担当区分：最終著者   掲載種別：研究論文（学術雑誌）   出版者・発行元：Public Library of Science (PLoS)  

DOI： 10.1371/journal.pgen.1009091

researchmap

担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

Extreme overproduction of gratuitous proteins can overload cellular protein production resources, leading to growth defects, a phenomenon known as the protein burden/cost effect. Genetic screening in the budding yeast Saccharomyces cerevisiae has isolated several dubious ORFs whose deletions mitigated the protein burden effect, but individual characterization thereof has yet to be delineated. We found that deletion of the YJL175W ORF yielded an N-terminal deletion of Swi3, a subunit of the SWI/SNF chromatin remodeling complex, and partial loss of function of Swi3. The deletion mutant showed a reduction in transcription of genes encoding highly expressed, secreted proteins and an overall reduction in translation. Mutations in the chromatin remodeling complex could thus mitigate the protein burden effect, likely by reallocating residual cellular resources used to overproduce proteins. This cellular state might also be related to cancer cells, as they frequently harbor mutations in the SWI/SNF complex.

DOI： 10.1038/s41598-020-66307-z

PubMed

researchmap

掲載種別：研究論文（学術雑誌）   出版者・発行元：Springer Science and Business Media LLC  

DOI： 10.1007/s10126-020-09976-1

PubMed

researchmap

その他リンク： https://link.springer.com/article/10.1007/s10126-020-09976-1/fulltext.html

担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

Protein overexpression sometimes causes cellular defects, although the underlying mechanism is still unknown. A protein's expression limit, which triggers cellular defects, is a useful indication of the underlying mechanism. In this study, we developed an experimental method of estimating the expression limits of target proteins in the human embryonic kidney cell line HEK293 by measuring the proteins' expression levels in cells that survived after the high-copy introduction of plasmid DNA by which the proteins were expressed under a strong cytomegalovirus promoter. The expression limits of nonfluorescent target proteins were indirectly estimated by measuring the levels of green fluorescent protein (GFP) connected to the target proteins with the self-cleaving sequence P2A. The expression limit of a model GFP was ~5.0% of the total protein, and sustained GFP overexpression caused cell death. The expression limits of GFPs with mitochondria-targeting signals and endoplasmic reticulum localization signals were 1.6% and 0.38%, respectively. The expression limits of four proteins involved in vesicular trafficking were far lower compared to a red fluorescent protein. The protein expression limit estimation method developed will be valuable for defining toxic proteins and consequences of protein overexpression.

DOI： 10.1038/s41598-020-61646-3

PubMed

researchmap

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Benzyl isothiocyanate (BITC) is a naturally-occurring isothiocyanate derived from cruciferous vegetables. BITC has been reported to inhibit the proliferation of various cancer cells, which is believed to be important for the inhibition of tumorigenesis. However, the detailed mechanisms of action remain unclear. In this study, we employed a budding yeast Saccharomyces cerevisiae as a model organism for screening. Twelve genes including MTW1 were identified as the overexpression suppressors for the antiproliferative effect of BITC using the genome-wide multi-copy plasmid collection for S. cerevisiae. Overexpression of the kinetochore protein Mtw1 counteracts the antiproliferative effect of BITC in yeast. The inhibitory effect of BITC on the proliferation of human colon cancer HCT-116 cells was consistently suppressed by the overexpression of Mis12, a human orthologue of Mtw1, and enhanced by the knockdown of Mis12. We also found that BITC increased the phosphorylated and ubiquitinated Mis12 level with consequent reduction of Mis12, suggesting that BITC degrades Mis12 through an ubiquitin-proteasome system. Furthermore, cell cycle analysis showed that the change in the Mis12 level affected the cell cycle distribution and the sensitivity to the BITC-induced apoptosis. These results provide evidence that BITC suppresses cell proliferation through the post-transcriptional regulation of the kinetochore protein Mis12.

DOI： 10.1038/s41598-019-45248-2

PubMed

researchmap

担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

The ultimate overexpression of a protein could cause growth defects, which are known as the protein burden. However, the expression limit at which the protein-burden effect is triggered is still unclear. To estimate this limit, we systematically measured the overexpression limits of glycolytic proteins in Saccharomyces cerevisiae. The limits of some glycolytic proteins were up to 15% of the total cellular protein. These limits were independent of the proteins' catalytic activities, a finding that was supported by an in silico analysis. Some proteins had low expression limits that were explained by their localization and metabolic perturbations. The codon usage should be highly optimized to trigger the protein-burden effect, even under strong transcriptional induction. The S-S-bond-connected aggregation mediated by the cysteine residues of a protein might affect its expression limit. Theoretically, only non-harmful proteins could be expressed up to the protein-burden limit. Therefore, we established a framework to distinguish proteins that are harmful and non-harmful upon overexpression.

DOI： 10.7554/eLife.34595

PubMed

researchmap

その他リンク： http://orcid.org/0000-0001-7638-3640

記述言語：英語   掲載種別：研究論文（学術雑誌）  

The yeast S. cerevisiae senses glucose through Snf3 and Rgt2, transmembrane proteins that generate an intracellular signal in response to glucose that leads to inhibition of the Rgt1 transcriptional repressor and consequently to derepression of HXT genes encoding glucose transporters. Snf3 and Rgt2 are thought to be glucose receptors because they are similar to glucose transporters. In contrast to glucose transporters, they have unusually long C-terminal tails that bind to Mth1 and Std1, paralogous proteins that regulate function of the Rgt1 transcription factor. We show that the C-terminal tail of Rgt2 is not responsible for its inability to transport glucose. To gain insight into how the glucose sensors generate an intracellular signal, we identified RGT2 mutations that cause constitutive signal generation. Most of the mutations alter evolutionarily-conserved amino acids in the transmembrane spanning regions of Rgt2 that are predicted to be involved in maintaining an outward-facing conformation or to be in the substrate binding site. Our analysis of these mutations suggests they cause Rgt2 to adopt inward-facing or occluded conformations that generate the glucose signal. These results support the idea that Rgt2 and Snf3 are glucose receptors that signal in response to binding of extracellular glucose and inform the basis of their signaling.

DOI： 10.1534/g3.118.200338

PubMed

researchmap

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：OXFORD UNIV PRESS  

We used cells of the yeast Saccharomyces cerevisiae expressing green fluorescent protein (GFP) as fluorescently labelled prey to assess the phagocytic activities of the mixotrophic ciliate Paramecium bursaria, which harbours symbiotic Chlorella-like algae. Because of different fluorescence spectra of GFP and algal chlorophyll, ingested GFP-expressing yeast cells can be distinguished from endosymbiotic algal cells and directly counted in individual P. bursaria cells using fluorescence microscopy. By using GFP-expressing yeast cells, we found that P. bursaria altered ingestion activities under different physiological conditions, such as different growth phases or the presence/absence of endosymbionts. Use of GFP-expressing yeast cells allowed us to estimate the digestion rates of live prey of the ciliate. In contrast to the ingestion activities, the digestion rate within food vacuoles was not affected by the presence of endosymbionts, consistent with previous findings that food and perialgal vacuoles are spatially and functionally separated in P. bursaria. Thus, GFP-expressing yeast may provide a valuable tool to assess both ingestion and digestion activities of ciliates that feed on eukaryotic organisms.

DOI： 10.1093/femsle/fnx117

Web of Science

PubMed

researchmap

担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：PUBLIC LIBRARY SCIENCE  

Understanding buffering mechanisms for various perturbations is essential for understanding robustness in cellular systems. Protein-level dosage compensation, which arises when changes in gene copy number do not translate linearly into protein level, is one mechanism for buffering against genetic perturbations. Here, we present an approach to identify genes with dosage compensation by increasing the copy number of individual genes using the genetic tug-of-war technique. Our screen of chromosome I suggests that dosage-compensated genes constitute approximately 10% of the genome and consist predominantly of sub-units of multi-protein complexes. Importantly, because subunit levels are regulated in a stoichiometry-dependent manner, dosage compensation plays a crucial role in maintaining subunit stoichiometries. Indeed, we observed changes in the levels of a complex when its subunit stoichiometries were perturbed. We further analyzed compensation mechanisms using a proteasome-defective mutant as well as ribosome profiling, which provided strong evidence for compensation by ubiquitin-dependent degradation but not reduced translational efficiency. Thus, our study provides a systematic understanding of dosage compensation and highlights that this post-translational regulation is a critical aspect of robustness in cellular systems.

DOI： 10.1371/journal.pgen.1006554

Web of Science

researchmap

その他リンク： http://orcid.org/0000-0001-7638-3640

担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：NATURE PUBLISHING GROUP  

High-level expression of a protein localized to an intracellular compartment is expected to cause cellular defects because it overloads localization processes. However, overloads of localization processes have never been studied systematically. Here, we show that the expression levels of green fluorescent proteins (GFPs) with localization signals were limited to the same degree as a toxic misfolded GFP in budding yeast cells, and that their high-level expression caused cellular defects associated with localization processes. We further show that limitation of the exportin Crm1 determined the expression limit of GFP with a nuclear export signal. Although misfolding of GFP with a vesicle-mediated transport signal triggered endoplasmic reticulum stress, it was not the primary determinant of its expression limit. The precursor of GFP with a mitochondrial targeting signal caused a cellular defect. Finally, we estimated the residual capacities of localization processes. High-level expression of a localized protein thus causes cellular defects by overloading the capacities of localization processes.

DOI： 10.1038/srep31774

Web of Science

PubMed

researchmap

担当区分：筆頭著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

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 overexpression.

DOI： 10.1091/mbc.E15-07-0512

PubMed

researchmap

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Aneuploidy-the gain or loss of one or more whole chromosome-typically has an adverse impact on organismal fitness, manifest in conditions such as Down syndrome. A central question is whether aneuploid phenotypes are the consequence of copy number changes of a few especially harmful genes that may be present on the extra chromosome or are caused by copy number alterations of many genes that confer no observable phenotype when varied individually. We used the proliferation defect exhibited by budding yeast strains carrying single additional chromosomes (disomes) to distinguish between the "few critical genes" hypothesis and the "mass action of genes" hypothesis. Our results indicate that subtle changes in gene dosage across a chromosome can have significant phenotypic consequences. We conclude that phenotypic thresholds can be crossed by mass action of copy number changes that, on their own, are benign.

DOI： 10.1101/gad.261743.115

PubMed

researchmap

その他リンク： http://orcid.org/0000-0001-7638-3640

担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

In a previous study, we found an unknown element that caused growth inhibition after its copy number increased in the 3' 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 transcriptional response and multicopy suppression of the growth inhibition caused by sORF2 overexpression 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.

DOI： 10.1371/journal.pone.0120678

PubMed

J-GLOBAL

researchmap

DOI： 10.1038/nchembio.1366

Web of Science

researchmap

担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：BIOMED CENTRAL LTD  

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.

DOI： 10.1186/1752-0509-8-2

Web of Science

PubMed

J-GLOBAL

researchmap

記述言語：英語   掲載種別：研究論文（国際会議プロシーディングス）   出版者・発行元：Springer Verlag  

This study considers formal methods for finding unknown interactions of incomplete molecular networks using microarray profiles. In systems biology, a challenging problem lies in the growing scale and complexity of molecular networks. Along with high-throughput experimental tools, it is not straightforward to reconstruct huge and complicated networks using observed data by hand. Thus, we address the completion problem of our target networks represented by a standard markup language, called SBGN (in particular, Activity Flow). Our proposed method is based on logic-based hypothesis finding techniques
given an input SBGN network and its profile data, missing interactions can be logically generated as hypotheses by the proposed method. In this paper, we also show empirical results that demonstrate how the proposed method works with a real network involved in the glucose repression of S. cerevisiae. © Springer International Publishing Switzerland 2014.

DOI： 10.1007/978-3-319-10398-3_14

Scopus

researchmap

DOI： 10.1038/nchembio.1366

PubMed

researchmap

担当区分：責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1021/sb300116y

Web of Science

PubMed

J-GLOBAL

researchmap

担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT  

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.

DOI： 10.1101/gr.146662.112

Web of Science

PubMed

J-GLOBAL

researchmap

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：JAPAN SOC ANALYTICAL CHEMISTRY  

A microchip-based real-time polymerase chain reaction (PCR) device has been developed for the genetic tug-of-war (gTOW) method that provides quantitative data for research on biorobustness and systems biology. The device was constructed of a silicon glass chip, a temperature controlling Peltier element, and a microscope. A parallel real-time amplification process of target genes on the plasmids and the housekeeping genes in a model eukaryote Saccharomyces cerevisiae were detected simultaneously, and the copy number of the target genes were estimated. The device provides unique quantitative data that can be used to augment understanding of the system-level properties of living cells.

DOI： 10.2116/analsci.29.367

Web of Science

PubMed

J-GLOBAL

researchmap

担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：PUBLIC LIBRARY SCIENCE  

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.

DOI： 10.1371/journal.pone.0073319

Web of Science

PubMed

J-GLOBAL

researchmap

担当区分：筆頭著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ROYAL SOC CHEMISTRY  

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.

DOI： 10.1039/c2mb25100k

Web of Science

PubMed

J-GLOBAL

researchmap

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：WILEY-V C H VERLAG GMBH  

Fungal genome sequencing has revealed many genes coding for biosynthetic enzymes, including polyketide synthases and nonribosomal peptide synthetases. However, characterizing these enzymes and identifying the compounds they synthesize remains a challenge, whether the genes are expressed in their original hosts or in more tractable heterologous hosts, such as yeast. Here, we developed a streamlined method for isolating biosynthetic genes from fungal sources and producing bioactive molecules in an engineered Saccharomyces cerevisiae host strain. We used overlap extension PCR and yeast homologous recombination to clone desired fungal polyketide synthase or a nonribosomal peptide synthetase genes (5-20 kb) into a yeast expression vector quickly and efficiently. This approach was used successfully to clone five polyketide synthases and one nonribosomal peptide synthetase, from various fungal species. Subsequent detailed chemical characterizations of the resulting natural products identified six polyketide and two nonribosomal peptide products, one of which was a new compound. Our system should facilitate investigating uncharacterized fungal biosynthetic genes, identifying novel natural products, and rationally engineering biosynthetic pathways for the production of enzyme analogues possessing modified bioactivity.

DOI： 10.1002/cbic.201100798

Web of Science

PubMed

researchmap

担当区分：筆頭著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：WILEY  

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 >100. 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.

DOI： 10.1038/msb.2011.91

Web of Science

PubMed

J-GLOBAL

researchmap

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：NATURE PUBLISHING GROUP  

With the accumulation of data on complex molecular machineries coordinating cell-cycle dynamics, coupled with its central function in disease patho-physiologies, it is becoming increasingly important to collate the disparate knowledge sources into a comprehensive molecular network amenable to systems-level analyses. In this work, we present a comprehensive map of the budding yeast cell-cycle, curating reactions from ∼600 original papers. Toward leveraging the map as a framework to explore the underlying network architecture, we abstract the molecular components into three planes--signaling, cell-cycle core and structural planes. The planar view together with topological analyses facilitates network-centric identification of functions and control mechanisms. Further, we perform a comparative motif analysis to identify around 194 motifs including feed-forward, mutual inhibitory and feedback mechanisms contributing to cell-cycle robustness. We envisage the open access, comprehensive cell-cycle map to open roads toward community-based deeper understanding of cell-cycle dynamics.

DOI： 10.1038/msb.2010.73

Web of Science

PubMed

J-GLOBAL

researchmap

担当区分：責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：PUBLIC LIBRARY SCIENCE  

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.

DOI： 10.1371/journal.pgen.1000919

Web of Science

PubMed

J-GLOBAL

researchmap

担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：PUBLIC LIBRARY SCIENCE  

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.
Methodology/Principal Findings: We investigated GRC efficiency in S. pombe in this study. We first showed that GRC was feasible in S. pombe by constructing a plasmid that contained the LEU2 auxotrophic marker gene in vivo and showed sufficient efficiency with short homology sequences (>25 bp). No preference was shown for the sequence length from the cut site in the vector plasmid. We next showed that plasmids could be constructed in a proper way using 3 DNA fragments with 70% efficiency without any specific selections being made. The GRC efficiency with 3 DNA fragments was dramatically increased >95% in lig4 Delta mutant cell, where non-homologous end joining is deficient. Following this approach, we successfully constructed plasmid vectors with leu1+, ade6+, his5+, and lys1+ markers with the low-copy stable plasmid pDblet as a backbone by applying GRC in S. pombe.
Conclusions/Significance: We concluded that GRC was sufficiently feasible in S. pombe for genome-wide gene functional analysis as well as for regular plasmid construction. Plasmids with different markers constructed in this research are available from NBRP-yeast (http://yeast.lab.nig.ac.jp/).

DOI： 10.1371/journal.pone.0009652

Web of Science

PubMed

J-GLOBAL

researchmap

記述言語：英語   掲載種別：研究論文（国際会議プロシーディングス）  

A micro-chip based real-time PCR device has been developed for biorobustness research and systems biology. The device is constructed of a silicon-glass chip, a Peltier element and a microscopy. With the device, amplification of target gene on a plasmid and housekeeping gene were detected simultaneously, and the copy number of the plasmid could be estimated. We had optimized the device, and achieved detection of fluorescent intensity increase in reaction channels. © 2009 CBMS.

Scopus

researchmap

担当区分：筆頭著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：PUBLIC LIBRARY SCIENCE  

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.

DOI： 10.1371/journal.pgen.0020111

Web of Science

PubMed

J-GLOBAL

researchmap

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC MICROBIOLOGY  

Expression of the HXT genes encoding glucose transporters in the budding yeast Saccharomyces cerevisiae is regulated by two interconnected glucose-signaling pathways: the Snf3/Rgt2-Rgt1 glucose induction pathway and the Snf1-Mig1 glucose repression pathway. The Snf3 and Rgt2 glucose sensors in the membrane generate a signal in the presence of glucose that inhibits the functions of Std1 and Mth1, paralogous proteins that regulate the function of the Rgt1 transcription factor, which binds to the HXT promoters. It is well established that glucose induces degradation of Mth1, but the fate of its paralogue Std1 has been less clear. We present evidence that glucose-induced degradation of Std1 via the SCFGrr1 ubiquitin-protein ligase and the 26S proteasome is obscured by feedback regulation of STD1 expression. Disappearance of Stdl in response to glucose is accelerated when glucose induction of STD1 expression due to feedback regulation by Rgt1 is prevented. The consequence of relieving feedback regulation of STD1 expression is that reestablishment of repression of HXT1 expression upon removal of glucose is delayed. In contrast, degradation of Mth1 is reinforced by glucose repression of MTH1 expression: disappearance of Mth1 is slowed when glucose repression of MTH1 expression is prevented, and this results in a delay in induction of HXT3 expression in response to glucose. Thus, the cellular levels of Std1 and Mth1, and, as a consequence, the kinetics of induction and repression of HXT gene expression, are closely regulated by interwoven transcriptional and posttranslational controls mediated by two different glucose-sensing pathways.

DOI： 10.1128/EC.5.1.167-173.2006

Web of Science

PubMed

J-GLOBAL

researchmap

記述言語：英語   掲載種別：研究論文（国際会議プロシーディングス）   出版者・発行元：IEEE  

This paper discusses a robustness analysis of eukaryotic cell cycle and focuses on understanding functions of Cdc25 and Wee1 proteins. The robustness of the eukaryotic cell cycle is analyzed based on the sensitivity analysis for a mathematical model. From the first analysis result, it was shown that Cdc2 and Cyclin proteins have main roles for eukaryotic cell cycle in this model but the robustness is not high against perturbation on its parameters. By introducing Cdc25 and Weel proteins to the mathematical model, it was verified by the sensitivity analysis that the modified has higher level of robustnesses; than the original model does. Numerical examples are shown to demonstrate the original model and the modified model have almost identical cell cycle behaviors leaving robustness as a salient difference.

Web of Science

researchmap

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：MOSBY, INC  

Objectives: Ischemia-reperfusion injury after lung transplantation is associated with significant morbidity and mortality. The activation of the transcription factor nuclear factor kappa 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 kappa B inhibition on experimental lung transplant ischemia-reperfusion injury with gene transfer of the nuclear factor kappa B inhibitor I kappa B in a superrepressor form (I kappa BSR).
Methods: An orthotopic left lung transplant model in isogeneic rats was used, with 18 hours of prolonged cold storage of donor lung grafts used to create severe ischemia-reperfusion injury. Donor rats underwent endobronchial gene transfection with saline alone or adenovirus encoding either beta-galactosidase control or I kappa BSR 48 hours before harvest. The function of transplanted lung grafts was assessed on the basis of isolated graft oxygenation, wet/dry lung weight ratio, and myeloperoxidase activity. Nuclear factor kappa B activation was assessed by means of enzyme-linked immunosorbent assay. Apoptotic cell death was assessed by evaluating the levels of histone-associated DNA fragments and caspase-3 activity.
Results: Treatment of donor lung grafts with I kappa BSR resulted in significantly improved oxygenation compared with that seen in control tissue 24 hours after transplantation. I kappa BSR-treated lungs also demonstrated less pulmonary edema and reduced neutrophil infiltration 24 hours after reperfusion. Nuclear factor kappa B activation and apoptotic cell death induction 2 hours after transplantation was significantly reduced in I kappa BSR-treated lungs compared with in control lungs.
Conclusions: Inhibition of nuclear factor kappa B activation by I kappa BSR gene transfer improves transplanted lung graft oxygenation, decreases pulmonary edema and neutrophil sequestration, and reduces apoptotic cell death after experimental lung transplantation.

DOI： 10.1016/j.jtcvs.2005.02.040

Web of Science

PubMed

J-GLOBAL

researchmap

担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：NATL ACAD SCIENCES  

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 HXTgenes 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 phosphorylation sites in Mth1 and Std1 are required for normal glucose regulation of HXT1 expression, and Yck1 catalyzes phosphorylation 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 Yckl in the cell membrane, which then phosphorylates Mthl and Stdl 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.

DOI： 10.1073/pnas.0305901101

Web of Science

PubMed

J-GLOBAL

researchmap

担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：COLD SPRING HARBOR LAB PRESS  

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 barfly detectable in a yak1 Delta 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.

DOI： 10.1101/gad.884001

Web of Science

researchmap

担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：WILEY-BLACKWELL  

The DHH1 gene of Saccharomyces cerevisiae belongs to a family of genes that encode highly conserved DEAD-box proteins commonly present in various eukaryotic organisms. Its precise function in yeast has not yet been well documented. To investigate its role in vivo, we constructed a DHH1 disruptant, characterized it genetically and searched for genes the mutations in which would cause synthetic lethality in combination with the DHH1 disruption. CDC28, ELM1 and SSD1 were thus found to be such candidates and we subsequently analysed their interactions. Mutations in ELM1 were previously reported to result in the elongation of cells. We confirmed this phenotype and observed in addition elongated bud formation in an Elm1p overproducing strain. Also, Elm1p fused with the green fluorescent protein (GFP) was found to be localized at the bud neck. These and other observations seem to suggest that Elm1p plays a role during cytokinesis in S. cerevisiae. The phenotypes of strains harbouring either Delta dhh1 Delta elm1 or ssd1-d Delta elm1 were very similar to each other, showing abnormal cellular morphology and defects in cytokinesis and mitosis. Furthermore, DHH1 and SSD1 could functionally complement each other in the ade2 red colour pigment formation, hypersensitivity to SDS, growth on synthetic media and at high temperature. A triple mutant, Delta dhh1 ssd1-d Delta elm1, apparently had very fragile cell walls and could grow only in a medium supplemented with 1 M sorbitol. Copyright (C) 1999 John Wiley & Sons, Ltd.

DOI： 10.1002/(SICI)1097-0061(199904)15:6<481::AID-YEA391>3.0.CO;2-M

Web of Science

researchmap

担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：OXFORD UNIV PRESS UNITED KINGDOM  

During the course of systematic nucleotide sequence analysis of the terC region of E.coli K-12 by using the ordered lambda phage clones, we found the presence of a gene, termed hrpA, that showed a high degree of sequence similarity to the PRP2, PRP16 and PRP22 genes of Saccharomyces cerevisiae, The products of these yeast genes are known to play their roles in mRNA splicing, and belong to a group of proteins collectively called the DEAH family, The hrpA gene is the first example of a DEAR family gene in prokaryotes, The N-terminal region of the protein it encodes contains conserved sequence stretches characteristic of an RNA helicase, Its molecular mass is calculated to be 146 kDa, Previously, a 135 kDa protein was identified by Moir at al, [J. Bacteriol, (1992) 174, 2102-2110] in this region which is most likely identical to that encoded by hrpA. The C-terminal region of the hrpA gene product seems to contain an RNA binding motif weakly resembling that of ribosomal protein S1 of E.coli, Disruption of the hrpA gene suggested that it is not essential for the growth of E.coli.

DOI： 10.1093/nar/23.4.595

Web of Science

researchmap

記述言語：日本語   出版者・発行元：学研メディカル秀潤社 ; 1982-  

CiNii Article

CiNii Books

researchmap

記述言語：日本語   出版者・発行元：日本プロテオーム学会（日本ヒトプロテオーム機構）  

DOI： 10.14889/jhupo.2014.0.32.0

CiNii Article

researchmap

記述言語：日本語   出版者・発行元：学研メディカル秀潤社 ; 1982-  

CiNii Article

CiNii Books

researchmap

記述言語：日本語   出版者・発行元：一般社団法人 日本生物物理学会  

DOI： 10.2142/biophys.53.323

CiNii Article

CiNii Books

researchmap

記述言語：日本語  

J-GLOBAL

researchmap

記述言語：日本語   出版者・発行元：学研メディカル秀潤社  

CiNii Article

CiNii Books

J-GLOBAL

researchmap

記述言語：日本語   出版者・発行元：日本農芸化学会  

近年，天然物の生合成遺伝子の情報をもとに，有用天然物を獲得する取り組みが行なわれるようになってきた．中でも植物および真菌といった真核生物から単離された生物活性物質の生物合成に関する報告がなされてきた．ここでは，特に真核生物由来の天然物生合成遺伝子を出芽酵母の異種発現系を用いて発現させ，有用物質の生産に挑戦する取り組みについて解説する．

DOI： 10.1271/kagakutoseibutsu.50.163

CiNii Article

CiNii Books

researchmap

記述言語：日本語   出版者・発行元：社団法人 日本農芸化学会  

DOI： 10.1271/kagakutoseibutsu.47.269

CiNii Article

CiNii Books

researchmap

記述言語：日本語   出版者・発行元：東京化学同人  

CiNii Article

CiNii Books

researchmap

記述言語：英語   出版者・発行元：PUBLIC LIBRARY SCIENCE  

DOI： 10.1371/journal.pgen.0020218

Web of Science

researchmap

記述言語：日本語   出版者・発行元：システム制御情報学会  

DOI： 10.11509/isciesci.50.8_309

CiNii Article

CiNii Books

researchmap

記述言語：日本語   出版者・発行元：バイオインダストリー協会  

CiNii Article

CiNii Books

researchmap