened a large collection of S. cerevisiae natural isolates and identified one strain that is resistant to high concentrations of ammonium. This strain, K12, was isolated in sake breweries. When the K12 strain was crossed to the standard laboratory strain (FY4), the resulting tetrads displayed 2:2 segregation of the resistance phenotype, suggesting a single gene trait. Using a bulk segregant analysis strategy, we mapped this trait to a 150-kb region on chromosome X containing the TRK1 gene. This gene encodes a transporter required for high-affinity potassium transport in S. cerevisiae. Data from reciprocal hemizygosity experiments with TRK1 deletion strains in K12 and BY backgrounds, as well as analysis of the deletion of this gene in the K12 strain, demonstrate that the K12 allele of TRK1 is responsible for ammonium toxicity resistance. Furthermore, we determined the minimal amount of potassium required for both the K12 and laboratory strain needed for growth. These results demonstrate that the gene encoded by the K12 allele of TRK1 has a greater affinity for potassium than the standard allele of TRK1 found in Saccharomyces strains. We hypothesize that this greater-affinity allele of the potassium transporter reduces the flux of ammonium into the yeast cells under conditions of ammonium toxicity. These findings further refine our understanding of ammonium toxicity in yeast and provide an example of using natural variation to understand cellular processes.
doi:10.1534/g3.113.005884
PMCID: PMC3618360  PMID: 23550140
8.  TOR and RAS pathways regulate desiccation tolerance in Saccharomyces cerevisiae 
Molecular Biology of the Cell  2013;24(2):115-128.
Desiccation is thought to impose many stresses. Which of these stresses is responsible for desiccation-induced death and how the stress response is regulated are unknown, however. Here we use Saccharomyces cerevisiae to show that reduction of a 60S biogenesis intermediate via RAS or TOR down-regulation increases desiccation tolerance.
Tolerance to desiccation in cultures of Saccharomyces cerevisiae is inducible; only one in a million cells from an exponential culture survive desiccation compared with one in five cells in stationary phase. Here we exploit the desiccation sensitivity of exponentially dividing cells to understand the stresses imposed by desiccation and their stress response pathways. We found that induction of desiccation tolerance is cell autonomous and that there is an inverse correlation between desiccation tolerance and growth rate in glucose-, ammonia-, or phosphate-limited continuous cultures. A transient heat shock induces a 5000–fold increase in desiccation tolerance, whereas hyper-ionic, -reductive, -oxidative, or -osmotic stress induced much less. Furthermore, we provide evidence that the Sch9p-regulated branch of the TOR and Ras-cAMP pathway inhibits desiccation tolerance by inhibiting the stress response transcription factors Gis1p, Msn2p, and Msn4p and by activating Sfp1p, a ribosome biogenesis transcription factor. Among 41 mutants defective in ribosome biogenesis, a subset defective in 60S showed a dramatic increase in desiccation tolerance independent of growth rate. We suggest that reduction of a specific intermediate in 60S biogenesis, resulting from conditions such as heat shock and nutrient deprivation, increases desiccation tolerance.
doi:10.1091/mbc.E12-07-0524
PMCID: PMC3541959  PMID: 23171550
9.  Decoupling nutrient signaling from growth rate causes aerobic glycolysis and deregulation of cell size and gene expression 
Molecular Biology of the Cell  2013;24(2):157-168.
The nutrition and the growth rate of a cell are two interacting factors with pervasive physiological effects. Our experiments decouple these factors and demonstrate the role of a growth rate signal, independent of the actual rate of biomass increase, on gene regulation, the cell division cycle, and the switch to a respiro-fermentative metabolism.
To survive and proliferate, cells need to coordinate their metabolism, gene expression, and cell division. To understand this coordination and the consequences of its failure, we uncoupled biomass synthesis from nutrient signaling by growing, in chemostats, yeast auxotrophs for histidine, lysine, or uracil in excess of natural nutrients (i.e., sources of carbon, nitrogen, sulfur, and phosphorus), such that their growth rates (GRs) were regulated by the availability of their auxotrophic requirements. The physiological and transcriptional responses to GR changes of these cultures differed markedly from the respective responses of prototrophs whose growth-rate is regulated by the availability of natural nutrients. The data for all auxotrophs at all GRs recapitulated the features of aerobic glycolysis, fermentation despite high oxygen levels in the growth media. In addition, we discovered wide bimodal distributions of cell sizes, indicating a decoupling between the cell division cycle (CDC) and biomass production. The aerobic glycolysis was reflected in a general signature of anaerobic growth, including substantial reduction in the expression levels of mitochondrial and tricarboxylic acid genes. We also found that the magnitude of the transcriptional growth-rate response (GRR) in the auxotrophs is only 40–50% of the magnitude in prototrophs. Furthermore, the auxotrophic cultures express autophagy genes at substantially lower levels, which likely contributes to their lower viability. Our observations suggest that a GR signal, which is a function of the abundance of essential natural nutrients, regulates fermentation/respiration, the GRR, and the CDC.
doi:10.1091/mbc.E12-09-0670
PMCID: PMC3541962  PMID: 23135997
10.  Why we need more basic biology research, not less 
Molecular Biology of the Cell  2012;23(21):4160-4161.
Much of the spectacular progress in biomedical science over the last half-century is the direct consequence of the work of thousands of basic scientists whose primary goal was understanding of the fundamental working of living things. Despite this, many politicians, funders, and even scientists have come to believe that the pace of successful applications to medical diagnosis and therapy is limited by our willingness to focus directly on human health, rather than a continuing deficit of understanding. By this theory, curiosity-driven research, aimed at understanding, is no longer important or even useful. What is advocated instead is “translational” research aimed directly at treating disease. I believe this idea to be deeply mistaken. Recent history suggests instead that what we have learned in the last 50 years is only the beginning. The way forward is to invest more in basic science, not less.
doi:10.1091/mbc.E12-05-0406
PMCID: PMC3484092  PMID: 23112227
11.  Synthetic gene expression perturbation systems with rapid, tunable, single-gene specificity in yeast 
Nucleic Acids Research  2012;41(4):e57.
A general method for the dynamic control of single gene expression in eukaryotes, with no off-target effects, is a long-sought tool for molecular and systems biologists. We engineered two artificial transcription factors (ATFs) that contain Cys2His2 zinc-finger DNA-binding domains of either the mouse transcription factor Zif268 (9 bp of specificity) or a rationally designed array of four zinc fingers (12 bp of specificity). These domains were expressed as fusions to the human estrogen receptor and VP16 activation domain. The ATFs can rapidly induce a single gene driven by a synthetic promoter in response to introduction of an otherwise inert hormone with no detectable off-target effects. In the absence of inducer, the synthetic promoter is inactive and the regulated gene product is not detected. Following addition of inducer, transcripts are induced >50-fold within 15 min. We present a quantitative characterization of these ATFs and provide constructs for making their implementation straightforward. These new tools allow for the elucidation of regulatory network elements dynamically, which we demonstrate with a major metabolic regulator, Gcn4p.
doi:10.1093/nar/gks1313
PMCID: PMC3575806  PMID: 23275543
12.  Perturbation-based analysis and modeling of combinatorial regulation in the yeast sulfur assimilation pathway 
Molecular Biology of the Cell  2012;23(15):2993-3007.
Here we establish the utility of a recently described perturbative method to study complex regulatory circuits in vivo. By combining rapid modulation of single TFs under physiological conditions with genome-wide expression analysis, we elucidate several novel regulatory features within the pathways of sulfur assimilation and beyond.
In yeast, the pathways of sulfur assimilation are combinatorially controlled by five transcriptional regulators (three DNA-binding proteins [Met31p, Met32p, and Cbf1p], an activator [Met4p], and a cofactor [Met28p]) and a ubiquitin ligase subunit (Met30p). This regulatory system exerts combinatorial control not only over sulfur assimilation and methionine biosynthesis, but also on many other physiological functions in the cell. Recently we characterized a gene induction system that, upon the addition of an inducer, results in near-immediate transcription of a gene of interest under physiological conditions. We used this to perturb levels of single transcription factors during steady-state growth in chemostats, which facilitated distinction of direct from indirect effects of individual factors dynamically through quantification of the subsequent changes in genome-wide patterns of gene expression. We were able to show directly that Cbf1p acts sometimes as a repressor and sometimes as an activator. We also found circumstances in which Met31p/Met32p function as repressors, as well as those in which they function as activators. We elucidated and numerically modeled feedback relationships among the regulators, notably feedforward regulation of Met32p (but not Met31p) by Met4p that generates dynamic differences in abundance that can account for the differences in function of these two proteins despite their identical binding sites.
doi:10.1091/mbc.E12-03-0232
PMCID: PMC3408425  PMID: 22696683
13.  Combinatorial control of diverse metabolic and physiological functions by transcriptional regulators of the yeast sulfur assimilation pathway 
Molecular Biology of the Cell  2012;23(15):3008-3024.
The sulfur assimilation pathway is used to understand how combinatorial transcription coordinates cellular processes. Global gene expression was measured in yeast lacking different combinations of transcription factors in order to determine how these factors coordinate sulfur assimilation with diverse metabolic and physiological processes.
Methionine abundance affects diverse cellular functions, including cell division, redox homeostasis, survival under starvation, and oxidative stress response. Regulation of the methionine biosynthetic pathway involves three DNA-binding proteins—Met31p, Met32p, and Cbf1p. We hypothesized that there exists a “division of labor” among these proteins that facilitates coordination of methionine biosynthesis with diverse biological processes. To explore combinatorial control in this regulatory circuit, we deleted CBF1, MET31, and MET32 individually and in combination in a strain lacking methionine synthase. We followed genome-wide gene expression as these strains were starved for methionine. Using a combination of bioinformatic methods, we found that these regulators control genes involved in biological processes downstream of sulfur assimilation; many of these processes had not previously been documented as methionine dependent. We also found that the different factors have overlapping but distinct functions. In particular, Met31p and Met32p are important in regulating methionine metabolism, whereas p functions as a “generalist” transcription factor that is not specific to methionine metabolism. In addition, Met31p and Met32p appear to regulate iron–sulfur cluster biogenesis through direct and indirect mechanisms and have distinguishable target specificities. Finally, CBF1 deletion sometimes has the opposite effect on gene expression from MET31 and MET32 deletion.
doi:10.1091/mbc.E12-03-0233
PMCID: PMC3408426  PMID: 22696679
14.  Loss of a 20S Proteasome Activator in Saccharomyces cerevisiae Downregulates Genes Important for Genomic Integrity, Increases DNA Damage, and Selectively Sensitizes Cells to Agents With Diverse Mechanisms of Action 
G3: Genes|Genomes|Genetics  2012;2(8):943-959.
Cytoprotective functions of a 20S proteasome activator were investigated. Saccharomyces cerevisiae Blm10 and human 20S proteasome activator 200 (PA200) are homologs. Comparative genome-wide analyses of untreated diploid cells lacking Blm10 and growing at steady state at defined growth rates revealed downregulation of numerous genes required for accurate chromosome structure, assembly and repair, and upregulation of a specific subset of genes encoding protein-folding chaperones. Blm10 loss or truncation of the Ubp3/Blm3 deubiquitinating enzyme caused massive chromosomal damage and cell death in homozygous diploids after phleomycin treatments, indicating that Blm10 and Ubp3/Blm3 function to stabilize the genome and protect against cell death. Diploids lacking Blm10 also were sensitized to doxorubicin, hydroxyurea, 5-fluorouracil, rapamycin, hydrogen peroxide, methyl methanesulfonate, and calcofluor. Fluorescently tagged Blm10 localized in nuclei, with enhanced fluorescence after DNA replication. After DNA damage that caused a classic G2/M arrest, fluorescence remained diffuse, with evidence of nuclear fragmentation in some cells. Protective functions of Blm10 did not require the carboxyl-terminal region that makes close contact with 20S proteasomes, indicating that protection does not require this contact or the truncated Blm10 can interact with the proteasome apart from this region. Without its carboxyl-terminus, Blm10(−339aa) localized to nuclei in untreated, nonproliferating (G0) cells, but not during G1 S, G2, and M. The results indicate Blm10 functions in protective mechanisms that include the machinery that assures proper assembly of chromosomes. These essential guardian functions have implications for ubiquitin-independent targeting in anticancer therapy. Targeting Blm10/PA200 together with one or more of the upregulated chaperones or a conventional treatment could be efficacious.
doi:10.1534/g3.112.003376
PMCID: PMC3411250  PMID: 22908043
20S proteasome activator; BLM10/PA200; UBP3/BLM3; DNA damage; molecular chaperones
15.  A conserved cell growth cycle can account for the environmental stress responses of divergent eukaryotes 
Molecular Biology of the Cell  2012;23(10):1986-1997.
Transitions between the two phases of the cell growth cycle can account for the environmental stress response, the growth-rate response, and the cross-protection between slow growth and various types of stress factors. It is suggested that this mechanism is conserved across budding and fission yeast and normal human cells.
The respiratory metabolic cycle in budding yeast (Saccharomyces cerevisiae) consists of two phases that are most simply defined phenomenologically: low oxygen consumption (LOC) and high oxygen consumption (HOC). Each phase is associated with the periodic expression of thousands of genes, producing oscillating patterns of gene expression found in synchronized cultures and in single cells of slowly growing unsynchronized cultures. Systematic variation in the durations of the HOC and LOC phases can account quantitatively for well-studied transcriptional responses to growth rate differences. Here we show that a similar mechanism—transitions from the HOC phase to the LOC phase—can account for much of the common environmental stress response (ESR) and for the cross-protection by a preliminary heat stress (or slow growth rate) to subsequent lethal heat stress. Similar to the budding yeast metabolic cycle, we suggest that a metabolic cycle, coupled in a similar way to the ESR, in the distantly related fission yeast, Schizosaccharomyces pombe, and in humans can explain gene expression and respiratory patterns observed in these eukaryotes. Although metabolic cycling is associated with the G0/G1 phase of the cell division cycle of slowly growing budding yeast, transcriptional cycling was detected in the G2 phase of the division cycle in fission yeast, consistent with the idea that respiratory metabolic cycling occurs during the phases of the cell division cycle associated with mass accumulation in these divergent eukaryotes.
doi:10.1091/mbc.E11-11-0961
PMCID: PMC3350561  PMID: 22456505
16.  Fast-acting and nearly gratuitous induction of gene expression and protein depletion in Saccharomyces cerevisiae 
Molecular Biology of the Cell  2011;22(22):4447-4459.
We developed systems to rapidly express any yeast gene or to specifically degrade any protein, each with minimal untargeted disturbance of cell physiology. We illustrate applications of these new tools for elucidating the architecture and dynamics of genetic regulatory networks.
We describe the development and characterization of a system that allows the rapid and specific induction of individual genes in the yeast Saccharomyces cerevisiae without changes in nutrients or temperature. The system is based on the chimeric transcriptional activator Gal4dbd.ER.VP16 (GEV). Upon addition of the hormone β-estradiol, cytoplasmic GEV localizes to the nucleus and binds to promoters containing Gal4p consensus binding sequences to activate transcription. With galactokinase Gal1p and transcriptional activator Gal4p absent, the system is fast-acting, resulting in readily detectable transcription within 5 min after addition of the inducer. β-Estradiol is nearly a gratuitous inducer, as indicated by genome-wide profiling that shows unintended induction (by GEV) of only a few dozen genes. Response to inducer is graded: intermediate concentrations of inducer result in production of intermediate levels of product protein in all cells. We present data illustrating several applications of this system, including a modification of the regulated degron method, which allows rapid and specific degradation of a specific protein upon addition of β-estradiol. These gene induction and protein degradation systems provide important tools for studying the dynamics and functional relationships of genes and their respective regulatory networks.
doi:10.1091/mbc.E11-05-0466
PMCID: PMC3216669  PMID: 21965290
17.  Coordinated regulation of sulfur and phospholipid metabolism reflects the importance of methylation in the growth of yeast 
Molecular Biology of the Cell  2011;22(21):4192-4204.
The sulfur assimilation and phospholipid biosynthesis pathways interact metabolically and transcriptionally. Genetic analysis, genome-wide sequencing, and expression microarrays show that regulators of these pathways, Met4p and Opi1p, control cellular methylation capacity that can limit the growth rate.
A yeast strain lacking Met4p, the primary transcriptional regulator of the sulfur assimilation pathway, cannot synthesize methionine. This apparently simple auxotroph did not grow well in rich media containing excess methionine, forming small colonies on yeast extract/peptone/dextrose plates. Faster-growing large colonies were abundant when overnight cultures were plated, suggesting that spontaneous suppressors of the growth defect arise with high frequency. To identify the suppressor mutations, we used genome-wide single-nucleotide polymorphism and standard genetic analyses. The most common suppressors were loss-of-function mutations in OPI1, encoding a transcriptional repressor of phospholipid metabolism. Using a new system that allows rapid and specific degradation of Met4p, we could study the dynamic expression of all genes following loss of Met4p. Experiments using this system with and without Opi1p showed that Met4 activates and Opi1p represses genes that maintain levels of S-adenosylmethionine (SAM), the substrate for most methyltransferase reactions. Cells lacking Met4p grow normally when either SAM is added to the media or one of the SAM synthetase genes is overexpressed. SAM is used as a methyl donor in three Opi1p-regulated reactions to create the abundant membrane phospholipid, phosphatidylcholine. Our results show that rapidly growing cells require significant methylation, likely for the biosynthesis of phospholipids.
doi:10.1091/mbc.E11-05-0467
PMCID: PMC3204079  PMID: 21900497
18.  A Test of the Coordinated Expression Hypothesis for the Origin and Maintenance of the GAL Cluster in Yeast 
PLoS ONE  2011;6(9):e25290.
Metabolic gene clusters—functionally related and physically clustered genes—are a common feature of some eukaryotic genomes. Two hypotheses have been advanced to explain the origin and maintenance of metabolic gene clusters: coordinated gene expression and genetic linkage. Here we test the hypothesis that selection for coordinated gene expression underlies the clustering of GAL genes in the yeast genome. We find that, although clustering coordinates the expression of GAL1 and GAL10, disrupting the GAL cluster does not impair fitness, suggesting that other mechanisms, such as genetic linkage, drive the origin and maintenance metabolic gene clusters.
doi:10.1371/journal.pone.0025290
PMCID: PMC3178652  PMID: 21966486
19.  Coupling among growth rate response, metabolic cycle, and cell division cycle in yeast 
Molecular Biology of the Cell  2011;22(12):1997-2009.
We discovered that the relative durations of the phases of the yeast metabolic cycle change with the growth rate. These changes can explain mechanistically the transcriptional growth-rate responses of all yeast genes (25% of the genome) that we find to be the same across all studied nutrient limitations in either ethanol or glucose media.
We studied the steady-state responses to changes in growth rate of yeast when ethanol is the sole source of carbon and energy. Analysis of these data, together with data from studies where glucose was the carbon source, allowed us to distinguish a “universal” growth rate response (GRR) common to all media studied from a GRR specific to the carbon source. Genes with positive universal GRR include ribosomal, translation, and mitochondrial genes, and those with negative GRR include autophagy, vacuolar, and stress response genes. The carbon source–specific GRR genes control mitochondrial function, peroxisomes, and synthesis of vitamins and cofactors, suggesting this response may reflect the intensity of oxidative metabolism. All genes with universal GRR, which comprise 25% of the genome, are expressed periodically in the yeast metabolic cycle (YMC). We propose that the universal GRR may be accounted for by changes in the relative durations of the YMC phases. This idea is supported by oxygen consumption data from metabolically synchronized cultures with doubling times ranging from 5 to 14 h. We found that the high oxygen consumption phase of the YMC can coincide exactly with the S phase of the cell division cycle, suggesting that oxidative metabolism and DNA replication are not incompatible.
doi:10.1091/mbc.E11-02-0132
PMCID: PMC3113766  PMID: 21525243
20.  Genetic Variation and the Fate of Beneficial Mutations in Asexual Populations 
Genetics  2011;188(3):647-661.
The fate of a newly arising beneficial mutation depends on many factors, such as the population size and the availability and fitness effects of other mutations that accumulate in the population. It has proved difficult to understand how these factors influence the trajectories of particular mutations, since experiments have primarily focused on characterizing successful clones emerging from a small number of evolving populations. Here, we present the results of a massively parallel experiment designed to measure the full spectrum of possible fates of new beneficial mutations in hundreds of experimental yeast populations, whether these mutations are ultimately successful or not. Using strains in which a particular class of beneficial mutation is detectable by fluorescence, we followed the trajectories of these beneficial mutations across 592 independent populations for 1000 generations. We find that the fitness advantage provided by individual mutations plays a surprisingly small role. Rather, underlying “background” genetic variation is quickly generated in our initially clonal populations and plays a crucial role in determining the fate of each individual beneficial mutation in the evolving population.
doi:10.1534/genetics.111.128942
PMCID: PMC3176544  PMID: 21546542
21.  Comparison of the Complete Protein Sets of Worm and Yeast: Orthology and Divergence 
Science (New York, N.Y.)  1998;282(5396):2022-2028.
Comparative analysis of predicted protein sequences encoded by the genomes of Caenorhabditis elegans and Saccharomyces cerevisiae suggests that most of the core biological functions are carried out by orthologous proteins (proteins of different species that can be traced back to a common ancestor) that occur in comparable numbers. The specialized processes of signal transduction and regulatory control that are unique to the multicellular worm appear to use novel proteins, many of which re-use conserved domains. Major expansion of the number of some of these domains seen in the worm may have contributed to the advent of multicellularity. The proteins conserved in yeast and worm are likely to have orthologs throughout eukaryotes; in contrast, the proteins unique to the worm may well define metazoans.
PMCID: PMC3057080  PMID: 9851918
22.  Genetic and physical maps of Saccharomyces cerevisiae 
Nature  1997;387(6632 Suppl):67-73.
Genetic and physical maps for the 16 chromosomes of Saccharomyces cerevisiae are presented. The genetic map is the result of 40 years of genetic analysis. The physical map was produced from the results of an international systematic sequencing effort. The data for the maps are accessible electronically from the Saccharomyces Genome Database (SGD: http://genome-www.stanford.edu/Saccharomyces/).
PMCID: PMC3057085  PMID: 9169866
23.  Yeast as a Model Organism 
Science (New York, N.Y.)  1997;277(5330):1259-1260.
PMCID: PMC3039837  PMID: 9297238
24.  Saccharomyces cerevisiae S288C genome annotation: a working hypothesis 
Yeast (Chichester, England)  2006;23(12):857-865.
The S. cerevisiae genome is the most well-characterized eukaryotic genome and one of the simplest in terms of identifying open reading frames (ORFs), yet its primary annotation has been updated continually in the decade since its initial release in 1996 (Goffeau et al., 1996). The Saccharomyces Genome Database (SGD; www.yeastgenome.org) (Hirschman et al., 2006), the community-designated repository for this reference genome, strives to ensure that the S. cerevisiae annotation is as accurate and useful as possible. At SGD, the S. cerevisiae genome sequence and annotation are treated as a working hypothesis, which must be repeatedly tested and refined. In this paper, in celebration of the tenth anniversary of the completion of the S. cerevisiae genome sequence, we discuss the ways in which the S. cerevisiae sequence and annotation have changed, consider the multiple sources of experimental and comparative data on which these changes are based, and describe our methods for evaluating, incorporating and documenting these new data.
doi:10.1002/yea.1400
PMCID: PMC3040122  PMID: 17001629
S. cerevisiae; genome sequence; genome annotation; comparative genomics; exon/intron boundaries
25.  GO::TermFinder—open source software for accessing Gene Ontology information and finding significantly enriched Gene Ontology terms associated with a list of genes 
Bioinformatics (Oxford, England)  2004;20(18):3710-3715.
Summary
GO::TermFinder comprises a set of object-oriented Perl modules for accessing Gene Ontology (GO) information and evaluating and visualizing the collective annotation of a list of genes to GO terms. It can be used to draw conclusions from microarray and other biological data, calculating the statistical significance of each annotation. GO::TermFinder can be used on any system on which Perl can be run, either as a command line application, in single or batch mode, or as a web-based CGI script.
Availability
The full source code and documentation for GO::TermFinder are freely available from http://search.cpan.org/dist/GO-TermFinder/
Contact
sherlock@genome.stanford.edu
doi:10.1093/bioinformatics/bth456
PMCID: PMC3037731  PMID: 15297299

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