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To determine how polyploidy affects the rate of adaptation, we performed hundreds of independent passaging experiments in a poor carbonsource medium (raffinose, Fig. 1a), comparing isogenic haploid (1N), diploid (2N), [...], Polyploidy is observed across the tree of life, yet its influence on evolution remains incompletely understood (1-4). Polyploidy, usually whole-genome duplication, is proposed to alter the rate of evolutionary adaptation. This could occur through complex effects on the frequency or fitness of beneficial mutations (2, 5-7). For example, in diverse cell types and organisms, immediately after a whole-genome duplication, newly formed polyploids missegregate chromosomes and undergo genetic instability (8-13). The instability following whole-genome duplications is thought to provide adaptive mutations in microorganisms (13, 14) and can promote tumorigenesis in mammalian cells (11, 15). Polyploidy may also affect adaptation independently of beneficial mutations through ploidy-specific changes in cell physiology (16). Here we perform in vitro evolution experiments to test directly whether polyploidy can accelerate evolutionary adaptation. Compared with haploids and diploids, tetraploids undergo significantly faster adaptation. Mathematical modelling suggests that rapid adaptation of tetraploids is driven by higher rates of beneficial mutations with stronger fitness effects, which is supported by whole-genome sequencing and phenotypic analyses of evolved clones. Chromosome aneuploidy, concerted chromosome loss, and point mutations all provide large fitness gains. We identify several mutations whose beneficial effects are manifest specifically in the tetraploid strains. Together, these results provide direct quantitative evidence that in some environments polyploidy can accelerate evolutionary adaptation.

Bacterial pathogens have evolved specific effector proteins that, by interfacing with host kinase signalling pathways, provide a mechanism to evade immune responses during infection (1,2). Although these effectors contribute to [...]

In the budding yeast Saccharomyces cerevisiae, self-recognition and the thereby promoted aggregation of thousands of cells into protective flocs is mediated by a family of cell-surface adhesins, the flocculins (Flo). Based on this social behavior FLO genes fulfill the definition of 'greenbeard' genes, which direct cooperation toward other carriers of the same gene. The process of flocculation plays an eminent role in the food industry for the production of beer and wine. However, the precise mode of flocculin-mediated surface recognition and the exact structure of cognate ligands have remained elusive. Here, we present structures of the adhesion domain of a flocculin complexed to its cognate ligands derived from yeast high-mannose oligosaccharides at resolutions up to 0.95 [Angstrom]. Besides a PA14-like architecture, the Flo5A domain reveals a previously undescribed lectin fold that utilizes a unique DcisD calcium-binding motif for carbohydrate binding and that is widely spread among pro- and eukaryotes. Given the high abundance of high-mannose oligosaccharides in yeast cell walls, the Flo5A structure suggests a model for recognition, where social non-self-instead of unsocial self-interactions are favored. altruism | molecular recognition | fungal development | atomic resolution doi: 10.1073/pnas.1013210108.

How dividing cells monitor the effective transmission of genomes during mitosis is poorly understood. Budding yeast use a signaling pathway known as the spindle position checkpoint (SPC) to ensure the arrival of one end of the mitotic spindle in the nascent daughter cell. An important question is how SPC activity is coordinated with mother-daughter polarity. We sought to identify factors at the bud neck, the junction between mother and bud, which contribute to checkpoint signaling. In this paper, we show that the protein kinase Elm1 is an obligate regulator of the SPC, and this function requires localization of Elm1 to the bud neck. Furthermore, we show that Elm1 promotes the activity of the checkpoint kinase Kin4. These findings reveal a novel function for Elm1 in the SPC and suggest how checkpoint activity may be linked to cellular organization. doi/ 10.1083/jcb.201006092

Yeast prion [[PSI.sup.+]] is caused by aggregated structures of the Sup35 protein. Although Sup35 forms typical amyloid fibrils in vitro, there is no direct evidence for the fibrillar structures of Sup35 in vivo. We analyzed [[PSI.sup.+]] cells in which Sup35 fused with green fluorescent protein (GFP) formed aggregates visible by fluorescence microscopy using thin-section electron microscopy (EM). Rapid-freeze EM combined with an immunogold-labeling technique as well as correlative light EM, which allows high-resolution imaging by EM of the same structure observed by light (fluorescence) microscopy, shows that the aggregates contain bundled fibrillar structures of Sup35-GFP. Additional biochemical and fluorescent correlation spectroscopy results suggest that the Sup35 oligomers diffused in the [[PSI.sup.+]] lysates adopt fibril-like shapes. Our findings demonstrate that [[PSI.sup.+]] cells contain Sup35 fibrillar structures closely related to those formed in vitro and provide insight into the molecular mechanism by which Sup35 aggregates are assembled and remodeled in [[PSI.sup.+]] cells. doi/ 10.1083/jcb.201002149

The interactions of protein kinases and phosphatases with their regulatory subunits and substrates underpin cellular regulation. We identified a kinase and phosphatase interaction (KPI) network of 1844 interactions in budding yeast by mass spectrometric analysis of protein complexes. The KPI network contained many dense local regions of interactions that suggested new functions. Notab|y, the cell cycle phosphatase Cdc14 associated with muttiple kinases that revealed roles for Cdc14 in mitogen-activated protein kinase signaling, the DNA damage response, and metabolism, whereas interactions of the target of rapamycin comptex 1 (TORCl) uncovered new effector kinases in nitrogen and carbon metabolism. An extensive backbone of kinase-kinase interactions cross-connects the proteome and may serve to coordinate diverse cellular responses. doi: 10.1126/science.1176495

X-ray diffraction microscopy complements other x-ray microscopy methods by being free of lens-imposed radiation dose and resolution limits, and it allows for high-resolution imaging of biological specimens too thick to be viewed by electron microscopy. We report here the highest resolution (11-13 nm) x-ray diffraction micrograph of biological specimens, and a demonstration of molecular-specific gold labeling at different depths within cells via through-focus propagation of the reconstructed wavefield. The lectin concanavalin A conjugated to colloidal gold particles was used to label the [alpha]-mannan sugar in the cell wall of the yeast Saccharomyces cerevisiae. Cells were plunge-frozen in liquid ethane and freeze-dried, after which they were imaged whole using x-ray diffraction microscopy at 750 eV photon energy. coherent imaging | immunogold labeling doi: /10.1073/pnas.0910874107

A systematic approach to the discovery of conformation-specific antibodies or those that recognize activation-induced neoepitopes in signaling molecules and enzymes will be a powerful tool in developing antibodies for basic science and therapy. Here, we report the isolation of antibody antagonists that preferentially bind activated integrin Mac-1 ([[alpha].sub.M][[beta].sub.2]) and are potent in blocking neutrophil adhesion and migration. A novel strategy was developed for this task, consisting of yeast surface display of Mac-1 inserted (I) domain library, directed evolution to isolate active mutants of the I domain, and screening of phage display of human antibody library against the active I domain in yeast. Enriched antibody library was then introduced into yeast surface two-hybrid system for final quantitative selection of antibodies from monomeric antigen--antibody interaction. This led to highly efficient isolation of intermediate to high affinity antibodies, which preferentially reacted with the active I domain, antagonized the I domain binding to intercellular adhesion molecule (ICAM)-I, complement C3 fragment iC3b, and fibronectin, and potently inhibited neutrophil migration on fibrinogen. The strategy demonstrated herein can be broadly applicable to developing antibodies against modular domains that switch between inactive and active conformations, particularly toward the discovery of antibody antagonists in therapeutic and diagnostic applications. antibody antagonist | mac-1 integrin | phage display | yeast display doi/10.1073/pnas.0914358107

Schizosaccharomyces species are currently the only known organisms with two types of genes encoding U DP-glucose/-galactose 4-epimerase, [uge1.sup.+] and [gal10.sup.+]. A strain deleted for [uge1.sup.+] exhibited a severe galactosylation defect and a decrease in activity and in UDP-galactose content when grown in glucose-rich medium (2 % glucose), indicating that Ugel p is a major UDPglucose/-galactose 4-epimerase under these growth conditions. In contrast, [gal10.sup.+] was efficiently expressed and involved in galactosylation of cell-surface proteins in low-glucose medium (0.1% glucose and 2 % glycerol), but not in galactose-containing medium. In a uge1[DELTA]gal10[DELTA] strain, the galactosylation defect was suppressed and UDP-galactose content restored to wild-type levels in galactose-containing medium. Disruption of [gal7.sup.+], encoding galactose-1-phosphate uridylyltransferase, in the uge1[DELTA]gal10[DELTA] strain reversed suppression of the galactosylation defect and reduced levels of UDP-galactose, indicating that galactose is transported from the medium to the cytosol and is converted into UDP-galactose via galactose 1-phosphate by Gal7p in Sch. pombe. DOI 10.1099/mic.0.035279-0

Molecular biology has several distinct origins, but especially important are those contributed by fungal and yeast physiology, biochemistry and genetics. From the first gene action studies that became the basis of our understanding of the relationship between genes and proteins, through chromosome structure, mitochondrial genetics and membrane biogenesis, gene silencing and circadian clocks, studies with these organisms have yielded basic insight into these processes applicable to all eukaryotes. Examples are cited of pioneering studies with fungi that have stimulated new research in clinical medicine and agriculture; these studies include sexual interactions, cell stress responses, the cytoskeleton and pathogenesis. Studies with the yeasts and fungi have been effective in applying the techniques and insights gained from other types of experimental systems to research in fungal cell signalling, cell development and hyphal morphogenesis. DOI 10.1099/mic.0.035238-0

Faithful segregation of sister chromatids requires the attachment of each kinetochore (Kt) to microtubules (MTs) that extend from opposite spindle poles. Merotelic Kt orientation is a Kt--MT misattachment in which a single Kt binds MTs from both spindle poles rather than just one. Genetic induction of merotelic Kt attachment during anaphase in fission yeast resulted in intra-Kt stretching followed by either correction or Kt disruption. Laser ablation of spindle MTs revealed that intra-Kt stretching and merotelic correction were dependent on MT forces. The presence of multiple merotelic chromosomes linearly antagonized the spindle elongation rate, and this phenomenon could be solved numerically using a simple force balance model. Based on the predictions of our mechanical model, we provide in vivo evidence that correction of merotelic attachment in anaphase is tension dependent and requires an Ase1/Prc1-dependent mechanism that prevents spindle collapse and thus asymmetric division and/or the appearance of the cut phenotype. doi/10.1083/jcb.200902093

Nodal and Notch signaling pathways play essential roles in vertebrate development. Through a yeast two-hybrid screening, we identified Notch3 as a candidate binding partner of the Nodal coreceptor Cripto-1. Coimmunoprecipitation analysis confirmed the binding of Cripto-1 with all four mammalian Notch receptors. Deletion analyses revealed that the binding of Cripto-1 and Notch1 is mediated by the Cripto-1/ FRL-1/Cryptic domain of Cripto-1 and the C-terminal region of epidermal growth factor-like repeats of Notch1. Binding of Cripto-1 to Notch1 occurred mainly in the endoplasmic reticulum--Golgi network. Cripto-1 expression resulted in the recruitment of Notch1 protein into lipid raft microdomains and enhancement of the furin-like protein convertase-mediated proteolytic maturation of Notch1 (S1 cleavage). Enhanced S1 cleavage resulted in the sensitization to ligand-induced activation of Notch signaling. In addition, knockdown of Cripto-1 expression in human and mouse embryonal carcinoma cells desensitized the ligand-induced Notch signaling activation. These results suggest a novel role of Cripto-1 in facilitating the posttranslational maturation of Notch receptors. doi/10.1083/jcb.200905105

Mei4 is a key sporulation-specific transcription factor in fission yeast. Ectopic expression of Mei4 in vegetative cells caused formation of nucleated membranous compartments, which shared common features with normal forespore membranes, thereby perturbing nuclear division. These results suggest why expression of development-specific transcription factors must be strictly controlled.

Lipophilic yeasts of the genus Malassezia are associated with several skin diseases, such as pityriasis versicolor, Malassezia folliculitis, seborrhoeic dermatitis and atopic dermatitis, and are also increasingly associated with catheter-related fungaemia. The cell wall components of pathogenic micro-organisms behave as an antigen and/or ligand of the innate immune response. Live cells of Malassezia furfur and Malassezia pachydermatis did not react with an anti-[alpha]-1,2-mannoside antibody. However, they showed a strong hydrophobicity and reactivity with an anti-[beta]-1,3-glucan antibody compared to those of C. albicans. The cell wall polysaccharides of M. furfur and M. pachydermatis were isolated and their structures analysed by [sup.1]H and [sup.13]C NMR experiments. Both polysaccharides were shown to be [beta]-1,6-linked linear galactofuranosyl polymers with a small amount of mannan. The presence of galactomannan on cells of Malassezia species has not been described previously. The galactomannan did not react with an anti-Aspergillus fumigatus monoclonal antibody which has specificity for [beta]-1,5-linked galactofuranosyl residues. An anti-M, furfur antibody strongly reacted with the galactomannans of M. furfur and M. pachydermafis, but did not react with the galactomannans of Trichophyton rubrum, A. fumigatus or Fonsecaea pedrosoi. The characteristics of the anti-M, furfur antibody suggest that there is potential for diagnosis of Malassezia infections by antigen detection. DOI 10.1099/mic.0.029967-0

During meiosis, homologues become juxtaposed and synapsed along their entire length. Mutations in the cohesin complex disrupt not only sister chromatid cohesion but also homologue pairing and synaptonemal complex formation. In this study, we report that Pds5, a cohesin-associated protein known to regulate sister chromatid cohesion, is required for homologue pairing and synapsis in budding yeast. Pds5 colocalizes with cohesin along the length of meiotic chromosomes. In the absence of Pds5, the meiotic cohesin subunit Rec8 remains bound to chromosomes with only minor defects in sister chromatid cohesion, but sister chromatids synapse instead of homologues. Double-strand breaks (DSBs) are formed but are not repaired efficiently. In addition, meiotic chromosomes undergo hypercondensation. When the mitotic cohesin subunit Mcd1 is substituted for Rec8 in Pds5-depleted cells, chromosomes still hypercondense, but synapsis of sister chromatids is abolished. These data suggest that Pds5 modulates the Rec8 activity to facilitate chromosome morphological changes required for homologue synapsis, DSB repair, and meiotic chromosome segregation.

To clarify the budding pattern of Wickerhamomyces pijperi, the vegetative cells were observed by scanning electron microscopy. The cells grew by bipolar budding, but cells that budded from the shoulder of a mother cell were occasionally observed. We examined the cell morphology and phylogeny of five strains of Wickerhamomyces sp. isolated in Thailand as well as seven W. pijperi and three Wickerhamomyces sp. strains that were preserved in culture collections. Phylogenetic analysis based on three different nucleotide sequences (D1/D2 domain of 26S rDNA, the actin gene ACT1 and the elongation factor 2 gene EF2) indicated that all the strains belonged to the genus Wickerhamomyces and were neighbours of the type strain W. pijperi NBRC [1290.sup.T]. The strains fell into two groups in this analysis. The budding patterns of the strains were carefully observed by staining the bud scars, and these patterns were categorized into three groups: types I-III. Type I included cells that grew by bipolar budding and formed multiple scars, type III included cells that grew by multilateral budding and formed a single scar, and type II included cells that exhibited a mixture of type I and type III patterns. Among the 15 strains, 12 strains, including W. pijperi NBRC [1290.sup.T], mainly exhibited type I or type II budding patterns; these strains belonged to group 1 of the phylogenetic analysis. The remaining three strains, which belonged to group 2, exhibited either type II or type III patterns. Thus the phylogenetic relationship and budding patterns are related. Moreover, some cells also exhibited budding characteristics that were intermediate between bipolar and multilateral budding.

Paracoccidioides brasiliensis is a thermally dimorphic fungus, and causes the most prevalent systemic mycosis in Latin America. Infection is initiated by inhalation of conidia or mycelial fragments by the host, followed by further differentiation into the yeast form. Information regarding gene expression by either form has rarely been addressed with respect to multiple time points of growth in culture. Here, we report on the construction of a genomic DNA microarray, covering approximately 25 % of the genome of the organism, and its utilization in identifying genes and gene expression patterns during growth in vitro. Cloned, amplified inserts from randomly sheared genomic DNA (gDNA) and known control genes were printed onto glass slides to generate a microarray of over 12 000 elements. To examine gene expression, mRNA was extracted and amplified from mycelial or yeast cultures grown in semi-defined medium for 5, 8 and 14 days. Principal components analysis and hierarchical clustering indicated that yeast gene expression profiles differed greatly from those of mycelia, especially at earlier time points, and that mycelial gene expression changed less than gene expression in yeasts over time. Genes upregulated in yeasts were found to encode proteins shown to be involved in methionine/cysteine metabolism, respiratory and metabolic processes (of sugars, amino acids, proteins and lipids), transporters (small peptides, sugars, ions and toxins), regulatory proteins and transcription factors. Mycelial genes involved in processes such as cell division, protein catabolism, nucleotide biosynthesis and toxin and sugar transport showed differential expression. Sequenced clones were compared with Histoplasma capsulatum and Coccidioides posadasii genome sequences to assess potentially common pathways across species, such as sulfur and lipid metabolism, amino acid transporters, transcription factors and genes possibly related to virulence. We also analysed gene expression with time in culture and found that while transposable elements and components of respiratory pathways tended to increase in expression with time, genes encoding ribosomal structural proteins and protein catabolism tended to sharply decrease in expression over time, particularly in yeast. These findings expand our knowledge of the different morphological forms of P. brasiliensis during growth in culture.

Transition from growth to the stationary phase in yeast is still poorly understood. Previously, we identified a group of yeast genes that are universally upregulated upon starvation for different macronutrients. Here, we demonstrate that the Gis1 transcription factor and the Rim1 5 kinase are responsible for the upregulation of many of these genes. In chemostat cultures, gisl or rim15 mutant cells are outcompeted by their wild-type parents under conditions resembling the later stages of diauxie (glucose-limiting) and post-diauxie (ethanol as a carbon source). Whilst Gis1 p and Rim1 5p have distinct functions in gene repression, the growth defects of gis1 or rim15 deletants can be accounted for by the overlapping functions of their protein products in promoting the expression of genes involved in glutamate biosynthesis, the glyoxylate cycle, the pentose phosphate pathway and the stress response. Further, we show that the sets of GIS1-and RIM15-dependent genes and the degree of their regulation change in response to the identity of the carbon source, suggesting the likely dynamics of gene regulation exerted by Rim15p and Gis1 p during different phases of the transition into stationary phase. In particular, Rim15p is required for the expression of genes involved in gluconeogenesis/glycolysis and glycerol biosynthesis only when ethanol is used as the carbon source. In agreement with this, Rim15p is shown to act in parallel with Hog1 p to defend cells against osmotic stress.

Methionine residues and iron-sulphur (FeS) clusters are primary targets of reactive oxygen species in the proteins of micro-organisms. Here, we show that methionine redox modifications help to preserve essential FeS cluster activities in yeast. Mutants defective for the highly conserved methionine sulphoxide reductases (MSRs; which re-reduce oxidized methionines) are sensitive to many pro-oxidants, but here exhibited an unexpected copper resistance. This phenotype was mimicked by methionine sulphoxide supplementation. Microarray analyses highlighted several Cu and Fe homeostasis genes that were upregulated in the mxr[DELTA] double mutant, which lacks both of the yeast MSRs. Of the upregulated genes, the Cu-binding Fe transporter Fet3p proved to be required for the Cu-resistance phenotype. FET3 is known to be regulated by the Aft1 transcription factor, which responds to low mitochondrial FeS-cluster status. Here, constitutive Aft1p expression in the wild-type reproduced the Cu-resistance phenotype, and FeS-cluster functions were found to be defective in the mxr[DELTA] mutant. Genetic perturbation of FeS activity also mimicked FET3-dependent Cu resistance. [sup.55]Fe-labelling studies showed that FeS clusters are turned over more rapidly in the mxr[DELTA] mutant than the wild-type, consistent with elevated oxidative targeting of the clusters in MSR-deficient cells. The potential underlying molecular mechanisms of this targeting are discussed. Moreover, the results indicate an important new role for cellular MSR enzymes in helping to protect the essential function of FeS clusters in aerobic settings.

The effects of octyl gallate on Ustilago maydis yeast cells were analysed in relation to its capacity to oxidize compounds (pro-oxidant actions). All phenolic compounds tested inhibited the alternative oxidase (AOX). However, only octyl gallate induced a morphological change in yeast cells and collapsed the mitochondrial membrane potential. In contrast to octyl gallate, propyl gallate and nordihydroguaiaretic acid caused only a negligible cell change and the membrane potential was not affected. Our findings show that structurally related phenolic compounds do not necessarily exert similar actions on target cells. Preincubation of U. maydis cells with trolox inhibited the change to pseudohyphal growth produced by octyl gallate. These results suggest that in addition to the inhibitory action of octyl gallate on the AOX, this compound induces a switch from yeast to a mycelium, probably through the formation of lipid peroxides.

Our understanding of the genetic basis of phenotypic diversity is limited by the paucity of examples in which multiple, interacting loci have been identified. We show that natural variation in the efficiency of sporulation, the program in yeast that initiates the sexual phase of the life cycle, between oak tree and vineyard strains is due to allelic variation between four nucleotide changes in three transcription factors: IME1, RME1, and RSF1. Furthermore, we identified that selection has shaped quantitative variation in yeast sporulation between strains. These results illustrate how genetic interactions between transcription factors are a major source of phenotypic diversity within species.

The ecology of nectarivorous microbial communities remains virtually unknown, which precludes elucidating whether these organisms play some role in plantpollinator mutualisms beyond minor commensalism. We simultaneously assessed microbial abundance and nectar composition at the individual nectary level in flowers of three southern Spanish bumble bee-pollinated plants (Hellehorus foetidus, Aquilegia vulgaris, and Aquilegia pyrenaica cazorlensis). Yeasts were frequent and abundant in nectar of all species, and variation in yeast density was correlated with drastic changes in nectar sugar concentration and composition. Yeast communities built up in nectar from early to late floral stages, at which time all nectaries contained yeasts, often at densities between [10.sup.4] and [10.sup.5] cells/m[m.sup.3]. Total sugar concentration and percentage sucrose declined, and percentage fructose increased, with increasing density of yeast cells in nectar. Among-nectary variation in microbial density accounted for 65% (H.foetidus and A. vulgaris) and 35% (A. p. cazorlensis) of intra-specific variance in nectar sugar composition, and 60% (H. foetidus) and 38% (A. vulgaris) of variance in nectar concentration. Our results provide compelling evidence that nectar microbial communities can have detrimental effects on plants and/or pollinators via extensive nectar degradation and also call for a more careful interpretation of nectar traits in the future, if uncontrolled for yeasts. Key words: Aquilegia pyrenaica cazorlensis; Aquilegia vulgaris; humble bee pollitation; floral microbiology; Helleborus foetidus; mutualism exploitation; nectar concentration; nectar sugar composition; yeast communities.

The mutation process ultimately defines the genetic features of all populations and, hence, has a bearing on a wide range of issues involving evolutionary genetics, inheritance, and genetic disorders, including the predisposition to cancer. Nevertheless, formidable technical barriers have constrained our understanding of the rate at which mutations arise and the molecular spectrum of their effects. Here, we report on the use of complete-genome sequencing in the characterization of spontaneously arising mutations in the yeast Saccharomyces cerevisiae. Our results confirm some findings previously obtained by indirect methods but also yield numerous unexpected findings, in particular a very high rate of point mutation and skewed distribution of base-substitution types in the mitochondrion, a very high rate of segmental duplication and deletion in the nuclear genome, and substantial deviations in the mutational profile among various model organisms. chromosomal instability | mitochondrion | mutation rate | mutational spectrum | Saccharomyces cerevisiae

Large, multisubunit Ccr4-Not complexes are evolutionarily conserved global regulators of gene expression. Deletion of CCR4 or several components of Ccr4-Not complexes results in abnormally large cells. Since yeast must attain a critical cell size at Start to commit to division, the large size of ccr4[DELTA] cells implies that they may have a size-specific proliferation defect. Overexpression of CLN1, CLN2, CLN3, and SWI4 reduces the size of ccr4[DELTA] cells, suggesting that ccr4[DELTA] cells have a Grphase cyclin deficiency. In support of this, we find that CLN1 and CLN2 expression and budding are delayed in ccr4[DELTA] cells. Moreover, overexpression of CCR4 advances the timing of CLN1 expression, promotes premature budding, and reduces cell size. Genetic analyses suggest that Ccr4 functions independently of Cln3 and downstream of Bck2. Thus, like cln3[DELTA]bck2[DELTA] double deletions, cln3[DELTA]ccr4[DELTA] cells are also inviable. However, deletion of Whi5, a transcriptional repressor of CLN1 and CLN2, restores viability. We find that Ccr4 negatively regulates the half-life of WHI5 mRNAs, and we conclude that, by modulating the stability of WHI5 mRNAs, Ccr4 influences the size-dependent timing of [G.sub.1]-phase cyclin transcription.

The yeast TEL1 and MEC1 genes (homologous to the mammalian ATM and ATR genes, respectively) serve partially redundant roles in the detection of DNA damage and in the regulation of telomere length. Haploid yeast tell mec1 strains were subcultured nonselectively for ~200 cell divisions. The subcultured strains had very high rates of chromosome aberrations: duplications, deletions, and translocations. The breakpoints of the rearranged chromosomes were within retrotransposons (Ty or &-repeats), and these chromosome aberrations nonrandomly involved chromosome III. In addition, we showed that strains with the hypomorphic mec1-21 allele often became disomic for chromosome VIII. This property of the mec1-21 strains is suppressed by a plasmid containing the DNA2 gene (located on chromosome VIII) that encodes an essential nuclease/helicase involved in DNA replication and DNA repair.

DNA double-strand breaks (DSB) are repaired through two different pathways, homologous recombination (HR) and nonhomologous end joining (NHEJ). Yeast Xrs2, a homolog of human Nbsl, is a component of the Mrel1-Rad50-Xrs2 (MRX) complex required tot both HR and NHEJ. Previous studies showed that the N-terminal forkhead-associated (FHA) domain of Xrs2/Nbs1 in yeast is not involved in HR, but is likely to be in NHEJ. In this study, we showed that the FHA domain of Xrs2 plays a critical role in efficient DSB repair by NHEJ. The FHA domain of Xrs2 specifically interacts with Lif1, a component of the ligase IV complex, Dn14-Nej1-Lif1 (DNL). Lif1, which is phosphorylated in vivo, contains two Xrs2-binding regions. Serine 383 of Lif1 plays an important role in the interaction with Xrs2 as well as in NHEJ. interestingly, the phospho-mimetic substitutions of serine 383 enhance the NHEJ activity of Lif1. Our results suggest that the phosphorylation of Lif1 at serine 383 is recognized by the Xrs2 FHA domain, which in turn may promote recruitment of the DNL complex to DSB for NHEJ. The interaction between Xrs2 and Lift through the FHA domain is conserved in humans; the FHA domain Nbs1 interacts with Xrcc4, a Lif1 homolog of human.

In both animal and yeast cells, reactive oxygen species (ROS) are produced as byproducts of metabolism and upon exposure to diverse environmental stresses. Cellular defense systems operate to avoid molecular damage caused by ROS, but the redox balance is disturbed under excessive stress. Cells of the budding yeast Saccharomyces cerevisiae undergo apoptotic-like cell death upon exposure to hydrogen peroxide ([H.sub.2][O.sub.2]). Here, we report that the Rho5 GTPase of budding yeast is necessary for [H.sub.2][O.sub.2]-induced cell death, which accompanies ROS accumulation and DNA fragmentation. Unlike WT, a rho5 deletion mutant (rh05[DELTA]) exhibits little cell death, whereas the constitutively active rho[5.sup.G12V] mutant exhibits excess ROS accumulation and increased cell death upon [H.sub.2][O.sub.2] treatment. Consistent with a role in the oxidative stress response, Rho5 interacts with the thioredoxin reductase Trr1, a key component of the cytoplasmic thioredoxin antioxidant system, in a GTP-dependent manner. This interaction occurs on the vacuolar membrane before exposure to [H.sub.2][O.sub.2] but also in the vacuolar lumen after [H.sub.2][O.sub.2] treatment, Trr1 levels are elevated in rho5[DELTA] cells but are elevated only slightly in WT and not in the rho[5.sup.G12V] cells after [H.sub.2][O.sub.2] treatment. Taken together, these data suggest that Rho5 mediates [H.sub.2][O.sub.2]-induced cell death by regulating the level of Trr1 or by excluding Trr1 from its cytoplasmic substrate. thioredoxin reductase | apoptosis | reactive oxygen species

The urea-induced unfolding of the dimeric Ste11 SAM domain of MAPKKK from the budding yeast is investigated using NMR and other optical spectroscopic methods. The results demonstrate the crucial involvement of the interfacial residues of the Ste11 SAM domain in its structural stability and binding to the Ste50 SAM domain.

Transient receptor potential (TRP) channels found in animals, protists, and fungi are primary chemo-, thermo-, or mechanosensors. Current research emphasizes the characteristics of individual channels in each animal TRP subfamily but not the mechanisms common across subfamilies. A forward genetic screen of the TrpY1, the yeast TRP channel, recovered gain-of-function (GOF) mutations with phenotype in vivo and in vitro. Single-channel patch-clamp analyses of these GOF-mutant channels show prominent aberrations in open probability and channel kinetics. These mutations revealed functionally important aromatic amino acid residues in four locations: at the intracellular end of the fifth transmembrane helix (TM5), at both ends of TM6, and at the immediate extension of TM6. These aromatics have counterparts in most TRP subfamilies. The one in TM5 (F380L) aligns precisely with an exceptional Drosophila mutant allele (F550I) that causes constitutive activity in the canonical TRP channel, resulting in rapid and severe retinal degeneration beyond mere loss of phototaxis. Thus, this phenylalanine maintains the balance of various functional states (conformations) of a channel for insect phototransduction as well as one for fungal mechanotransduction. This residue is among a small cluster of phenylalanines found in all known subfamilies of TRP channels. This unique case illustrates that GOF mutations can reveal structure-function principles that can be generalized across different TRP subfamilies. It appears that the conserved aromatics in the four locations have conserved functions in most TRP channels. The possible mechanistic roles of these aromatics and the further use of yeast genetics to dissect TRP channels are discussed. mechanosensitivity | mechanosensor | aromatic residues | single channel

The spoilage yeast Saccharomyces cerevisiae degrades the food preservative sorbic acid with the help of the gene identified as PAD1. This decarboxylation is assessed as a possible mechanism of resistance in spoilage yeasts.

The differential transcriptome of a Pichia pastoris strain overexpressing human trypsinogen is analyzed in comparison to that of a nonexpressing strain.

Transformation of both prokaryotes and eukaryotes with single-stranded oligonucleotides can transfer sequence information from the oligonucleotide to the chromosome. We have studied this process using oligonucleotides that correct a--1 frameshift mutation in the LYS2 gene of Saccharomyces cerevisiae. We demonstrate that transformation by oligonucleotides occurs preferentially on the lagging strand of replication and is strongly inhibited by the mismatch-repair system. These results are consistent with a mechanism in which oligonucleotides anneal to single-stranded regions of DNA at a replication fork and serve as primers for DNA synthesis. Because the mispairs the primers create are efficiently removed by the mismatch-repair system, single-stranded oligonucleotides can be used to probe mismatch-repair function in a chromosomal context. Removal of mispairs created by annealing of the single-stranded oligonucleotides to the chromosomal DNA is as expected, with 7-nt loops being recognized solely by MutS~ and 1-nt loops being recognized by both MutS[alpha] and MutS[beta]. We also find evidence for Mlh1-independent repair of 7-nt, but not 1-nt, loops. Unexpectedly, we find a strand asymmetry of mismatchrepair function; transformation is blocked more efficiently by MutS[alpha] on the lagging strand of replication, whereas MutS[beta] does not show a significant strand bias. These results suggest an inherent strand-related difference in how the yeast MutS[alpha] and MutS[beta] complexes access and/or repair mismatches that arise in the context of DNA replication.

Studies of the role of sex in evolution typically involve a longitudinal comparison of a single ancestor to several intermediate descendants and to one terminally evolved descendant after many generations of adaptation under a given selective regime. Here we take a complementary, statistical approach to sex in evolution, by describing the distribution of phenotypic similarity in a population of yeast [F.sub.1] meiotic recombinants. By applying graph theory to fitness measurements of thousands of Saccharomyces cerevisiae recombinants treated with 10 mechanistically distinct, growth-inhibitory small-molecule perturbagens (SMPs), we show that the network of phenotypic similarity among [F.sub.1] recombinants exhibits a scale-free degree distribution. [F.sub.1] recombinants are often phenotypically unique and sometimes exceptional, and their fitness strengths are unevenly distributed across the 10 compound treatments. By contrast, highly phenotypically similar [F.sub.1] recombinants constitute failing hubs that display below-average fitness across all compound treatments and are candidate substrates for purifying selection. Comparison of the [F.sub.1] generation with the parental strains reveals that (i) there is a specialist more fit in any given single condition than any of the parents but (ii) only rarely are there generalists that exhibit greater fitness than both parental strains across a majority of conditions. This analysis allows us to evaluate and to gain better theoretical understanding of the costs and benefits of sex in the [F.sub.1] generation. graph theory | meiotic recombination

A study was conducted to investigate the occurrence of sublethal injury after the pulsed electric field (PEF) treatment of yeast cells and the effect of combination of PEF and sorbic acid. It was found that PEF caused sublethal injury in both yeasts Dekeera bruxellensis and Saccharomyces cervisiae and combination of PEF and sorbic acid achieved higher level of yeast inactivation.

cis-SNARE complexes (anchored in one membrane) are disassembled by Sec17p ([alpha]-SNAP) and Sec18p (NSF), permitting the unpaired SNAREs to assemble in trans. We now report a direct assay of trans-SNARE complex formation during yeast vacuole docking. SNARE complex assembly and fusion is promoted by high concentrations of the SNARE Vam7p or Nyvlp or by addition of HOPS (homotypic fusion and vacuole protein sorting), a Ypt7p (Rab)effector complex with a Sec1/Munc18-family subunit. Inhibitors that target Ypt7p, HOPS, or key regulatory lipids prevent trans-SNARE complex assembly and ensuing fusion. Strikingly, the lipid ligand MED (myristoylated alanine-rich C kinase substrate effector domain) or elevated concentrations of Sec17p, which can displace HOPS from SNARE complexes, permit full trans-SNARE pairing but block fusion. These findings suggest that efficient fusion requires trans-SNARE complex associations with factors such as HOPS and subsequent regulated lipid rearrangements. homotypic fusion and vacuole protein sorting | Rab/Ypt

Noncoding, or intergenic, transcription by RNA polymerase II (RNA-PII) is remarkably widespread in eukaryotic organisms, but the effects of such transcription remain poorly understood. Here we show that noncoding transcription plays a role in activation, but not repression, of the Saccharomyces cerevisiae PHO5 gene. Histone eviction from the PHO5 promoter during activation occurs with normal kinetics even in the absence of the PHO5 TATA box, showing that transcription of the gene itself is not required for promoter remodeling. Nevertheless, we find that mutations that impair transcript elongation by RNAPII affect the kinetics of histone eviction from the PHO5 promoter. Most dramatically, inactivation of RNAPII itself abolishes eviction completely. Under repressing conditions, an [approximately equal to] 2.4-kb noncoding exosome-degraded transcript is detected that originates near the PHO5 termination site and is transcribed in the antisense direction. Abrogation of this transcript delays chromatin remodeling and subsequent RNAPII recruitment to PHO5 upon activation. We propose that noncoding transcription through positioned nucleosomes can enhance chromatin plasticity so that chromatin remodeling and activation of traversed genes occur in a timely manner. elongation | intergenic transcription | RNA polymerase II

The Ccr4-Not complex consists of nine subunits and acts as a regulator of mRNA biogenesis in Saccharomyces cerevisiae. The human ortholog of yeast NOT4, CNOT4, displays UbcH5B-dependent ubiquitin-protein ligase (E3 ligase) activity in a reconstituted in vitro system. However, an in vivo role for this enzymatic activity has not been identified. Site-directed mutagenesis of the RING finger of yeast Not4p identified residues required for interaction with Ubc4p and Ubc5p, the yeast orthologs of UbcH5B. Subsequent in vitro assays with purified Ccr4-Not complexes showed Not4p-mediated E3 ligase activity, which was dependent on the interaction with Ubc4p. To investigate the in vivo relevance of this activity, we performed synthetic genetic array (SGA) analyses using not4[DELTA] and not4L35A alleles. This indicates involvement of the RING finger of Not4p in transcription, ubiquitylation, and DNA damage responses. In addition, we found a phenotypic overlap between deletions of UBC4 and mutants encoding single-aminoacid substitutions of the RING finger of Not4p. Together, our results show that Not4p functions as an E3 ligase by modulating Ubc4p/Ubc5p-mediated stress responses in vivo.

There are many types of DNA damage that are repaired by a multiplicity of different repair pathways. All damage and repair occur in the context of chromatin, and histone modifications are involved in many repair processes. We have analyzed the roles of H2A and its modifications in repair by mutagenizing modifiable residues in the N- and C-terminal tails of yeast H2A and by testing strains containing these mutations in multiple DNA repair assays. We show that residues in both tails are important for homologous recombination and nonhomologous end-joining pathways of double-strand break repair, as well as for survival of UV irradiation and oxidative damage. We show that H2A serine 129 is important for repair and/or survival in each of these assays. We also observe a complex pattern of H2A phosphorylation at residues S122, T126, and S129 in response to different damage conditions. We find that overlapping but nonidentical groups of H2A residues in both tails are involved in different pathways of repair. These data suggest the presence of a set of H2A 'damage codes' in which distinct patterns of modifications on both tails of H2A may be used to identify specific types of damage or to promote specific repair pathways.

The solid-state magnetic angle spinning (MAS) nuclear magnetic resonance (NMR) methods are used for comparing nanocrystals and fibrils formed by the peptide fragment GNNQQNY, which is Sup35p prion protein found in yeast. The results have shown that various structures assumed by the peptide are correlated to the steric zipper observed in the monoclinic crystals.