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Onouchi Hitoshi

Research Faculty of Agriculture　Fundamental AgriScience Research　Applied BioscienceProfessor

Researcher basic information

■ Degree
  • 博士（理学）, 名古屋大学
  • 修士（理学）, 名古屋大学
■ URL
researchmap URLホームページURL■ Various IDs
J-Global ID■ Research Keywords and Fields
Research Keyword
  • 分子生物学
  • 植物生理学
  • Plant physiology
  • Molecular genetics
  • Molecular biology
Research Field
  • Life Science, Molecular biology
  • Life Science, Plant molecular biology and physiology
■ Educational Organization

Career

■ Career
Career
  • Jul. 2021 - Present
    Hokkaido University, Graduate School of Agriculture Research Faculty of Agriculture, Professor, Professor
  • Apr. 2007 - Jun. 2021
    Hokkaido University, Graduate School of Agriculture Research Faculty of Agriculture, Associate Professor, 准教授
  • Apr. 2006 - Mar. 2007
    Hokkaido University, Graduate School of Agriculture Research Faculty of Agriculture, Associate Professor, Associate Professor
  • 2005
    - 北海道大学大学院農学研究科・助教授 助教授
  • 2005
    - Associate Professor
  • 2000
    - 北海道大学大学院農学研究科・助手 助手
  • 2000
    - Research Associate
  • 1996
    -
  • 1996
    - Postdoctoral Fellowships of Japan Society for the Promotion of Science
Educational Background
  • 1995, Nagoya University, 理学研究科, 生物学専攻博士後期過程, Japan
  • 1995, Nagoya University, Graduate School, Division of Natural Science
  • 1992, Nagoya University, 理学研究科, 生物学専攻博士前期過程, Japan
  • 1992, Nagoya University, Graduate School, Division of Natural Science
  • 1990, Nagoya University, School of Science, 生物学科, Japan
  • 1990, Nagoya University, Faculty of Science

Research activity information

■ Papers
  • Ribosome stalling-induced NIP5;1 mRNA decay triggers ARGONAUTE1-dependent transcription downregulation.
    Mayuki Tanaka; Naoyuki Sotta; Susan Duncan; Yukako Chiba; Hitoshi Onouchi; Athanasius F M Marée; Satoshi Naito; Verônica A Grieneisen; Toru Fujiwara
    Nucleic acids research, 53, 5, 27 Feb. 2025, [Peer-reviewed], [International Magazine]
    English, Scientific journal, In eukaryotes, messenger RNA (mRNA) accumulation is regulated through the levels of transcription, processing, and degradation. Here, we uncover the multi-level regulatory mechanism governing the expression of NIP5;1, a boron (B) diffusion facilitator in Arabidopsis. B-dependent NIP5;1 mRNA degradation is triggered by ribosome stalling at an AUGUAA sequence in its 5'-untranslated region. We showed that deletion of ATGTAA also abolishes B-dependent transcriptional downregulation, revealing a dual role of this sequence in both mRNA degradation and transcriptional control. Small RNAs (sRNAs) and ARGONAUTE1 (AGO1) are implicated in mRNA-degradation-mediated B-dependent transcriptional downregulation: a 5'-3' exonuclease mutant, xrn4, presents both elevated levels of NIP5;1 mRNA degradation intermediates and transcriptional downregulation; AGO1-associated sRNA-sequencing reveals the presence of sRNAs with sequences upstream of NIP5;1 AUGUAA; and nascent mRNA profiling by global run-on sequencing demonstrates RNA polymerase II pausing at ATGTAA, a phenomenon diminished in the ago1 mutant that lacks B-dependent transcriptional downregulation. These findings point to multi-level coordination of NIP5;1 expression with the AUGUAA sequence at its core: ribosome stalling orchestrates translational inhibition, mRNA degradation and transcriptional downregulation in response to B. The fast response resulting from this synergy suggests that similar mechanisms may exist in other eukaryotic systems for efficient and rapid regulation of gene expression.
  • Phylogeny-linked occurrence of ribosome stalling on the mRNAs of Arabidopsis unfolded protein response factor bZIP60 orthologs in divergent plant species.
    Tomoya Imamichi; Nao Kusumoto; Haruka Aoyama; Seidai Takamatsu; Yugo Honda; Shiori Muraoka; Yuka Hagiwara-Komoda; Yukako Chiba; Hitoshi Onouchi; Yui Yamashita; Satoshi Naito
    Nucleic acids research, 16 Feb. 2024, [Peer-reviewed], [Corresponding author], [International Magazine]
    English, Scientific journal, The bZIP60, XBP1 and HAC1 mRNAs encode transcription factors that mediate the unfolded protein response (UPR) in plants, animals and yeasts, respectively. Upon UPR, these mRNAs undergo unconventional cytoplasmic splicing on the endoplasmic reticulum (ER) to produce active transcription factors. Although cytoplasmic splicing is conserved, the ER targeting mechanism differs between XBP1 and HAC1. The ER targeting of HAC1 mRNA occurs before translation, whereas that of XBP1 mRNA involves a ribosome-nascent chain complex that is stalled when a hydrophobic peptide emerges from the ribosome; the corresponding mechanism is unknown for bZIP60. Here, we analyzed ribosome stalling on bZIP60 orthologs of plants. Using a cell-free translation system, we detected nascent peptide-mediated ribosome stalling during the translation elongation of the mRNAs of Arabidopsis, rice and Physcomitrium (moss) orthologs, and the termination-step stalling in the Selaginella (lycopod) ortholog, all of which occurred ∼50 amino acids downstream of a hydrophobic region. Transfection experiments showed that ribosome stalling contributes to cytoplasmic splicing in bZIP60u orthologs of Arabidopsis and Selaginella. In contrast, ribosome stalling was undetectable for liverwort, Klebsormidium (basal land plant), and green algae orthologs. This study highlights the evolutionary diversity of ribosome stalling and its contribution to ER targeting in plants.
  • In vitro co-expression chromatin assembly and remodeling platform for plant histone variants.
    Petra Banko; Kei-Ichi Okimune; Szilvia K Nagy; Akinori Hamasaki; Ryo Morishita; Hitoshi Onouchi; Taichi E Takasuka
    Scientific reports, 14, 1, 936, 936, 10 Jan. 2024, [Peer-reviewed], [International Magazine]
    English, Scientific journal, Histone variants play a central role in shaping the chromatin landscape in plants, yet, how their distinct combinations affect nucleosome properties and dynamics is still largely elusive. To address this, we developed a novel chromatin assembly platform for Arabidopsis thaliana, using wheat germ cell-free protein expression. Four canonical histones and five reported histone variants were used to assemble twelve A. thaliana nucleosome combinations. Seven combinations were successfully reconstituted and confirmed by supercoiling and micrococcal nuclease (MNase) assays. The effect of the remodeling function of the CHR11-DDR4 complex on these seven combinations was evaluated based on the nucleosome repeat length and nucleosome spacing index obtained from the MNase ladders. Overall, the current study provides a novel method to elucidate the formation and function of a diverse range of nucleosomes in plants.
  • Upstream open reading frame-mediated upregulation of ANAC082 expression in response to nucleolar stress in Arabidopsis
    Shun Sasaki; Toru Murakami; Miharu Yasumuro; Ayaka Makita; Yutaro Oi; Yuta Hiragori; Shun Watanabe; Rin Kudo; Noriya Hayashi; Iwai Ohbayashi; Munetaka Sugiyama; Yui Yamashita; Satoshi Naito; Hitoshi Onouchi
    Plant Biotechnology, 40, 1, 21, 30, Japanese Society for Plant Cell and Molecular Biology, 25 Mar. 2023, [Last author, Corresponding author]
    Scientific journal
  • Genome-wide identification of Arabidopsis non-AUG-initiated upstream ORFs with evolutionarily conserved regulatory sequences that control protein expression levels.
    Yuta Hiragori; Hiro Takahashi; Taihei Karino; Atsushi Kaido; Noriya Hayashi; Shun Sasaki; Kodai Nakao; Taichiro Motomura; Yui Yamashita; Satoshi Naito; Hitoshi Onouchi
    Plant molecular biology, 31 Aug. 2022, [Peer-reviewed], [Last author, Corresponding author], [International Magazine]
    English, Scientific journal, KEY MESSAGE: This study identified four novel regulatory non-AUG-initiated upstream ORFs (uORFs) with evolutionarily conserved sequences in Arabidopsis and elucidated the mechanism by which a non-AUG-initiated uORF promotes main ORF translation. Upstream open reading frames (uORFs) are short ORFs found in the 5'-untranslated regions (5'-UTRs) of eukaryotic transcripts and can influence the translation of protein-coding main ORFs (mORFs). Recent genome-wide ribosome profiling studies have revealed that hundreds or thousands of uORFs initiate translation at non-AUG start codons. However, the physiological significance of these non-AUG uORFs has so far been demonstrated for only a few of them. In this study, to identify physiologically important regulatory non-AUG uORFs in Arabidopsis, we took an approach that combined bioinformatics and experimental analysis. Since physiologically important non-AUG uORFs are likely to be conserved across species, we first searched the Arabidopsis genome for non-AUG-initiated uORFs with evolutionarily conserved sequences. Then, we examined the effects of the conserved non-AUG uORFs on the expression of the downstream mORFs using transient expression assays. As a result, three inhibitory and one promotive non-AUG uORFs were identified. Among the inhibitory non-AUG uORFs, two exerted repressive effects on mORF expression in an amino acid sequence-dependent manner. These two non-AUG uORFs are likely to encode regulatory peptides that cause ribosome stalling, thereby enhancing their repressive effects. In contrast, one of the identified regulatory non-AUG uORFs promoted mORF expression by alleviating the inhibitory effect of a downstream AUG-initiated uORF. These findings provide insights into the mechanisms that enable non-AUG uORFs to play regulatory roles despite their low translation initiation efficiencies.
  • De novo reconstitution of chromatin using wheat germ cell‐free protein synthesis
    Yaeta Endo; Nobuaki Takemori; Szilvia K. Nagy; Kei‐ichi Okimune; Rohinton Kamakaka; Hitoshi Onouchi; Taichi E. Takasuka
    FEBS Open Bio, 11, 6, 1552, 1564, Wiley, Jun. 2021, [Peer-reviewed], [International Magazine]
    English, Scientific journal, DNA is packaged with histones to form chromatin that impinges on all nuclear processes, including transcription, replication and repair, in the eukaryotic nucleus. A complete understanding of these molecular processes requires analysis of chromatin context in vitro. Here, Drosophila four core histones were produced in a native and unmodified form using wheat germ cell-free protein synthesis. In the assembly reaction, four unpurified core histones and three chromatin assembly factors (dNAP-1, dAcf1 and dISWI) were incubated with template DNA. We then assessed stoichiometry with the histones, nucleosome arrays, supercoiling and the ability of the chromatin to serve as a substrate for histone-modifying enzymes. Overall, our method provides a new avenue to produce chromatin that can be useful in a wide range of chromatin research.
  • Exhaustive identification of conserved upstream open reading frames with potential translational regulatory functions from animal genomes
    Hiro Takahashi; Shido Miyaki; Hitoshi Onouchi; Taichiro Motomura; Nobuo Idesako; Anna Takahashi; Masataka Murase; Shuichi Fukuyoshi; Toshinori Endo; Kenji Satou; Satoshi Naito; Motoyuki Itoh
    Scientific Reports, 10, 1, Springer Science and Business Media LLC, Oct. 2020, [Peer-reviewed], [Lead author]
    English, Scientific journal
  • Comprehensive genome-wide identification of angiosperm upstream ORFs with peptide sequences conserved in various taxonomic ranges using a novel pipeline, ESUCA.
    Hiro Takahashi; Noriya Hayashi; Yuta Hiragori; Shun Sasaki; Taichiro Motomura; Yui Yamashita; Satoshi Naito; Anna Takahashi; Kazuyuki Fuse; Kenji Satou; Toshinori Endo; Shoko Kojima; Hitoshi Onouchi
    BMC genomics, 21, 1, 260, 260, 30 Mar. 2020, [Peer-reviewed], [Last author, Corresponding author], [International Magazine]
    English, Scientific journal, BACKGROUND: Upstream open reading frames (uORFs) in the 5'-untranslated regions (5'-UTRs) of certain eukaryotic mRNAs encode evolutionarily conserved functional peptides, such as cis-acting regulatory peptides that control translation of downstream main ORFs (mORFs). For genome-wide searches for uORFs with conserved peptide sequences (CPuORFs), comparative genomic studies have been conducted, in which uORF sequences were compared between selected species. To increase chances of identifying CPuORFs, we previously developed an approach in which uORF sequences were compared using BLAST between Arabidopsis and any other plant species with available transcript sequence databases. If this approach is applied to multiple plant species belonging to phylogenetically distant clades, it is expected to further comprehensively identify CPuORFs conserved in various plant lineages, including those conserved among relatively small taxonomic groups. RESULTS: To efficiently compare uORF sequences among many species and efficiently identify CPuORFs conserved in various taxonomic lineages, we developed a novel pipeline, ESUCA. We applied ESUCA to the genomes of five angiosperm species, which belong to phylogenetically distant clades, and selected CPuORFs conserved among at least three different orders. Through these analyses, we identified 89 novel CPuORF families. As expected, ESUCA analysis of each of the five angiosperm genomes identified many CPuORFs that were not identified from ESUCA analyses of the other four species. However, unexpectedly, these CPuORFs include those conserved across wide taxonomic ranges, indicating that the approach used here is useful not only for comprehensive identification of narrowly conserved CPuORFs but also for that of widely conserved CPuORFs. Examination of the effects of 11 selected CPuORFs on mORF translation revealed that CPuORFs conserved only in relatively narrow taxonomic ranges can have sequence-dependent regulatory effects, suggesting that most of the identified CPuORFs are conserved because of functional constraints of their encoded peptides. CONCLUSIONS: This study demonstrates that ESUCA is capable of efficiently identifying CPuORFs likely to be conserved because of the functional importance of their encoded peptides. Furthermore, our data show that the approach in which uORF sequences from multiple species are compared with those of many other species, using ESUCA, is highly effective in comprehensively identifying CPuORFs conserved in various taxonomic ranges.
  • Reverse genetics-based biochemical studies of the ribosomal exit tunnel constriction region in eukaryotic ribosome stalling: spatial allocation of the regulatory nascent peptide at the constriction
    Seidai Takamatsu; Yubun Ohashi; Noriyuki Onoue; Yoko Tajima; Tomoya Imamichi; Shinya Yonezawa; Kyoko Morimoto; Hitoshi Onouchi; Yui Yamashita; Satoshi Naito
    Nucleic Acids Research, 48, 4, 1985, 1999, Oxford University Press (OUP), 28 Feb. 2020, [Peer-reviewed], [International Magazine]
    English, Scientific journal, AbstractA number of regulatory nascent peptides have been shown to regulate gene expression by causing programmed ribosome stalling during translation. Nascent peptide emerges from the ribosome through the exit tunnel, and one-third of the way along which β-loop structures of ribosomal proteins uL4 and uL22 protrude into the tunnel to form the constriction region. Structural studies have shown interactions between nascent peptides and the exit tunnel components including the constriction region. In eukaryotes, however, there is a lack of genetic studies for the involvement of the constriction region in ribosome stalling. Here, we established transgenic Arabidopsis lines that carry mutations in the β-loop structure of uL4. Translation analyses using a cell-free translation system derived from the transgenic Arabidopsis carrying the mutant ribosome showed that the uL4 mutations reduced the ribosome stalling of four eukaryotic stalling systems, including those for which stalled structures have been solved. Our data, which showed differential effects of the uL4 mutations depending on the stalling systems, explained the spatial allocations of the nascent peptides at the constriction that were deduced by structural studies. Conversely, our data may predict allocation of the nascent peptide at the constriction of stalling systems for which structural studies are not done.
  • バイオインフォマティクスによる配列データベース横断解析に基づく機能上流ORFの推定
    髙橋広夫; 伊藤素行; 尾之内均
    生物工学会誌, 97, 8, 496, 499, (公社)日本生物工学会, Aug. 2019, [Invited], [Last author]
    Japanese
  • Boron-dependent translational suppression of the borate exporter BOR1 contributes to the avoidance of boron toxicity
    Izumi Aibara; Tatsuya Hirai; Koji Kasai; Junpei Takano; Hitoshi Onouchi; Satoshi Naito; Toru Fujiwara; Kyoko Miwa
    Plant Physiology, 177, 2, 759, 774, American Society of Plant Biologists, 01 Jun. 2018, [Peer-reviewed]
    English, Scientific journal
  • Identification of Arabidopsis thaliana upstream open reading frames encoding peptide sequences that cause ribosomal arrest
    Noriya Hayashi; Shun Sasaki; Hiro Takahashi; Yui Yamashita; Satoshi Naito; Hitoshi Onouchi
    NUCLEIC ACIDS RESEARCH, 45, 15, 8844, 8858, Sep. 2017, [Peer-reviewed], [Last author, Corresponding author]
    English, Scientific journal
  • The Minimum Open Reading Frame, AUG-Stop, Induces Boron-Dependent Ribosome Stalling and mRNA Degradation.
    Mayuki Tanaka; Naoyuki Sotta; Yusuke Yamazumi; Yui Yamashita; Kyoko Miwa; Katsunori Murota; Yukako Chiba; Masami Yokota Hirai; Tetsu Akiyama; Hitoshi Onouchi; Satoshi Naito; Toru Fujiwara
    The Plant cell, 28, 11, 2830, 2849, Nov. 2016, [Peer-reviewed], [International Magazine]
    English, Scientific journal, Upstream open reading frames (uORFs) are often translated ahead of the main ORF of a gene and regulate gene expression, sometimes in a condition-dependent manner, but such a role for the minimum uORF (hereafter referred to as AUG-stop) in living organisms is currently unclear. Here, we show that AUG-stop plays an important role in the boron (B)-dependent regulation of NIP5;1, encoding a boric acid channel required for normal growth under low B conditions in Arabidopsis thaliana High B enhanced ribosome stalling at AUG-stop, which was accompanied by the suppression of translation and mRNA degradation. This mRNA degradation was promoted by an upstream conserved sequence present near the 5'-edge of the stalled ribosome. Once ribosomes translate a uORF, reinitiation of translation must take place in order for the downstream ORF to be translated. Our results suggest that reinitiation of translation at the downstream NIP5;1 ORF is enhanced under low B conditions. A genome-wide analysis identified two additional B-responsive genes, SKU5 and the transcription factor gene ABS/NGAL1, which were regulated by B-dependent ribosome stalling through AUG-stop. This regulation was reproduced in both plant and animal transient expression and cell-free translation systems. These findings suggest that B-dependent AUG-stop-mediated regulation is common in eukaryotes.
  • 止まって働くリボソーム 新生ペプチドが司る植物の細胞内恒常性維持機構
    山下由衣; 尾之内均; 内藤哲
    化学と生物, 54, 3, 191, 197, Mar. 2016, [Invited]
    Japanese
  • 4-2-1 シロイヌナズナのホウ素輸送体、NIP5;1のホウ素に応答したmRNA蓄積のmRNA分解と転写の制御機構の解析（4-2 植物の微量栄養素，2016年度佐賀大会）
    田中 真幸; 反田 直之; 千葉 由佳子; 尾之内 均; 内藤 哲; 藤原 徹
    日本土壌肥料学会講演要旨集, 62, 57, 57, 一般社団法人 日本土壌肥料学会, 2016
    Japanese
  • シロイヌナズナCGS1 mRNAの新生ペプチドによる翻訳アレストへのリボソーム出口トンネルの関与
    高松 世大; 大橋 悠文; 尾上 典之; 山下 由衣; 尾之内 均; 内藤 哲
    日本生化学会大会・日本分子生物学会年会合同大会講演要旨集, 88回・38回, [3T25, 07(3P0741)], (公社)日本生化学会, Dec. 2015
    Japanese
  • An upstream open reading frame represses expression of a tomato homologue of Arabidopsis ANAC096, a NAC domain transcription factor gene, in a peptide sequence-dependent manner
    Abdul Latif Noh; Shun Watanabe; Hiro Takahashi; Satoshi Naito; Hitoshi Onouchi
    PLANT BIOTECHNOLOGY, 32, 2, 157, U126, Jun. 2015, [Peer-reviewed], [Last author, Corresponding author]
    English, Scientific journal
  • Identification of novel Arabidopsis thaliana upstream open reading frames that control expression of the main coding sequences in a peptide sequence-dependent manner
    Isao Ebina; Mariko Takemoto-Tsutsumi; Shun Watanabe; Hiroaki Koyama; Yayoi Endo; Kaori Kimata; Takuya Igarashi; Karin Murakami; Rin Kudo; Arisa Ohsumi; Abdul Latif Noh; Hiro Takahashi; Satoshi Naito; Hitoshi Onouchi
    NUCLEIC ACIDS RESEARCH, 43, 3, 1562, 1576, Feb. 2015, [Peer-reviewed], [Last author, Corresponding author]
    English, Scientific journal
  • A U-system approach for predicting metabolic behaviors and responses based on an alleged metabolic reaction network
    Kansuporn Sriyudthsak; Yuji Sawada; Yukako Chiba; Yui Yamashita; Shigehiko Kanaya; Hitoshi Onouchi; Toru Fujiwara; Satoshi Naito; Ebernard O. Voit; Fumihide Shiraishi; Masami Yokota Hirai
    BMC SYSTEMS BIOLOGY, 8, S4, Dec. 2014, [Peer-reviewed]
    English, Scientific journal
  • The N-Terminal Cleavable Pre-Sequence Encoded in the First Exon of Cystathionine gamma-Synthase Contains Two Different Functional Domains for Chloroplast Targeting and Regulation of Gene Expression
    Yuka Hagiwara-Komoda; Tomoya Sugiyama; Yui Yamashita; Hitoshi Onouchi; Satoshi Naito
    PLANT AND CELL PHYSIOLOGY, 55, 10, 1779, 1792, Oct. 2014, [Peer-reviewed]
    English, Scientific journal
  • Polyamine-responsive ribosomal arrest at the stop codon of an upstream open reading frame of the AdoMetDC1 gene triggers nonsense-mediated mRNA decay in Arabidopsis thaliana.
    Naoko Uchiyama-Kadokura; Karin Murakami; Mariko Takemoto; Naoto Koyanagi; Katsunori Murota; Satoshi Naito; Hitoshi Onouchi
    Plant & cell physiology, 55, 9, 1556, 67, Sep. 2014, [Peer-reviewed], [Last author, Corresponding author], [Domestic magazines]
    English, Scientific journal, During mRNA translation, nascent peptides with certain specific sequences cause arrest of ribosomes that have synthesized themselves. In some cases, such ribosomal arrest is coupled with mRNA decay. In yeast, mRNA quality control systems have been shown to be involved in mRNA decay associated with ribosomal arrest. However, a link between ribosomal arrest and mRNA quality control systems has not been found in multicellular organisms. In this study, we aimed to explore the relationship between ribosomal arrest and mRNA decay in plants. For this purpose, we used an upstream open reading frame (uORF) of the Arabidopsis thaliana AdoMetDC1 gene, in which the uORF-encoded peptide is involved in polyamine-responsive translational repression of the main coding sequence. Our in vitro analyses revealed that the AdoMetDC1 uORF-encoded peptide caused ribosomal arrest at the uORF stop codon in response to polyamine. Using transgenic calli harboring an AdoMetDC1 uORF-containing reporter gene, we showed that polyamine promoted mRNA decay in a uORF sequence-dependent manner. These results suggest that the polyamine-responsive ribosomal arrest mediated by the uORF-encoded peptide is coupled with mRNA decay. Our results also showed that the polyamine-responsive acceleration of mRNA decay was compromised by defects in factors that are essential for nonsense-mediated mRNA decay (NMD), an mRNA quality control system that degrades mRNAs with premature stop codons, suggesting that NMD is involved in AdoMetDC1 uORF peptide-mediated mRNA decay. Collectively, these findings suggest that AdoMetDC1 uORF peptide-mediated ribosomal arrest at the uORF stop codon induces NMD.
  • Ribosomes in a Stacked Array
    Yui Yamashita; Yoshitomo Kadokura; Naoyuki Sotta; Toru Fujiwara; Ichigaku Takigawa; Akiko Satake; Hitoshi Onouchi; Satoshi Naito
    JOURNAL OF BIOLOGICAL CHEMISTRY, 289, 18, 12693, 12704, May 2014, [Peer-reviewed]
    English, Scientific journal
  • Translation elongation arrest induced by S-adenosyl-l-methionine-sensing nascent peptide in plants
    Yui Yamashita; Noriyuki Onoue; Katsunori Murota; Hitoshi Onouchi; Satoshi Naito
    Regulatory Nascent Polypeptides, 187, 201, Apr. 2014, [Peer-reviewed], [Invited]
    In book
  • A halt in poly(A) shortening during S-adenosyl-L-methionine-induced translation arrest in CGS1 mRNA of Arabidopsis thaliarta
    Yui Yamashita; Ingrid Lambein; Soko Kobayashi; Hitoshi Onouchi; Yukako Chiba; Satoshi Naito
    GENES & GENETIC SYSTEMS, 88, 4, 241, 249, Aug. 2013, [Peer-reviewed]
    English, Scientific journal
  • SS-mPMG and SS-GA: Tools for finding pathways and dynamic simulation of metabolic networks
    Tetsuo Katsuragi; Naoaki Ono; Keiichi Yasumoto; Md. Altaf-Ul-Amin; Masami Y. Hirai; Kansuporn Sriyudthsak; Yuji Sawada; Yui Yamashita; Yukako Chiba; Hitoshi Onouchi; Toru Fujiwara; Satoshi Naito; Fumihide Shiraishi; Shigehiko Kanaya
    Plant and Cell Physiology, 54, 5, 728, 739, May 2013, [Peer-reviewed]
    English, Scientific journal
  • Changes in mRNA stability associated with cold stress in arabidopsis cells
    Yukako Chiba; Katsuhiko Mineta; Masami Y. Hirai; Yuya Suzuki; Shigehiko Kanaya; Hiro Takahashi; Hitoshi Onouchi; Junji Yamaguchi; Satoshi Naito
    Plant and Cell Physiology, 54, 2, 181, 194, Oxford University Press, 2013, [Peer-reviewed]
    English, Scientific journal
  • BAIUCAS: a novel BLAST-based algorithm for the identification of upstream open reading frames with conserved amino acid sequences and its application to the Arabidopsis thaliana genome
    Hiro Takahashi; Anna Takahashi; Satoshi Naito; Hitoshi Onouchi
    BIOINFORMATICS, 28, 17, 2231, 2241, Sep. 2012, [Peer-reviewed], [Last author, Corresponding author]
    English, Scientific journal
  • Boron-Dependent Degradation of NIP5;1 mRNA for Acclimation to Excess Boron Conditions in Arabidopsis
    Mayuki Tanaka; Junpei Takano; Yukako Chiba; Fabien Lombardo; Yuki Ogasawara; Hitoshi Onouchi; Satoshi Naito; Toru Fujiwara
    PLANT CELL, 23, 9, 3547, 3559, Sep. 2011, [Peer-reviewed]
    English, Scientific journal
  • Arabidopsis cell-free extract, ACE, a new in vitro translation system derived from Arabidopsis callus cultures.
    Katsunori Murota; Yuka Hagiwara-Komoda; Keisuke Komoda; Hitoshi Onouchi; Masayuki Ishikawa; Satoshi Naito
    Plant & cell physiology, 52, 8, 1443, 53, Aug. 2011, [Peer-reviewed], [Domestic magazines]
    English, Scientific journal, The analysis of post-transcriptional regulatory mechanisms in plants has benefited greatly from the use of cell-free extract systems. Arabidopsis as a model system provides extensive genetic resources; however, to date a suitable cell-free translation system from Arabidopsis has not been available. In this study, we devised an Arabidopsis cell-free extract (ACE) to be used for in vitro translation studies. Protoplasts were prepared from callus cultures derived from Arabidopsis seedlings, and cell-free extracts were prepared after evacuolation of the protoplasts by Percoll gradient centrifugation. The new ACE system exhibits translation activity comparable with that of the wheat germ extract system. We demonstrated that ACE prepared from the 5'-3' exoribonuclease-deficient mutant of Arabidopsis, xrn4-5, exhibited increased stability of an uncapped mRNA as compared with that from wild-type Arabidopsis. We applied the ACE system to study post-transcriptional regulation of AtCGS1. AtCGS1 codes for cystathionine γ-synthase (CGS) that catalyzes the first committed step of methionine and S-adenosyl-l-methionine (AdoMet) biosynthesis in plants, and is feedback regulated by mRNA degradation coupled with translation elongation arrest. The ACE system was capable of reproducing translation elongation arrest and subsequent AtCGS1 mRNA degradation that are induced by AdoMet. The ACE system described here can be prepared in a month after seed sowing and will make it possible to study post-transcriptional regulation of plant genes while taking advantage of the genetics of Arabidopsis.
  • S-Adenosyl-L-methionine Induces Compaction of Nascent Peptide Chain inside the Ribosomal Exit Tunnel upon Translation Arrest in the Arabidopsis CGS1 Gene
    Noriyuki Onoue; Yui Yamashita; Nobuhiro Nagao; Derek B. Goto; Hitoshi Onouchi; Satoshi Naito
    JOURNAL OF BIOLOGICAL CHEMISTRY, 286, 17, 14903, 14912, Apr. 2011, [Peer-reviewed]
    English, Scientific journal
  • Analysis of boron-dependent regulatory mechanism of NIP5;1, transcript accumulation in Arabidopsis thaliana
    Tanaka Mayuki; Takano Junpei; Chiba Yukako; Onouchi Hitoshi; Naito Satoshi; Fujiwara Toru
    Plant and Cell Physiology Supplement, 2011, 0, 540, 540, The Japanese Society of Plant Physiologists, 2011
    NIP5;1 encodes s a boron (B) channel required for normal plant growth under low B conditions and its transcript accumulation is upregulated under low B conditions. At the annual meeting last year, we reported that at least +182-+200 region of 5'untranlated region (5'UTR) is important for NIP5;1 transcript accumulation in response to low B.
    To elucidate mechanisms of NIP5;1 regulation in response to B, the half-life of NIP5;1 mRNA was estimated by determining time course of mRNA decay after exposure to an RNA synthesis inhibitor under the high and low B conditions. We established constructs carrying either intact 5'UTR or 5'UTR without the sequence from +182 to +200 between the GUS reporter gene and the cauliflower mosaic virus 35S RNA promoter. In transgenic plants carrying the intact 5'UTR, the GUS mRNA level under the high B condition more rapidly decreased compared to the low B condition. In transgenic plants carrying the 5'UTR without +182-+200 construct, GUS mRNA level was not significantly different between B treatments. These results indicated that the sequence from +182 to +200 is required for B-dependent mRNA degradation.
  • Comparative metabolomics charts the impact of genotype-dependent methionine accumulation in Arabidopsis thaliana
    Miyako Kusano; Atsushi Fukushima; Henning Redestig; Makoto Kobayashi; Hitomi Otsuki; Hitoshi Onouchi; Satoshi Naito; Masami Yokota Hirai; Kazuki Saito
    AMINO ACIDS, 39, 4, 1013, 1021, Oct. 2010, [Peer-reviewed]
    English, Scientific journal
  • Polar localization and degradation of Arabidopsis boron transporters through distinct trafficking pathways
    Junpei Takano; Mayuki Tanaka; Atsushi Toyoda; Kyoko Miwa; Koji Kasai; Kentaro Fuji; Hitoshi Onouchi; Satoshi Naito; Toru Fujiwara
    Proceedings of the National Academy of Sciences of the United States of America, 107, 11, 5220, 5225, 16 Mar. 2010, [Peer-reviewed]
    English, Scientific journal
  • Nascent peptide-mediated translation elongation arrest of Arabidopsis thaliana CGS1 mRNA occurs autonomously.
    Hitoshi Onouchi; Yuhi Haraguchi; Mari Nakamoto; Daisuke Kawasaki; Yoko Nagami-Yamashita; Katsunori Murota; Azusa Kezuka-Hosomi; Yukako Chiba; Satoshi Naito
    Plant & cell physiology, 49, 4, 549, 56, Apr. 2008, [Peer-reviewed], [Lead author], [Domestic magazines]
    English, Scientific journal, The Arabidopsis thaliana CGS1 gene encodes cystathionine gamma-synthase, the first committed enzyme of methionine biosynthesis in higher plants. Expression of CGS1 is feedback-regulated at the step of mRNA degradation in response to S-adenosyl-L-methionine (AdoMet). A short stretch of amino acid sequence, termed the MTO1 region, encoded within the first exon of CGS1 itself acts in cis in the regulation. In vitro analyses using wheat germ extract (WGE) revealed that AdoMet induces temporal translation arrest of CGS1 mRNA prior to mRNA degradation. This translational pausing occurs immediately downstream of the MTO1 region and is mediated by the nascent MTO1 peptide. In order to elucidate further the nature of this unique regulatory mechanism, we have examined whether a non-plant system also contains the post-transcriptional regulation activity. Despite the fact that mammals do not carry cystathionine gamma-synthase, AdoMet was able to induce the MTO1 sequence-dependent translation elongation arrest in rabbit reticulocyte lysate (RRL) in a similar manner to that observed in WGE. This result suggests that MTO1 peptide-mediated translation arrest does not require a plant-specific factor and rather most probably occurs via a direct interaction between the nascent MTO1 peptide and the ribosome that has translated it. In contrast, decay intermediates of CGS1 mRNA normally observed upon induction of CGS1 mRNA decay in plant systems were not detected in RRL, raising the possibility that CGS1 mRNA degradation involves a plant-specific mechanism.
  • Ribosome stacking defines CGS1 mRNA degradation sites during nascent peptide-mediated translation arrest
    Yuhi Haraguchi; Yoshitomo Kadokura; Mari Nakamoto; Hitoshi Onouchi; Satoshi Naito
    PLANT AND CELL PHYSIOLOGY, 49, 3, 314, 323, Mar. 2008, [Peer-reviewed]
    English, Scientific journal
  • Promoter region of the beta subunit gene of beta-conglycinin responds to methionine and glutathione in transient assays using Arabidopsis protoplasts
    Naoko Ohkama-Ohtsu; Azusa Kezuka; Hitoshi Onouchi; Toru Fujiwara; Satoshi Naito
    SOIL SCIENCE AND PLANT NUTRITION, 54, 1, 128, 132, Feb. 2008, [Peer-reviewed]
    English, Scientific journal
  • Nascent peptide-mediated translation elongation arrest coupled with mRNA degradation in the CGS1 gene of Arabidopsis
    H Onouchi; Y Nagami; Y Haraguchi; M Nakamoto; Y Nishimura; R Sakurai; N Nagao; D Kawasaki; Y Kadokura; S Naito
    GENES & DEVELOPMENT, 19, 15, 1799, 1810, Aug. 2005, [Peer-reviewed], [Lead author]
    English, Scientific journal
  • Mizoguchi T, Wright L, Fujiwara S, Cremer F, Lee K, Onouchi H, Mouradov A, Fowler S, Kamada H, Putterill J, Coupland G. : "Distinct roles of GIGANTEA in promoting flowering and regulating circadian rhythms in Arabidopsis" Plant Cell, 17:2255-2270 (2005)*
    2005, [Peer-reviewed]
  • Autoregulated MRNA stability of the gene for cystathionine gamma-synthase, the key-step enzyme of methionine biosynthesis in Arabidopsis
    Y Nagami; H Onouchi; Y Haraguchi; R Sakurai; M Nakamoto; N Nagao; S Naito
    Sulfur Transport and Assimilation in Plants in the Post Genomic Era, 73, 80, 2005, [Invited]
    English, International conference proceedings
  • Dynamics of methionine biosynthesis
    Derek B. Goto; Hitoshi Onouchi; Satoshi Naito
    Plant Biotechnology, 22, 5, 379, 388, Japanese Society for Plant Cell and Molecular Biology, 2005, [Peer-reviewed], [Invited]
    English, Scientific journal, Methionine biosynthesis in plants provides the primary source of this essential amino acid. Early pioneering works characterised many of the steps involved in the methionine biosynthetic pathway and properties of the enzymes involved. They also showed that methionine biosynthesis was strictly controlled in higher plants and part of a larger, complex pathway that involves the biosynthesis of lysine, threonine and isoleucine. The adoption of the model plant Arabidopsis thaliana has enabled us to isolate mto mutants that over-accumulate soluble methionine. Through the analysis of these mto mutants, valuable insight has been gained into the regulation and in vivo dynamics of the methionine biosynthetic pathway in higher plants. Each of the three mutant mto loci identified to date (mto1, mto2, mto3) disrupt different processes within the pathway and display distinct phenotypic profiles. They have also revealed that, in addition to the common feedback controls, the pathway is subject to changing temporal and spatial regulation over the course of the plant life cycle.
  • Autoregulation of the gene for cystathionine gamma-synthase in arabidopsis: post-transcriptional regulation induced by S-adenosylmethionine
    H Onouchi; Lambein, I; R Sakurai; A Suzuki; Y Chiba; S Naito
    BIOCHEMICAL SOCIETY TRANSACTIONS, 32, 597, 600, Aug. 2004, [Invited], [Lead author]
    English, Scientific journal
  • Decay kinetics of autogenously regulated CGS1 mRNA that codes for cystathionine gamma-synthase in Arabidopsis thaliana
    Lambein, I; Y Chiba; H Onouchi; S Naito
    PLANT AND CELL PHYSIOLOGY, 44, 9, 893, 900, Sep. 2003, [Peer-reviewed]
    English, Scientific journal
  • S-adenosyl-L-methionine is an effector in the posttranscriptional autoregulation of the cystathionine gamma-synthase gene in Arabidopsis
    Y Chiba; R Sakurai; M Yoshino; K Ominato; M Ishikawa; H Onouchi; S Naito
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 100, 18, 10225, 10230, Sep. 2003, [Peer-reviewed]
    English, Scientific journal
  • 細胞内シグナル伝達の機構 新生ペプチドによる遺伝子発現制御 (植物の形づくり--遺伝子からみた分子メカニズム) -- (細胞分化を促すシグナル伝達系)
    尾之内 均; 千葉 由佳子; 内藤 哲
    蛋白質核酸酵素, 47, 12, 1730, 1734, 共立出版, Sep. 2002, [Invited], [Lead author]
    Japanese
  • Identification of a short highly conserved amino acid sequence as the functional region required for Posttranscriptional autoregulation of the cystathionine gamma-synthase gene in Arabidopsis
    K Ominato; H Akita; A Suzuki; F Kijima; T Yoshino; M Yoshino; Y Chiba; H Onouchi; S Naito
    JOURNAL OF BIOLOGICAL CHEMISTRY, 277, 39, 36380, 36386, Sep. 2002, [Peer-reviewed]
    English, Scientific journal
  • [Regulation of gene expression by nascent peptide].
    Onouchi H; Chiba Y; Naito S
    Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme, 47, 12 Suppl, 1730, 1734, 12 Suppl, Sep. 2002, [Invited], [Lead author]
  • The ASYMMETRIC LEAVES2 gene of Arabidopsis thaliana, required for formation of a symmetric flat leaf lamina, encodes a member of a novel family of proteins characterized by cysteine repeats and a leucine zipper
    H Iwakawa; Y Ueno; E Semiarti; H Onouchi; S Kojima; H Tsukaya; M Hasebe; T Soma; M Ikezaki; C Machida; Y Machida
    PLANT AND CELL PHYSIOLOGY, 43, 5, 467, 478, May 2002, [Peer-reviewed]
    English, Scientific journal
  • A single-nucleotide mutation in a gene encoding S-adenosylmethionine synthetase is associated with methionine over-accumulation phenotype in Arabidopsis thaliana
    DB Goto; M Ogi; F Kijima; T Kumagai; F van Werven; H Onouchi; S Naito
    GENES & GENETIC SYSTEMS, 77, 2, 89, 95, Apr. 2002, [Peer-reviewed]
    English, Scientific journal
  • The first exon coding region of cystathionine gamma-synthase gene is necessary and sufficient for downregulation of its own mRNA accumulation in transgenic Arabidopsis thaliana
    A Suzuki; Y Shirata; H Ishida; Y Chiba; H Onouchi; S Naito
    PLANT AND CELL PHYSIOLOGY, 42, 10, 1174, 1180, Oct. 2001, [Peer-reviewed]
    English, Scientific journal
  • The transposition pattern of the Ac element and its use for targeted transposition in Arabidopsis thaliana
    Machida Chiyoko; Onouchi Hitoshi; Tanaka Hirokazu; Hamada Susumu; Ishikawa Takaaki; Semiarti Endang; Iwakawa Hidekazu; Nomura Kiyohito; Machida Yasunori
    Annual report of Research Institute for Biological Function, 1, 0, 9, 16, Chubu University, 31 Mar. 2001, [Invited]
    English, In order to evaluate feasibility of the gene tagging by the maize transposable element Ac in heterologous plant systems, we have investigated physical distances and directions of transposition of the element in Arabidopsis thaliana and tobacco cultured cell line BY-2. We prepared a T-DNA construct that carried a non-autonomous derivative of Ac with a site for cleavage by endonuclease I-SceI (designated dAc-I-RS element). Another cleavage site was also introduced into the T-DNA region outside dAc-I-RS. A number of transgenic Arabidopsis plants were generated, each of which had a single copy ...
  • Suarez-Lopez P, Wheatley K, Robson F, Onouchi H, Valverde F and Coupland G.:"CONSTANS mediates between the circadian clock and the control of flowering in Arabidopsis", Nature, 410(6832):1116-1120 (2001).*
    2001, [Peer-reviewed]
    English, Scientific journal
  • A subtilisin-like serine protease is required for epidermal surface formation in Arabidopsis embryos and juvenile plants.
    Tanaka, H; Onouchi, H; Kondo, M; Hara-Nishimura, I; Nishimura, M; Machida, C; Machida, Y
    Development, 128, 4681, 4689, 2001, [Peer-reviewed]
  • The ASYMMETRIC LEAVES2 gene of Arabidopsis thaliana regulates formation of a symmetric lamina, establishment of venation and repression of meristem-related homeobox genes in leaves.
    Semiarti, E; Onouchi, H; Torikai, S; Ishikawa, T; Machida, Y; Machida, C
    Development, 128, 1771, 1783, 2001, [Peer-reviewed]
  • The transposition pattern of the Ac element in tobacco cultured cells.
    Endang Semiarti; Hitoshi Onouchi; Satomi Torikai; Takaaki Ishikawa; Yasunori Machida; Chiyoko Machida
    Genes Genet. Syst., 76, 2, 131, 139, The Genetics Society of Japan, 2001, [Peer-reviewed]
    English, We investigated physical distances and directions of transposition of the maize transposable element Ac in tobacco cultured cells. We introduced a T-DNA construct that carried a non-autonomous derivative of Ac (designated dAc-I-RS) that included sites for cleavage by restriction endonuclease MluI. Another cleavage site was also introduced into the T-DNA region outside of the dAc-I-RS transposable element. The tobacco cultured cell line BY-2 was transformed with the T-DNA and several transformed lines that had a single copy of the T-DNA at a different chromosomal location were isolated. These lines were co-cultured with Agrobacterium tumefaciens cells that carried a cDNA for the Ac transposase gene under the control of various promoters. Sublines of cultured cells in which dAc-I-RS had been transposed, were isolated. The genomic DNAs of these sublines were isolated and digested with MluI. Sizes of DNA segments generated by digestion were determined by pulse-field gel electrophoresis. Our results showed that 20 to 70% of transposition events had occurred within several hundreds kilo-base pairs (kb) on the same chromosome. These results demonstrate that the Ac-Ds element preferentially transposed to regions near the original site in a tobacco chromosome. In addition, the present results are an example of asymmetric transposition as demonstrated by the distance of transposition on the chromosome.
  • Mutations in the WUSCHEL gene of Arabidopsis thaliana result in the development of shoots without juvenile leaves
    Susumu Hamada; Hitoshi Onouchi; Hirokazu Tanaka; Mitsuko Kudo; Yao‐Guang Liu; Daisuke Shibata; Chiyoko Machida; Yasunori Machida
    The Plant Journal, 24, 1, 91, 101, Wiley, Oct. 2000
    Scientific journal, Summary

    The vegetative growth of Arabidopsis thaliana can be divided into two phases. The transition from the juvenile (early) phase to the adult (later) phase is associated with changes in several morphological features of leaves, such as the shape of leaf blades, the number of trichomes and patterns of venation. In a screening of mutants with altered morphological identities of leaves, we found one which we named juvenile leafless and misshapen shoot apical meristem (jam). The mutation represented a new allele of the WUSCHEL (WUS) gene, and, in its presence, plants produced no juvenile leaves. Analysis of the morphology of mutant plants revealed that all the rosette leaves had characteristics of adult leaves. The formation of the first rosette leaf in the wus(jam) mutant was markedly delayed, and occurred at the almost same time as formation of the third or fourth leaf in wild‐type plants. In the wild‐type, these leaves correspond to the first adult leaves. Analysis by RT–PCR showed that transcripts of WUS accumulated in shoot apices and roots, but not in cotyledons and leaves. The present results suggest that the WUS gene controls the morphological traits of rosette leaves either directly or indirectly. In view of the predicted function of the WUS gene, namely maintenance of stem cells within the shoot apical meristem, we suggest that the lack of juvenile leaves in the mutant might have been caused by interruption of leaf initiation during the juvenile phase or by halting of an entire process of formation of juvenile leaves.
  • Mutagenesis of plants overexpressing CONSTANS demonstrates novel interactions among Arabidopsis flowering-time genes
    H Onouchi; MI Igeno; C Perilleux; K Graves; G Coupland
    PLANT CELL, 12, 6, 885, 900, Jun. 2000, [Peer-reviewed], [Lead author]
    English, Scientific journal
  • Hamada S, Onouchi H, Tanaka H, Kudo M, Liu Y, Shibata D, Machida C and Machida M.: "Mutations in the WUSCHEL gene of Arabidopsis thaliana result in the development of shoots without juvenile leaves", Plant J., 24:1-13(2000)*
    2000, [Peer-reviewed]
    English, Scientific journal
  • Samach A, Onouchi H, Gold SE, Ditta GS, Schwarz-Sommer Z, Yanofsky MF, Coupland G.: "Distinct roles of CONSTANS target genes in reproductive development of Arabidopsis", Science, 288:1613-1616 (2000)*
    2000, [Peer-reviewed], [Lead author]
    English, Scientific journal
  • MOLECULAR-GENETIC ANALYSIS OF THE AS2 GENE ON THE MECHANISM OF SYMMETRICAL LEAF DEVEL OPMENT IN Arabidopsis thaliana
    SEMIARTI Endang; IWAKAWA Hidekazu; TSUKAYA Hirokazu; ONOUCHI Hitoshi; MACHIDA Chiyoko; MACHIDA Yasunori
    Plant and cell physiology, 41, 0, s212, 日本植物生理学会, 2000, [Peer-reviewed]
    English
  • GIGANTEA : a circadian clock-controlled gene that regulates photoperiodic flowering in Arabidopsis and encodes a protein with several possible membrane-spanning domains
    FOWLER S.
    EMBO J., 18, 4679, 4688, 1999, [Peer-reviewed]
    English, Scientific journal, Fowler S, Lee K, Onouchi H, Samach A, Richardson K, Morris B, Coupland G, Putterill J. "GIGANTEA: a circadian clock-controlled gene that regulates photoperiodic flowering in Arabidopsis and encodes a protein with several possible membrane-spanning domains", EMBO J. , 18, 4679-88(1999)*
  • Six Arabidopsis thaliana homologues of the human respiratory burst oxidase (gp91(phox))
    MA Torres; H Onouchi; S Hamada; C Machida; KE Hammond-Kosack; JDG Jones
    PLANT JOURNAL, 14, 3, 365, 370, May 1998, [Peer-reviewed]
    English, Scientific journal
  • Torres MA, Onouchi H, Hamada S, Machida C, Hammond-Kosack KE, Jones JD. :"Six Arabidopsis thaliana homologues of the human respiratory burst oxidase (gp91phox)". Plant J., 14(3):365-370 (1998)*
    1998, [Peer-reviewed]
    English, Scientific journal
  • Characterization of the transposition pattern of the Ac element in Arabidopsis thaliana using endonuclease I-SceI
    C Machida; H Onouchi; J Koizumi; S Hamada; E Semiarti; S Torikai; Y Machida
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 94, 16, 8675, 8680, Aug. 1997, [Peer-reviewed]
    English, Scientific journal
  • トランスポソンタギング
    町田 千代子; 尾之内 均; 浜田 進
    化学と生物, 34, 9, 617, 623, 学会出版センタ-, 25 Sep. 1996, [Invited]
    Japanese
  • A new method of manipulation of chromosomes in higher plant
    MACHIDA Chiyoko; ONOUCHI Hitoshi
    Bioscience & industry, 54, 8, 16, 22, バイオインダストリ-協会, 01 Aug. 1996, [Invited]
    Japanese
  • Manipulation of plant chromosomes using the Ac transposale element and R-RS site-specific recombination system from Z. rouxii.
    MACHIDA C; ONOUCHI H; KOIZUMI J; KUDO M; MACHIDA Y
    育種学最近の進歩, 37, 0, 13, 16, Nov. 1995, [Invited]
    Japanese
  • VISUALIZATION OF SITE-SPECIFIC RECOMBINATION CATALYZED BY A RECOMBINASE FROM ZYGOSACCHAROMYCES-ROUXII IN ARABIDOPSIS-THALIANA
    H ONOUCHI; R NISHIHAMA; M KUDO; Y MACHIDA; C MACHIDA
    MOLECULAR & GENERAL GENETICS, 247, 6, 653, 660, 1995, [Peer-reviewed], [Lead author]
    English, Scientific journal
  • Analysis of Plant Genes Using T-DNA Transfer from Agrobacterium.
    MACHIDA Chiyoko; ONOUCHI Hitoshi; MACHIDA Yasunori
    Nippon Nōgeikagaku Kaishi, 67, 4, 711, 715, Japan Society for Bioscience, Biotechnology, and Agrochemistry, 1993
    Japanese
  • Onouchi H, Yokoi K, Machida C, Matsuzaki H, Oshima Y, Matsuoka K, Nakamura K, Machida Y. "Operation of an efficient site-specific recombination system of Zygosaccharomyces rouxii in tobacco cells." Nucleic Acids Res., 19(23):6373-6378(1991)*
    1991, [Peer-reviewed], [Lead author]
    English, Scientific journal
■ Other Activities and Achievements
■ Books and other publications
  • 植物ゲノム科学辞典
    尾之内 均
    朝倉書店, 2009, [Contributor]
  • RNA実験ノート
    羊土社, 2008, [Contributor]
  • 生化学辞典（第４版）
    尾之内 均
    東京化学同人, 2007, [Contributor]
■ Lectures, oral presentations, etc.
  • 維管束形成を司るLONESOME HIGHWAY遺伝子の上流ORFが介する翻訳制御とmRNA分解制御
    梅原俊一; 木俣薫織; 戸田智美; 遠洞弥生; 大角有里沙; 蝦名績; 内藤 哲; 尾之内均
    第59回日本植物生理学会年会, 29 Mar. 2018, Japanese, Oral presentation
    [Domestic Conference]
  • 核小体ストレスに応答して翻訳を制御するシロイヌナズナANAC082遺伝子の上流ORF
    佐々木 駿; 工藤 凛; 渡部 俊; 大林 祝; 杉山 宗隆; 刑部 祐里子; 刑部 敬史; 内藤 哲; 尾之内 均
    第59回日本植物生理学会年会, 29 Mar. 2018, Japanese, Oral presentation
    [Domestic Conference]
  • リボソームの停滞を引き起こす新規被子植物uORFの同定
    林憲哉; 佐々木駿; Zhihang Feng; 藤原徹; 内藤哲; 尾之内均
    第58回日本植物生理学会年会, 17 Mar. 2017, Japanese, Oral presentation
    [Domestic Conference]
  • ペプチド配列依存的に遺伝子発現を制御するシロイヌナズナの上流ORFの探索と制御機構の解析
    尾之内均; 林憲哉; 梅原俊一; 木俣薫織; 高橋広夫; 内藤哲
    日本遺伝学会第88回大会ワークショップ, 07 Sep. 2016, Japanese, Nominated symposium
    [Invited], [Domestic Conference]
  • 維管束形成を制御するLONESOME HIGHWAY遺伝子のuORFペプチドが介する翻訳制御機構
    大角有里沙; 木俣薫織; 梅原俊一; 戸田智美; 遠洞弥生; 蝦名績; 内藤哲; 尾之内均
    第57回植物生理学会年会, 18 Mar. 2016, Japanese, Oral presentation
    [Domestic Conference]
  • リボソームアレストを引き起こす被子植物uORFの探索
    林憲哉; 高橋広夫; 内藤哲; 尾之内均
    第57回植物生理学会年会, 18 Mar. 2016, Japanese, Oral presentation
    [Domestic Conference]
  • Bioinformatic identification method of evolutional ranges for potential functional non-coding molecules on genomes
    Takahashi H; Takahashi A; Hayashi N; Satoshi N; Onouchi H
    International Meeting "Non-coding DNA and Chromosomal Integrity -toward the finding of Intermeres-", 07 Aug. 2015, English, Poster presentation
    [International presentation]
  • リボソームアレストを引き起こす被子植物のCPuORFの探索と機能解析
    林憲哉; 高橋広夫; 内藤哲; 尾之内均
    第17回RNA学会年会, 16 Jul. 2015, Japanese, Poster presentation
    [Domestic Conference]
  • 翻訳制御に関わる種間保存性uORFペプチドの網羅的探索・進化保存性評価手法の開発と動植物ゲノムへの応用
    髙橋広夫; 髙橋アンナ; 林憲哉; 内藤哲; 尾之内均
    第17回RNA学会年会, 15 Jul. 2015, Japanese, Poster presentation
    [Domestic Conference]
  • 制御ペプチドをコードする二つのuORFが関与する翻訳制御機構
    工藤凜; 小山博彰; 大谷美紗都; 高橋広夫; 内藤哲; 尾之内均
    第17回RNA学会年会, 15 Jul. 2015, Japanese, Poster presentation
    [Domestic Conference]
  • Identification of novel Arabidopsis upstream open reading frames that control translation of the main coding sequences in a peptide sequence-dependent manner
    ONOUCHI Hitoshi
    Frontiers in Plant RNA Research, 17 Oct. 2012, English, Invited oral presentation
    [Invited], [International presentation]
  • 植物の uORF にコードされるペプチドが関与する翻訳制御機構の多様性
    尾之内 均
    第14回日本RNA学会年会, 20 Jul. 2012, Japanese, Oral presentation
    [Domestic Conference]
  • uORFがコードするペプチドにより発現が制御されるシロイヌナズナ遺伝子の探索
    尾之内 均
    第53回日本植物生理学会年会, 17 Mar. 2012, Japanese, Oral presentation
    [Domestic Conference]
  • Nascent peptide-mediated control of gene expression in Arabidopsis: Feedback regulation of methionine biosynthesis and beyond
    ONOUCHI Hitoshi
    12th International Congress on Amino Acids, Peptides and Protein, 04 Aug. 2011, English, Invited oral presentation
    [Invited], [International presentation]
■ Syllabus
  • 応用分子生物学特論, 2024年, 修士課程, 農学院
  • 応用分子生物学特論演習, 2024年, 修士課程, 農学院
  • 農学フロンティア総論, 2024年, 修士課程, 農学院
  • 分子細胞生物学, 2024年, 学士課程, 農学部
  • 応用生命科学実験, 2024年, 学士課程, 農学部
  • 応用生命科学概論, 2024年, 学士課程, 農学部
  • 分子生物学, 2024年, 学士課程, 農学部
■ Affiliated academic society
  • 日本分子生物学会
  • 日本植物生理学会
  • 日本植物学会
  • RNA学会
■ Research Themes
  • 上流ORFと偽リボソームタンパク質によるストレスや栄養欠乏に応答した翻訳制御
    科学研究費助成事業
    01 Apr. 2023 - 31 Mar. 2026
    尾之内 均
    1. ストレス応答性uORFと栄養欠乏応答性uORFの探索
    植物においてストレスや栄養欠乏に応答した翻訳制御に関与するuORFを探索するために、植物間で保存されたuORFを含む5′非翻訳領域の下流にルシフェラーゼ(LUC)遺伝子をつないだレポーターコンストラクトを作製した。各レポーターコンストラクトをシロイヌナズナのプロトプラストに導入し、ストレス条件や栄養欠乏条件で細胞を培養した後、一過的発現解析を行った。その結果、栄養欠乏に応答してuORF依存的に主要ORFの翻訳が促進される遺伝子を２つ、抑制される遺伝子を１つ見出した。そのうちの翻訳促進がみられた遺伝子はそれぞれの5′非翻訳領域にuORFを1つのみ持つが、翻訳抑制がみられた遺伝子の5′非翻訳領域には２つのuORFが存在する。その２つのuORFのうち少なくとも下流側のuORFが栄養欠乏に応答した翻訳抑制に必要であることを明らかにした。


    2. 偽リボソームタンパク質の解析
    研究代表者のグループはこれまでに、リボソームタンパク質と似たアミノ酸配列を持ちストレスに応答して発現が誘導される「偽リボソームタンパク質」をシロイヌナズヌナにおいて同定した。ストレス応答に関与するある転写制御因子の過剰発現によってその偽リボソームタンパク質の発現が促進されることをこれまでに見出していたが、令和５年度の研究ではその転写制御因子の欠損変異株を用いた解析により、ストレスに応答した偽リボソームタンパク質の発現誘導がその転写制御因子に依存することを明らかにした。さらに、栄養欠乏によってもその偽リボソームタンパク質の発現が誘導されることを見出した。
    日本学術振興会, 基盤研究(C), 北海道大学, 23K05799
  • 上流ORFが関与する細胞環境感知機構の解明とゲノム育種への応用
    科学研究費助成事業
    01 Apr. 2019 - 31 Mar. 2023
    尾之内 均
    研究代表者のグループはこれまでに、進化的に保存された配列を持つシロイヌナズナのuORFの中から、下流の主要ORFの翻訳に影響を与えるものを同定した。その中には、AUG以外の開始コドンで始まるuORF（非AUG開始型uORF）が４つ含まれる。それらの非AUG開始型uORFの１つを持つ遺伝子はストレスに応答して転写が促進されるが、その遺伝子の発現がポリアミンに応答して翻訳段階で抑制され、その翻訳制御に非AUG開始型uORFが関与することを本年度の研究において見出した。さらに、非AUG開始型uORFの開始コドンの下流に存在するRNA二次構造がこの翻訳制御に必要であることを明らかにした。この翻訳制御は、植物細胞内でポリアミンが過剰に蓄積した場合に、ポリアミン濃度を適切な範囲内に維持する役割を担っていると考えられる。
    他の３つの非AUG開始型uORFのうちの２つでは、uORFのペプチド配列が植物で広く保存されている。これらの２つのuORFのペプチド配列の中で翻訳制御に重要なアミノ酸残基の同定を行い、それぞれC末端側の8アミノ酸および15アミノ酸の領域が重要であることを見出した。
    また、これまでに生理学的役割を明らかにしたAUG開始型uORF及び非AUG開始型uORFについて、それらが介する翻訳制御に関与するトランス因子を同定するために、トランス因子として予想される候補タンパク質が欠損したシロイヌナズナ変異体をゲノム編集技術を用いて作出した。
    日本学術振興会, 基盤研究(B), 北海道大学, 19H02917
  • ゲノム編集による5´非翻訳領域の改変によりタンパク質発現量を増加させる技術の開発
    科学研究費助成事業
    28 Jun. 2019 - 31 Mar. 2022
    尾之内 均
    近年、ゲノム編集技術が作物の品種改良に活用されるようになってきている。しかし、遺伝子組換えにあたらない範囲でのゲノム編集技術の作物育種への利用はほとんど遺伝子破壊に限られており、転写や翻訳を抑制する配列が同定されている一部の遺伝子以外は、外来導入DNAを残さずに特定のタンパク質の発現量を増加させることは現状では困難である。そこで本研究では、より多くの作物遺伝子において遺伝子組換えに該当せずにタンパク質の発現量を増加させることを可能にするために、mRNAの翻訳効率に影響を与える5´非翻訳領域配列をゲノム編集を用いて改変することでタンパク質発現量を増加させる方法の開発を目指す。
    2019年度の研究において、CRISPR/Cas9システムを用いてイネの遺伝子の5´非翻訳領域に変異を導入し、5´非翻訳領域に様々な変異が生じた変異体を分離した。2020年度の研究では、それらの変異がタンパク質発現量にどのような影響を与えるかを調べるために、変異が生じた5´非翻訳領域をクローニングしてルシフェラーゼ遺伝子につなぎ、一過的発現解析により変異の影響を検討した。その結果、5´非翻訳領域に生じた変異によりルシフェラーゼの発現量が様々なレベルに変化し、それらの変異の中でルシフェラーゼの発現量を増加させるものも見つかった。さらに、それらの変異のヘテロ接合体の次世代の個体の中から、ゲノム編集用コンストラクトが除かれた個体や変異をホモ接合体として持つ個体をそれぞれ分離した。
    日本学術振興会, 挑戦的研究(萌芽), 北海道大学, 19K22299
  • Elucidation of translation-arresting sequences in genomes using computer-based intersectional analyses of life science databases
    Grants-in-Aid for Scientific Research
    2018 - 2020
    高橋 広夫; 伊藤 素行; 尾之内 均; 佐々木 博己; 町田 泰則
    Upstream Open Reading Frame (uORF)は、AUG以外の開始コドンから翻訳が始まる非AUG開始型uORFが存在する。そのような非AUG開始型uORFの中で翻訳アレストを起こすものを同定するために、ESUCAパイプラインを改変して進化的に保存された配列を持つ非AUG開始型uORFを動植物のゲノムから探索した。その結果、そのような非AUG開始型uORFが14個同定された。それらの中から、主要ORFの翻訳制御に関与するものを一過的発現解析により4個同定した。また、胃がん発症機構解明のため、本年度は、高度遺伝子増幅領域内の遺伝子発現がプロモーターのメチル化と関連しているか、メチロームデータとの3層オミクス統合解析を行い、遺伝子の発現量とメチル化の間に、強い相関があることを見出した。遺伝子増幅に伴って過剰発現する遺伝子にはGATA6のような新たながん遺伝子が多く、発現が低下している遺伝子にはGSDMAのようながん抑制遺伝子が含まれていた。これらにより、3層オミクス統合解析によって、腹膜転移性胃がんの増幅遺伝子の全体像が明らかになった。
    Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), Kanazawa University, 18H03330
  • uORFペプチドによる翻訳制御の新たな役割と機構の解明
    科学研究費補助金（基盤研究(C)）
    Apr. 2016 - Mar. 2018
    尾之内 均
    文部科学省, Principal investigator, Competitive research funding
  • uORFにコードされるペプチドによる翻訳制御の植物における多様な役割の解明
    科学研究費補助金（基盤研究(C)）
    Apr. 2013 - Apr. 2015
    尾之内 均
    文部科学省, Principal investigator, Competitive research funding
  • 植物アミノ酸代謝のオミクス統合解析による解明
    戦略的基礎研究推進事業 (CREST)
    Sep. 2007 - Mar. 2013
    平井優美
    科学技術振興事業団, Competitive research funding
  • 植物における翻訳アレストにより誘導されるmRNA分解機構
    科学研究費補助金(基盤研究(C))
    2009 - 2011
    尾之内 均
    植物において翻訳アレストとmRNA分解の共役の普遍的性について検討する目的で、新生ペプチドによって翻訳アレストが引き起こされる可能性が考えられるuORFの系を用いて解析を行った。前年度に新たに同定したペプチド配列依存的に翻訳抑制を起こす2つのuORFについて、試験管内翻訳系において翻訳アレストが起こるかの検討を行ったが、通常の翻訳条件では翻訳アレストは観察されなかった。細胞内に存在する何らかの代謝産物が翻訳アレストを誘導するために必要である可能性も考えられるため、今後、どのような条件で翻訳抑制が誘導されるかについての詳細な検討を行う。また、それらのuORFにおいて翻訳抑制と共役したmRNA分解の促進が見られるかを検討するために、各遺伝子の翻訳抑制に必要な野生型配列と翻訳抑制が起こらない変異型配列をそれぞれ導入した形質転換植物を作出した。次年度に、これらの植物を用いて、このuORFによる翻訳抑制と共役してmRNA分解が誘導されるかを検討する予定である。今年度に新たに同定したペプチド配列依存的に翻訳抑制を起こすuORFの中に、翻訳阻害剤であるシクロヘキシミド処理によってmRNA蓄積量が増加するものを見いだした。このuORFでは、翻訳と共役してmRNA分解が起こる可能性が考えられる。このuORFについて、アミノ酸置換解析を行うことによって、uORFにコードされるペプチド中の翻訳抑制...
    文部科学省, 基盤研究(C), 北海道大学, Principal investigator, Competitive research funding, 21570032
  • 植物の発生分化におけるuORFによる翻訳制御機構
    科学研究費補助金(特定領域研究)
    2009 - 2010
    尾之内 均; 内藤 哲
    これまでに同定したシロイヌナズナのuORFペプチドが関与する発現制御の中で、複数のuORFが関与するものを2つ見いだした。それらの系では、ペプチド配列依存的に翻訳を抑制するuORFのさらに上流に別のuORFが存在し、上流側のuORFを削除した場合に主要なORFの発現が強く抑制され、その効果は下流側のuORFのアミノ酸配列に依存することが示された。これらのことから、上流側のuORFは下流のuORFの翻訳効率を調節する働きがあることが示唆された。また、シロイヌナズナにおいてアミノ酸配列依存的な発現制御がみられなかったuORFの中に、アブラナ科植物では他の科の植物と比べてアミノ酸配列の保存性が低いものがいくつか見いだされた。そのうちの1つの遺伝子について、アミノ酸配列がより高度に保存されているポプラのオルソログのuORFを用いて、ペプチド配列依存的な発現制御がみられるかを検討した。その結果、ポプラのオルソログのuORFはアミノ酸配列依存的に主要なORFの発現を抑制することを見いだした。この遺伝子の5'UTRにも複数のuORFが存在し、ポプラにおいてペプチド配列依存的な発現制御がみられた下流側のuORFだけでなく、上流側のuORFのアミノ酸配列も双子葉植物間で高度に保存されている。上流側のuORFの役割について解析したところ、シロイヌナズナとポプラのいずれにおいても上流側のuORFは...
    文部科学省, 特定領域研究, 北海道大学, Principal investigator, Competitive research funding, 21027001
  • 新生ペプチドによる翻訳アレストと連携したRNA制御機構
    科学研究費補助金(新学術領域研究(研究領域提案型))
    2009 - 2010
    尾之内 均
    前年度にバイオインフォマティクス解析によって同定した植物間で保存されたアミノ酸配列を持つ14個のuORFの中から、uORFにコードされるペプチドが実際に翻訳制御に関与するものを同定する目的で、フレームシフト変異やアミノ酸置換によってアミノ酸配列を変化させ場合の下流のレポーター遺伝子の発現への影響を検討した。シロイヌナズナ培養細胞MM2dから調製したプロトプラストを用いた一過的発現解析の結果、アミノ酸配列依存的に下流ORFの発現に影響を与えるuORFを新たに5つ同定した。新たに同定したuORFについて、発現制御に重要な領域を同定するために、アミノ酸置換解析を行った。制御に重要な領域のアミノ酸配列に特に共通性はみられなかったが、C末端の15-20残基が発現制御に重要であるという傾向がみられた。昨年度の解析により、LONESOME HIGHWAY(LHW)遺伝子のuORFペプチドによる発現制御にはuORFの終止コドンが必要であるのに対し、ANAC082遺伝子のuORFペプチドによる発現制御は終止コドンに依存しないことが示された。今年度、新たに同定したuORFについても発現制御の終止コドン依存性を検討したところ、そのうちの一つのuORFでは発現制御が部分的に終止コドンに依存することが示された。これらの結果から、遺伝子によってuORFペプチドによる発現制御のメカニズムに違いがあることが...
    文部科学省, 新学術領域研究(研究領域提案型), 北海道大学, Principal investigator, Competitive research funding, 21112501
  • 葉の形態形成におけるリボソームによる制御
    科学研究費補助金(特定領域研究)
    2008 - 2008
    尾之内 均; 内藤 哲
    シロイヌナズナのリボソームタンパク質RRL17の変異が葉の形態形成に影響を与える原因を明らかにするために、まずrp117変異の影響によってリボソームの翻訳活性が低下した可能性を検討した。そのために、いくつかの遺伝子についてポリソームプロファイル解析を行った。その結果、いずれの遺伝子についても野生型株とrp117変異株の間で顕著な差はみられず、rp117変異はリボソームの翻訳活性に大きな影響を与えないことが示唆された。また、リボソームタンパク質の変異の新生ペプチドによる制御への影響と葉の形態異常との関連について調べる目的で、rp117b変異株と類似の葉の形態異常を示すrp14d変異株において、CGS1遺伝子の転写後制御への影響を解析した。その結果、rp117b変異とは異なり、rp14d変異はCG51遺伝子の転写後制御に影響を与えないことが示された。このことから、リボソームタンパク質変異株の葉の形態異常は、新生ペプチドによる制御への影響によるのではなく、リボソームの変異による他の影響によるものであることが示唆された。また、形態形成に関わる遺伝子のuORF配列による下流ORFの翻訳への影響を検討し、いくつかの遺伝子においてuORFの配列が下流ORFの翻訳制御に関与することを見いだした。そのうちの一つの遺伝子は、uORFペプチドによる翻訳抑制と選択的スプライシングの組み合わせにより制...
    文部科学省, 特定領域研究, 北海道大学, Principal investigator, Competitive research funding, 20061001
  • Molecular genetic analysis and screen for nascent-peptide mediated regulation in Arabidopsis
    Grants-in-Aid for Scientific Research(基盤研究(C))
    2006 - 2007
    Hitoshi NOUCHI
    In the course of study on the posttranscriptional regulation of the Arabidopsis CGS1 gene, which encodes the key enzyme of methionine biosynthesis, we have found that the nascent CGS1 peptide mediates its own translation elongation arrest in response to S-adenosylmethionine, a metabolite of methionine. Although several examples of nascent peptide-mediated ribosomal regulations have been reported in eukaryotes, all other regulatory nascent peptides are encoded by upstream ORFs (uORF) located in the 5'UTR, except for the CGS1 gene. In these systems, uORF-encoded peptides cause ribosome stalli...
    Ministry of Education, Culture, Sports, Science and Technology, 基盤研究(C), 北海道大学, Principal investigator, Competitive research funding, 18570032
  • Molecular mechanism of translation arrest and mRNA degradation in cystathionine gamma-synthase, the key-step enzyme of methionine biosynthesis.
    Grants-in-Aid for Scientific Research(基盤研究(B))
    2004 - 2006
    Satoshi NAITO; 尾之内 均
    Cystathionine gamma-synthase (CGS) of Arabidopsis catalyzes the key step in methionine biosynthesis. Unlike many of the key-step enzymes in biosynthetic pathways, CGS is not an allosteric enzyme. Expression of CGS is feedback-regulated at the step of mRNA degradation and S-adenosylmethionine (SAM) acts as the effector. Upon mRNA degradation, 5'-truncated RNA species are formed as degradation intermediates. A short stretch of amino acids, termed the MTO1 region, located within the first exon-coding region of CGS itself, functions as the cis element in this regulation. CGS regulation occurs d...
    Ministry of Education, Culture, Sports, Science and Technology, 基盤研究(B), 北海道大学, Coinvestigator not use grants, Competitive research funding, 16370016
  • 翻訳伸長段階の制御を網羅的に検索する方法の開発
    科学研究費補助金(若手研究(B))
    2004 - 2005
    尾之内 均
    翻訳伸長段階の制御を検索するための方法として、翻訳の伸長が途中で停止することによってリボソームが5'側に偏在するようなmRNAを検索する方法の開発を試みた。そのために、ポリソームをマイクロコッカルヌクレアーゼで部分消化し、そのあとショ糖密度勾配遠心分離を行うことによってリボソームのmRNA上の偏在を検出することを計画した。しかし、翻訳伸長停止によってリボソームが偏在することが示されているシロイヌナズナのシスタチオニンγ-シンターゼ(CGS)mRNAにおいても、上述のような方法で顕著なリボソームの偏在は検出されなかった。したがって、この方法で翻訳伸長が途中で停止したmRNAを検索することは困難であると考えられたため、別の方法として翻訳停止したリボソームと翻訳伸長中のリボソームの翻訳阻害剤に対する感受性の違いを利用する方法を考えた。そのための予備実験として、CGSmRNA上で翻訳停止したリボソームの様々な翻訳伸長阻害剤に対する感受性を調べた。その結果、通常ではピューロマイシン処理後5分以内に大部分のポリペプチドがリボソームから解離するのに対し、CGS mRNA上で翻訳停止したリボソームではピューロマイシン処理30分後も半分近いポリペプチドが解離せずに残った。このようなピューロマイシンに対する感受性の違いを利用して、翻訳伸長停止を起こしたmRNAの検索を進めている。また、原核生物で...
    文部科学省, 若手研究(B), 北海道大学, Principal investigator, Competitive research funding, 16770026
  • RNaseによるRNA分解制御
    科学研究費補助金(特定領域研究)
    2002 - 2004
    正木 春彦; 尾之内 均
    <代表者>1.コリシンDのtRNase高活性ドメインをC末端103残基(D-CRD595)に特定し、D-CRD595単独およびImmDとの複合体の構造をX線結晶解析で決定し、先にImmDとの複合体構造を決定していたC末端94残基(D-CRD604)と構造と活性を比較した。D-CRD595に比べD-CRD604はN末端9残基を欠き、活性を大きく損なうが、ImmD複合体構造はほとんど同一なので、9残基の特異的重要性が示唆された。ImmDはD-CRD595の活性中心に結合し、またImmDの結合前後でD-CRD595は構造をほとんど変えていなかった。2.D-CRD595領域のさまざまな点変異体を作製して各残基の寄与を評価した。予想に反しN末端9アミノ酸は個別には重要でなかった。Hisと二つのLysが触媒中心と考えられ、先のコリシンE5とも異なる新しいRNase反応機構が推定できた。3.D-CRD遺伝子を出芽酵母に導入し、E5-CRDの場合と同様に、CRDの誘導発現に伴ってコロニー形成が停止した。しかし、大腸菌と異なり、tRNA感受性は同等なのに殺菌的でなく静菌的に見え、細胞毒としての作用が細菌と真核細胞とで異なる可能性が生じた。<分担者>1.植物のメチオニン生合成系の鍵酵素であるシスタチオニンγ-シンターゼ(CGS)の遺伝子発現制御では、S-アデノシルメチオニンに応答して翻訳伸長が停...
    文部科学省, 特定領域研究, 東京大学, Coinvestigator not use grants, Competitive research funding, 14035208
  • Molecular genetic studies of control of mRNA stability in the gene for the key enzyme of methionine biosynthesis
    Grants-in-Aid for Scientific Research(基盤研究(B))
    2001 - 2003
    Satoshi NAITO; 尾之内 均
    Expression of the gene for cystathionine gamma-synthase (CGS), the key enzyme of methionine biosynthesis in higher plants is feedback-regulated at the step of mRNA stability and the amino acid sequence in the exon 1 of CGS itself is involved in this regulation. In order to identity the factors that are involved in this regulation, a chimeric gene carrying green fluorescent protein gene (GFP) fused to the exon 1 of CGS gene and placed under the control of a cauliflower mosaic virus 35S promoter was constructed and introduced into wild-type Arabidopsis. The transgenic line termed GFPc4 was us...
    Ministry of Education, Culture, Sports, Science and Technology, 基盤研究(B), 北海道大学, Coinvestigator not use grants, Competitive research funding, 13440233
  • 分裂組織からの器官分化過程におけるシスタチオニンγ-シンターゼmRNA安定性制御
    科学研究費補助金(特定領域研究(A))
    2001 - 2001
    尾之内 均; 内藤 哲
    シスタチオニンγ-シンターゼ(CGS) mRNAの安定性制御機構を解明する目的で、この制御のエフェクター分子の同定を行った。小麦胚芽由来のin vitro翻訳系を用いたアッセイによって、メチオニンの代謝産物についてエフェクターとしての効果を調べたところ、S-アデノシルメチオニン(SAM)のみが効果を示し、SAMがCGS mRNA安定性制御のエフェクター分子であることが強く示唆された。また、SAMを基質とするメチル基転位酵素の競争阻害剤であるS-アデノシルホモシステインは、SAMの効果を阻害しなかった。このことから、SAMの効果はメチル基転位反応によるものではないことが示唆された。また、SAMのアナログについても、同じin vitroアッセイを用いて、CGS mRNA安定性制御のエフェクターとしての効果を調べた。その結果、SAMのメチル基をエチル基に置換したS-アデノシルエチオニン(SAE)も、SAMと同様の効果を示した。一方、SAMからメチル基を除いたS-アデノシルホモシステインは効果を示さなかった。したがって、SAMとSAEに共通する構造的特徴あるいは硫黄原子にアルキル基が付加することによって生じる正電荷が重要である可能性が考えられる。また、in vitro翻訳系を用いて、翻訳反応後のRNA分解中間体の解析を行った。これまでにin vivoの実験系でみられたのと同様の5'側...
    文部科学省, 特定領域研究(A), 北海道大学, Principal investigator, Competitive research funding, 13017201
  • 植物における遺伝子発現の転写後制御
    Competitive research funding
  • Post-transcriptional regulation of gene expression in plant
    Competitive research funding