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Systematic screening of polyphosphate (poly P) levels in yeast mutant cells reveals strong interdependence with primary metabolism
Florian M Freimoser, Hans Hürlimann, Claude A Jakob, Thomas P Werner, Nikolaus Amrhein
Genome Biology , 2006, DOI: 10.1186/gb-2006-7-11-r109
Abstract: After three consecutive screens, 255 genes (almost 4% of the yeast genome) were found to be involved in the maintenance of normal poly P content. Many of these genes encoded proteins functioning in the cytoplasm, the vacuole or in transport and transcription. Besides reduced poly P content, many strains also exhibited reduced total phosphate content, showed altered ATP and glycogen levels and were disturbed in the secretion of acid phosphatase.Cellular energy and phosphate homeostasis is suggested to result from the equilibrium between poly P, ATP and free phosphate within the cell. Poly P serves as a buffer for both ATP and free phosphate levels and is, therefore, the least essential and consequently most variable component in this network. However, strains with reduced poly P levels are not only affected in their ATP and phosphate content, but also in other components that depend on ATP or free phosphate content, such as glycogen or secreted phosphatase activity.Inorganic polyphosphate (poly P) is a linear polymer that consists of phosphoanhydride linked phosphate residues and occurs ubiquitously in all organisms and living cells [1]. The functions of poly P range from its role as a phosphate store and buffer [2-4] to the activation of enzymes [5,6] and regulation of chromatin condensation, gene expression and translation [1,7,8]. Poly P is also involved in bacterial pathogenicity [9,10], survival during stationary phase in bacteria and yeast [9,11,12], or the adaptation to alkaline and osmotic stress [13-15]. In the slime mold Dictyostelium discoideum, poly P regulates development and predation behavior [16], and in humans blood coagulation is accelerated and fibrinolysis is delayed by poly P [17]. At the cellular level, poly P has been detected in the cytosol, the nucleus, mitochondria, as well as the endoplasmic reticulum [7,18,19]. Poly P is particularly prominent in the acidocalcisomes of trypanosomatids [20], and in Saccharomyces cerevisiae almost the entire
Genome-Wide Functional Profiling Reveals Genes Required for Tolerance to Benzene Metabolites in Yeast  [PDF]
Matthew North, Vickram J. Tandon, Reuben Thomas, Alex Loguinov, Inna Gerlovina, Alan E. Hubbard, Luoping Zhang, Martyn T. Smith, Chris D. Vulpe
PLOS ONE , 2011, DOI: 10.1371/journal.pone.0024205
Abstract: Benzene is a ubiquitous environmental contaminant and is widely used in industry. Exposure to benzene causes a number of serious health problems, including blood disorders and leukemia. Benzene undergoes complex metabolism in humans, making mechanistic determination of benzene toxicity difficult. We used a functional genomics approach to identify the genes that modulate the cellular toxicity of three of the phenolic metabolites of benzene, hydroquinone (HQ), catechol (CAT) and 1,2,4-benzenetriol (BT), in the model eukaryote Saccharomyces cerevisiae. Benzene metabolites generate oxidative and cytoskeletal stress, and tolerance requires correct regulation of iron homeostasis and the vacuolar ATPase. We have identified a conserved bZIP transcription factor, Yap3p, as important for a HQ-specific response pathway, as well as two genes that encode putative NAD(P)H:quinone oxidoreductases, PST2 and YCP4. Many of the yeast genes identified have human orthologs that may modulate human benzene toxicity in a similar manner and could play a role in benzene exposure-related disease.
Transcriptome-Wide Mapping of 5-methylcytidine RNA Modifications in Bacteria, Archaea, and Yeast Reveals m5C within Archaeal mRNAs  [PDF]
Sarit Edelheit equal contributor,Schraga Schwartz equal contributor,Maxwell R. Mumbach,Omri Wurtzel,Rotem Sorek
PLOS Genetics , 2013, DOI: 10.1371/journal.pgen.1003602
Abstract: The presence of 5-methylcytidine (m5C) in tRNA and rRNA molecules of a wide variety of organisms was first observed more than 40 years ago. However, detection of this modification was limited to specific, abundant, RNA species, due to the usage of low-throughput methods. To obtain a high resolution, systematic, and comprehensive transcriptome-wide overview of m5C across the three domains of life, we used bisulfite treatment on total RNA from both gram positive (B. subtilis) and gram negative (E. coli) bacteria, an archaeon (S. solfataricus) and a eukaryote (S. cerevisiae), followed by massively parallel sequencing. We were able to recover most previously documented m5C sites on rRNA in the four organisms, and identified several novel sites in yeast and archaeal rRNAs. Our analyses also allowed quantification of methylated m5C positions in 64 tRNAs in yeast and archaea, revealing stoichiometric differences between the methylation patterns of these organisms. Molecules of tRNAs in which m5C was absent were also discovered. Intriguingly, we detected m5C sites within archaeal mRNAs, and identified a consensus motif of AUCGANGU that directs methylation in S. solfataricus. Our results, which were validated using m5C-specific RNA immunoprecipitation, provide the first evidence for mRNA modifications in archaea, suggesting that this mode of post-transcriptional regulation extends beyond the eukaryotic domain.
Yeast fitness and protein evolution  [cached]
Reiner Veitia
Genome Biology , 2001, DOI: 10.1186/gb-2001-2-9-reports0030
Abstract: Hirsh and Fraser obtained reliable estimates of fitness for 548 homozygous single-gene deletants. The evolutionary distances di (number of substitutions per amino acid site) could be estimated for 119 mutants out of the 548 using two different methods. They found a statistically significant relationship between di and fi, showing that proteins with a lower fitness effect are more divergent. But, the di values represent evolutionary change not only in the yeast sequences but also in the corresponding nematode orthologs. To estimate evolutionary change only in the lineage leading to yeast, di measures were split into two components, one between yeast and the hypothetical most recent common ancestor (MRCA) of fungi and animals and another between the MRCA and C. elegans: the outgroup sequences were orthologs present in other completely sequenced genomes. Hirsh and Fraser analyzed 48 yeast proteins in this way and again a significant relationship between fi and the 'new' di values was obtained. Most interestingly, statistically significant results were obtained when they plotted fi (from yeast) against the di between the MRCA and the nematode. This suggests that non-essential proteins that have an impact on the fitness of yeast might also have a proportional effect on worm fitness. No differences were detected when the essential genes were subject to the same analysis and compared with the non-essential ones, which is consistent with the results of analyses in mouse. When the comparison was carried out against the most dispensable 60 proteins (smallest fi values), however, a highly significant difference was detected.This work provides the long awaited (at least preliminary) confirmation of a fundamental prediction about protein evolution. It is an example of how genomic data can be exploited from different perspectives to address important biological questions. In fact, the starting point of the present study was the results of a high-throughput parallel analysis of ye
Genome-wide characterisation of the Gcn5 histone acetyltransferase in budding yeast during stress adaptation reveals evolutionarily conserved and diverged roles
Yongtao Xue-Franzén, Anna Johnsson, David Brodin, Johan Henriksson, Thomas R Bürglin, Anthony PH Wright
BMC Genomics , 2010, DOI: 10.1186/1471-2164-11-200
Abstract: We show that Gcn5 is important for a common set of stress responses in evolutionarily diverged yeast species and that the activity of the conserved histone acetyltransferase domain is required. We define a group of KCl stress response genes in S. cerevisiae that are specifically dependent on Gcn5. Gcn5 is localised to many Gcn5-dependent genes including Gcn5 repressed targets such as FLO8. Gcn5 regulates divergent sets of KCl responsive genes in S. cerevisiae and S. pombe. Genome-wide localization studies showed a tendency for redistribution of Gcn5 during KCl stress adaptation in S. cerevisiae from short genes to the transcribed regions of long genes. An analogous redistribution was not observed in S. pombe.Gcn5 is required for the regulation of divergent sets of KCl stress-response genes in S. cerevisiae and S. pombe even though it is required a common group of stress responses, including the response to KCl. Genes that are physically associated with Gcn5 require its activity for their repression or activation during stress adaptation, providing support for a role of Gcn5 as a corepressor as well as a coactivator. The tendency of Gcn5 to re-localise to the transcribed regions of long genes during KCl stress adaptation suggests that Gcn5 plays a specific role in the expression of long genes under adaptive conditions, perhaps by regulating transcriptional elongation as has been seen for Gcn5 in S. pombe. Interestingly an analogous redistribution of Gcn5 is not seen in S. pombe. The study thus provides important new insights in relation to why coregulators like Gcn5 are required for the correct expression of some genes but not others.Evolutionary changes frequently involve modifications to gene regulatory programs. Several lines of evidence indicate the importance of transcriptional regulation in evolution. First, comparative studies have shown that cis-acting regulatory elements that are binding sites for regulatory transcription factors evolve rapidly [1-3]. Second
A Systematic Analysis of Cell Cycle Regulators in Yeast Reveals That Most Factors Act Independently of Cell Size to Control Initiation of Division  [PDF]
Scott A. Hoose,Jeremy A. Rawlings,Michelle M. Kelly,M. Camille Leitch,Qotaiba O. Ababneh,Juan P. Robles,David Taylor,Evelyn M. Hoover,Bethel Hailu,Kayla A. McEnery,S. Sabina Downing,Deepika Kaushal,Yi Chen,Alex Rife,Kirtan A. Brahmbhatt,Roger Smith III,Michael Polymenis
PLOS Genetics , 2012, DOI: 10.1371/journal.pgen.1002590
Abstract: Upstream events that trigger initiation of cell division, at a point called START in yeast, determine the overall rates of cell proliferation. The identity and complete sequence of those events remain unknown. Previous studies relied mainly on cell size changes to identify systematically genes required for the timely completion of START. Here, we evaluated panels of non-essential single gene deletion strains for altered DNA content by flow cytometry. This analysis revealed that most gene deletions that altered cell cycle progression did not change cell size. Our results highlight a strong requirement for ribosomal biogenesis and protein synthesis for initiation of cell division. We also identified numerous factors that have not been previously implicated in cell cycle control mechanisms. We found that CBS, which catalyzes the synthesis of cystathionine from serine and homocysteine, advances START in two ways: by promoting cell growth, which requires CBS's catalytic activity, and by a separate function, which does not require CBS's catalytic activity. CBS defects cause disease in humans, and in animals CBS has vital, non-catalytic, unknown roles. Hence, our results may be relevant for human biology. Taken together, these findings significantly expand the range of factors required for the timely initiation of cell division. The systematic identification of non-essential regulators of cell division we describe will be a valuable resource for analysis of cell cycle progression in yeast and other organisms.
The Fitness Consequences of Aneuploidy Are Driven by Condition-Dependent Gene Effects  [PDF]
Anna B. Sunshine?,Celia Payen?,Giang T. Ong?,Ivan Liachko?,Kean Ming Tan?,Maitreya J. Dunham
PLOS Biology , 2015, DOI: 10.1371/journal.pbio.1002155
Abstract: Aneuploidy is a hallmark of tumor cells, and yet the precise relationship between aneuploidy and a cell’s proliferative ability, or cellular fitness, has remained elusive. In this study, we have combined a detailed analysis of aneuploid clones isolated from laboratory-evolved populations of Saccharomyces cerevisiae with a systematic, genome-wide screen for the fitness effects of telomeric amplifications to address the relationship between aneuploidy and cellular fitness. We found that aneuploid clones rise to high population frequencies in nutrient-limited evolution experiments and show increased fitness relative to wild type. Direct competition experiments confirmed that three out of four aneuploid events isolated from evolved populations were themselves sufficient to improve fitness. To expand the scope beyond this small number of exemplars, we created a genome-wide collection of >1,800 diploid yeast strains, each containing a different telomeric amplicon (Tamp), ranging in size from 0.4 to 1,000 kb. Using pooled competition experiments in nutrient-limited chemostats followed by high-throughput sequencing of strain-identifying barcodes, we determined the fitness effects of these >1,800 Tamps under three different conditions. Our data revealed that the fitness landscape explored by telomeric amplifications is much broader than that explored by single-gene amplifications. As also observed in the evolved clones, we found the fitness effects of most Tamps to be condition specific, with a minority showing common effects in all three conditions. By integrating our data with previous work that examined the fitness effects of single-gene amplifications genome-wide, we found that a small number of genes within each Tamp are centrally responsible for each Tamp’s fitness effects. Our genome-wide Tamp screen confirmed that telomeric amplifications identified in laboratory-evolved populations generally increased fitness. Our results show that Tamps are mutations that produce large, typically condition-dependent changes in fitness that are important drivers of increased fitness in asexually evolving populations.
Genome-Wide Analysis of Heteroduplex DNA in Mismatch Repair–Deficient Yeast Cells Reveals Novel Properties of Meiotic Recombination Pathways  [PDF]
Emmanuelle Martini ,Valérie Borde,Matthieu Legendre,Stéphane Audic,Béatrice Regnault,Guillaume Soubigou,Bernard Dujon,Bertrand Llorente
PLOS Genetics , 2011, DOI: 10.1371/journal.pgen.1002305
Abstract: Meiotic DNA double-strand breaks (DSBs) initiate crossover (CO) recombination, which is necessary for accurate chromosome segregation, but DSBs may also repair as non-crossovers (NCOs). Multiple recombination pathways with specific intermediates are expected to lead to COs and NCOs. We revisited the mechanisms of meiotic DSB repair and the regulation of CO formation, by conducting a genome-wide analysis of strand-transfer intermediates associated with recombination events. We performed this analysis in a SK1 × S288C Saccharomyces cerevisiae hybrid lacking the mismatch repair (MMR) protein Msh2, to allow efficient detection of heteroduplex DNAs (hDNAs). First, we observed that the anti-recombinogenic activity of MMR is responsible for a 20% drop in CO number, suggesting that in MMR–proficient cells some DSBs are repaired using the sister chromatid as a template when polymorphisms are present. Second, we observed that a large fraction of NCOs were associated with trans–hDNA tracts constrained to a single chromatid. This unexpected finding is compatible with dissolution of double Holliday junctions (dHJs) during repair, and it suggests the existence of a novel control point for CO formation at the level of the dHJ intermediate, in addition to the previously described control point before the dHJ formation step. Finally, we observed that COs are associated with complex hDNA patterns, confirming that the canonical double-strand break repair model is not sufficient to explain the formation of most COs. We propose that multiple factors contribute to the complexity of recombination intermediates. These factors include repair of nicks and double-stranded gaps, template switches between non-sister and sister chromatids, and HJ branch migration. Finally, the good correlation between the strand transfer properties observed in the absence of and in the presence of Msh2 suggests that the intermediates detected in the absence of Msh2 reflect normal intermediates.
A Genome-Wide Systematic Analysis Reveals Different and Predictive Proliferation Expression Signatures of Cancerous vs. Non-Cancerous Cells  [PDF]
Yedael Y. Waldman ,Tamar Geiger,Eytan Ruppin
PLOS Genetics , 2013, DOI: 10.1371/journal.pgen.1003806
Abstract: Understanding cell proliferation mechanisms has been a long-lasting goal of the scientific community and specifically of cancer researchers. Previous genome-scale studies of cancer proliferation determinants have mainly relied on knockdown screens aimed to gauge their effects on cancer growth. This powerful approach has several limitations such as off-target effects, partial knockdown, and masking effects due to functional backups. Here we employ a complementary approach and assign each gene a cancer Proliferation Index (cPI) that quantifies the association between its expression levels and growth rate measurements across 60 cancer cell lines. Reassuringly, genes found essential in cancer gene knockdown screens exhibit significant positive cPI values, while tumor suppressors exhibit significant negative cPI values. Cell cycle, DNA replication, splicing and protein production related processes are positively associated with cancer proliferation, while cellular migration is negatively associated with it – in accordance with the well known “go or grow” dichotomy. A parallel analysis of genes' non-cancerous proliferation indices (nPI) across 224 lymphoblastoid cell lines reveals surprisingly marked differences between cancerous and non-cancerous proliferation. These differences highlight genes in the translation and spliceosome machineries as selective cancer proliferation-associated proteins. A cross species comparison reveals that cancer proliferation resembles that of microorganisms while non-cancerous proliferation does not. Furthermore, combining cancerous and non-cancerous proliferation signatures leads to enhanced prediction of patient outcome and gene essentiality in cancer. Overall, these results point to an inherent difference between cancerous and non-cancerous proliferation determinants, whose understanding may contribute to the future development of novel cancer-specific anti-proliferative drugs.
A genome wide analysis of the response to uncapped telomeres in budding yeast reveals a novel role for the NAD+ biosynthetic gene BNA2 in chromosome end protection
Amanda Greenall, Guiyuan Lei, Daniel C Swan, Katherine James, Liming Wang, Heiko Peters, Anil Wipat, Darren J Wilkinson, David Lydall
Genome Biology , 2008, DOI: 10.1186/gb-2008-9-10-r146
Abstract: The genome-wide response to uncapped telomeres in yeast cdc13-1 strains, which have telomere capping defects at temperatures above approximately 27°C, was determined. Telomere uncapping in cdc13-1 strains is associated with the differential expression of over 600 transcripts. Transcripts affecting responses to DNA damage and diverse environmental stresses were statistically over-represented. BNA2, required for the biosynthesis of NAD+, is highly and significantly up-regulated upon telomere uncapping in cdc13-1 strains. We find that deletion of BNA2 and NPT1, which is also involved in NAD+ synthesis, suppresses the temperature sensitivity of cdc13-1 strains, indicating that NAD+ metabolism may be linked to telomere end protection.Our data support the hypothesis that the response to telomere uncapping is related to, but distinct from, the response to non-telomeric double-strand breaks. The induction of environmental stress responses may be a conserved feature of the eukaryotic response to telomere damage. BNA2, which is involved in NAD+ synthesis, plays previously unidentified roles in the cellular response to telomere uncapping.Telomeres are the specialized structures at the ends of linear eukaryotic chromosomes [1,2]. Their fundamental configuration is conserved in most eukaryotes and consists of repetitive DNA elements with single-stranded (ss) 3' G-rich overhangs. Telomeres are bound by numerous proteins with specificity for both double-stranded DNA (dsDNA) and the ss overhangs [3] and telomere 'capping' function is critical in preventing the cell from recognizing the chromosome ends as double-strand breaks (DSBs) [1,3]. Telomeres also need to circumvent the 'end replication problem', which is due to the inability of DNA polymerases to fully replicate chromosome ends [1]. In the presence of telomerase, a reverse transcriptase that uses an RNA template to add telomeric DNA, chromosome ends are maintained by the addition of DNA repeats [4]. In budding yeast and mamm
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