| 1. |
- Molin, Mikael, 1973, et al.
(författare)
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Protein kinase A controls yeast growth in visible light
- 2020
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Ingår i: BMC Biology. - : Springer Science and Business Media LLC. - 1741-7007. ; 18:1
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Tidskriftsartikel (refereegranskat)abstract
- Background: A wide variety of photosynthetic and non-photosynthetic species sense and respond to light, having developed protective mechanisms to adapt to damaging effects on DNA and proteins. While the biology of UV light-induced damage has been well studied, cellular responses to stress from visible light (400–700 nm) remain poorly understood despite being a regular part of the life cycle of many organisms. Here, we developed a high-throughput method for measuring growth under visible light stress and used it to screen for light sensitivity in the yeast gene deletion collection. Results: We found genes involved in HOG pathway signaling, RNA polymerase II transcription, translation, diphthamide modifications of the translational elongation factor eEF2, and the oxidative stress response to be required for light resistance. Reduced nuclear localization of the transcription factor Msn2 and lower glycogen accumulation indicated higher protein kinase A (cAMP-dependent protein kinase, PKA) activity in many light-sensitive gene deletion strains. We therefore used an ectopic fluorescent PKA reporter and mutants with constitutively altered PKA activity to show that repression of PKA is essential for resistance to visible light. Conclusion: We conclude that yeast photobiology is multifaceted and that protein kinase A plays a key role in the ability of cells to grow upon visible light exposure. We propose that visible light impacts on the biology and evolution of many non-photosynthetic organisms and have practical implications for how organisms are studied in the laboratory, with or without illumination.
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| 2. |
- Ahmadpour, Doryaneh, 1973, et al.
(författare)
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Robustness analysis of HOG pathway genes in Saccharomyces cerevisiae
- 2006
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Ingår i: YSBN Meeting Nov. 14-16, 2006- Vienna- Austria.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Robustness analysis of HOG pathway genes in Saccharomyces cerevisiae Doryaneh Ahmadpour1, Lars-Göran Ottosson1, Markus Krantz2, Jonas Warringer1, Anders Blomberg1 and Stefan Hohmann1* 1Department of Cell and Molecular Biology/Microbiology, Göteborg University, S-405 30 Göteborg, Sweden 2 The Systems Biology Institute (SBI), Shibuya, Tokyo, Japan E-mail: doryaneh.ahmadpour@gmm.gu.se Robustness is a fundamental property of biological systems and crucial for their effective function under internal or external perturbations. For instance, it has been proposed that internal parameters such as gene expression have been optimized during evolution such that a given system has the observed robustness. The permissible ranges of internal parameters in the cells are not comprehensively understood since there has not been a technique to measure such parameters. “Genetic tug-of-war” (gTOW) [1] is a genetic screening method that allows the investigation of the upper limit copy number of genes, and thereby the upper permissible range of gene expression level. This method is based on a 2-micron plasmid vector containing the leu2d allele with a very weak complementation activity and the gene of interest inserted as target gene. When the leu2ura3 deletion yeast cells transformed with pTOW plasmid are cultured under leucine-limiting conditions, there will be a bias toward increasing the plasmid copy number to compensate for the lack of leucine. On the other hand there will be an opposing bias toward decreasing the plasmid copy number if the target gene inhibits growth or has a toxic effect when a certain copy number is exceeded (it reaches to its upper limit). Eventually as a result of the “tug-of-war” between these two selection biases cells with optimized plasmid copy number will be concentrated. In this study we have applied the gTOW method on 29 HOG pathway related genes in Saccharomyces cerevisiae. The high osmolarity glycerol (HOG) MAPK pathway is essential for yeast survival in high osmolarity condition and consists of two branches that activate a MAPK (Hog1) via a MAPKK (Pbs2) to orchestrate part of the transcriptional response. The HOG pathway is the best understood osmoresponsive system in eukaryotes and the quantitative data provided by the gTOW method collating with the existing computational models could be used to analyze the robustness and fragility of the pathway. 1. Hisao Moriya, Yuki Shimizu-Yoshida and Hiroaki Kitano, 2006, PLoS Genetics, 2:7
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| 3. |
- Ahmadpour, Doryaneh, 1973, et al.
(författare)
-
Robustness analysis of HOG pathway related genes in Saccharomyces cerevisiae
- 2007
-
Ingår i: FEBS-SysBio March 10-16, 2007- Gosau, Austria.
-
Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Robustness analysis of HOG pathway related genes in Saccharomyces cerevisiae Doryaneh Ahmadpour1, Lars-Göran Ottosson1, Markus Krantz2, Jonas Warringer1, Anders Blomberg1 and Stefan Hohmann1* 1Department of Cell and Molecular Biology/Microbiology, Göteborg University, S-405 30 Göteborg, Sweden 2 The Systems Biology Institute (SBI), Shibuya, Tokyo, Japan E-mail: doryaneh.ahmadpour@gmm.gu.se Robustness is a fundamental property of biological systems and crucial for their effective function under internal or external perturbations. For instance, it has been proposed that internal parameters such as gene expression have been optimized during evolution such that a given system has the observed robustness. The permissible ranges of internal parameters in the cells are not comprehensively understood since there has not been a technique to measure such parameters. “Genetic tug-of-war” (gTOW) [1] is a genetic screening approach that allows the determination of the upper limit copy number of genes, and thereby the upper permissible range of the level of gene expression. This method is based on a 2-micron plasmid vector containing the LEU2d allele with a very weak complementation activity and the gene of interest inserted as target gene. When the leu2 ura3 mutant yeast transformed with pTOW plasmids is cultured under leucine-limiting conditions, there will be a bias toward increasing the plasmid copy number to satisfy the requirement for leucine. On the other hand there will be an opposing bias toward decreasing the plasmid copy number if the target gene inhibits growth when a certain copy number is exceeded (i.e. it reaches its upper limit). Eventually as a result of the “tug-of-war” between these two selection biases cells with optimized plasmid copy number will accumulate. In this study we have applied the gTOW method on 29 HOG pathway genes in S. cerevisiae. The high osmolarity glycerol (HOG) MAPK pathway is essential for yeast survival in high osmolarity condition [2]. It consists of two branches that activate a MAPK (Hog1) to orchestrate part of the transcriptional response. The HOG pathway is the best understood osmoresponsive system in eukaryotes. The quantitative data provided by the gTOW method collating with the existing computational models [3] could be used to analyze the robustness and fragility of the pathway. 1. Moriya H, et al., (2006), PLoS Genet 2(7): e111 2. Hohmann S (2002), Microbiol Mol Biol Rev 66:300 3. Klipp E, et al., (2005), Nat Biotechnol 23:975
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| 4. |
- Alalam, Hanna, et al.
(författare)
-
A High-Throughput Method for Screening for Genes Controlling Bacterial Conjugation of Antibiotic Resistance.
- 2020
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Ingår i: mSystems. - 2379-5077. ; 5:6
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Tidskriftsartikel (refereegranskat)abstract
- The rapid horizontal transmission of antibiotic resistance genes on conjugative plasmids between bacterial host cells is a major cause of the accelerating antibiotic resistance crisis. There are currently no experimental platforms for fast and cost-efficient screening of genetic effects on antibiotic resistance transmission by conjugation, which prevents understanding and targeting conjugation. We introduce a novel experimental framework to screen for conjugation-based horizontal transmission of antibiotic resistance between >60,000 pairs of cell populations in parallel. Plasmid-carrying donor strains are constructed in high-throughput. We then mix the resistance plasmid-carrying donors with recipients in a design where only transconjugants can reproduce, measure growth in dense intervals, and extract transmission times as the growth lag. As proof-of-principle, we exhaustively explore chromosomal genes controlling F-plasmid donation within Escherichia coli populations, by screening the Keio deletion collection in high replication. We recover all seven known chromosomal gene mutants affecting conjugation as donors and identify many novel mutants, all of which diminish antibiotic resistance transmission. We validate nine of the novel genes' effects in liquid mating assays and complement one of the novel genes' effect on conjugation (rseA). The new framework holds great potential for exhaustive disclosing of candidate targets for helper drugs that delay resistance development in patients and societies and improve the longevity of current and future antibiotics. Further, the platform can easily be adapted to explore interspecies conjugation, plasmid-borne factors, and experimental evolution and be used for rapid construction of strains.IMPORTANCE The rapid transmission of antibiotic resistance genes on conjugative plasmids between bacterial host cells is a major cause of the accelerating antibiotic resistance crisis. There are currently no experimental platforms for fast and cost-efficient screening of genetic effects on antibiotic resistance transmission by conjugation, which prevents understanding and targeting conjugation. We introduce a novel experimental framework to screen for conjugation-based horizontal transmission of antibiotic resistance between >60,000 pairs of cell populations in parallel. As proof-of-principle, we exhaustively explore chromosomal genes controlling F-plasmid donation within E. coli populations. We recover all previously known and many novel chromosomal gene mutants that affect conjugation efficiency. The new framework holds great potential for rapid screening of compounds that decrease transmission. Further, the platform can easily be adapted to explore interspecies conjugation, plasmid-borne factors, and experimental evolution and be used for rapid construction of strains.
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| 5. |
- Barré, Benjamin P., et al.
(författare)
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Intragenic repeat expansion in the cell wall protein gene HPF1 controls yeast chronological aging
- 2020
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Ingår i: Genome Research. - : Cold Spring Harbor Laboratory. - 1088-9051 .- 1549-5469. ; 30:5, s. 697-710
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Tidskriftsartikel (refereegranskat)abstract
- Aging varies among individuals due to both genetics and environment, but the underlying molecular mechanisms remain largely unknown. Using a highly recombined Saccharomyces cerevisiae population, we found 30 distinct quantitative trait loci (QTLs) that control chronological life span (CLS) in calorie-rich and calorie-restricted environments and under rapamycin exposure. Calorie restriction and rapamycin extended life span in virtually all genotypes but through different genetic variants. We tracked the two major QTLs to the cell wall glycoprotein genes FLO11 and HPF1. We found that massive expansion of intragenic tandem repeats within the N-terminal domain of HPF1 was sufficient to cause pronounced life span shortening. Life span impairment by HPF1 was buffered by rapamycin but not by calorie restriction. The HPF1 repeat expansion shifted yeast cells from a sedentary to a buoyant state, thereby increasing their exposure to surrounding oxygen. The higher oxygenation altered methionine, lipid, and purine metabolism, and inhibited quiescence, which explains the life span shortening. We conclude that fast-evolving intragenic repeat expansions can fundamentally change the relationship between cells and their environment with profound effects on cellular lifestyle and longevity.
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| 6. |
- Caesar, Robert, 1973, et al.
(författare)
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Physiological importance and identification of novel targets for the N-terminal acetyltransferase NatB
- 2006
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Ingår i: Eukaryotic Cell. - 1535-9778. ; 5:2, s. 368-378
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Tidskriftsartikel (refereegranskat)abstract
- The N-terminal acetyltransferase NatB in Saccharomyces cerevisiae consists of the catalytic subunit Nat3p and the associated subunit Mdm20p. We here extend our present knowledge about the physiological role of NatB by a combined proteomics and phenomics approach. We found that strains deleted for either NAT3 or MDM20 displayed different growth rates and morphologies in specific stress conditions, demonstrating that the two NatB subunits have partly individual functions. Earlier reported phenotypes of the nat3{Delta} strain have been associated with altered functionality of actin cables. However, we found that point mutants of tropomyosin that suppress the actin cable defect observed in nat3{Delta} only partially restores wild-type growth and morphology, indicating the existence of functionally important acetylations unrelated to actin cable function. Predicted NatB substrates were dramatically overrepresented in a distinct set of biological processes, mainly related to DNA processing and cell cycle progression. Three of these proteins, Cac2p, Pac10p, and Swc7p, were identified as true NatB substrates. To identify N-terminal acetylations potentially important for protein function, we performed a large-scale comparative phenotypic analysis including nat3{Delta} and strains deleted for the putative NatB substrates involved in cell cycle regulation and DNA processing. By this procedure we predicted functional importance of the N-terminal acetylation for 31 proteins.
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| 7. |
- Ericson, Elke, 1973, et al.
(författare)
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Genetic pleiotropy in Saccharomyces cerevisiae quantified by high-resolution phenotypic profiling
- 2006
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Ingår i: Molecular Genetics and Genomics. - : Springer Science and Business Media LLC. - 1617-4615 .- 1617-4623. ; 275:6, s. 605-614
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Tidskriftsartikel (refereegranskat)abstract
- Genetic pleiotropy, the ability of a mutation in a single gene to give rise to multiple phenotypic outcomes, constitutes an important but incompletely understood biological phenomenon. We used a highresolution and high-precision phenotypic profiling approach to quantify the fitness contribution of genes on the five smallest yeast chromosomes during different forms of environmental stress, selected to probe a wide diversity of physiological features. We found that the extent of pleiotropy is much higher than previously claimed; 17% of the yeast genes were pleiotropic whereof one-fifth were hyper-pleiotropic. Pleiotropic genes preferentially participate in functions related to determination of protein fate, cell growth and morphogenesis, signal transduction and transcription. Contrary to what has earlier been proposed we did not find experimental evidence for slower evolutionary rate of pleiotropic genes/proteins. We also refute the existence of phenotypic islands along chromosomes but report on a remarkable loss both of pleiotropy and of phenotypic penetrance towards chromosomal ends. Thus, the here reported features of pleiotropy both have implications on our understanding of evolutionary processes as well as the mechanisms underlying disease.
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| 8. |
- Fernandez-Ricaud, Luciano, 1975, et al.
(författare)
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PROPHECY - a database for high-resolution phenomics
- 2005
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 33
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Tidskriftsartikel (refereegranskat)abstract
- The rapid recent evolution of the field phenomics—the genome-wide study of gene dispensability by quantitative analysis of phenotypes—has resulted in an increasing demand for new data analysis and visualization tools. Following the introduction of a novel approach for precise, genome-wide quantification of gene dispensability in Saccharomyces cerevisiae we here announce a public resource for mining, filtering and visualizing phenotypic data—the PROPHECY database. PROPHECY is designed to allow easy and flexible access to physiologically relevant quantitative data for the growth behaviour of mutant strains in the yeast deletion collection during conditions of environmental challenges. PROPHECY is publicly accessible at http://prophecy.lundberg.gu.se.
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| 9. |
- Fernandez-Ricaud, Luciano, 1975, et al.
(författare)
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PROPHECY—a yeast phenome database, update 2006
- 2007
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 35
-
Tidskriftsartikel (refereegranskat)abstract
- Connecting genotype to phenotype is fundamental in biomedical research and in our understanding of disease. Phenomics—the large-scale quantitative phenotypic analysis of genotypes on a genome-wide scale—connects automated data generation with the development of novel tools for phenotype data integration, mining and visualization. Our yeast phenomics database PROPHECY is available at http://prophecy.lundberg.gu.se. Via phenotyping of 984 heterozygous diploids for all essential genes the genotypes analysed and presented in PROPHECY have been extended and now include all genes in the yeast genome. Further, phenotypic data from gene overexpression of 574 membrane spanning proteins has recently been included. To facilitate the interpretation of quantitative phenotypic data we have developed a new phenotype display option, the Comparative Growth Curve Display, where growth curve differences for a large number of mutants compared with the wild type are easily revealed. In addition, PROPHECY now offers a more informative and intuitive first-sight display of its phenotypic data via its new summary page. We have also extended the arsenal of data analysis tools to include dynamic visualization of phenotypes along individual chromosomes. PROPHECY is an initiative to enhance the growing field of phenome bioinformatics
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| 10. |
- Forsmark, Annabelle, 1973, et al.
(författare)
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Quantitative Proteomics of Yeast Post-Golgi Vesicles Reveals a Discriminating Role for Sro7p in Protein Secretion
- 2011
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Ingår i: Traffic. - : John Wiley & Sons. - 1398-9219 .- 1600-0854. ; 12:6, s. 740-753
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Tidskriftsartikel (refereegranskat)abstract
- We here report the first comparative proteomics of purified yeast post-Golgi vesicles (PGVs). Vesicle samples isolated from PGV-accumulating sec6-4 mutants were treated with isobaric tags (iTRAQ) for subsequent quantitative tandem mass spectrometric analysis of protein content. After background subtraction, a total of 66 vesicle-associated proteins were identified, including known or assumed vesicle residents as well as a fraction not previously known to be PGV associated. Vesicles isolated from cells lacking the polarity protein Sro7p contained essentially the same catalogue of proteins but showed a reduced content of a subset of cargo proteins, in agreement with a previously shown selective role for Sro7p in cargo sorting.
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| 11. |
- Gombault, Aurélie, et al.
(författare)
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A phenotypic study of TFS1 mutants differentially altered in the inhibition of Ira2p or CPY.
- 2009
-
Ingår i: FEMS yeast research. - : Oxford University Press (OUP). - 1567-1364 .- 1567-1356. ; 9:6, s. 867-74
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Tidskriftsartikel (refereegranskat)abstract
- The Saccharomyces cerevisiae protein Tfs1p is known as a dual protein. On the one hand, it inhibits the carboxypeptidase Y protease, and on the other, it inhibits Ira2p, a GTPase-activating protein of Ras. We managed to dissect precise areas of Tfs1p specifically involved in only one of those functions. Based on these data, specific Tfs1p point mutants affected in only one of these two functions were constructed. In order to obtain insights on the physiological role of these functions, systematic phenotypic tests were performed on strains expressing these specific Tfs1p mutants. The results obtained demonstrate that the inhibition of Ira2p by Tfs1p is the predominant function under the conditions tested.
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| 12. |
- Jansson, Kristina, 1969, et al.
(författare)
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A role for Myh1 in DNA repair after treatment with strand-breaking and crosslinking chemotherapeutic agents
- 2013
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Ingår i: Environmental and Molecular Mutagenesis. - : Wiley. - 0893-6692 .- 1098-2280. ; 54:5, s. 327-337
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Tidskriftsartikel (refereegranskat)abstract
- The highly conserved DNA glycosylase MutY is implicated in repair of oxidative DNA damage, in particular in removing adenines misincorporated opposite 7,8-dihydro-8-oxoguanine (8-oxo-G). The MutY homologues (MutYH) physically associate with proteins implicated in replication, DNA repair, and checkpoint signaling, specifically with the DNA damage sensor complex 9-1-1 proteins. Here, we ask whether MutYH could have a broader function in sensing and repairing different types of DNA damage induced by conventional chemotherapeutics. Thus, we examined if deletion of the Schizosaccharomyces pombe MutY homologue, Myh1, alone or in combination with deletion of either component of the 9-1-1 sensor complex, influences survival after exposure to different classes of DNA damaging chemotherapeutics that do not act primarily by causing 8-oxoG lesions. We show that Myh1 contributes to survival on genotoxic stresses induced by the oxidizing, DNA double strand break-inducing, bleomycins, or the DNA crosslinking platinum compounds, particularly in a rad1 mutant background. Exposure of cells to cisplatin leads to a moderate overall accumulation of Myh1 protein. Interestingly, we found that DNA damage induced by phleomycin results in increased chromatin association of Myh1. Further, we demonstrate that Myh1 relocalizes to the nucleus after exposure to hydrogen peroxide or chemotherapeutics, most prominently seen after phleomycin treatment. These observations indicate a wider role of Myh1 in DNA repair and DNA damage-induced checkpoint activation than previously thought
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| 13. |
- Kohler, A., et al.
(författare)
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High-throughput biochemical fingerprinting of Saccharomyces cerevisiae by Fourier transform infrared spectroscopy
- 2015
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 10:2
-
Tidskriftsartikel (refereegranskat)abstract
- Single-channel optical density measurements of population growth are the dominant large scale phenotyping methodology for bridging the gene-function gap in yeast. However, a substantial amount of the genetic variation induced by single allele, single gene or double gene knock-out technologies fail to manifest in detectable growth phenotypes under conditions readily testable in the laboratory. Thus, new high-throughput phenotyping technologies capable of providing information about molecular level consequences of genetic variation are sorely needed. Here we report a protocol for high-throughput Fourier transform infrared spectroscopy (FTIR) measuring biochemical fingerprints of yeast strains. It includes high-throughput cultivation for FTIR spectroscopy, FTIR measurements and spectral pretreatment to increase measurement accuracy.We demonstrate its capacity to distinguish not only yeast genera, species and populations, but also strains that differ only by a single gene, its excellent signal-to-noise ratio and its relative robustness to measurement bias. Finally, we illustrated its applicability by determining the FTIR signatures of all viable Saccharomyces cerevisiae single gene knock-outs corresponding to lipid biosynthesis genes. Many of the examined knock-out strains showed distinct, highly reproducible FTIR phenotypes despite having no detectable growth phenotype. These phenotypes were confirmed by conventional lipid analysis and could be linked to specific changes in lipid composition. We conclude that the introduced protocol is robust to noise and bias, possible to apply on a very large scale, and capable of generating biologically meaningful biochemical fingerprints that are strain specific, even when strains lack detectable growth phenotypes. Thus, it has a substantial potential for application in the molecular functionalization of the yeast genome.
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| 14. |
- Krantz, Marcus, 1975, et al.
(författare)
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Robustness and fragility in the high osmolarity glycerol (HOG) pathway in S. cerevisiae
- 2009
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Ingår i: 10th International Conference on Systems Biology (ICSB2009) proceedings, 10th International Conference on Systems Biology (ICSB2009), Aug 30 - Sep 4, Stanford, California, USA.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Cellular signalling networks integrate environmental stimuli with information on cellular status. These networks must be robust against stochastic fluctuations in external stimuli as well as in the amounts of signalling components. Here [1], we challenge the yeast HOG signal transduction pathway with systematic perturbations in components’ expression levels implemented by a “genetic tug-of-war” methodology under various external conditions in search of nodes of fragilities. We observe a substantially higher frequency of fragile nodes in this signal transduction pathway than has been observed for other cellular processes. These fragilities disperse without any clear pattern over biochemical functions or location in pathway topology, with the most sensitive nodes being the proteins PBS2 and SSK1. They are also largely independent of pathway activation by external stimuli. However, the strongest toxicities are caused by pathway hyperactivation. We studied the influence of seven regulatory motifs around these HOG pathway components in silico through ODE models. Based on the SLN1 and the MAPK modules of a mathematical model of osmoregulation in budding yeast by Klipp et al. [2] we included new motifs and fitted the affected parameters to time courses of dually phosphorylated Hog1p generated by the original model under stress and stress-free conditions. The regulations taken into account by our analysis include Pbs2p scaffolding, Ssk1p and Pbs2p autoactivation, and the formation of a stable dimer between Ssk2p and Ssk1p. A subsequent sensitivity analysis identified Pbs2's role as a scaffold protein and Ssk1p-Ssk2p dimerization as the important contributors to the observed robustness pattern in silico. Thus, in vivo robustness data can be used to discriminate and improve mathematical models.
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| 15. |
- Krantz, Marcus, 1975, et al.
(författare)
-
Robustness and fragility in the yeast high osmolarity glycerol (HOG) signal-transduction pathway.
- 2009
-
Ingår i: Molecular systems biology. - : EMBO. - 1744-4292. ; 5
-
Tidskriftsartikel (refereegranskat)abstract
- Cellular signalling networks integrate environmental stimuli with the information on cellular status. These networks must be robust against stochastic fluctuations in stimuli as well as in the amounts of signalling components. Here, we challenge the yeast HOG signal-transduction pathway with systematic perturbations in components' expression levels under various external conditions in search for nodes of fragility. We observe a substantially higher frequency of fragile nodes in this signal-transduction pathway than that has been observed for other cellular processes. These fragilities disperse without any clear pattern over biochemical functions or location in pathway topology and they are largely independent of pathway activation by external stimuli. However, the strongest toxicities are caused by pathway hyperactivation. In silico analysis highlights the impact of model structure on in silico robustness, and suggests complex formation and scaffolding as important contributors to the observed fragility patterns. Thus, in vivo robustness data can be used to discriminate and improve mathematical models.
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| 16. |
- Krantz, Marcus, 1975, et al.
(författare)
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Robustness and fragility in the yeast High Osmolarity signal transduction pathway
- 2008
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Ingår i: 2008 Yeast Genetics and Molecular Biology Meeting Program and Abstract Book, 2008 Yeast Genetics and Molecular Biology Meeting, July 22-27, 2008.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- The cellular signalling networks that integrate various environmental stimuli with information on cellular status must be robust to stimuli fluctuations as well as to stochastic differences in the amounts of signalling components. Here, we challenge the Hog signal transduction pathway with systematic disturbances in components’ expression levels implemented by a “genetic tug-of-war”, or gToW, methodology. The disturbances were performed under various external perturbations, including pathway activation by osmotic shock. Ideally, the obtained sensitivity profiles will allow us to impose parameter constraints. However, a more important aspect is the qualitative improvement of model structures, when local fragilities cannot be explained by the model structure. The resulting phenotypes in this particular study reflect a wide range of sensitivities, and disperse without any clear pattern over biochemical functions and pathway modules alike, with the most sensitive nodes being PBS2 and SSK1. Surprisingly, the “neighbouring” nodes HOG1 and SSK2 were affected to a much lesser extent, questioning our current understanding.
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| 17. |
- Krantz, Marcus, 1975, et al.
(författare)
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Robustness and fragility in the yeast High Osmolarity signal transduction pathway
- 2008
-
Ingår i: 9th International Conference on Systems Biology (ICSB2008) proceedings, 9th International Conference on Systems Biology (ICSB2008), August 22-28, Gothenburg.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Objective: The cellular signalling networks that integrate various environmental stimuli with information on cellular status must be robust to stimuli fluctuations as well as to stochastic differences in the amounts of signalling components. Here, we challenge the high osmolarity glycerol response (HOG) signal transduction pathway in the yeast Saccharomyces cerevisiae with systematic disturbances in components’ expression levels implemented by a “genetic tug-of-war”, or gTOW, methodology. Results: The disturbances were performed under various external perturbations, including pathway activation by osmotic shock. The resulting phenotypes in this particular study reflect a wide range of sensitivities, and disperse without any clear pattern over biochemical functions and pathway modules alike, with the most sensitive nodes being PBS2 and SSK1. Conclusions: Ideally, the obtained sensitivity profiles will allow us to impose parameter constraints. However, a more important aspect is the qualitative improvement of model structures, when local fragilities cannot be explained by the model structure. Surprisingly, the “neighboring” nodes HOG1 and SSK2 were affected to a much lesser extent, questioning our current understanding.
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| 18. |
- Ottosson, Lars-Göran, et al.
(författare)
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Robustness analysis of HOG pathway related genes in budding yeast
- 2007
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Ingår i: ICSB 2007 Conference Proceedings, 8th International Conference on Systems Biology (ICSB2007), October 1-6, 2007.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- We applied a novel genetic screening method, named “genetic tug-of-war” (gTOW) to estimate the upper limit of gene copy numbers in Saccharomyces cerevisiae. The study involved 29 HOG pathway related genes which included kinases, phosphatases and transcription factors to cover different set of players in the signal transduction system. In addition a phenotypic profiling was conducted in four different growth conditions with three outputs: lag phase, growth phase and efficiency of growth. A number of interesting hits were identified, including PBS2 which had low numbers of gene copies. It will be of interest to expand the study to encompass the entire known signal transduction system in yeast to search for sensitive nodes.
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| 19. |
- Ottosson, Lars-Göran, et al.
(författare)
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Robustness and fragility in the yeast High Osmolarity Glycerol (HOG) signal transduction pathway
- 2009
-
Ingår i: Abstracts of the 24th International Conference on Yeast Genetics and Molecular Biology (Supplement to Yeast Volume 26 Issue S1), 25th International Conference on Yeast Genetics & Molecular Biology, July 19-24, Manchester, UK. ; 26:Issue S1
-
Konferensbidrag (refereegranskat)abstract
- Cellular signalling networks integrate environmental stimuli with information on cellular status. These networks must be robust against stochastic fluctuations in stimuli as well as in the amounts of signalling components. Here, we challenge the yeast HOG signal transduction pathway with systematic perturbations in components’ expression levels implemented by a “genetic tug-of-war” methodology under various external conditions in search of nodes of fragilities. We observe a substantially higher frequency of fragile nodes in this signal transduction pathway than has been observed for other cellular processes. These fragilities disperse without any clear pattern over biochemical functions or location in pathway topology, with the most sensitive node being the scaffold protein PBS2. They are also largely independent of pathway activation by external stimuli. However, the strongest toxicities are caused by pathway hyperactivation. In silico analysis highlights the impact of model structure on in silico robustness, and suggests complex formation and scaffolding as important contributors to the observed fragility patterns. Thus, in vivo robustness data can be used to discriminate and improve mathematical models.
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| 20. |
- Parts, Leopold, et al.
(författare)
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Revealing the genetic structure of a trait by sequencing a population under selection.
- 2011
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Ingår i: Genome research. - : Cold Spring Harbor Laboratory. - 1549-5469 .- 1088-9051. ; 21:7, s. 1131-8
-
Tidskriftsartikel (refereegranskat)abstract
- One approach to understanding the genetic basis of traits is to study their pattern of inheritance among offspring of phenotypically different parents. Previously, such analysis has been limited by low mapping resolution, high labor costs, and large sample size requirements for detecting modest effects. Here, we present a novel approach to map trait loci using artificial selection. First, we generated populations of 10-100 million haploid and diploid segregants by crossing two budding yeast strains of different heat tolerance for up to 12 generations. We then subjected these large segregant pools to heat stress for up to 12 d, enriching for beneficial alleles. Finally, we sequenced total DNA from the pools before and during selection to measure the changes in parental allele frequency. We mapped 21 intervals with significant changes in genetic background in response to selection, which is several times more than found with traditional linkage methods. Nine of these regions contained two or fewer genes, yielding much higher resolution than previous genomic linkage studies. Multiple members of the RAS/cAMP signaling pathway were implicated, along with genes previously not annotated with heat stress response function. Surprisingly, at most selected loci, allele frequencies stopped changing before the end of the selection experiment, but alleles did not become fixed. Furthermore, we were able to detect the same set of trait loci in a population of diploid individuals with similar power and resolution, and observed primarily additive effects, similar to what is seen for complex trait genetics in other diploid organisms such as humans.
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| 21. |
- Stenberg, Simon, et al.
(författare)
-
Control of mitochondrial superoxide production includes programmed mtDNA deletion and restoration
- 2020
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Deletion of mitochondrial DNA in eukaryotes is mainly attributed to rare accidental events associated with mitochondrial replication or repair of double-strand breaks. We report the discovery that yeast cells arrest harmful intramitochondrial superoxide production by shutting down respiration through genetically controlled deletion of mitochondrial oxidative phosphorylation genes. We show that the regulatory circuitry underlying this editing critically involves the antioxidant enzyme superoxide dismutase 2 and two-way mitochondrial-nuclear communication. While mitochondrial DNA homeostasis is rapidly restored after cessation of a short-term superoxide stress, long-term stress causes maladaptive persistence of the deletion process, leading to complete annihilation of the cellular pool of intact mitochondrial genomes and irrevocable loss of respiratory ability. Our results may therefore be of etiological as well as therapeutic importance with regard to age-related mitochondrial impairment and disease.One-Sentence SummaryGenetically controlled editing of mitochondrial DNA is an integral part of the yeast’s defenses against oxidative damage.
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| 22. |
- Stenberg, Simon, et al.
(författare)
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Genetically controlled mtDNA deletions prevent ROS damage by arresting oxidative phosphorylation
- 2022
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Ingår i: eLife. - : eLife Sciences Publications, Ltd. - 2050-084X. ; 11
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Tidskriftsartikel (refereegranskat)abstract
- Deletion of mitochondrial DNA in eukaryotes is currently attributed to rare accidental events associated with mitochondrial replication or repair of double-strand breaks. We report the discovery that yeast cells arrest harmful intramitochondrial superoxide production by shutting down respiration through genetically controlled deletion of mitochondrial oxidative phosphorylation genes. We show that this process critically involves the antioxidant enzyme superoxide dismutase 2 and two-way mitochondrial-nuclear communication through Rtg2 and Rtg3. While mitochondrial DNA homeostasis is rapidly restored after cessation of a short-term superoxide stress, long-term stress causes maladaptive persistence of the deletion process, leading to complete annihilation of the cellular pool of intact mitochondrial genomes and irrevocable loss of respiratory ability. This shows that oxidative stress-induced mitochondrial impairment may be under strict regulatory control. If the results extend to human cells, the results may prove to be of etiological as well as therapeutic importance with regard to age-related mitochondrial impairment and disease.
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| 23. |
- Warringer, Jonas, 1973, et al.
(författare)
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Trait variation in yeast is defined by population history.
- 2011
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Ingår i: PLoS genetics. - : Public Library of Science (PLoS). - 1553-7404. ; 7:6
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Tidskriftsartikel (refereegranskat)abstract
- A fundamental goal in biology is to achieve a mechanistic understanding of how and to what extent ecological variation imposes selection for distinct traits and favors the fixation of specific genetic variants. Key to such an understanding is the detailed mapping of the natural genomic and phenomic space and a bridging of the gap that separates these worlds. Here we chart a high-resolution map of natural trait variation in one of the most important genetic model organisms, the budding yeast Saccharomyces cerevisiae, and its closest wild relatives and trace the genetic basis and timing of major phenotype changing events in its recent history. We show that natural trait variation in S. cerevisiae exceeds that of its relatives, despite limited genetic variation, and follows the population history rather than the source environment. In particular, the West African population is phenotypically unique, with an extreme abundance of low-performance alleles, notably a premature translational termination signal in GAL3 that cause inability to utilize galactose. Our observations suggest that many S. cerevisiae traits may be the consequence of genetic drift rather than selection, in line with the assumption that natural yeast lineages are remnants of recent population bottlenecks. Disconcertingly, the universal type strain S288C was found to be highly atypical, highlighting the danger of extrapolating gene-trait connections obtained in mosaic, lab-domesticated lineages to the species as a whole. Overall, this study represents a step towards an in-depth understanding of the causal relationship between co-variation in ecology, selection pressure, natural traits, molecular mechanism, and alleles in a key model organism.
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| 24. |
- Alalam, Hanna, et al.
(författare)
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Conjugation factors controlling F-plasmid antibiotic resistance transmission
- 2018
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Ingår i: BioRxiv. - : Cold Spring Harbor Laboratory.
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- The rapid horizontal transmission of many antibiotic resistance genes between bacterial host cells on conjugative plasmids is a major cause of the accelerating antibiotic resistance crisis. Preventing understanding and targeting conjugation, there currently are no experimental platforms for fast and cost-efficient screening of genetic effects on antibiotic resistance transmission by conjugation. We introduce a novel experimental framework to screen for conjugation based horizontal transmission of antibiotic resistance between >60,000 pairs of cell populations in parallel. Plasmid-carrying donor strains are constructed in high throughput. We then mix the resistance plasmid carrying donors with recipients in a design where only transconjugants can reproduce, measure growth in dense intervals and extract transmission times as the growth lag. As proof-of-principle, we exhaustively explored chromosomal genes controlling F plasmid donation within E. coli populations, by screening the Keio deletion collection at high replication. We recover all six known chromosomal gene mutants affecting conjugation and identify >50 novel factors, all of which diminish antibiotic resistance transmission. We verify 10 of the novel genes' effects in a liquid mating assay. The new framework holds great potential for exhaustive disclosing of candidate targets for helper drugs that delay resistance development in patients and societies and improves the longevity of current and future antibiotics.
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| 25. |
- Babazadeh, Roja, et al.
(författare)
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The Ashbya gossypiiEF-1α promoter of the ubiquitously used MX cassettes is toxic to Saccharomyces cerevisiae.
- 2011
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Ingår i: FEBS letters. - : Wiley. - 1873-3468 .- 0014-5793.
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Tidskriftsartikel (refereegranskat)abstract
- Protein overexpression based on introduction of multiple gene copies is well established. To improve purification or quantification, proteins are typically fused to peptide tags. In Saccharomyces cerevisiae, this has been hampered by multicopy toxicity of the TAP and GFP cassettes used in the global strain collections. Here, we show that this effect is due to the EF-1α promoter in the HIS3MX marker cassette rather than the tags per se. This promoter is frequently used in heterologous marker cassettes, including HIS3MX, KanMX, NatMX, PatMX and HphMX. Toxicity could be eliminated by promoter replacement or exclusion of the marker cassette. To our knowledge, this is the first report of toxicity caused by introduction of a heterologous promoter alone.
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