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西田 敬二先端バイオ工学研究センター教授
研究者基本情報
■ 学位■ 研究ニュース
- 2022年08月08日, 最高精度かつ最小サイズの塩基編集技術の実現
- 2020年12月01日, トマトの複数遺伝子の同時ゲノム編集に成功 〜ゲノム編集技術Target-AIDによる効率的品種改良〜
- 2020年06月02日, 狙ったDNA配列にC→TとA→Gの同時塩基置換を誘導する新ゲノム編集技術
- 2019年10月04日, 西田敬二教授が第43回井植文化賞を受賞しました
- 2017年11月30日, 西田敬二教授が「科学技術への顕著な貢献2017（ナイスステップな研究者）」に選定されました
- 2017年09月22日, 西田 敬二（NISHIDA Keiji）――「研究と起業の両面で世の中の役に立つ！」ゲノム編集の最前線を切り開く
- 2017年03月28日, DNAを切らずに書き換える新たなゲノム編集技術を作物に応用
- 2016年08月05日, DNAを切らずに書き換える新たなゲノム編集技術「Target-AID」の開発に成功
研究活動情報
■ 受賞- 2019年 神戸大学, 第11回学長表彰（財務貢献者）
- 2019年 井植記念会, 第43回井植文化賞（科学技術部門）
- 2018年 神戸大学, 第10回学長表彰（財務貢献者）
- 2017年 神戸大学, 第9回学長表彰（財務貢献者）
- 2017年 科学技術・学術政策研究所, 科学技術への顕著な貢献 2017, DNA 塩基書き換えによる切らないゲノム編集(Target-AID)
- 2016年 International Botanical Congress, Excellent Scholar Award
- 2007年 日本植物学会, 若手奨励賞
- 2005年 日本植物形態学会, 奨励賞
- We identified LsMybW as the allele responsible for the shift in color from black to white seeds in wild ancestors of lettuce to modern cultivars. Successfully selected white seeds are a key agronomic trait for lettuce cultivation and breeding; however, the mechanism underlying the shift from black-in its wild ancestor-to white seeds remains uncertain. We aimed to identify the gene/s responsible for white seed trait in lettuce. White seeds accumulated less proanthocyanidins than black seeds, similar to the phenotype observed in Arabidopsis TT2 mutants. Genetic mapping of a candidate gene was performed with double-digest RAD sequencing using an F2 population derived from a cross between "ShinanoPower" (white) and "Escort" (black). The white seed trait was controlled by a single recessive locus (48.055-50.197 Mbp) in linkage group 7. Using five PCR-based markers and numerous cultivars, eight candidate genes were mapped in the locus. Only the LG7_v8_49.251Mbp_HinfI marker, employing a single-nucleotide mutation in the stop codon of Lsat_1_v5_gn_7_35020.1, was completely linked to seed color phenotype. In addition, the coding region sequences for other candidate genes were identical in the resequence analysis of "ShinanoPower" and "Escort." Therefore, we proposed Lsat_1_v5_gn_7_35020.1 as the candidate gene and designated it as LsMybW (Lactuca sativa Myb White seeds), an ortholog encoding the R2R3-MYB transcription factor in Arabidopsis. When we validated the role of LsMybW through genome editing, LsMybW knockout mutants harboring an early termination codon showed a change in seed color from black to white. Therefore, LsMybW was the allele responsible for the shift in seed color. The development of a robust marker for marker-assisted selection and identification of the gene responsible for white seeds have implications for future breeding technology and physiological analysis.2024年01月, Plant cell reports, 43(2) (2), 35 - 35, 英語, 国際誌研究論文（学術雑誌）
- Clonal heterogeneity underlies diverse biological processes, including cancer progression, cell differentiation, and microbial evolution. Cell tagging strategies with DNA barcodes have recently enabled analysis of clone size dynamics and clone-restricted transcriptomic landscapes of heterogeneous populations. However, isolating a target clone that displays a specific phenotype from a complex population remains challenging. Here, we present a new multi-kingdom genetic barcoding system, CloneSelect, in which a target cell clone can be triggered to express a reporter gene for isolation through barcode-specific CRISPR base editing. In CloneSelect, cells are first barcoded and propagated so their subpopulation can be subjected to a given experiment. A clone that shows a phenotype or genotype of interest at a given time can then be isolated from the initial or subsequent cell pools stored throughout the experimental timecourse. This novel CRISPR-barcode genetics platform provides many new ways of analyzing and manipulating mammalian, yeast, and bacterial systems. Teaser A multi-kingdom CRISPR-activatable barcoding system enables the precise isolation of target barcode-labeled clones from a complex cell population.Cold Spring Harbor Laboratory, 2023年01月
- Target activation-induced cytidine deaminase (Target-AID), a novel CRISPR/Cas9-based genome-editing tool, confers the base-editing capability on the Cas9 genome-editing system. It involves the fusion of cytidine deaminase (CDA), which catalyzes cytidine (C) to uridine (U) substitutions, to the mutated nickase-type nCas9 or deactivated-type dCas9. To confirm and extend the applicability of the Target-AID genome-editing system in tomatoes (Solanum lycopersicum L.), we transformed the model tomato cultivar "Micro-Tom" and commercial tomato cultivars using this system by targeting SlDELLA, which encodes a negative regulator of the plant phytohormone gibberellic acid (GA) signaling pathway. We confirmed that the nucleotide substitutions were induced by the Target-AID system, and we isolated mutants showing high GA sensitivity in both "Micro-Tom" and the commercial cultivars. Moreover, by successfully applying this system to ETHYLENE RECEPTOR 1 (SlETR1) with single sgRNA targeting, double sgRNA targeting, as well as dual-targeting of both SlETR1 and SlETR2 with a single sgRNA, we demonstrated that the Target-AID genome-editing system is a promising tool for molecular breeding in tomato crops. This study highlights an important aspect of the scientific and agricultural potential of the combinatorial use of the Target-AID and other base-editing systems.2022年01月, Horticulture research, 9, 英語, 国際誌研究論文（学術雑誌）
- Our previous study demonstrated that Target-AID which is the modified CRISPR/Cas9 system enabling base-editing is an efficient tool for targeting multiple genes. Three genes, SlDDB1, SlDET1, and SlCYC-B, responsible for carotenoid accumulation were targeted, and allelic variations were previously obtained by Target-AID. In this research, we characterized the effect of new alleles on plant growth and fruit development, as well as carotenoid accumulation, individually in segregating backcross populations or combined in null self-segregant lines. Only lines carrying homozygous substitutions in the three targeted genes and the segregating backcross population of individual mutations were characterized, resulting in the isolation of two allelic versions for SlDDB1, one associated with SlDET1 and the last one with SlCYC-B. All edited lines showed variations in carotenoid accumulation, with an additive effect for each single mutation. These results suggest that Target-AID base-editing technology is an effective tool for creating new allelic variations in target genes to improve carotenoid accumulation in tomato.2022年, Frontiers in plant science, 13, 848560 - 848560, 英語, 国際誌研究論文（学術雑誌）
- Springer Science and Business Media LLC, 2021年12月, Scientific Reports, 11(1) (1), 21534 - 21534, 英語, 国際誌
Abstract Sugar content is one of the most important quality traits of tomato. Cell wall invertase promotes sucrose unloading in the fruit by maintaining a gradient of sucrose concentration between source leaves and fruits, while invertase inhibitor (INVINH) regulates this process. In this study, knock-out of cell wallINVINH in tomato (SlINVINH1 ) was performed by genome editing using, CRISPR/Cas9 and Target-AID technologies. Most of the genome-edited lines set higher soluble solid content (SSC) fruit than the original cultivar ‘Suzukoma’, while fruit weight was different among the genome-edited lines. From these genome-edited lines, three lines (193–3, 199–2, and 247–2), whose SSC was significantly higher than ‘Suzukoma’ and fruit weight were almost the same as the original cultivar, were selected. The fruit weight and overall plant growth of the two lines were comparable to those of the original cultivar. In contrast, the fructose and glucose contents in the mature fruits of the two lines were significantly higher than those of the original cultivar. The mature fruits of genome edited line 193–3 showed the highest sugar content, and the fructose and glucose contents were 29% and 36% higher than that of the original cultivar, respectively. Whole genome sequence data showed no off-target mutations in the genome-edited lines. Non-target metabolome analysis of mature fruits revealed that fructose was the highest loading factor in principal component analysis (PCA) between the genome-edited line and the original cultivar, and no unexpected metabolites appeared in the genome-edited line. In this study, we succeeded in producing tomato lines with high sugar content without a decrease in fruit weight and deterioration of plant growth by knock-out ofSlINVINH1 using genome editing technology. This study showed that functional disruption ofSlINVINH1 is an effective approach to produce tomato cultivars with high sugar content.研究論文（学術雑誌） - 2021年08月, The CRISPR journal, 4(4) (4), 462 - 463, 英語, 国際誌研究論文（学術雑誌）
- Abstract Structure-based rational engineering of the cytosine base editing system Target-AID was performed to minimize its off-target effect and molecular size. By intensive and careful truncation, DNA-binding domain of its deaminase PmCDA1 was eliminated and additional mutations were introduced to restore enzyme function. The resulting tCDA1EQ was effective in N-terminal fusion (AID-2S) or inlaid architecture (AID-3S) with Cas9, showing minimized gRNA-independent off-targets, as assessed in yeast and human cells. Combining with the smaller Cas9 ortholog system, the smallest cytosine base editing system was created that is within the size limit of AAV vector.Research Square Platform LLC, 2021年04月, Nature communications, 13(1) (1), 4531 - 4531, 英語, 国際誌研究論文（学術雑誌）
- KEY MESSAGE: Base editing in tomatoes was achieved by transient expression. The Solanaceae plants, particularly the tomato (Solanum lycopersicum), is of huge economic value worldwide. The tomato is a unique model plant for studying the functions of genes related to fruit ripening. Deeper understanding of tomatoes is of great importance for both plant research and the economy. Genome editing technology, such as CRISPR/Cas9, has been used for functional genetic research. However, some challenges, such as low transformation efficiency, remain with this technology. Moreover, the foreign Cas9 and gRNA expression cassettes must be removed to obtain null-segregants In this study, we used a high-level transient expression system to improve the base editing technology. A high-level transient expression system has been established previously using geminiviral replication and a double terminator. The pBYR2HS vector was used for this transient expression system. nCas9-CDA and sgRNA-SlHWS were introduced into this vector, and the protein and RNA were then transiently expressed in tomato tissues by agroinfiltration. The homozygous mutant produced by base editing was obtained in the next generation with an efficiency of about 18%. nCas9-free next-generation plants were 71%. All the homozygous base-edited plants in next generation are nCas9-free. These findings show that the high-level transient expression system is useful for base editing in tomatoes.Springer Science and Business Media LLC, 2021年04月, Plant Cell Reports, 40(4) (4), 667 - 676, 英語, 国際誌研究論文（学術雑誌）
- In metabolic engineering, genome editing tools make it much easier to discover and evaluate relevant genes and pathways and construct strains. Clustered regularly interspaced palindromic repeats (CRISPR)-associated (Cas) systems now have become the first choice for genome engineering in many organisms includingindustrially relevant ones. Targeted DNA cleavage by CRISPR-Cas provides variousgenome engineering modes such as indels, replacements, large deletions, knock-in and chromosomal rearrangements, while host-dependent differences in repair pathways need to be considered. The versatility of the CRISPR system has given rise to derivative technologies that complement nuclease-based editing, which causes cytotoxicity especially in microorganisms. Deaminase-mediated base editing installs targeted point mutations with much less toxicity. CRISPRi and CRISPRa can temporarily control gene expression without changing the genomic sequence. Multiplex, combinatorial and large scale editing are made possible by streamlined design and construction of gRNA libraries to further accelerates comprehensive discovery, evaluation and building of metabolic pathways. This review summarizes the technical basis and recent advances in CRISPR-related genome editing tools applied for metabolic engineering purposes, with representative examples of industrially relevant eukaryotic and prokaryotic organisms.Elsevier BV, 2021年01月, Metabolic Engineering, 63, 141 - 147, 英語, 国際誌研究論文（学術雑誌）
- Springer Science and Business Media LLC, 2020年12月, Scientific Reports, 10(1) (1), 20471 - 20471, 英語, 国際誌
Abstract The use of Target activation-induced cytidine deaminase (Target-AID) base-editing technology with the CRISPR-Cas 9 system fused with activation-induced cytidine deaminase (AID) resulted in the substitution of a cytidine with a thymine. In previous experiments focusing on a single target gene, this system has been reported to work in several plant species, including tomato (Solanum lycopersicum L.). In this research, we used Target-AID technology to target multiple genes related to carotenoid accumulation in tomato. We selected 3 genes,SlDDB1 ,SlDET1 andSlCYC-B, for their roles in carotenoid accumulation. Among 12 edited T0 lines, we obtained 10 independent T0 lines carrying nucleotide substitutions in the three targeted genes, with several allelic versions for each targeted gene. The two edited lines showed significant differences in carotenoid accumulation. These results demonstrate that Target-AID technology is a highly efficient tool for targeting multiple genes with several allelic versions.研究論文（学術雑誌） - Fruit set is the process whereby ovaries develop into fruits after pollination and fertilization. The process is induced by the phytohormone gibberellin (GA) in tomatoes, as determined by the constitutive GA response mutant procera However, the role of GA on the metabolic behavior in fruit-setting ovaries remains largely unknown. This study explored the biochemical mechanisms of fruit set using a network analysis of integrated transcriptome, proteome, metabolome, and enzyme activity data. Our results revealed that fruit set involves the activation of central carbon metabolism, with increased hexoses, hexose phosphates, and downstream metabolites, including intermediates and derivatives of glycolysis, the tricarboxylic acid cycle, and associated organic and amino acids. The network analysis also identified the transcriptional hub gene SlHB15A, that coordinated metabolic activation. Furthermore, a kinetic model of sucrose metabolism predicted that the sucrose cycle had high activity levels in unpollinated ovaries, whereas it was shut down when sugars rapidly accumulated in vacuoles in fruit-setting ovaries, in a time-dependent manner via tonoplastic sugar carriers. Moreover, fruit set at least partly required the activity of fructokinase, which may pull fructose out of the vacuole, and this could feed the downstream pathways. Collectively, our results indicate that GA cascades enhance sink capacities, by up-regulating central metabolic enzyme capacities at both transcriptional and posttranscriptional levels. This leads to increased sucrose uptake and carbon fluxes for the production of the constituents of biomass and energy that are essential for rapid ovary growth during the initiation of fruit set.2020年09月, Proceedings of the National Academy of Sciences of the United States of America, 117(38) (38), 23970 - 23981, 英語, 国際誌研究論文（学術雑誌）
- When cultivated rice seed fall into fields, they may overwinter and spontaneously germinate the next spring. Such germinated plants are termed "volunteer rice." Volunteer grains originating from feed rice varieties may differ in certain traits, such as quality and taste, as compared with those of rice cultivated for human consumption, which may reduce the overall quality of the final harvested grain. Many rice varieties show resistance to benzobicyclon (BBC), a beta-triketone herbicide (bTH) that inhibits 4-hydroxyphenylpyruvate dioxygenase (HPPD). Recently, the rice gene HIS1 (HPPD INHIBITOR SENSITIVE 1) conferring resistance to BBC and other bTHs was identified. In this study, to suppress the occurrence of volunteer rice infestation, we attempted to generate a BBC-sensitive rice strain via the knockout of the HIS1 gene using genome editing techniques. The production of a his1 knockout line was carried out by the start-codon substitution or stop-codon creation using CRISPR-Cas9 cytidine deaminase fusion, which is useful as a novel amino acid sequence is not generated due to the shifting of the reading frame. The mutation frequencies of independent transgenic plants were 3.6, 13.5, 13.8, and 21.2% at four gRNAs for start-codon substitution and three stop-codon creations. The his1 knockout lines were conferred with sensitivity to BBC, re-confirming by genome editing that this is indeed the gene responsible for BBC resistance/sensitivity. The his1 knockout lines also exhibited a sensitive phenotype to other bTHs, including sulcotrione, mesotrione, tembotrione, and tefuryltrione, compared with the wild-type variety 'Nipponbare.' These results demonstrate the potential of herbicide-sensitive rice produced by genome editing technology as a material to control volunteer feed rice using pre-labeled herbicides for varieties consumed by humans.Frontiers Media SA, 2020年08月, Frontiers in Plant Science, 11, 925 - 925, 英語, 国際誌[査読有り]研究論文（学術雑誌）
- We describe base editors that combine both cytosine and adenine base-editing functions. A codon-optimized fusion of the cytosine deaminase PmCDA1, the adenosine deaminase TadA and a Cas9 nickase (Target-ACEmax) showed a high median simultaneous C-to-T and A-to-G editing activity at 47 genomic targets. On-target as well as DNA and RNA off-target activities of Target-ACEmax were similar to those of existing single-function base editors.2020年07月, Nature biotechnology, 38(7) (7), 865 - 869, 英語, 国際誌[査読有り]研究論文（学術雑誌）
- An amendment to this paper has been published and can be accessed via a link at the top of the paper.2020年07月, Nature biotechnology, 38(7) (7), 901 - 901, 英語, 国際誌[査読有り]
- Technologies harnessing CRISPR systems have been rapidly evolving and expanding the capacity of researchers for understanding of mammalian cell behavior and its underlying mechanisms through genome and epigenome manipulations. In this review, we summarized the recent developments of CRISPR-based technologies for genetic and epigenetic modifications that include engineering of Cas9 for PAM simplification, non-cleaving base editing tools and alteration of gene expression. Applications such as genome-wide screening methods or CRISPR-based DNA barcoding for cellular lineage tracking are highlighted. Anticipated and upcoming development for mammalian synthetic biology that includes organelle engineering is also discussed.2019年10月, Current Opinion in Chemical Biology, 52, 79 - 84, 英語, 国際誌[査読有り][招待有り]研究論文（学術雑誌）
- The Target-AID system, consisting of a complex of cytidine deaminase and deficient CRISPR/Cas9, enables highly specific genomic nucleotide substitutions without the need for template DNA. The Cas9-fused cytidine deaminase is guided by sgRNAs and catalyzes the conversion of cytosine to uracil. The resulting U-G DNA mismatches trigger nucleotide substitutions (C to T or G to A) through DNA replication and repair pathways. Target-AID also retains the benefits of conventional CRISPR/Cas9 including robustness in various organisms, high targeting efficiency, and multiplex simultaneous gene editing. Our research group recently developed plant-optimized Target-AID system and demonstrated targeted base editing in tomato and rice. In this chapter, we introduce methods for Target-AID application in tomato.2019年04月, Methods in molecular biology (Clifton, N.J.), 1917, 297 - 307, 英語, 国際誌[査読有り]研究論文（学術雑誌）
- Genome engineering in plants is highly dependent on the availability of effective molecular techniques. Despite vast quantities of research, genome engineering in plants is still limited in terms of gene delivery, which requires the use of infectious bacteria or harsh conditions owing to the difficulty delivering biomaterial into plant cells through the cell wall. Here, we describe a method that uses electroporation-mediated protein delivery into cultured Arabidopsis thaliana cells possessing an intact cell wall, and demonstrate Cre-mediated site-specific recombination. By optimizing conditions for the electric pulse, protein concentration, and electroporation buffer, we were able to achieve efficient and less-toxic protein delivery into Arabidopsis thaliana cells with 83% efficiency despite the cell wall. To the best of our knowledge, this is the first report demonstrating the electroporation-mediated protein delivery of Cre recombinase to achieve nucleic acid-free genome engineering in plant cells possessing an intact cell wall.2019年02月, Scientific Reports, 9(1) (1), 2163 - 2163, 英語, 国際誌[査読有り]研究論文（学術雑誌）
- Kluyveromyces marxianus is a thermotolerant, crabtree-negative yeast, which preferentially directs metabolism (e.g., from the tricarboxylic acid cycle) to aerobic alcoholic fermentation. Thus K. marxianus has great potential for engineering to produce various materials under aerobic cultivation conditions. In this study, we engineered K. marxianus to produce and secrete a single-chain antibody (scFv), a product that is highly valuable but has historically proven difficult to generate at large scale. scFv production was obtained with strains carrying either plasmid-borne or genomically integrated constructs using various combinations of promoters (PMDH1 or PACO1) and secretion signal peptides (KmINUss or Scα-MFss). As the wild-type K. marxianus secretes endogenous inulinase predominantly, the corresponding INU1 gene was disrupted using a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)—associated protein (CRISPR–Cas9) system to re-direct resources to scFv production. Genomic integration was used to replace INU1 with sequences encoding a fusion of the INU1 signal peptide to scFv the resulting construct yielded the highest scFv production among the strains tested. Optimization of growth conditions revealed that scFv production by this strain was enhanced by incubation at 30 °C in xylose medium containing 200 mM MgSO4. These results together demonstrate that K. marxianus has the potential to serve as a host strain for antibody production.Springer Verlag, 2018年12月, AMB Express, 8(1) (1), 56 - 56, 英語, 国際誌[査読有り]研究論文（学術雑誌）
- Acetolactate synthase (ALS) catalyzes the initial step in the biosynthesis of branched-chain amino acids, and is highly conserved from bacteria to higher plants. ALS is encoded by a single copy gene in rice genome and is a target enzyme of several classes of herbicides. Although ALS mutations conferring herbicide-resistance property to plants are well documented, effect of Imazamox (IMZ) on rice and the mutations in ALS correlated with IMZ tolerance were unclear. In this article, the effect of IMZ on rice calli and seedlings in tissue culture conditions were evaluated. Also, the ALSA96V mutation was confirmed to improve IMZ tolerance of rice calli. Based on these results, ALS-assisted multiplex targeted base editing in rice was demonstrated in combination with Target-AID, a CRISPR/Cas9-cytidine deaminase fusion system [1], [2].2018年08月, Data in Brief, 20, 1325 - 1331, 英語, 国際誌[査読有り]研究論文（学術雑誌）
- To date, several genome editing technologies have been developed and are widely utilized in many fields of biology. Most of these technologies, if not all, use nucleases to create DNA double-strand breaks (DSBs), raising the potential risk of cell death and/or oncogenic transformation. The risks hinder their therapeutic applications in humans. Here, we show that in vivo targeted single-nucleotide editing in zebrafish, a vertebrate model organism, can be successfully accomplished with the Target-AID system, which involves deamination of a targeted cytidine to create a nucleotide substitution from cytosine to thymine after replication. Application of the system to two zebrafish genes, chordin (chd) and one-eyed pinhead (oep), successfully introduced premature stop codons (TAG or TAA) in the targeted genomic loci. The modifications were heritable and faithfully produced phenocopies of well-known homozygous mutants of each gene. These results demonstrate for the first time that the Target-AID system can create heritable nucleotide substitutions in vivo in a programmable manner, in vertebrates, namely zebrafish.2018年07月, Scientific reports, 8(1) (1), 11423 - 11423, 英語, 国際誌研究論文（学術雑誌）
- Since the emergence of programmable RNA-guided nucleases based on clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) systems, genome editing technologies have become a simplified and versatile tool for genome editing in various organisms and cell types. Although genome editing enables efficient genome manipulations, such as gene disruptions, gene knockins, and chromosomal translocations via DNA double-strand break (DSB) repair in eukaryotes, DSBs induced by the CRISPR/Cas system are lethal or severely toxic to many microorganisms. Therefore, in many prokaryotes, including industrially useful microbes, the CRISPR/Cas system is often used as a negative selection component in combination with recombineering or other related strategies. Novel and revolutionary technologies have been recently developed to re-write targeted nucleotides (C:G to T:A and A:T to G:C) without DSBs and donor DNA templates. These technologies rely on the recruitment of deaminases at specific target loci using the nuclease-deficient CRISPR/Cas system. Here, the authors review and compare CRISPR-based genome editing, current base editing platforms and their spectra. The authors discuss how these technologies can be applied in various aspects of microbial metabolic engineering to overcome barriers to cellular regulation in prokaryotes.2018年06月, Biotechnology journal, 13(9) (9), e1700596, 英語, 国際誌[査読有り]研究論文（学術雑誌）
- In eukaryotes, the CRISPR-Cas9 system has now been widely used as a revolutionary genome engineering tool1, 2. However, in prokaryotes, the use of nuclease-mediated genome editing tools has been limited to negative selection for the already modified cells because of its lethality 3, 4. Here, we report on deaminase-mediated targeted nucleotide editing (Target-AID)5 adopted in Escherichia coli. Cytidine deaminase PmCDA1 fused to the nuclease-deficient CRISPR-Cas9 system achieved specific point mutagenesis at the target sites in E. coli by introducing cytosine mutations without compromising cell growth. The cytosine-to-thymine substitutions were induced mainly within an approximately five-base window of target sequences on the protospacer adjacent motif-distal side, which can be shifted depending on the length of the single guide RNA sequence. Use of a uracil DNA glycosylase inhibitor6 in combination with a degradation tag (LVA tag)7 resulted in a robustly high mutation efficiency, which allowed simultaneous multiplex editing of six different genes. The major multi-copy transposase genes that consist of at least 41 loci were also simultaneously edited by using four target sequences. As this system does not rely on any additional or host-dependent factors, it may be readily applicable to a wide range of bacteria.Nature Publishing Group, 2018年04月, Nature Microbiology, 3(4) (4), 423 - 429, 英語, 国際誌[査読有り]研究論文（学術雑誌）
- In the production of useful microbial secondary metabolites, the breeding of strains is generally performed by random mutagenesis. However, because random mutagenesis introduces many mutations into genomic DNA, the causative mutations leading to increased productivity are mostly unknown. Therefore, although gene targeting is more efficient for breeding than random mutagenesis, it is difficult to apply. In this study, a wild-type strain and randomly mutagenized strains of fungal sp. No. 14919, a filamentous fungus producing the HMG-CoA reductase inhibitor polyketide FR901512, were subjected to point mutation analysis based on whole genome sequencing. Among the mutated genes found, mutation of the sterol regulatory element-binding protein (SREBP) cleavage-activating protein (SCAP) had a positive effect on increasing FR901512 productivity. By complementing the SCAP gene in the SCAP-mutated strain, productivity was decreased to the level of the SCAP-intact strain. Conversely, when either the SCAP or SREBP gene was deleted, the productivity was significantly increased. By genomic transcriptional analysis, the expression levels of three enzymes in the ergosterol biosynthesis pathway were shown to be decreased by SCAP mutation. These findings led to the hypothesis that raw materials of polyketides, such as acetyl-CoA and malonyl-CoA, became more available for FR901512 biosynthesis due to depression in sterol biosynthesis caused by knockout of the SREBP system. This mechanism was confirmed in Aspergillus terreus producing the polyketide lovastatin, which is structurally similar to FR901512. Thus, knockout of the SREBP system should be considered significant for increasing the productivities of polyketides, such as HMG-CoA reductase inhibitors, by filamentous fungi.Springer Verlag, 2018年02月, Applied Microbiology and Biotechnology, 102(3) (3), 1393 - 1405, 英語[査読有り]研究論文（学術雑誌）
- The CRISPR/Cas9 system is a revolutionary genome-editing tool for directed gene editing in various organisms. Cas9 variants can be applied as molecular homing devices when combined with various functional effectors such as transcriptional activators or DNA modification enzymes. Target-AID is a synthetic complex of nuclease deficient Cas9 fused to an activation-induced cytidine deaminase (AID) that enables targeted nucleotide substitution (C to T or G to A). We previously demonstrated that the introduction of desired point mutations into target genes by Target-AID confers herbicide tolerance to rice callus. Inheritance of the introduced mutations, as well as the removal of transgenes, are key issues that must be addressed in order to fully develop Target-AID as a plant breeding technique. Here we report the transmission of such mutations from the callus to regenerants and their progenies, leading to a generation of selectable marker-free (SMF) herbicide tolerant rice plants with simultaneous multiplex nucleotide substitutions. These findings demonstrate that Target-AID can be developed into novel plant breeding technology which enables improvement of multiplex traits at one time in combination with sophisticated targeted base editing with the simplicity and versatility of CRISPR/Cas9 system.Elsevier Masson SAS, 2018年, Plant Physiology and Biochemistry, 131, 78 - 83, 英語, 国際誌[査読有り]研究論文（学術雑誌）
- Mitochondria, which evolved from a free-living bacterial ancestor, contain their own genomes and genetic systems and are produced from preexisting mitochondria by binary division. The mitochondrion-dividing (MD) ring is the main skeletal structure of the mitochondrial division machinery. However, the assembly mechanism and molecular identity of the MD ring are unknown. Multi-omics analysis of isolated mitochondrial division machinery from the unicellular alga Cyanidioschyzon merolae revealed an uncharacterized glycosyltransferase, MITOCHONDRION-DIVIDING RING1 (MDR1), which is specifically expressed during mitochondrial division and forms a single ring at the mitochondrial division site. Nanoscale imaging using immunoelectron microscopy and componential analysis demonstrated that MDR1 is involved in MD ring formation and that the MD ring filaments are composed of glycosylated MDR1 and polymeric glucose nanofilaments. Down-regulation of MDR1 strongly interrupted mitochondrial division and obstructed MD ring assembly. Taken together, our results suggest that MDR1 mediates the synthesis of polyglucan nanofilaments that assemble to form the MD ring. Given that a homolog of MDR1 performs similar functions in chloroplast division, the establishment of MDR1 family proteins appears to have been a singular, crucial event for the emergence of endosymbiotic organelles.NATL ACAD SCIENCES, 2017年12月, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 114(50) (50), 13284 - 13289, 英語, 国際誌[査読有り]研究論文（学術雑誌）
- Clustered regularly interspaced short palindromic repeats (CRISPR)-mediated manipulation of genomic information is becoming more versatile by combining nuclease-deficient CRISPR systems with a wide variety of effectors including base-editing deaminases, transcriptional regulators, and epigenetic modifiers. The programmable binding ability of CRISPR systems is essential when the systems are employed as targeting domains to recruit the effectors to specific genomic loci. The discovery of a variety of Cas9 orthologs and engineered variants enables high-fidelity genome editing and a wider selection of genomic targets, and CRISPR-mediated deaminases enable more precise and predictable genome editing compared with CRISPR nuclease-based editing. Finally, combining transcriptional regulators with CRISPR systems can control expression of specific genes in a genome. Some applications and future challenges of CRISPR-derived tools are also discussed.ELSEVIER SCIENCE LONDON, 2017年10月, TRENDS IN BIOTECHNOLOGY, 35(10) (10), 983 - 996, 英語, 国際誌[査読有り]研究論文（学術雑誌）
- Kluyveromyces marxianus, a non-conventional thermotolerant yeast, is potentially useful for production of ethanol and other products. This species has a strong tendency to randomly integrate transforming DNA fragments, making necessary the development of more precise methods for gene targeting. In this study, we first demonstrated that K. marxianus NBRC1777 is cold-tolerant, and then established a highly efficient and precise technique for gene editing by introducing genes encoding deaminase-mediated targeted point mutagenesis (Target-AID) and clustered regularly interspaced short palindromic repeats (CRISPR) associated proteins (CRISPR-Cas9). We used Target-AID to introduce targeted point mutations that disrupted Nej1 or Dnl4, genes that are involved in non-homologous end-joining (NHEJ). Both of the resulting mutant strains showed enhanced proportions of homology-mediated integration compared to the wild-type parent. In combination with target cleavage by CRISPR-Cas9, markerless integration was performed using short (-50 bp) flanking homologous sequences. Together, these tools render this species fully tractable for gene manipulation, permitting targeted genetic changes in the cold- and thermo-tolerant yeast K. marxianus.NATURE PUBLISHING GROUP, 2017年08月, SCIENTIFIC REPORTS, 7(1) (1), 8993 - 8993, 英語, 国際誌[査読有り]研究論文（学術雑誌）
- 2017年06月, Bio-protocol, 7, 11, 英語Targeted nucleotide substitution in mammalian cell by Target-AID[査読有り]研究論文（学術雑誌）
- We applied a fusion of CRISPR-Cas9 and activation-induced cytidine deaminase (Target-AID) for point mutagenesis at genomic regions specified by single guide RNAs (sgRNAs) in two crop plants. In rice, we induced multiple herbicide-resistance point mutations by multiplexed editing using herbicide selection, while in tomato we generated marker-free plants with homozygous heritable DNA substitutions, demonstrating the feasibility of base editing for crop improvement.NATURE PUBLISHING GROUP, 2017年05月, NATURE BIOTECHNOLOGY, 35(5) (5), 441 - +, 英語, 国際誌[査読有り]研究論文（学術雑誌）
- The generation of genetic variation (somatic hypermutation) is an essential process for the adaptive immune system in vertebrates. We demonstrate the targeted single-nucleotide substitution of DNA using hybrid vertebrate and bacterial immune systems components. Nuclease-deficient type II CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated) and the activation-induced cytidine deaminase (AID) ortholog PmCDA1 were engineered to form a synthetic complex (Target-AID) that performs highly efficient target-specific mutagenesis. Specific point mutation was induced primarily at cytidines within the target range of five bases. The toxicity associated with the nuclease-based CRISPR/Cas9 system was greatly reduced. Although combination of nickase Cas9(D10A) and the deaminase was highly effective in yeasts, it also induced insertion and deletion (indel) in mammalian cells. Use of uracil DNA glycosylase inhibitor suppressed the indel formation and improved the efficiency.AMER ASSOC ADVANCEMENT SCIENCE, 2016年09月, SCIENCE, 353(6305) (6305), aaf8729, 英語, 国際誌[査読有り]研究論文（学術雑誌）
- Glutathione is a valuable tripeptide widely used in the pharmaceutical, food, and cosmetic industries. In industrial fermentation, glutathione is currently produced primarily using the yeast Saccharomyces cerevisiae. Intracellular glutathione exists in two forms; the majority is present as reduced glutathione (GSH) and a small amount is present as oxidized glutathione (GSSG). However, GSSG is more stable than GSH and is a more attractive form for the storage of glutathione extracted from yeast cells after fermentation. In this study, intracellular GSSG content was improved by engineering thiol oxidization metabolism in yeast. An engineered strain producing high amounts of glutathione from over-expression of glutathione synthases and lacking glutathione reductase was used as a platform strain. Additional over-expression of thiol oxidase (1.8.3.2) genes ERV1 or ERO1 increased the GSSG content by 2.9-fold and 2.0-fold, respectively, compared with the platform strain, without decreasing cell growth. However, over-expression of thiol oxidase gene ERV2 showed almost no effect on the GSSG content. Interestingly, ERO1 over-expression did not decrease the GSH content, raising the total glutathione content of the cell, but ERV1 over-expression decreased the GSH content, balancing the increase in the GSSG content. Furthermore, the increase in the GSSG content due to ERO1 over-expression was enhanced by additional over-expression of the gene encoding Pdi1, whose reduced form activates Ero1 in the endoplasmic reticulum. These results indicate that engineering the thiol redox metabolism of S. cerevisiae improves GSSG and is critical to increasing the total productivity and stability of glutathione.SPRINGER, 2015年11月, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 99(22) (22), 9771 - 9778, 英語, 国際誌[査読有り]研究論文（学術雑誌）
- Background: Measurement of mitochondrial ATP synthesis is a critical way to compare cellular energetic performance. However, fractionation of mitochondria requires large amounts of cells, lengthy purification procedures, and an extreme caution to avoid damaging intact mitochondria, making it the highest barrier to high-throughput studies of mitochondrial function. To evaluate 45 genes involved in oxidative phosphorylation in Saccharomyces cerevisiae, we aimed to develop a simple and rapid method to measure mitochondrial ATP synthesis. Results: To obtain functional mitochondria, S. cerevisiae cells were lysed with zymolyase followed by two-step, low-then high-speed centrifugation. Using a firefly luciferin-luciferase assay, the ATP synthetic activity of the mitochondria was determined. Decreasing the ATP synthesis in the presence of mitochondrial inhibitors confirmed functionality of the isolated crude mitochondria. Deletion of genes encoding mitochondrial ATP synthesis-related protein showed their dependency on the oxidative phosphorylation in S. cerevisiae. Conclusions: Compared with conventional procedures, this measurement method for S. cerevisiae Mitochondrial ATP Synthetic activity in High-throughput (MASH method) is simple and requires a small amount of cells, making it suitable for high-throughput analyses. To our knowledge, this is the first report on a rapid purification process for yeast mitochondria suitable for high-throughput screening.BIOMED CENTRAL LTD, 2015年04月, Microbial Cell Factories, 14, 56 - 56, 英語, 国際誌[査読有り]研究論文（学術雑誌）
- The Golgi body has important roles in modifying, sorting, and transport of proteins and lipids. Eukaryotic cells have evolved in various ways to inherit the Golgi body from mother to daughter cells, which allows the cells to function properly immediately after mitosis. Here we used Cyanidioschyzon merolae, one of the most suitable systems for studies of organelle dynamics, to investigate the inheritance of the Golgi. Two proteins, Sed5 and Got1, were used as Golgi markers. Using immunofluorescence microscopy, we demonstrated that C. merolae contains one to two Golgi bodies per cell. The Golgi body was localized to the perinuclear region during the G1 and S phases and next to the spindle poles in a microtubule-dependent manner during M phase. It was inherited together with spindle poles upon cytokinesis. These observations suggested that Golgi inheritance is dependent on microtubules in C. merolae. © 2012 Springer-Verlag Wien.2013年08月, Protoplasma, 250(4) (4), 943 - 948, 英語, 国際誌[査読有り]研究論文（学術雑誌）
- Peroxisomes (microbodies) are ubiquitous single-membrane-bounded organelles and fulfill essential roles in the cellular metabolism. They are found in virtually all eukaryotic cells and basically multiply by division. However, the mechanochemical machinery involved in peroxisome division remains elusive. Here, we first identified the peroxisome- dividing (POD) machinery. We isolated the POD machinery from Cyanidioschyzon merolae, a unicellular red alga containing a single peroxisome. Peroxisomal division in C. merolae can be highly synchronized by light/dark cycles and the microtubule-disrupting agent oryzalin. By proteomic analysis based on the complete genome sequence of C. merolae, we identified a dynamin-related protein 3 (DRP3) ortholog, CmDnm1 (Dnm1), that predominantly accumulated with catalase in the dividing-peroxisome fraction. Immunofluorescence microscopy demonstrated that Dnm1 formed a ring at the division site of the peroxisome. The outlines of the isolated dynamin rings were dimly observed by phase-contrast microscopy and clearly stained for Dnm1. Electron microscopy revealed that the POD machinery was formed at the cytoplasmic side of the equator. Immunoelectron microscopy showed that the POD machinery consisted of an outer dynamin-based ring and an inner filamentous ring. Down-regulation of Dnm1 impaired peroxisomal division. Surprisingly, the same Dnm1 serially controlled peroxisomal division after mitochondrial division. Because genetic deficiencies of Dnm1 orthologs in multiperoxisomal organisms inhibited both mitochondrial and peroxisomal proliferation, it is thought that peroxisomal division by contraction of a dynamin-based machinery is universal among eukaryotes. These findings are useful for understanding the fundamental systems in eukaryotic cells.NATL ACAD SCIENCES, 2013年06月, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 110(23) (23), 9583 - 9588, 英語, 国際誌[査読有り]研究論文（学術雑誌）
- Endoplasmic reticulum (ER) is a major site for secretory protein folding and lipid synthesis. Since ER cannot be synthesized de novo, it must be inherited during the cell cycle. Studying ER inheritance can however be difficult because the ER of typical plant and animal cells is morphologically complex. Therefore, our study used Cyanidioschyzon merolae, a species that has a simple ER structure, to investigate the inheritance of this organelle. Using immunofluorescence microscopy, we demonstrated that C. merolae contains a nuclear ER (nuclear envelope) and a small amount of peripheral ER extending from the nuclear ER. During mitosis, the nuclear ER became dumbbell-shaped and underwent division. Peripheral ER formed ring-like structures during the G1 and S phases, and extended toward the mitochondria and cell division planes during the M phase. These observations indicated that C. merolae undergoes closed mitosis, whereby the nuclear ER does not diffuse, and the peripheral ER contains cell cycle-specific structures.SPRINGER WIEN, 2012年10月, PROTOPLASMA, 249(4) (4), 1129 - 1135, 英語, 国際誌[査読有り]研究論文（学術雑誌）
- Most organisms are simply diamagnetic, while magnetotactic bacteria and migratory animals are among organisms that exploit magnetism. Biogenic magnetization not only is of fundamental interest, but also has industrial potential. However, the key factor(s) that enable biogenic magnetization in coordination with other cellular functions and metabolism remain unknown. To address the requirements for induction and the application of synthetic bio-magnetism, we explored the creation of magnetism in a simple model organism. Cell magnetization was first observed by attraction towards a magnet when normally diamagnetic yeast Saccharomyces cerevisiae were grown with ferric citrate. The magnetization was further enhanced by genetic modification of iron homeostasis and introduction of ferritin. The acquired magnetizable properties enabled the cells to be attracted to a magnet, and be trapped by a magnetic column. Superconducting quantum interference device (SQUID) magnetometry confirmed and quantitatively characterized the acquired paramagnetism. Electron microscopy and energy-dispersive X-ray spectroscopy showed electron-dense iron-containing aggregates within the magnetized cells. Magnetization-based screening of gene knockouts identified Tco89p, a component of TORC1 (Target of rapamycin complex 1), as important for magnetization; loss of TCO89 and treatment with rapamycin reduced magnetization in a TCO89-dependent manner. The TCO89 expression level positively correlated with magnetization, enabling inducible magnetization. Several carbon metabolism genes were also shown to affect magnetization. Redox mediators indicated that TCO89 alters the intracellular redox to an oxidized state in a dose-dependent manner. Taken together, we demonstrated that synthetic induction of magnetization is possible and that the key factors are local redox control through carbon metabolism and iron supply.PUBLIC LIBRARY SCIENCE, 2012年02月, PLOS BIOLOGY, 10(2) (2), e1001269, 英語, 国際誌[査読有り]研究論文（学術雑誌）
- Involvement of Elongation Factor-1α in Cytokinesis without Actomyosin Contractile Ring in the Primitive Red Alga Cyanidioschyzon merolaeCytokinesis is a pivotal event in cell division in all living organisms. In mammals, cytokinesis progresses via constriction of the contractile ring, a bundle of actin filaments and myosin that is located on the equatorial plane. The eukaryotic elongation factor 1 alpha (eEF-1 alpha) is associated with the contractile ring in sea urchin eggs. Although eEF-1 alpha is a ubiquitous protein translation factor and is highly conserved in eukaryotes, archaea, and prokaryotes, the archaebacterial EF-1 alpha and the prokaryotic EF-1 alpha ortholog EF-Tu do not function in cytokinesis, suggesting that the association between the contractile ring and EF-1 alpha appeared at about the same time as the establishment of eukaryotic cells. However, the role of EF-1 alpha in cytokinesis in primitive cells is unclear. In this study, we show that the primitive alga Cyanidioschyzon merolae elongation factor-1 alpha (CmEF-1 alpha) is localized in the contractile division plane, but not in the actomyosin contractile ring. The genome of C. merolae contains 2 unexpressed actin genes and lacks a myosin gene. Immunoblotting analyses revealed that the protein level of CmEF-1 alpha remained constant during the G1 to M phase, and then peaked during cytokinesis. Immunofluorescent microscopy revealed 2 patterns of localization of CmEF-1 alpha during the cell cycle: it was dispersed throughout the cytoplasm from G1 to early M phase, and then localized to the contractile region during cytokinesis. From the results of current study on cytokinesis in the primitive red algae C. merolae, our findings led us to hypothesize that when first established in the lower eukaryotes, cytokinesis fundamentally relied on eEF-1 alpha, and the actomyosin system was acquired later during evolution.UNIV TOKYO CYTOLOGIA, 2011年12月, Cytologia, 76(4) (4), 431 - 437, 英語[査読有り]研究論文（学術雑誌）
- 2011年, Cytologia, 76, 431 - 437, 英語Involvement of elongation[査読有り]研究論文（学術雑誌）
- The Vacuole Binding to Mitochondria by VIG1 Contributes an Equal Inheritance of the Vacuoles in Cyanidioschyzon merolaeVacuoles function in endocytosis, storage and digestion of metabolites in eukaryotic cells. They are inherited by the daughter cells. However, the mechanisms of vacuole inheritance are poorly understood because the cells contain multiple vacuoles that behave randomly. Cyanidioschyzon merolae cell has a minimum set of organelles. The vacuoles were equally inherited by the daughter cells by binding to dividing mitochondria. The binding was mediated by VIG1. However, the role of the binding in the vacuoles inheritance was poorly understood. We examined it by inhibiting the binding cytochemically. The vacuoles, which were not bound to mitochondria, were not equally inherited by the daughter cells. As the results, vacuole-less daughter cells were generated. These results suggested that the binding contributed the equal inheritance of vacuoles and ensured the permanence of vacuoles in daughter cells.UNIV TOKYO CYTOLOGIA, 2010年06月, CYTOLOGIA, 75(2) (2), 189 - 194, 英語[査読有り]研究論文（学術雑誌）
- 2010年06月, Cytologia, 69(1) (1), 97 - 100, 英語The Vacuole Binding to Mitochondria by VIG1 Contributes an Equal Inheritance of the Vacuoles in Cyanidioschyzon merolae[査読有り]研究論文（学術雑誌）
- 2010年03月, Plant Cell, 22(3) (3), 772 - 781, 英語The coiled-coil protein VIG1 is essential for tethering vacuoles to mitochondria during vacuole inheritance of Cyanidioschyzon merolae[査読有り]研究論文（学術雑誌）
- P>Plant vacuoles are organelles bound by a single membrane, and involved in various functions such as intracellular digestion, metabolite storage, and secretion. To understand their evolution and fundamental mechanisms, characterization of vacuoles in primitive plants would be invaluable. Algal cells often contain polyphosphate-rich compartments, which are thought to be the counterparts of seed plant vacuoles. Here, we developed a method for isolating these vacuoles from Cyanidioschyzon merolae, and identified their proteins by MALDI TOF-MS. The vacuoles were of unexpectedly high density, and were highly enriched at the boundary between 62 and 80% w/v iodixanol by density-gradient ultracentrifugation. The vacuole-containing fraction was subjected to SDS-PAGE, and a total of 46 proteins were identified, including six lytic enzymes, 13 transporters, six proteins for membrane fusion or vesicle trafficking, five non-lytic enzymes, 13 proteins of unknown function, and three miscellaneous proteins. Fourteen proteins were homologous to known vacuolar or lysosomal proteins from seed plants, yeasts or mammals, suggesting functional and evolutionary relationships between C. merolae vacuoles and these compartments. The vacuolar localization of four novel proteins, namely CMP249C (metallopeptidase), CMJ260C (prenylated Rab receptor), CMS401C (ABC transporter) and CMT369C (o-methyltransferase), was confirmed by labeling with specific antibodies or transient expression of hemagglutinin-tagged proteins. The results presented here provide insights into the proteome of C. merolae vacuoles and shed light on their functions, as well as indicating new features.WILEY-BLACKWELL PUBLISHING, INC, 2009年12月, PLANT JOURNAL, 60(5) (5), 882 - 893, 英語, 国際誌[査読有り]研究論文（学術雑誌）
- The ability of the primitive red alga Cyanidioschyzon merolae to adapt to high temperatures was utilized to produce thermotolerant transgenic plants. C. merolae inhabits an extreme environment (42 degrees C, pH 2.5) and the nuclear, mitochondrial, and plastid genomes have been sequenced. We analyzed expressed sequence tag (EST) data to reveal mechanisms of tolerance to high temperatures. The stromal ascorbate peroxidase (CmstAPX) that scavenges reactive oxygen species (ROS) was expressed at high levels (4th of 4,479 entries), thus, it offers clues to understanding high-temperature tolerance. CmstAPX has a chloroplast transit peptide (cTP) and a peroxidase domain. The peroxidase domain of CmstAPX has deletions and insertions when compared with that of Arabidopsis thaliana stromal APX (AtstAPX). To clarify aspects of tolerance to oxidative and high-temperature stress, we produced transgenic A. thaliana plants overexpressing CmstAPX and AtstAPX. CmstAPX plants showed higher activities of soluble APX than those of wildtype and AtstAPX plants. Fluorescence signals of a GFP fusion protein, immuno-fluorescence, and immunogold electron microscopy showed that CmstAPX was localized in the stroma of chloroplasts. Compared with wild-type plants and AtstAPX plants, CmstAPX plants were more tolerant to oxidative stress induced by methylviologen (MV, 0.4 mu M) and high-temperature stress (33 degrees C). CmstAPX plants retained the highest chlorophyll content when treated with MV and high temperature, and their stroma and chloroplasts remained intact in their chloroplasts, whereas they disintegrated in wild-type plants. Our results suggest that the increased activity of APX in the chloroplasts of CmstAPX plants increased thermotolerance by increasing ROS-scavenging capacity at high temperatures.SPRINGER, 2009年12月, PLANT CELL REPORTS, 28(12) (12), 1881 - 1893, 英語, 国際誌[査読有り]研究論文（学術雑誌）
- 2009年02月, DNA Research, 16(1) (1), 59 - 72, 英語Periodic gene expression patterns during the highly synchronized cell nucleus and organelle division cycles in the unicellular red alga Cyanidioschyzon merolae[査読有り]研究論文（学術雑誌）
- Previous cell cycle studies have been based on cell-nuclear proliferation only. Eukaryotic cells, however, have double membranes-bound organelles, such as the cell nucleus, mitochondrion, plastids and single-membrane-bound organelles such as ER, the Golgi body, vacuoles (lysosomes) and microbodies. Organelle proliferations, which are very important for cell functions, are poorly understood. To clarify this, we performed a microarray analysis during the cell cycle of Cyanidioschyzon merolae. C. merolae cells contain a minimum set of organelles that divide synchronously. The nuclear, mitochondrial and plastid genomes were completely sequenced. The results showed that, of 158 genes induced during the S or G2-M phase, 93 were known and contained genes related to mitochondrial division, ftsZ1-1, ftsz1-2 and mda1, and plastid division, ftsZ2-1, ftsZ2-2 and cmdnm2. Moreover, three genes, involved in vesicle trafficking between the single-membrane organelles such as vps29 and the Rab family protein, were identified and might be related to partitioning of single-membrane-bound organelles. In other genes, 46 were hypothetical and 19 were hypothetical conserved. The possibility of finding novel organelle division genes from hypothetical and hypothetical conserved genes in the S and G2-M expression groups is discussed.2009年, DNA Res., 74(1) (1), 1 - 72, 英語, 国際誌[査読有り]研究論文（学術雑誌）
- Novel Dynamics of FtsZ Ring Before Plastid AbscissionPlastids were derived from free-living cyanobacterium ancestors that were engulfed by a primary non-photosynthetic eukaryotic host cell and subsequently evolved into plastids. Consistent with their bacterial origin, plastids use the bacterial FtsZ ring, the gene of which was transferred to the host eukaryotic nucleus over evolutionary time. However, recent genome sequencing projects show that most of the other proteins once involved in bacterial division have been lost. Here we show, highly sensitive immunofluorescence analysis using plastid FtsZ antiserum revealed that plastid FtsZ forms two types of ring structures during plastid division: (i) the 1st FtsZ ring is formed at the division site to constitute the foundation of the plastid division machinery and (ii) the 2nd FtsZ ring is formed at a right angle with the 1st FtsZ ring. One notable point is that the 2nd FtsZ ring disappears at the end of plastid division. This result suggests that the nuclear encoded plastid FtsZ may function in daughter plastids, in addition to foundation of the plastid division machinery.UNIV TOKYO CYTOLOGIA, 2008年06月, CYTOLOGIA, 73(2) (2), 197 - 201, 英語[査読有り]研究論文（学術雑誌）
- SPRINGER TOKYO, 2008年03月, JOURNAL OF PLANT RESEARCH, 121(2) (2), 251 - 251, 英語[査読有り]
- Algae play a more important role than land plants in the maintenance of the global environment and productivity. Progress in genome analyses of these organisms means that we can now obtain information on algal genomes, global annotation and gene expression. The full genome information for several algae has already been analyzed. Whole genomes of the red alga Cyanidioschyzon [corrected] merolae, the green algae Ostreococcus tauri and Chlamydomonas reinhardtii, and the diatom Thalassiosira pseudonana have been sequenced. Genome composition and the features of cells among the four algae were compared. Each alga maintains basic genes as photosynthetic eukaryotes and possesses additional gene groups to represent their particular characteristics. This review discusses and introduces the latest research that makes the best use of the particular features of each organism and the significance of genome analysis to study biological phenomena. In particular, examples of post-genome studies of organelle multiplication in C. merolae based on analyzed genome information are presented.Springer Japan, 2008年, Journal of Plant Research, 121(1) (1), 1 - 17, 英語, 国内誌[査読有り]研究論文（学術雑誌）
- Cyanidioschyzon merolae is considered as a suitable model system for studies of organelle differentiation, proliferation and partitioning. Here, we have identified and characterized vacuoles in this organism and examined the partitioning of vacuoles using fluorescence and electron microscopy. Vacuoles were stained with the fluorescent aminopeptidase substrate 7-amino-4-chloromethylcoumarin L-arginine amide, acidotrophic dyes quinacrine and LysoTracker, and 4', 6-diamidino-2-phenyl indole, which, at a high concentration, stains polyphosphate. Vacuoles have been shown to be approximately 500 nm in diameter with a mean of around five per interphase cell. The vacuolar H+-ATPase inhibitor concanamycin A blocked the accumulation of quinacrine in the vacuoles, suggesting the presence of the enzyme on these membranes. Electron microscopy revealed that the vacuoles were single membrane-bound organelles with an electron-dense substance, often containing a thick layer surrounding the membrane. Immunoelectron microscopy using an anti-vacuolar-H+-pyrophosphatase antibody revealed the presence of the enzyme on these membranes. In interphase cells, vacuoles were distributed in the cytoplasm, while in mitotic cells they were localized adjacent to the mitochondria. Filamentous structures were observed between vacuoles and mitochondria. Vacuoles were distributed almost evenly to daughter cells and redistributed in the cytoplasm after cytokinesis. The change in localization of vacuoles also happened in microtubule-disrupted cells. Since no actin protein or filaments have been detected in C. merolae, this result suggests an intrinsic mechanism for the movement of vacuoles that differs from commonly known mechanisms mediated by microtubules and actin filaments.SPRINGER, 2007年09月, PLANTA, 226(4) (4), 1017 - 1029, 英語, 国際誌[査読有り]研究論文（学術雑誌）
- Background: All previously reported eukaryotic nuclear genome sequences have been incomplete, especially in highly repeated units and chromosomal ends. Because repetitive DNA is important for many aspects of biology, complete chromosomal structures are fundamental for understanding eukaryotic cells. Our earlier, nearly complete genome sequence of the hot-spring red alga Cyanidioschyzon merolae revealed several unique features, including just three ribosomal DNA copies, very few introns, and a small total number of genes. However, because the exact structures of certain functionally important repeated elements remained ambiguous, that sequence was not complete. Obviously, those ambiguities needed to be resolved before the unique features of the C. merolae genome could be summarized, and the ambiguities could only be resolved by completing the sequence. Therefore, we aimed to complete all previous gaps and sequence all remaining chromosomal ends, and now report the first nuclear-genome sequence for any eukaryote that is 100% complete. Results: Our present complete sequence consists of 16546747 nucleotides covering 100% of the 20 linear chromosomes from telomere to telomere, representing the simple and unique chromosomal structures of the eukaryotic cell. We have unambiguously established that the C. merolae genome contains the smallest known histone-gene cluster, a unique telomeric repeat for all chromosomal ends, and an extremely low number of transposons. Conclusion: By virtue of these attributes and others that we had discovered previously, C. merolae appears to have the simplest nuclear genome of the non-symbiotic eukaryotes. These unusually simple genomic features in the 100% complete genome sequence of C. merolae are extremely useful for further studies of eukaryotic cells.BMC, 2007年07月, BMC BIOLOGY, 5, 28 - 28, 英語, 国際誌[査読有り]研究論文（学術雑誌）
- Mitochondria are not produced de novo but are maintained by division. Mitochondrial division is a coordinated process of positioning and constriction of the division site and fission of double membranes, in which dynamin-related protein is believed to mediate outer membrane fission. Part of the mitochondrial division machinery was purified from M phase-arrested Cyanidioschyzon merolae cells through biochemical fractionation. The dynamin-related protein Dnm1 was one of the two major proteins in the purified fraction and was accompanied by a newly identified protein CMR185C, named Mda1. Mda1 contained a predictable coiled-coil region and WD40 repeats, similarly to Mdv1 and Caf4 in yeasts. Immunofluorescence and immunoelectron microscopy showed that Mda1 localizes as a medial belt or ring on the mitochondrial outer surface throughout the division. The ring formation of Mda1 followed the plane of the ring of FtsZ, a protein that resides in the matrix. Dnm1 consistently colocalized with Mda1 only in the late stages of division. Mda1 protein was expressed through S to M phases and was phosphorylated specifically in M phase when Mda1 transformed from belt into foci and became colocalizing with Dnm1. Dephosphorylation of Mda1 in vitro increased its sedimentation coefficient, suggesting conformational changes of the macromolecule. Disassembly of the purified mitochondrial division machinery was performed by adding GTP to independently release Dnm1, suggesting that Mda1 forms a stable homo-oligomer by itself as a core structure of the mitochondrial division machinery.NATL ACAD SCIENCES, 2007年03月, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 104(11) (11), 4736 - 4741, 英語, 国際誌[査読有り]研究論文（学術雑誌）
- Isolated chloroplast division machinery can actively constrict after stretchingChloroplast division involves plastid-dividing, dynamin, and FtsZ (PDF) rings. We isolated intact supertwisted (or spiral) and circular PDF machineries from chloroplasts of the red alga Cyanidioschyzon merolae. After individual intact PDF machineries were stretched to four times their original lengths with optical tweezers, they spontaneously returned to their original sizes. Dynamin-released PDF machineries did not retain the spiral structure and could not be stretched. Thus, dynamin may generate the motive force for contraction by filament sliding in dividing chloroplasts, in addition to pinching-off the membranes.2006年09月, Science, 313(5792) (5792), 1435 - 1438, 英語, 国際誌[査読有り]研究論文（学術雑誌）
- Mitochondria are derived from free-living alpha-proteobacteria that were engulfed by eukaryotic host cells through the process of endosymbiosis, and therefore have their own DNA which is organized using basic proteins to form organelle nuclei (nucleoids). Mitochondria divide and are split amongst the daughter cells during cell proliferation. Their division can be separated into two main events: division of the mitochondrial nuclei and division of the matrix (the so-called mitochondrial division, or mitochondriokinesis). In this review, we first focus on the cytogenetical relationships between mitochondrial nuclear division and mitochondriokinesis. Mitochondriokinesis occurs after mitochondrial nuclear division, similar to bacterial cytokinesis. We then describe the fine structure and dynamics of the mitochondrial division ring (MD ring) as a basic morphological background for mitochondriokinesis. Electron microscopy studies first identified a small electron-dense MD ring in the cytoplasm at the constriction sites of dividing mitochondria in the slime mold Physarum polycephalum, and then two large MD rings (with outer cytoplasmic and inner matrix sides) in the red alga Cyanidioschyzon merolae. Now MD rings have been found in all eukaryotes. In the third section, we describe the relationships between the MD ring and the FtsZ ring descended from ancestral bacteria. Other than the GTPase, FtsZ, mitochondria have lost most of the proteins required for bacterial cytokinesis as a consequence of endosymbiosis. The FtsZ protein forms an electron transparent ring (FtsZ or Z ring) in the matrix inside the inner MD ring. For the fourth section, we describe the dynamic association between the outer MD ring with a ring composed of the eukaryote-specific GTPase dynamin. Recent studies have revealed that eukaryote-specific GTPase dynamins form an electron transparent ring between the outer membrane and the MD ring. Thus, mitochondriokinesis is thought to be controlled by a mitochondrial division (MD) apparatus including a dynamic trio, namely the FtsZ, MD and dynamin rings, which consist of a chimera of rings from bacteria and eukaryotes in primitive organisms. Since the genes for the MD ring and dynamin rings are not found in the prokaryotic genome, the host genomes may make these rings to actively control mitochondrial division. In the fifth part, we focus on the dynamic changes in the formation and disassembly of the FtsZ, MD and dynamin rings. FtsZ rings are digested during a later period of mitochondrial division and then finally the MD and dynamin ring apparatuses pinched off the daughter mitochondria, supporting the idea that the host genomes are responsible for the ultimate control of mitochondrial division. We discuss the evolution, from the original vesicle division (VD) apparatuses to VD apparatuses including classical dynamin rings and MD apparatuses. It is likely that the MD apparatuses involving the dynamic trio evolved into the plastid division (PD) apparatus in Bikonta, while in Opisthokonta, the MD apparatus was simplified during evolution and may have branched into the mitochondrial fusion apparatus. Finally, we describe the possibility of intact isolation of large MD/PD apparatuses, the identification of all their proteins and their related genes using C. merolae genome information and TOF-MS analyses. These results will assist in elucidating the universal mechanism and evolution of MD, PD and VD apparatuses.2006年05月, Biochimica et biophysica acta, 1763(5-6) (5-6), 510 - 521, 英語, 国際誌[査読有り]研究論文（学術雑誌）
- Cyanidioschyzon merolae is considered to be a suitable model system for cytologial studies of organelle proliferation and partitioning because these unicellular cells contain each organelle singly. However, lysosomes of C. merolae have yet to be identified. Polyphosphate have been known to be accumulated in the vacuoles of many microorganisms including alga. The cells stained with Neisser staining method, which visualizes polyphosphate bodies, showed the lysosome-like structures. They were about 500 nm in diameter and usually found as four copies in a single cell. The structures changed their localization dynamically during the cell cycle. During interphase, they were observed in the cytosol. At the beginning of mitosis, they moved over the mitochondria. During cytokinesis, they were inherited to the daughter cells almost evenly, suggesting the existence of mechanisms for the ordered partitioning. © 2005 The Japan Mendel Society.2005年09月, Cytologia, 70(3) (3), 351 - 354, 英語[査読有り]研究論文（学術雑誌）
- Mitochondrial and chloroplast division controls the number and morphology of organelles, but how cells regulate organelle division remains to be clarified. Here, we show that each step of mitochondrial and chloroplast division is closely associated with the cell cycle in Cyanidioschyzon merolae. Electron microscopy revealed direct associations between the spindle pole bodies and mitochondria, suggesting that mitochondrial distribution is physically coupled with mitosis. Interconnected organelles were fractionated under microtubule-stabilizing condition. Immunoblotting analysis revealed that the protein levels required for organelle division increased before microtubule changes upon cell division, indicating that regulation of protein expression for organelle division is distinct from that of cytokinesis. At the mitochondrial division site, dynamin stuck to one of the divided mitochondria and was spatially associated with the tip of a microtubule stretching from the other one. Inhibition of microtubule organization, proteasome activity or DNA synthesis, respectively, induced arrested cells with divided but shrunk mitochondria, with divided and segregated mitochondria, or with incomplete mitochondrial division restrained at the final severance, and repetitive chloroplast division. The results indicated that mitochondrial morphology and segregation but not division depend on microtubules and implied that the division processes of the two organelles are regulated at distinct checkpoints.AMER SOC CELL BIOLOGY, 2005年05月, MOLECULAR BIOLOGY OF THE CELL, 16(5) (5), 2493 - 2502, 英語, 国際誌[査読有り]研究論文（学術雑誌）
- The ultrasmall unicellular red alga Cyanidioschyzon merolae lives in the extreme environment of acidic hot springs and is thought to retain primitive features of cellular and genome organization. We determined the 16.5-Mb nuclear genome sequence of C. merolae 10D as the first complete algal genome. BLASTs and annotation results showed that C. merolae has a mixed gene repertoire of plants and animals, also implying a relationship with prokaryotes, although its photosynthetic components were comparable to other phototrophs. The unicellular green alga Chlamydomonas reinhardtii has been used as a model system for molecular biology research on, for example, photosynthesis, motility, and sexual reproduction. Though both algae are unicellular, the genome size, number of organelles, and surface structures are remarkably different. Here, we report the characteristics of double membrane- and single membrane-bound organelles and their related genes in C. merolae and conduct comparative analyses of predicted protein sequences encoded by the genomes of C. merolae and C. reinhardtii. We examine the predicted proteins of both algae by reciprocal BLASTP analysis, KOG assignment, and gene annotation. The results suggest that most core biological functions are carried out by orthologous proteins that occur in comparable numbers. Although the fundamental gene organizations resembled each other, the genes for organization of chromatin, cytoskeletal components, and flagellar movement remarkably increased in C. reinhardtii. Molecular phylogenetic analyses suggested that the tubulin is close to plant tubulin rather than that of animals and fungi. These results reflect the increase in genome size, the acquisition of complicated cellular structures, and kinematic devices in C. reinhardtii.AMER SOC PLANT BIOLOGISTS, 2005年02月, PLANT PHYSIOLOGY, 137(2) (2), 567 - 585, 英語, 国際誌[査読有り]研究論文（学術雑誌）
- OXFORD UNIV PRESS, 2005年, PLANT AND CELL PHYSIOLOGY, 46, S68 - S68, 英語Identification and behavior of lysosome-like structures in unicellular red algae Cyanidioschyzon merolae[査読有り]研究論文（学術雑誌）
- Triple immunofluorescent labeling of FtsZ, dynamin, and EF-Tu reveals a loose association between the inner and outer membrane mitochondrial division machinery in the red alga Cyanidioschyzon merolaeIn the mitochondria of primitive eukaryotes, FtsZ and dynamin are part of the machinery involved in division of the inner and outer membranes, respectively. These genes also commonly function in the same manner during chloroplast division. In this study, a relationship between the localization of the inner and outer division machinery was directly shown for the first time. Triple immunofluorescent labeling was performed in the red alga Cyanidioschyzon merolae by a device using narrow bandpass filter sets and bright photostable dyes. FtsZ (CmFtsZ1) and dynamin (CmDnm1) localizations were examined simultaneously throughout the mitochondrial division cycle with an alternative mitochondrial marker protein, the mitochondrial translation elongation factor EF-Tu, whose localization was also shown to be identical to the mitochondrial matrix. FtsZ and dynamin did not necessarily co-localize when both were recruited to the mitochondrial constriction site, indicating that inner and outer dividing machineries are not in tight association during the late stage of division.2004年07月, Journal of Histochemistry & Cytochemistry, 52(7) (7), 843 - 849, 英語, 国際誌[査読有り]研究論文（学術雑誌）
- Small, compact genomes of ultrasmall unicellular algae provide information on the basic and essential genes that support the lives of photosynthetic eukaryotes, including higher plants(1,2). Here we report the 16,520,305-base-pair sequence of the 20 chromosomes of the unicellular red alga Cyanidioschyzon merolae 10D as the first complete algal genome. We identified 5,331 genes in total, of which at least 86.3% were expressed. Unique characteristics of this genomic structure include: a lack of introns in all but 26 genes; only three copies of ribosomal DNA units that maintain the nucleolus; and two dynamin genes that are involved only in the division of mitochondria and plastids. The conserved mosaic origin of Calvin cycle enzymes in this red alga and in green plants supports the hypothesis of the existence of single primary plastid endosymbiosis. The lack of a myosin gene, in addition to the unexpressed actin gene, suggests a simpler system of cytokinesis. These results indicate that the C. merolae genome provides a model system with a simple gene composition for studying the origin, evolution and fundamental mechanisms of eukaryotic cells.NATURE PUBLISHING GROUP, 2004年04月, NATURE, 428(6983) (6983), 653 - 657, 英語, 国際誌[査読有り]研究論文（学術雑誌）
- The ancestors of plastids and mitochondria were once free-living bacteria that became organelles as a result of endosymbiosis. According to this theory, a key bacterial division protein, FtsZ, plays a role in plastid division in algae and plants as well as in mitochondrial division in lower eukaryotes. Recent studies have shown that organelle division is a process that combines features derived from the bacterial division system with features contributed by host eukaryotic cells. Two nonredundant versions of FtsZ, FtsZ1 and FtsZ2, have been identified in green-lineage plastids, whereas most bacteria have a single ftsZ gene. To examine whether there is also more than one type of FtsZ in red-lineage chloroplasts (red algal chloroplasts and chloroplasts that originated from the secondary endosymbiosis of red algae) and in mitochondria, we obtained FtsZ sequences from the complete sequence of the primitive red alga Cyanidioschyzon merolae and the draft sequence of the stramenopile (heterokont) Thalassiosira pseudonana. Phylogenetic analyses that included known FtsZ proteins identified two types of chloroplast FtsZ in red algae (FtsZA and FtsZB) and stramenopiles (FtsZA and FtsZC). These analyses also showed that FtsZB emerged after the red and green lineages diverged, while FtsZC arose by the duplication of an ftsZA gene that in turn descended from a red alga engulfed by the ancestor of stramenopiles. A comparison of the predicted proteins showed that like bacterial FtsZ and green-lineage FtsZ2, FtsZA has a short conserved C-termmal sequence (the C-terminal core domain), whereas FtsZB and FtsZC, like the green-lineage FtsZ1, lack this sequence. In addition, the Cyanidioschyzon and Dictyostelium genomes encode two types of mitochondrial FtsZ proteins, one of which lacks the C-terminal variable domain. These results suggest that the acquisition of an additional FtsZ protein with a modified C terminus was common to the primary and secondary endosymbioses that produced plastids and that this also occurred during the establishment of mitochondria, presumably to regulate the multiplication of these organelles.SPRINGER-VERLAG, 2004年03月, JOURNAL OF MOLECULAR EVOLUTION, 58(3) (3), 291 - 303, 英語, 国際誌[査読有り]研究論文（学術雑誌）
- Cyanidioschyzon merolae is a unicellular red alga that lives in acidic hot springs. The genome sequence of C. merolae has been completely read, but a lack of transformation systems still limits its application in genetics. To choose an appropriate drug for use in selectable media, we examined the effects of seven antibiotics on the growth of C. merolae. Only cycloheximide, an inhibitor of protein synthesis, effectively inhibited the growth. We noticed that there was a population that could survive in the presence of cycloheximide and succeeded in isolating six cycloheximide-resistant clones. All these clones have the same single mutation in the ribosomal protein L29 gene that encodes a ribosomal protein.2004年03月, Cytologia, 69(1) (1), 97 - 100, 英語[査読有り]研究論文（学術雑誌）
- Consistent with their bacterial origin, chloroplasts and primitive mitochondria retain a FtsZ ring for division. However, chloroplasts and mitochondria have lost most of the proteins required for bacterial division other than FtsZ and certain homologues of the Min proteins, but they do contain plastid and mitochondrion dividing rings, which were recently shown to be distinct from the FtsZ ring. Moreover, recent studies have revealed that rings of the eukaryote-specific dynamin-related family of GTPases regulate the division of chloroplasts and mitochondria, and these proteins emerged early in eukaryotic evolution. These findings suggest that the division of chloroplasts and primitive mitochondria involve very similar systems, consisting of an amalgamation of rings from bacteria and eukaryotes.ELSEVIER SCIENCE LONDON, 2003年09月, TRENDS IN PLANT SCIENCE, 8(9) (9), 432 - 438, 英語, 国際誌[査読有り]研究論文（学術雑誌）
- Chloroplasts have retained the bacterial FtsZ for division, whereas mitochondria lack FtsZ except in some lower eukaryotes. Instead, mitochondrial division involves a dynamin-related protein, suggesting that chloroplasts retained the bacterial division system, whereas a dynamin-based system replaced the bacterial system in mitochondria during evolution. In this study, we identified a novel plant-specific group of dynamins from the primitive red alga Cyanidioschyzon merolae. Synchronization of chloroplast division and immunoblot analyses showed that the protein (CmDnm2) associates with the chloroplast only during division. Immunocytochemical analyses showed that CmDnm2 appears in cytoplasmic patches just before chloroplast division and is recruited to the cytosolic side of the chloroplast division site to form a ring in the late stage of division. The ring constricts until division is complete, after which it disappears. These results show that a dynamin-related protein also participates in chloroplast division and that its behavior differs from that of FtsZ and plastid-dividing rings that form before constriction at the site of division. Combined with the results of a recent study of mitochondrial division in Cyanidioschyzon, our findings led us to hypothesize that when first established in lower eukaryotes, mitochondria and chloroplasts divided using a very similar system that included the FtsZ ring, the plastid-dividing/mitochondrion-dividing ring, and the dynamin ring.AMER SOC PLANT BIOLOGISTS, 2003年03月, PLANT CELL, 15(3) (3), 655 - 665, 英語, 国際誌[査読有り]研究論文（学術雑誌）
- Dynamins are a eukaryote-specific family of GTPases. Some family members are involved in diverse and varied cellular activities. Here, we report that the primitive red alga Cyanidioschyzon merolae retains only one dynamin homolog, CmDnm1, belonging to the mitochondrial division subfamily. Previously, the bacterial cell division protein, FtsZ, was shown to localize at the mitochondrial division site in the alga. We showed that FtsZ and dynamin coexist as mitochondrial division-associated proteins that act during different phases of division. CmDnm1 was recruited from 10-20 cytoplasmic patches (dynamin patches) to the midpoint of the constricted mitochondrion-dividing ring (MD ring), which was observed as an electron-dense structure on the cytoplasmic side. CmDnm1 is probably not required for early constriction; it forms a ring or spiral when the outer mitochondrial membrane is finally severed, whereas the FtsZ and MD rings are formed before constriction. It is thought that the FtsZ, MD, and dynamin rings are involved in scaffolding, constriction, and final separation, respectively. In eukaryotes, mitochondrial severance is probably the most conserved role for the dynamin family.NATL ACAD SCIENCES, 2003年02月, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 100(4) (4), 2146 - 2151, 英語, 国際誌[査読有り]研究論文（学術雑誌）
- 2019年11月, Frontiers in Genome Engineering 2019A COMPUTATIONAL TOOL TO DESIGN UNIQUE GUIDE RNAS FOR TARGET-AID MEDIATED GENOME-WIDE BASE EDITING[査読有り]研究発表ペーパー・要旨（国際会議）
- 2019年09月, 日本植物学会第83回大会植物ミトコンドリアにおけるゲノム編集の成功
- 2019年06月, 第19回日本蛋白質科学会年会・第71回日本細胞生物学会大会 合同年次大会MitoTALENによる植物ミトコンドリアゲノム編集の成功研究発表ペーパー・要旨（全国大会，その他学術会議）
- 2018年01月, 現代化学, 1月号(562) (562), 26 - 29, 日本語進展するゲノム編集（＜特集＞生命をつくる：ゲノム編集の時代）記事・総説・解説・論説等（学術雑誌）
- 生命科学系学会合同年次大会運営事務局, 2017年12月, 生命科学系学会合同年次大会, 2017年度, [2P - 1314], 英語哺乳動物の全細胞系譜追跡にむけたDNAバーコード技術の開発
- 2017年06月, 植物の生長調節, 52(1) (1), 15 - 18, 日本語デアミナーゼを用いたゲノム編集記事・総説・解説・論説等（学術雑誌）
- 2017年, 日本原生生物学会大会講演要旨集, 50th, 20, 日本語ミトコンドリア分裂リングを形成する新規ミトコンドリア分裂遺伝子MDR1の同定と機能解析
- 裳華房, 2016年, ゲノム編集入門, 第3章, 40 - 55, 日本語微生物でのゲノム編集の利用と拡大技術[査読有り]記事・総説・解説・論説等（学術雑誌）
- 2014年05月15日, 日本細胞生物学会大会要旨集, 66th, 103, 日本語単膜系オルガネラ分裂リングの同定‐ゲノム科学を基盤としたペルオキシソーム分裂装置(POD machinery)の微細構造と分子機構の解析
- 2014年04月, Plant Morphol, 26(1) (1), 72, 日本語原始紅藻シゾンにおけるペルオキシソーム分裂装置(Pod‐machinery)の構造と分子機構の解明
- 2013年08月20日, 日本植物学会大会研究発表記録, 77th, 201, 日本語ポストゲノミクスを基盤としたペルオキシソーム分裂装置(Pod‐machinery)の構造同定と分子機構の解明
- 2013年04月, Plant Morphol, 25(1) (1), 112, 日本語ミトコンドリア‐マイクロボディ複合体分裂後における非収縮環依存型細胞質分裂機構の解明
- 2012年09月14日, 日本植物学会大会研究発表記録, 76th, 139, 日本語ミトコンドリア‐マイクロボディ複合体の分裂分配後における非アクトミオシン依存型細胞質分裂機構の解明
- Vacuoles/lysosomes function in endocytosis and in storage and digestion of metabolites. These organelles are inherited by the daughter cells in eukaryotes. However, the mechanisms of this inheritance are poorly understood because the cells contain multiple vacuoles that behave randomly. The primitive red alga Cyanidioschyzon merolae has a minimum set of organelles. Here, we show that C. merolae contains about four vacuoles that are distributed equally between the daughter cells by binding to dividing mitochondria. Binding is mediated by VIG1, a 30-kD coiled-coil protein identified by microarray analyses and immunological assays. VIG1 appears on the surface of free vacuoles in the cytosol and then tethers the vacuoles to the mitochondria. The vacuoles are released from the mitochondrion in the daughter cells following VIG1 digestion. Suppression of VIG1 by antisense RNA disrupted the migration of vacuoles. Thus, VIG1 is essential for tethering vacuoles to mitochondria during vacuole inheritance in C. merolae.AMER SOC PLANT BIOLOGISTS, 2010年03月, PLANT CELL, 22(3) (3), 772 - 781, 英語, 国際誌
- 2009年04月, Plant Morphol, 21(1) (1), 98, 日本語原始紅藻Cyanidioschyzon merolaeにおける液胞タンパク質の同定
- 2009年, Plant J, 60, 882 - 892
- 2008年09月25日, 日本植物学会大会研究発表記録, 72nd, 165, 日本語原子紅藻Cyanidioschyzon merolaeにおける液胞タンパク質の同定
- The original eukaryotic cells contained at least one set of double-membranebounded organelles (cell nucleus and mitochondria) and single-membranebounded organelles [endoplasmic reticulum, Golgi apparatus, lysosomes (vacuoies), and microbodies (peroxisomes)]. An increase in the number of organelles accompanied the evolution of these cells into Amoebozoa and Opisthokonta. Furthermore, the basic cells, containing mitochondria, engulfed photosynthetic Cyanobacteria, which were converted to plastids, and the cells thereby evolved into cells characteristic of the Bikonta. How did basic single-and double-membrane-bounded organelles originate from bacteria-like cells during early eukaryotic evolution? To answer this question, the important roles of the GTPase dynamin- and electron-dense rings in the promotion of diverse cellular activities in eukaryotes, including endocytosis, vesicular transport, mitochondrial division, and plastid division, must be considered. In this review, vesicle division, mitochondriat division, and plastid division machineries, including the dynamin- and electron-dense rings, and their roles in the origin and biogenesis of organelles in eukaryote cells are summarized.ELSEVIER ACADEMIC PRESS INC, 2008年, INTERNATIONAL REVIEW OF CELL AND MOLECULAR BIOLOGY, VOL 271, 271, 97 - 152, 英語
- 2007年09月06日, 日本植物学会大会研究発表記録, 71st, 146, 日本語細胞周期によるミトコンドリア分裂の制御機構
- 2007年09月06日, 日本植物学会大会研究発表記録, 71st, 145, 日本語原始紅藻におけるリソソーム様構造の解析
- 2007年09月06日, 日本植物学会大会研究発表記録, 71st, 146, 日本語葉緑体分裂(PDF)マシンのプロテオーム解析による構成タンパク質プロファイリングと機能解析
- 2007年, BMC Biology, 5, No28The first 100% complete eukaryotic genome sequences from the red alga Cyanidioschyzon merolae 10D.
- Mitochondria are derived from free-living alpha-proteobacteria that were engulfed by eukaryotic host cells through the process of endosymbiosis, and therefore have their own DNA which is organized using basic proteins to form organelle nuclei (nucleoids). Mitochondria divide and are split amongst the daughter cells during cell proliferation. Their division can be separated into two main events: division of the mitochondrial nuclei and division of the matrix (the so-called mitochondrial division, or mitochondriokinesis). In this review, we first focus on the cytogenetical relationships between mitochondrial nuclear division and mitochondriokinesis. Mitochondriokinesis occurs after mitochondrial nuclear division, similar to bacterial cytokinesis. We then describe the fine structure and dynamics of the mitochondrial division ring (MD ring) as a basic morphological background for mitochondriokinesis. Electron microscopy studies first identified a small electron-dense MD ring in the cytoplasm at the constriction sites of dividing mitochondria in the slime mold Physarum polycephalum, and then two large MD rings (with outer cytoplasmic and inner matrix sides) in the red alga Cyanidioschyzon merolae. Now MD rings have been found in all eukaryotes. In the third section, we describe the relationships between the MD ring and the FtsZ ring descended from ancestral bacteria. Other than the GTPase, FtsZ, mitochondria have lost most of the proteins required for bacterial cytokinesis as a consequence of endosymbiosis. The FtsZ protein forms an electron transparent ring (FtsZ or Z ring) in the matrix inside the inner MD ring. For the fourth section, we describe the dynamic association between the outer MD ring with a ring composed of the eukaryote-specific GTPase dynamin. Recent studies have revealed that eukaryote-specific GTPase dynamins form an electron transparent ring between the outer membrane and the MD ring. Thus, mitochondriokinesis is thought to be controlled by a mitochondrial division (MD) apparatus including a dynamic trio, namely the FtsZ, MD and dynamin rings, which consist of a chimera of rings from bacteria and eukaryotes in primitive organisms. Since the genes for the MD ring and dynamin rings are not found in the prokaryotic genome, the host genomes may make these rings to actively control mitochondrial division. In the fifth part, we focus on the dynamic changes in the formation and disassembly of the FtsZ, MD and dynamin rings. FtsZ rings are digested during a later period of mitochondrial division and then finally the MD and dynamin ring apparatuses pinched off the daughter mitochondria, supporting the idea that the host genomes are responsible for the ultimate control of mitochondrial division. We discuss the evolution, from the original vesicle division (VD) apparatuses to VD apparatuses including classical dynamin rings and MD apparatuses. It is likely that the MD apparatuses involving the dynamic trio evolved into the plastid division (PD) apparatus in Bikonta, while in Opisthokonta, the MD apparatus was simplified during evolution and may have branched into the mitochondrial fusion apparatus. Finally, we describe the possibility of intact isolation of large MD/PD apparatuses, the identification of all their proteins and their related genes using C. merolae genome information and TOF-MS analyses. These results will assist in elucidating the universal mechanism and evolution of MD, PD and VD apparatuses. (c) 2006 Elsevier B.V All rights reserved.ELSEVIER SCIENCE BV, 2006年05月, BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH, 1763(5-6) (5-6), 510 - 521, 英語書評論文,書評,文献紹介等
- 2005年03月, 日本植物生理学会年会要旨集, 46th, 145, 日本語単細胞紅藻Cyanidioschyzon merolaeにおけるリソソーム様構造の動態と同定
- 2005年03月, 日本植物生理学会年会要旨集, 46th, 145, 日本語原始紅藻におけるミトコンドリア分裂機構の解析
- OXFORD UNIV PRESS, 2005年, PLANT AND CELL PHYSIOLOGY, 46, S68 - S68, 英語Cell cycle regulation of mitochondrial and chloroplast division in Cyanisioschyzon merolae研究発表ペーパー・要旨（国際会議）
- 2005年, PLANT MORPHOLOGY, 17(1) (1), 51 - 55, 英語Cell cycle regulated organelles division in a primitive red alga記事・総説・解説・論説等（学術雑誌）
- In the mitochondria of primitive eukaryotes, FtsZ and dynamin are part of the machinery involved in division of the inner and outer membranes, respectively. These genes also commonly function in the same manner during chloroplast division. In this study, a relationship between the localization of the inner and outer division machinery was directly shown for the first time. Triple immunofluorescent labeling was performed in the red alga Cyanidioschyzon merolae by a device using narrow bandpass filter sets and bright photostable dyes. FtsZ (CmFtsZ1) and dynamin (CmDnm1) localizations were examined simultaneously throughout the mitochondrial division cycle with an alternative mitochondrial marker protein, the mitochondrial translation elongation factor EF-Tu, whose localization was also shown to be identical to the mitochondrial matrix. FtsZ and dynamin did not necessarily co-localize when both were recruited to the mitochondrial constriction site, indicating that inner and outer dividing machineries are not in tight association during the late stage of division.HISTOCHEMICAL SOC INC, 2004年07月, JOURNAL OF HISTOCHEMISTRY & CYTOCHEMISTRY, 52(7) (7), 843 - 849, 英語
- 2004年03月20日, 日本植物生理学会年会要旨集, 45th, 92, 日本語
- OXFORD UNIV PRESS, 2004年, PLANT AND CELL PHYSIOLOGY, 45, S20 - S20, 英語Three dividing rings common to mitochondria and plastid研究発表ペーパー・要旨（国際会議）
- OXFORD UNIV PRESS, 2004年, PLANT AND CELL PHYSIOLOGY, 45, S217 - S217, 英語Analysis on mitochondrial division mechanism in a primitive alga研究発表ペーパー・要旨（国際会議）
- 2002年09月20日, 日本植物学会大会研究発表記録, 66th, 168, 日本語FtsZリングおよび色素体分裂リングからなる葉緑体分裂装置複合体の構造解析
- 2002年09月20日, 日本植物学会大会研究発表記録, 66th, 166, 日本語細胞核はどのように色素体とミトコンドリアの増殖を制御してきたか
- 2002年09月20日, 日本植物学会大会研究発表記録, 66th, 167, 日本語細胞内共生のメカニズム,DynaminとFtsZが関わる原始紅藻のミトコンドリア分裂機構の解析
- 第2編 第5章 DNA二重鎖切断を伴わない塩基編集技術, 2021年02月最新のゲノム編集技術と用途展開
- 第3編 第9章 バクテリアでのゲノム編集の有用性, 2021年02月最新のゲノム編集技術と用途展開
- 裳華房, 2016年12月, 日本語, ISBN: 9784785358662ゲノム編集入門 : ZFN・TALEN・CRISPR-Cas9
- 単著, 羊土社, 2016年12月, 日本語『All About ゲノム編集』, 第1章 3) 切断を伴わないゲノム編集ツール開発（デアミナーゼ他）学術書
- 単著, 裳華房, 2016年12月, 日本語ゲノム編集入門, 第3章 微生物でのゲノム編集の利用と拡大技術学術書
- 2019 年植物科学シンポジウム 「SDGs に向けた植物科学の展開」, 2019年12月, 日本語, 国内会議塩基編集技術による次世代育種シンポジウム・ワークショップパネル（指名）
- 日本農芸化学会関西支部 支部例会（第511回講演会）, 2019年12月, 日本語, 国内会議塩基編集技術の開発と育種応用[招待有り]口頭発表（招待・特別）
- Frontiers in Genome Engineering 2019, 2019年11月, 英語, 国際会議Base editing, gene conversion and local diversification without DNA double strand break口頭発表（一般）
- Bio Japan 2019, 2019年10月, 日本語, 国際会議塩基編集技術Base editing の開発と展望[招待有り]口頭発表（招待・特別）
- 2019 Asian Synthetic Biology Association (ASBA) Meeting, 2019年10月, 英語, 国際会議Genome engineering by base editing in various organisms口頭発表（一般）
- 日本遺伝学会第91回大会, 2019年09月, 英語, 国際会議Development of base editing tools for efficient and precise genome engineering口頭発表（招待・特別）
- 第71回日本生物工学会大会, 2019年09月, 日本語, 岡山大学, 国内会議セルロース系バイオマスを原料とするカフェ酸発酵のためのゲノム編集による大腸菌代謝改変の作 製と培養法の検討口頭発表（一般）
- 第25回日本遺伝子細胞治療学会学術集会, 2019年07月, 日本語, 国内会議塩基編集技術の遺伝子治療への応用可能性[招待有り]口頭発表（招待・特別）
- 生物工学若手研究者の集い 夏のセミナー2019, 2019年07月, 日本語, 琵琶湖国定公園 近江白浜 政府登録旅館 白浜荘, 国内会議抗体生産性に関わる遺伝子探索のためのPichia pastorisゲノム編集技術の構築ポスター発表
- 協和発酵バイオ株式会社セミナー, 2019年05月, 日本語, 国内会議Target-AID[招待有り]公開講演，セミナー，チュートリアル，講習，講義等
- 2019年度東大医科研大学院セミナー, 2019年05月, 日本語, 国内会議塩基編集 Base editing の開発と応用公開講演，セミナー，チュートリアル，講習，講義等
- the 10th International Symposium of Innovative BioProduction Kobe (iBioK), 2019年01月, 英語, 国際会議Geneme engineering by base editing for various applications口頭発表（招待・特別）
- The 24th Symposium of Young Asian Biological Engineers’ Community (YABEC 2018), 2018年11月, 英語, National Taiwan University, 国際会議Caffeic acid production from lignocellulosic biomass by metabolically engineered Eshrichaia coli口頭発表（一般）
- 生命科学系フロンティアミーティング 2018, 2018年10月, 日本語, 国内会議新たなゲノム編集技術の開発口頭発表（一般）
- CRISPRing 学会, 2018年09月, 英語, 国際会議point mutagenesis via base-specific RRISPR-editing
- 第70回日本生物工学会大会, 2018年09月, 日本語, 関西大学 千里山キャンパス, 国内会議塩基編集技術Target-AIDの工学的応用口頭発表（一般）
- 第70回日本生物工学会大会, 2018年09月, 日本語, 関西大学 千里山キャンパス, 国内会議Target-AID を利用したゲノム編集による高収率ブタ ノール発酵性クロストリジウム属微生物の育種ポスター発表
- フロンティアサロン, 2018年07月, 日本語, 国内会議ゲノムを切らずに書き換える新たなゲノム編集技術の開発[招待有り]口頭発表（招待・特別）
- リンク公開フォーラム, 2018年07月, 日本語, 国内会議切らないゲノム編集技術の開発と産業応用の可能性[招待有り]シンポジウム・ワークショップパネル（指名）
- 第5回 Kobe Plant Science研究会, 2018年06月, 日本語, 国内会議塩基編集技術の開発
- 日本ゲノム編集学会第3回大会, 2018年06月, 日本語, 広島国際会議場, 国内会議塩基編集技術の開発と応用展開口頭発表（一般）
- Biosystems Design 4.0, 2018年05月, 英語, 国際会議Genome engineering by DNA base editing
- 日本農薬学会第43回大会, 2018年05月, 日本語, 国内会議ピンポイント塩基編集による次世代育種の可能性
- Cold Spring Harbor Asia, 2018年04月, 英語, 国際会議Base editing from Bacteria to Plants
- 第二回植物病理を紡ぐ会, 2018年03月, 日本語, 国内会議植物ゲノムの塩基編集技術[招待有り]口頭発表（招待・特別）
- Keystone Symposia, 2018年01月, 英語, 国際会議Genome Editing with Non-Nuclease Editors from Bacteria to Plants[招待有り]口頭発表（招待・特別）
- 第83回酵母研究会講演会, 2017年09月, 日本語, 京都大学, 国内会議点変異導入型のゲノム編集技術Target-AIDの開発口頭発表（一般）
- 第69回日本生物工学会大会, 2017年09月, 日本語, 早稲田大学 西早稲田キャンパス, 国内会議Target-AID を利用したゲノム編集による高収率ブタ ノール発酵性クロストリジウム属微生物の育種口頭発表（一般）
- 合成生物工学シンポジウム, 2017年08月, 日本語, 神戸大学百年記念館六甲ホール, 国内会議様々な生物に応用可能なピンポイントゲノム編集技術口頭発表（一般）
- BioKorea 2017, 2017年04月, 英語, COEX, Korea, 国際会議Development of a Targeted Nucleotide Editing Tool Target-AID and its Applications口頭発表（一般）
- 日本農芸化学2017年度大会シンポジウム, 2017年03月, 日本語, 京都女子大学, 国内会議塩基変換による切らないゲノム編集口頭発表（一般）
- 第2回デザイン生命工学研究会大会, 2017年03月, 日本語, 神戸大学統合研究拠点コンベンションホール, 国内会議より精密なゲノムデザイン改変を可能とする点変異ゲノム編集口頭発表（一般）
- 日本農芸化学2017年度大会, 2017年03月, 日本語, 日本農芸化学会, 京都女子大学, 国内会議ゲノム編集による Kluyveromyces marxianus の自在な 遺伝子改変口頭発表（一般）
- 日本農芸化学2017年度大会, 2017年03月, 日本語, 日本農芸化学会, 京都女子大学, 国内会議Target-AID を利用したゲノム編集による高収率ブタ ノール発酵性クロストリジウム属微生物の育種口頭発表（一般）
- the 8th International Symposium of Innovative BioProduction Kobe (iBioK), 2017年02月, 英語, 国際会議Targeted point mutagenesis in various organisms口頭発表（招待・特別）
- iBio-P, 2016年12月, 英語, Haeundae Grand Hotel, Pusan Korea, 国際会議Targeted nucleotide substitution as a new mode of genome editing口頭発表（一般）
- 第16回糸状菌分子生物学カンファレンス, 2016年11月, 日本語, 宇治おうばくプラザ, 国内会議麹菌におけるゲノムを切らずに書き換える新規ゲノム編集ツールの確立ポスター発表
- 第6回合成生物工学シンポジウム, 2016年07月, 日本語, 神戸大学百年記念館六甲ホール, 国内会議切らないゲノム編集の拡大応用口頭発表（一般）
- the 7th International Symposium of Innovative BioProduction Kobe (iBioK), 2016年01月, 英語, 国際会議Genome editing mediated by base-exchange without cleaving DNA口頭発表（招待・特別）
- 第67回日本生物工学会大会 国際シンポジウム, 2015年10月, 日本語, 国内会議ゲノムを切らずに書き換える新たなゲノム編集技術の開発口頭発表（一般）
- 第36回日本分子生物学会年会, 2013年12月, 日本語, 日本分子生物学会, 神戸市, 既存の生命は様々な化学的刺激や物理的刺激（熱、圧力、光など）を高度に感知し応答できるよう進化してきた。そのなかで磁場に関しては、極めて限られた利用しか行われていない。これは自然環境の中では主に地磁気でもって南北を知る以外に利用方法が無いからと思われる。逆に言えば現存の生命体に対しておおよそ無害、無反応であり、一方で人工的な環境において磁場は容易に作り出すことが出来、遮蔽物を透過できること、また周波数による特異性などエネルギーや情報の伝達手段として非常に使い勝手のよい物理特性がある。このような磁場を高度に感知するような生命体機能を構築することはできないか、新しい場を感知できる生命体を創り出すことは出来ないか、というところが本研究の発端である。実際にはすでに自然界にも磁場を感知できる生命が例外的に散在し、それらは磁性体を生体内に保持していることが知られて, 国内会議酵母で創って解析する第六感発現のメカニズム[招待有り]シンポジウム・ワークショップパネル（指名）
- 酵母ルネサンス前夜祭, 2013年12月, 日本語, 岡山市, 国内会議遺伝子特異的超進化の実現に向けて[招待有り]口頭発表（招待・特別）
- 科学研究費補助金／若手研究(A), 2017年04月 - 2021年03月, 研究代表者競争的資金
- 学術研究助成基金助成金／挑戦的萌芽研究, 2016年04月 - 2018年03月, 研究代表者競争的資金
- 国立研究開発法人科学技術振興機構, 研究成果展開事業 産学共創プラットフォーム共同研究推進プログラム（OPERA）, 2017年, 研究代表者【OPERA】ゲノム編集による革新的な有用細胞・生物作成技術の創出競争的資金
- 科学研究費補助金／新学術領域研究, 2014年04月 - 2016年03月, 研究代表者競争的資金
- 標的化したDNA配列の核酸塩基を特異的に変換するゲノム配列の改変方法及びそれに用いる分子複合体特願2017-164703, 2015年03月04日, 大学長, 特許6462069, 2019年01月11日特許権
- 標的化したDNA配列の核酸塩基を特異的に変換するゲノム配列の改変方法及びそれに用いる分子複合体 （シンガポール）11201609211V, 2015年03月04日, 大学長, 11201609211V, 2018年01月12日特許権
- 標的化したDNA配列の核酸塩基を特異的に変換するゲノム配列の改変方法及びそれに用いる分子複合体特願2016-506541, 2015年03月04日, 大学長, 特許6206893, 2017年09月15日特許権
- 脱塩基反応により標的化したDNA配列に特異的に変異を導入する、ゲノム配列の改変方法、並びにそれに用いる分子複合体特願2016-516997, 2015年11月02日, 大学長, 特許6153180, 2017年06月09日特許権
研究シーズ
■ 研究シーズ- 切らないゲノム編集シーズカテゴリ：ライフサイエンス研究キーワード：塩基編集, Target-AID, CRISPR, Base editing, ゲノム編集研究の背景と目的：CRISPRなどのゲノム編集技術は、これまで遺伝子操作が困難であった生物においても効率的な標的遺伝子改変を実現し、また外来DNA断片を残さず編集できるため遺伝子組み換え規制の回避も可能であることから幅広い応用可能性が見込まれます。一方、DNA切断による改変結果の不確実性や細胞毒性の課題が存在しました。本研究では、この切るゲノム編集の弱点を克服できる新たな技術の開発を目的としました。研究内容：私たちはDNAを切らずに塩基の脱アミノ化酵素によって文字変換する塩基編集（Base editing）法であるTarget-AIDの開発に成功し、DNA切断に関わる不確実性や毒性を回避しながら、一文字レベルの精密な配列改変を可能とすることができました。期待される効果や応用分野：ヒトの遺伝子治療を含む医療応用や、植物や微生物の改変による農業やバイオものづくりへの応用展開が期待されます。関係する業績：Nishida K et al. Science 353, aaf8729 (2016). Shimatani Z et al. Nature Biotechnology 35, 441-443 (2017). Banno S et al. Nature Microbiology 3, 423-429 (2018).
keiji_nishida
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