Intracellular biochemical parameters, such as the expression level of gene products, are consider... more Intracellular biochemical parameters, such as the expression level of gene products, are considered to be optimized so that a biological system, including the parameters, works effectively. Those parameters should have some permissible range so that the systems have robustness against perturbations, such as noise in gene expression. However, little is known about the permissible range in real cells because there has been no experimental technique to test it. In this study, we developed a genetic screening method, named ''genetic tug-of-war'' (gTOW) that evaluates upper limit copy numbers of genes in a model eukaryote Saccharomyces cerevisiae, and we applied it for 30 cell-cycle related genes (CDC genes). The experiment provided unique quantitative data that could be used to argue the system-level properties of the cell cycle such as robustness and fragility. The data were used to evaluate the current computational model, and refinements to the model were suggested.
The terminator regions of eukaryotes encode functional elements in the 3′ untranslated region (3′... more The terminator regions of eukaryotes encode functional elements in the 3′ untranslated region (3′-UTR) that influence the 3′-end processing of mRNA, mRNA stability, and translational efficiency, which can modulate protein production. However, the contribution of these terminator regions to gene expression remains unclear, and therefore their utilization in metabolic engineering or synthetic genetic circuits has been limited. Here, we comprehensively evaluated the activity of 5302 terminator regions from a total of 5880 genes in the budding yeast Saccharomyces cerevisiae by inserting each terminator region downstream of the P TDH3-green fluorescent protein (GFP) reporter gene and measuring the fluorescent intensity of GFP. Terminator region activities relative to that of the PGK1 standard terminator ranged from 0.036 to 2.52, with a mean of 0.87. We thus could isolate the most and least active terminator regions. The activities of the terminator regions showed a positive correlation with mRNA abundance, indicating that the terminator region is a determinant of mRNA abundance. The least active terminator regions tended to encode longer 3′-UTRs, suggesting the existence of active degradation mechanisms for those mRNAs. The terminator regions of ribosomal protein genes tended to be the most active, suggesting the existence of a common regulator of those genes. The ″terminatome″ (the genome-wide set of terminator regions) thus not only provides valuable information to understand the modulatory roles of terminator regions on gene expression but also serves as a useful toolbox for the development of metabolically and genetically engineered yeast.
Background: Identifying permissible limits of intracellular parameters such as protein expression... more Background: Identifying permissible limits of intracellular parameters such as protein expression provides important information for examining robustness. In this study, we used the TEV protease-mediated induction of protein instability (TIPI) in combination with the genetic Tug-of-War (gTOW) to develop a method to measure the lower limit of protein level. We first tested the feasibility of this method using ADE2 as a marker and then analyzed some cell cycle regulators to reveal genetic interactions. Results: Using TIPI-gTOW, we successfully constructed a strain in which GFP-TDegF Ade2 was expressed at the lower limit, just sufficient to support cellular growth under the −Ade condition by accelerating degradation by TEV protease. We also succeeded in constructing a strain in which the minimal level of GFP-TDegF Cdc20 was expressed by TIPI-gTOW. Using this strain, we studied genetic interactions between cell cycle regulators and CDC20, and the result was highly consistent with the previously identified interactions. Comparison of the experimental data with predictions of a mathematical model revealed some interactions that were not implemented into the current model. Conclusions: TIPI-gTOW is useful for estimating changes in the lower limit of a protein under different conditions, such as different genetic backgrounds and environments. TIPI-gTOW is also useful for analyzing genetic interactions of essential genes whose deletion mutants cannot be obtained.
The yeast Saccharomyces cerevisiae senses glucose through two transmembrane glucose sensors, Snf3... more The yeast Saccharomyces cerevisiae senses glucose through two transmembrane glucose sensors, Snf3 and Rgt2. Extracellular glucose causes these sensors to generate an intracellular signal that induces expression of HXT genes encoding glucose transporters by inhibiting the function of Rgt1, a transcriptional repressor of HXT genes. We present the following evidence that suggests that the glucose sensors are coupled to the membrane-associated protein kinase casein kinase I (Yck1). (i) Overexpression of Yck1 leads to constitutive HXT1 expression; (ii) Yck1 (or its paralogue Yck2) is required for glucose induction of HXT1 expression; (iii) Yck1 interacts with the Rgt2 glucose sensor; and (iv) attaching the C-terminal cytoplasmic tail of Rgt2 to Yck1 results in a constitutive glucose signal. The likely targets of Yck1 in this signal transduction pathway are Mth1 and Std1, which bind to and regulate function of the Rgt1 transcription factor and bind to the C-terminal cytoplasmic domain of glucose sensors. Potential casein kinase I phosphoryla-tion sites in Mth1 and Std1 are required for normal glucose regulation of HXT1 expression, and Yck1 catalyzes phosphoryla-tion of Mth1 and Std1 in vitro. These results support a model of glucose signaling in which glucose binding to the glucose sensors causes them to activate Yck1 in the cell membrane, which then phosphorylates Mth1 and Std1 bound to the cytoplasmic face of the glucose sensors, triggering their degradation and leading to the derepression of HXT gene expression. Our results add nutrient sensing to the growing list of processes in which casein kinase I is involved.
Intracellular biochemical parameters, such as the expression level of gene products, are consider... more Intracellular biochemical parameters, such as the expression level of gene products, are considered to be optimized so that a biological system, including the parameters, works effectively. Those parameters should have some permissible range so that the systems have robustness against perturbations, such as noise in gene expression. However, little is known about the permissible range in real cells because there has been no experimental technique to test it. In this study, we developed a genetic screening method, named ''genetic tug-of-war'' (gTOW) that evaluates upper limit copy numbers of genes in a model eukaryote Saccharomyces cerevisiae, and we applied it for 30 cell-cycle related genes (CDC genes). The experiment provided unique quantitative data that could be used to argue the system-level properties of the cell cycle such as robustness and fragility. The data were used to evaluate the current computational model, and refinements to the model were suggested. Citation: Moriya H, Shimizu-Yoshida Y, Kitano H (2006) In vivo robustness analysis of cell division cycle genes in Saccharomyces cerevisiae. PLoS Genet 2(7): e111.
Cellular systems are generally robust against fluctuations of intracellular parameters such as ge... more Cellular systems are generally robust against fluctuations of intracellular parameters such as gene expression level. However, little is known about expression limits of genes required to halt cellular systems. In this study, using the fission yeast Schizosaccharomyces pombe, we developed a genetic 'tug-of-war' (gTOW) method to assess the overexpression limit of certain genes. Using gTOW, we determined copy number limits for 31 cell-cycle regulators; the limits varied from 1 to 4100. Comparison with orthologs of the budding yeast Saccharomyces cerevisiae suggested the presence of a conserved fragile core in the eukaryotic cell cycle. Robustness profiles of networks regulating cytokinesis in both yeasts (septation-initiation network (SIN) and mitotic exit network (MEN)) were quite different, probably reflecting differences in their physiologic functions. Fragility in the regulation of GTPase spg1 was due to dosage imbalance against GTPase-activating protein (GAP) byr4. Using the gTOW data, we modified a mathematical model and successfully reproduced the robustness of the S. pombe cell cycle with the model.
Robustness is one of the principles of design inherent to biological systems. Cellular robustness... more Robustness is one of the principles of design inherent to biological systems. Cellular robustness can be measured as limits of intracellular parameters such as gene expression levels. We have recently developed an experimental approach coined as genetic Tug-Of-War (gTOW), which we used to perform robustness analysis in yeast. Using gTOW, we were able to measure the upper limit of expression of gene targets. In this review, we first elaborate on how the gTOW method compares to current mathematical simulation models prevalently used in the determination of robustness. We then explain the experimental principles underlying gTOW and its associated tools, and we provide concrete examples of robustness analysis using gTOW, i.e. cell cycle and HOG pathway gene expression analysis. Finally, we list a series of Q&As related to the experimental utilization of gTOW and we describe the potential impact of gTOW and its relevance to the understanding of biological systems.
Overexpression experiments are sometimes considered as qualitative experiments designed to identi... more Overexpression experiments are sometimes considered as qualitative experiments designed to identify novel proteins and study their function. However, in order to draw conclusions regarding protein overexpression through association analyses using large-scale biological data sets, we need to recognize the quantitative nature of overexpression experiments. Here I discuss the quantitative features of two different types of overexpression experiment: absolute and relative. I also introduce the four primary mechanisms involved in growth defects caused by protein overexpression: resource overload, stoichiometric imbalance , promiscuous interactions, and pathway modulation associated with the degree of over-expression.
POP2 protein of Saccharomyces cerevisiae is a component of a protein complex that regulates the t... more POP2 protein of Saccharomyces cerevisiae is a component of a protein complex that regulates the transcription of many genes. We found that the 97th threonine residue (Thr 97) of Pop2p was phosphorylated upon glucose limitation. The Thr 97 phosphorylation occurred within 2 min after removing glucose and was reversed within 1 min after the readdition of glucose. The effects of hexokinase mutations and glucose analogs indicate that this phosphorylation is dependent on glucose phosphorylating activity. We purified a protein kinase that phosphorylates a peptide containing Thr 97 of Pop2p and identified it as Yak1p, a DYRK family kinase. Phosphorylation of Pop2p was barely detectable in a yak1 strain. We found that Yak1p interacted with Bmh1p and Bmh2p only in the presence of glucose. A GFP-Yak1p fusion protein shuttled rapidly between the nucleus and the cytoplasm in response to glucose. A strain with alanine substituted for Thr 97 in Pop2p showed overgrowth in the postdiauxic transition and failed to stop the cell cycle at G 1 phase in response to glucose deprivation. Thus, Yak1p and Pop2p are part of a novel glucose-sensing system in yeast that is involved in growth control in response to glucose availability.
In a previous study, we found an unknown element that caused growth inhibition after its copy num... more In a previous study, we found an unknown element that caused growth inhibition after its copy number increased in the 3 0 region of DIE2 in Saccharomyces cerevisiae. In this study, we further identified this element and observed that overexpression of a small protein (sORF2) of 57 amino acids encoded in this region caused growth inhibition. The transcrip-tional response and multicopy suppression of the growth inhibition caused by sORF2 over-expression suggest that sORF2 overexpression inhibits the ergosterol biosynthetic pathway. sORF2 was not required in the normal growth of S. cerevisiae, and not conserved in related yeast species including S. paradoxus. Thus, sORF2 (designated as OTO1) is an orphan ORF that determines the specificity of this species.
Gene overexpression beyond a permissible limit causes defects in cellular functions. However, the... more Gene overexpression beyond a permissible limit causes defects in cellular functions. However, the permissible limits of most genes are unclear. Previously, we developed a genetic method designated genetic tug-of-war (gTOW) to measure the copy number limit of overexpression of a target gene. In the current study, we applied gTOW to the analysis of all protein-coding genes in the budding yeast Saccharomyces cerevisiae. We showed that the yeast cellular system was robust against an increase in the copy number by up to 100 copies in >80% of the genes. After frameshift and segmentation analyses, we isolated 115 dosage-sensitive genes (DSGs) with copy number limits of 10 or less. DSGs contained a significant number of genes involved in cytoskeletal organization and intracellular transport. DSGs tended to be highly expressed and to encode protein complex members. We demonstrated that the protein burden caused the dosage sensitivity of highly expressed genes using a gTOW experiment in which the open reading frame was replaced with GFP. Dosage sensitivities of some DSGs were rescued by the simultaneous increase in the copy numbers of partner genes, indicating that stoichiometric imbalances among complexes cause dosage sensitivity. The results obtained in this study will provide basic knowledge about the physiology of chromosomal abnormalities and the evolution of chromosomal composition. [Supplemental material is available for this article.]
Cells can maintain their functions despite fluctuations in intracellular parameters, such as prot... more Cells can maintain their functions despite fluctuations in intracellular parameters, such as protein activities and gene expression levels. This commonly observed biological property of cells is called robustness. On the other hand, these parameters have different limitations, each reflecting the property of the subsystem containing the parameter. The budding yeast cell cycle is quite fragile upon overexpression of CDC14, but is robust upon overexpression of ESP1. The gene products of both CDC14 and ESP1 are regulated by 1:1 binding with their inhibitors (Net1 and Pds1), and a mathematical model predicts the extreme fragility of the cell cycle upon overexpression of CDC14 and ESP1 caused by dosage imbalance between these genes. However, it has not been experimentally shown that dosage imbalance causes fragility of the cell cycle. In this study, we measured the quantitative genetic interactions of these genes by performing combinatorial ''genetic tug-of-war'' experiments. We first showed experimental evidence that dosage imbalance between CDC14 and NET1 causes fragility. We also showed that fragility arising from dosage imbalance between ESP1 and PDS1 is masked by CDH1 and CLB2. The masking function of CLB2 was stabilization of Pds1 by its phosphorylation. We finally modified Chen's model according to our findings. We thus propose that dosage imbalance causes fragility in biological systems.
Background: Construction of plasmids is crucial in modern genetic manipulation. As of now, the co... more Background: Construction of plasmids is crucial in modern genetic manipulation. As of now, the common method for constructing plasmids is to digest specific DNA sequences with restriction enzymes and to ligate the resulting DNA fragments with DNA ligase. Another potent method to construct plasmids, known as gap-repair cloning (GRC), is commonly used in the budding yeast Saccharomyces cerevisiae. GRC makes use of the homologous recombination activity that occurs within the yeast cells. Due to its flexible design and efficiency, GRC has been frequently used for constructing plasmids with complex structures as well as genome-wide plasmid collections. Although there have been reports indicating GRC feasibility in the fission yeast Schizosaccharomyces pombe, this species is not commonly used for GRC as systematic studies of reporting GRC efficiency in S. pombe have not been performed till date.
We previously determined the copy number limits of overexpression for cell division cycle (cdc) r... more We previously determined the copy number limits of overexpression for cell division cycle (cdc) regulatory genes in the fission yeast Schizosaccharomyces pombe using the ''genetic tug-of-war'' (gTOW) method. In this study, we measured the levels of tandem affinity purification (TAP)-tagged target proteins when their copy numbers are increased in gTOW. Twenty analyzed genes showed roughly linear correlations between increased protein levels and gene copy numbers, which suggested a general lack of compensation for gene dosage in S. pombe. Cdc16 and Sid2 protein levels but not their mRNA levels were much lower than that expected by their copy numbers, which suggested the existence of a post-transcriptional down regulation of these genes. The cyclin Cig1 protein level and its mRNA level were much higher than that expected by its copy numbers, which suggested a positive feedback mechanism for its expression. A higher Cdc10 protein level and its mRNA level, probably due to cloning its gene into a plasmid, indicated that Cdc10 regulation was more robust than that previously predicted.
Objectives: Ischemia-reperfusion injury after lung transplantation is associated with significant... more Objectives: Ischemia-reperfusion injury after lung transplantation is associated with significant morbidity and mortality. The activation of the transcription factor nuclear factor B is central to the 2 important pathways that characterize ischemia-reperfusion injury, namely the inflammatory response and apoptosis. The purpose of this study was to determine the effects of nuclear factor B inhibition on experimental lung transplant ischemia-reperfusion injury
Objectives: Ischemia-reperfusion injury after lung transplantation is associated with significant... more Objectives: Ischemia-reperfusion injury after lung transplantation is associated with significant morbidity and mortality. The activation of the transcription factor nuclear factor B is central to the 2 important pathways that characterize ischemia-reperfusion injury, namely the inflammatory response and apoptosis. The purpose of this study was to determine the effects of nuclear factor B inhibition on experimental lung transplant ischemia-reperfusion injury
Inhibition of nuclear factor Bb y IB superrepressor gene transfer ameliorates ischemia-reperfusion injury after experimental lung transplantation
Objectives: Ischemia-reperfusion injury after lung transplantation is associated with significant... more Objectives: Ischemia-reperfusion injury after lung transplantation is associated with significant morbidity and mortality. The activation of the transcription factor nuclear factor B is central to the 2 important pathways that characterize ischemia-reperfusion injury, namely the inflammatory response and apoptosis. The purpose of this study was to determine the effects of nuclear factor B inhibition on experimental lung transplant ischemia-reperfusion injury
Intracellular biochemical parameters, such as the expression level of gene products, are consider... more Intracellular biochemical parameters, such as the expression level of gene products, are considered to be optimized so that a biological system, including the parameters, works effectively. Those parameters should have some permissible range so that the systems have robustness against perturbations, such as noise in gene expression. However, little is known about the permissible range in real cells because there has been no experimental technique to test it. In this study, we developed a genetic screening method, named ''genetic tug-of-war'' (gTOW) that evaluates upper limit copy numbers of genes in a model eukaryote Saccharomyces cerevisiae, and we applied it for 30 cell-cycle related genes (CDC genes). The experiment provided unique quantitative data that could be used to argue the system-level properties of the cell cycle such as robustness and fragility. The data were used to evaluate the current computational model, and refinements to the model were suggested.
The terminator regions of eukaryotes encode functional elements in the 3′ untranslated region (3′... more The terminator regions of eukaryotes encode functional elements in the 3′ untranslated region (3′-UTR) that influence the 3′-end processing of mRNA, mRNA stability, and translational efficiency, which can modulate protein production. However, the contribution of these terminator regions to gene expression remains unclear, and therefore their utilization in metabolic engineering or synthetic genetic circuits has been limited. Here, we comprehensively evaluated the activity of 5302 terminator regions from a total of 5880 genes in the budding yeast Saccharomyces cerevisiae by inserting each terminator region downstream of the P TDH3-green fluorescent protein (GFP) reporter gene and measuring the fluorescent intensity of GFP. Terminator region activities relative to that of the PGK1 standard terminator ranged from 0.036 to 2.52, with a mean of 0.87. We thus could isolate the most and least active terminator regions. The activities of the terminator regions showed a positive correlation with mRNA abundance, indicating that the terminator region is a determinant of mRNA abundance. The least active terminator regions tended to encode longer 3′-UTRs, suggesting the existence of active degradation mechanisms for those mRNAs. The terminator regions of ribosomal protein genes tended to be the most active, suggesting the existence of a common regulator of those genes. The ″terminatome″ (the genome-wide set of terminator regions) thus not only provides valuable information to understand the modulatory roles of terminator regions on gene expression but also serves as a useful toolbox for the development of metabolically and genetically engineered yeast.
Background: Identifying permissible limits of intracellular parameters such as protein expression... more Background: Identifying permissible limits of intracellular parameters such as protein expression provides important information for examining robustness. In this study, we used the TEV protease-mediated induction of protein instability (TIPI) in combination with the genetic Tug-of-War (gTOW) to develop a method to measure the lower limit of protein level. We first tested the feasibility of this method using ADE2 as a marker and then analyzed some cell cycle regulators to reveal genetic interactions. Results: Using TIPI-gTOW, we successfully constructed a strain in which GFP-TDegF Ade2 was expressed at the lower limit, just sufficient to support cellular growth under the −Ade condition by accelerating degradation by TEV protease. We also succeeded in constructing a strain in which the minimal level of GFP-TDegF Cdc20 was expressed by TIPI-gTOW. Using this strain, we studied genetic interactions between cell cycle regulators and CDC20, and the result was highly consistent with the previously identified interactions. Comparison of the experimental data with predictions of a mathematical model revealed some interactions that were not implemented into the current model. Conclusions: TIPI-gTOW is useful for estimating changes in the lower limit of a protein under different conditions, such as different genetic backgrounds and environments. TIPI-gTOW is also useful for analyzing genetic interactions of essential genes whose deletion mutants cannot be obtained.
The yeast Saccharomyces cerevisiae senses glucose through two transmembrane glucose sensors, Snf3... more The yeast Saccharomyces cerevisiae senses glucose through two transmembrane glucose sensors, Snf3 and Rgt2. Extracellular glucose causes these sensors to generate an intracellular signal that induces expression of HXT genes encoding glucose transporters by inhibiting the function of Rgt1, a transcriptional repressor of HXT genes. We present the following evidence that suggests that the glucose sensors are coupled to the membrane-associated protein kinase casein kinase I (Yck1). (i) Overexpression of Yck1 leads to constitutive HXT1 expression; (ii) Yck1 (or its paralogue Yck2) is required for glucose induction of HXT1 expression; (iii) Yck1 interacts with the Rgt2 glucose sensor; and (iv) attaching the C-terminal cytoplasmic tail of Rgt2 to Yck1 results in a constitutive glucose signal. The likely targets of Yck1 in this signal transduction pathway are Mth1 and Std1, which bind to and regulate function of the Rgt1 transcription factor and bind to the C-terminal cytoplasmic domain of glucose sensors. Potential casein kinase I phosphoryla-tion sites in Mth1 and Std1 are required for normal glucose regulation of HXT1 expression, and Yck1 catalyzes phosphoryla-tion of Mth1 and Std1 in vitro. These results support a model of glucose signaling in which glucose binding to the glucose sensors causes them to activate Yck1 in the cell membrane, which then phosphorylates Mth1 and Std1 bound to the cytoplasmic face of the glucose sensors, triggering their degradation and leading to the derepression of HXT gene expression. Our results add nutrient sensing to the growing list of processes in which casein kinase I is involved.
Intracellular biochemical parameters, such as the expression level of gene products, are consider... more Intracellular biochemical parameters, such as the expression level of gene products, are considered to be optimized so that a biological system, including the parameters, works effectively. Those parameters should have some permissible range so that the systems have robustness against perturbations, such as noise in gene expression. However, little is known about the permissible range in real cells because there has been no experimental technique to test it. In this study, we developed a genetic screening method, named ''genetic tug-of-war'' (gTOW) that evaluates upper limit copy numbers of genes in a model eukaryote Saccharomyces cerevisiae, and we applied it for 30 cell-cycle related genes (CDC genes). The experiment provided unique quantitative data that could be used to argue the system-level properties of the cell cycle such as robustness and fragility. The data were used to evaluate the current computational model, and refinements to the model were suggested. Citation: Moriya H, Shimizu-Yoshida Y, Kitano H (2006) In vivo robustness analysis of cell division cycle genes in Saccharomyces cerevisiae. PLoS Genet 2(7): e111.
Cellular systems are generally robust against fluctuations of intracellular parameters such as ge... more Cellular systems are generally robust against fluctuations of intracellular parameters such as gene expression level. However, little is known about expression limits of genes required to halt cellular systems. In this study, using the fission yeast Schizosaccharomyces pombe, we developed a genetic 'tug-of-war' (gTOW) method to assess the overexpression limit of certain genes. Using gTOW, we determined copy number limits for 31 cell-cycle regulators; the limits varied from 1 to 4100. Comparison with orthologs of the budding yeast Saccharomyces cerevisiae suggested the presence of a conserved fragile core in the eukaryotic cell cycle. Robustness profiles of networks regulating cytokinesis in both yeasts (septation-initiation network (SIN) and mitotic exit network (MEN)) were quite different, probably reflecting differences in their physiologic functions. Fragility in the regulation of GTPase spg1 was due to dosage imbalance against GTPase-activating protein (GAP) byr4. Using the gTOW data, we modified a mathematical model and successfully reproduced the robustness of the S. pombe cell cycle with the model.
Robustness is one of the principles of design inherent to biological systems. Cellular robustness... more Robustness is one of the principles of design inherent to biological systems. Cellular robustness can be measured as limits of intracellular parameters such as gene expression levels. We have recently developed an experimental approach coined as genetic Tug-Of-War (gTOW), which we used to perform robustness analysis in yeast. Using gTOW, we were able to measure the upper limit of expression of gene targets. In this review, we first elaborate on how the gTOW method compares to current mathematical simulation models prevalently used in the determination of robustness. We then explain the experimental principles underlying gTOW and its associated tools, and we provide concrete examples of robustness analysis using gTOW, i.e. cell cycle and HOG pathway gene expression analysis. Finally, we list a series of Q&As related to the experimental utilization of gTOW and we describe the potential impact of gTOW and its relevance to the understanding of biological systems.
Overexpression experiments are sometimes considered as qualitative experiments designed to identi... more Overexpression experiments are sometimes considered as qualitative experiments designed to identify novel proteins and study their function. However, in order to draw conclusions regarding protein overexpression through association analyses using large-scale biological data sets, we need to recognize the quantitative nature of overexpression experiments. Here I discuss the quantitative features of two different types of overexpression experiment: absolute and relative. I also introduce the four primary mechanisms involved in growth defects caused by protein overexpression: resource overload, stoichiometric imbalance , promiscuous interactions, and pathway modulation associated with the degree of over-expression.
POP2 protein of Saccharomyces cerevisiae is a component of a protein complex that regulates the t... more POP2 protein of Saccharomyces cerevisiae is a component of a protein complex that regulates the transcription of many genes. We found that the 97th threonine residue (Thr 97) of Pop2p was phosphorylated upon glucose limitation. The Thr 97 phosphorylation occurred within 2 min after removing glucose and was reversed within 1 min after the readdition of glucose. The effects of hexokinase mutations and glucose analogs indicate that this phosphorylation is dependent on glucose phosphorylating activity. We purified a protein kinase that phosphorylates a peptide containing Thr 97 of Pop2p and identified it as Yak1p, a DYRK family kinase. Phosphorylation of Pop2p was barely detectable in a yak1 strain. We found that Yak1p interacted with Bmh1p and Bmh2p only in the presence of glucose. A GFP-Yak1p fusion protein shuttled rapidly between the nucleus and the cytoplasm in response to glucose. A strain with alanine substituted for Thr 97 in Pop2p showed overgrowth in the postdiauxic transition and failed to stop the cell cycle at G 1 phase in response to glucose deprivation. Thus, Yak1p and Pop2p are part of a novel glucose-sensing system in yeast that is involved in growth control in response to glucose availability.
In a previous study, we found an unknown element that caused growth inhibition after its copy num... more In a previous study, we found an unknown element that caused growth inhibition after its copy number increased in the 3 0 region of DIE2 in Saccharomyces cerevisiae. In this study, we further identified this element and observed that overexpression of a small protein (sORF2) of 57 amino acids encoded in this region caused growth inhibition. The transcrip-tional response and multicopy suppression of the growth inhibition caused by sORF2 over-expression suggest that sORF2 overexpression inhibits the ergosterol biosynthetic pathway. sORF2 was not required in the normal growth of S. cerevisiae, and not conserved in related yeast species including S. paradoxus. Thus, sORF2 (designated as OTO1) is an orphan ORF that determines the specificity of this species.
Gene overexpression beyond a permissible limit causes defects in cellular functions. However, the... more Gene overexpression beyond a permissible limit causes defects in cellular functions. However, the permissible limits of most genes are unclear. Previously, we developed a genetic method designated genetic tug-of-war (gTOW) to measure the copy number limit of overexpression of a target gene. In the current study, we applied gTOW to the analysis of all protein-coding genes in the budding yeast Saccharomyces cerevisiae. We showed that the yeast cellular system was robust against an increase in the copy number by up to 100 copies in >80% of the genes. After frameshift and segmentation analyses, we isolated 115 dosage-sensitive genes (DSGs) with copy number limits of 10 or less. DSGs contained a significant number of genes involved in cytoskeletal organization and intracellular transport. DSGs tended to be highly expressed and to encode protein complex members. We demonstrated that the protein burden caused the dosage sensitivity of highly expressed genes using a gTOW experiment in which the open reading frame was replaced with GFP. Dosage sensitivities of some DSGs were rescued by the simultaneous increase in the copy numbers of partner genes, indicating that stoichiometric imbalances among complexes cause dosage sensitivity. The results obtained in this study will provide basic knowledge about the physiology of chromosomal abnormalities and the evolution of chromosomal composition. [Supplemental material is available for this article.]
Cells can maintain their functions despite fluctuations in intracellular parameters, such as prot... more Cells can maintain their functions despite fluctuations in intracellular parameters, such as protein activities and gene expression levels. This commonly observed biological property of cells is called robustness. On the other hand, these parameters have different limitations, each reflecting the property of the subsystem containing the parameter. The budding yeast cell cycle is quite fragile upon overexpression of CDC14, but is robust upon overexpression of ESP1. The gene products of both CDC14 and ESP1 are regulated by 1:1 binding with their inhibitors (Net1 and Pds1), and a mathematical model predicts the extreme fragility of the cell cycle upon overexpression of CDC14 and ESP1 caused by dosage imbalance between these genes. However, it has not been experimentally shown that dosage imbalance causes fragility of the cell cycle. In this study, we measured the quantitative genetic interactions of these genes by performing combinatorial ''genetic tug-of-war'' experiments. We first showed experimental evidence that dosage imbalance between CDC14 and NET1 causes fragility. We also showed that fragility arising from dosage imbalance between ESP1 and PDS1 is masked by CDH1 and CLB2. The masking function of CLB2 was stabilization of Pds1 by its phosphorylation. We finally modified Chen's model according to our findings. We thus propose that dosage imbalance causes fragility in biological systems.
Background: Construction of plasmids is crucial in modern genetic manipulation. As of now, the co... more Background: Construction of plasmids is crucial in modern genetic manipulation. As of now, the common method for constructing plasmids is to digest specific DNA sequences with restriction enzymes and to ligate the resulting DNA fragments with DNA ligase. Another potent method to construct plasmids, known as gap-repair cloning (GRC), is commonly used in the budding yeast Saccharomyces cerevisiae. GRC makes use of the homologous recombination activity that occurs within the yeast cells. Due to its flexible design and efficiency, GRC has been frequently used for constructing plasmids with complex structures as well as genome-wide plasmid collections. Although there have been reports indicating GRC feasibility in the fission yeast Schizosaccharomyces pombe, this species is not commonly used for GRC as systematic studies of reporting GRC efficiency in S. pombe have not been performed till date.
We previously determined the copy number limits of overexpression for cell division cycle (cdc) r... more We previously determined the copy number limits of overexpression for cell division cycle (cdc) regulatory genes in the fission yeast Schizosaccharomyces pombe using the ''genetic tug-of-war'' (gTOW) method. In this study, we measured the levels of tandem affinity purification (TAP)-tagged target proteins when their copy numbers are increased in gTOW. Twenty analyzed genes showed roughly linear correlations between increased protein levels and gene copy numbers, which suggested a general lack of compensation for gene dosage in S. pombe. Cdc16 and Sid2 protein levels but not their mRNA levels were much lower than that expected by their copy numbers, which suggested the existence of a post-transcriptional down regulation of these genes. The cyclin Cig1 protein level and its mRNA level were much higher than that expected by its copy numbers, which suggested a positive feedback mechanism for its expression. A higher Cdc10 protein level and its mRNA level, probably due to cloning its gene into a plasmid, indicated that Cdc10 regulation was more robust than that previously predicted.
Objectives: Ischemia-reperfusion injury after lung transplantation is associated with significant... more Objectives: Ischemia-reperfusion injury after lung transplantation is associated with significant morbidity and mortality. The activation of the transcription factor nuclear factor B is central to the 2 important pathways that characterize ischemia-reperfusion injury, namely the inflammatory response and apoptosis. The purpose of this study was to determine the effects of nuclear factor B inhibition on experimental lung transplant ischemia-reperfusion injury
Objectives: Ischemia-reperfusion injury after lung transplantation is associated with significant... more Objectives: Ischemia-reperfusion injury after lung transplantation is associated with significant morbidity and mortality. The activation of the transcription factor nuclear factor B is central to the 2 important pathways that characterize ischemia-reperfusion injury, namely the inflammatory response and apoptosis. The purpose of this study was to determine the effects of nuclear factor B inhibition on experimental lung transplant ischemia-reperfusion injury
Inhibition of nuclear factor Bb y IB superrepressor gene transfer ameliorates ischemia-reperfusion injury after experimental lung transplantation
Objectives: Ischemia-reperfusion injury after lung transplantation is associated with significant... more Objectives: Ischemia-reperfusion injury after lung transplantation is associated with significant morbidity and mortality. The activation of the transcription factor nuclear factor B is central to the 2 important pathways that characterize ischemia-reperfusion injury, namely the inflammatory response and apoptosis. The purpose of this study was to determine the effects of nuclear factor B inhibition on experimental lung transplant ischemia-reperfusion injury
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Papers by Hisao Moriya