2022/10/01 更新

写真a

オオヤマ タカシ
大山 隆
所属
教育・総合科学学術院 教育学部
職名
教授

兼担

  • 理工学術院   大学院先進理工学研究科

  • 附属機関・学校   グローバルエデュケーションセンター

学内研究所等

  • 2020年
    -
    2022年

    理工学術院総合研究所   兼任研究員

学歴

  • 1982年04月
    -
    1985年03月

    名古屋大学   大学院理学研究科   分子生物学  

  • 1977年04月
    -
    1979年03月

    名古屋大学   大学院  

  • 1973年04月
    -
    1977年03月

    名古屋大学  

学位

  • 名古屋大学   理学博士

経歴

  • 2006年
    -
    継続中

    早稲田大学教育・総合科学学術院理学科 教授

  • 2006年
    -
    継続中

    Department of Biology, Waseda University

  • 1994年
    -
    2006年

    甲南大学理工学部生物学科 助教授・教授

  • 1994年
    -
    2006年

    Department of Biology, Konan University

  • 1994年
     
     

    スイス連邦工科大学細胞生物学研究所

  • 1994年
     
     

    Institut fur Zellbiologie, ETH Zurich

  • 1990年
    -
    1994年

    (財)地球環境産業技術研究機構(兼任)

  • 1990年
    -
    1994年

    Research Institute of Innovative Technology for the Earth

  • 1987年
    -
    1994年

    明治乳業ヘルスサイエンス研究所

  • 1987年
    -
    1994年

    Meiji Institute of Health Science

  • 1986年
    -
    1987年

    三菱化成生命科学研究所

  • 1986年
    -
    1987年

    Mitsubishi Kasei Institute of Life Science

  • 1979年
    -
    1981年

    民間企業

  • 1979年
    -
    1981年

    AICA Kogyo Company, Limited

  •  
     
     

    (2002,2003) Graduate School of Science, Hiroshima University (part-time lecturer)

  •  
     
     

    (2003,2005) Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University (part-time lecturer)

  •  
     
     

    (2005) Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University (part-time lecturer)

  •  
     
     

    (2010)Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University (part-time lecturer)

▼全件表示

所属学協会

  •  
     
     

    日本分子生物学会

  •  
     
     

    日本生化学会

  •  
     
     

    米国細胞生物学会

  •  
     
     

    食虫植物研究会

 

研究分野

  • 進化生物学

  • 遺伝学

  • ゲノム生物学

  • 分子生物学

研究キーワード

  • 自己集合・自己組織化

  • 必須金属

  • ES細胞

  • 食虫植物

  • エピジェネティクス

  • DNA物性

  • DNA高次構造

  • クロマチン

  • 染色体

  • 遺伝情報

  • 遺伝子発現

  • physical properties of DNA

  • DNA conformation

  • chromatin

  • chromosome

  • genetic information

  • gene expression

▼全件表示

論文

  • Cruciform formable sequences within Pou5f1 enhancer are indispensable for mouse ES cell integrity

    Yu Yamamoto, Osamu Miura, Takashi Ohyama

    Int. J. Mol. Sci.   22 ( 7 ) 3399  2021年  [査読有り]  [国際誌]

     概要を見る

    DNA can adopt various structures besides the B-form. Among them, cruciform structures are formed on inverted repeat (IR) sequences. While cruciform formable IRs (CFIRs) are sometimes found in regulatory regions of transcription, their function in transcription remains elusive, especially in eukaryotes. We found a cluster of CFIRs within the mouse Pou5f1 enhancer. Here, we demonstrate that this cluster or some member(s) plays an active role in the transcriptional regulation of not only Pou5f1, but also Sox2, Nanog, Klf4 and Esrrb. To clarify in vivo function of the cluster, we performed genome editing using mouse ES cells, in which each of the CFIRs was altered to the corresponding mirror repeat sequence. The alterations reduced the level of the Pou5f1 transcript in the genome-edited cell lines, and elevated those of Sox2, Nanog, Klf4 and Esrrb. Furthermore, transcription of non-coding RNAs (ncRNAs) within the enhancer was also upregulated in the genome-edited cell lines, in a similar manner to Sox2, Nanog, Klf4 and Esrrb. These ncRNAs are hypothesized to control the expression of these four pluripotency genes. The CFIRs present in the Pou5f1 enhancer seem to be important to maintain the integrity of ES cells.

    DOI PubMed

  • Organ-specific expression and epigenetic traits of genes encoding digestive enzymes in the lance-leaf sundew (Drosera adelae)

    Arai Naoki, Ohno Yusuke, Jumyo Shinya, Hamaji Yusuke, Ohyama Takashi

    J. Exp. Bot.   72   1946 - 1961  2020年  [査読有り]

  • A strong structural correlation between short inverted repeat sequences and the polyadenylation signal in yeast and nucleosome exclusion by these inverted repeats.

    Miura, O, Ogake, T, Yoneyama, H, Kikuchi, Y, Ohyama, T

    Curr. Genet.   65   575 - 590  2019年  [査読有り]

  • New aspects of magnesium function: A key regulator in nucleosome self-assembly, chromatin folding and phase separation.

    Ohyama, T

    Int. J. Mol. Sci.   20   4232  2019年  [査読有り]

  • Requirement or exclusion of inverted repeat sequences with cruciform-forming potential in Escherichia coli revealed by genome-wide analyses

    Osamu Miura, Toshihiro Ogake, Takashi Ohyama

    Curr. Genet.   64   1 - 14  2018年02月  [査読有り]

     概要を見る

    Inverted repeat (IR) sequences are DNA sequences that read the same from 5′ to 3′ in each strand. Some IRs can form cruciforms under the stress of negative supercoiling, and these IRs are widely found in genomes. However, their biological significance remains unclear. The aim of the current study is to explore this issue further. We constructed the first Escherichia coli genome-wide comprehensive map of IRs with cruciform-forming potential. Based on the map, we performed detailed and quantitative analyses. Here, we report that IRs with cruciform-forming potential are statistically enriched in the following five regions: the adjacent regions downstream of the stop codon-coding sites (referred to as the stop codons), on and around the positions corresponding to mRNA ends (referred to as the gene ends), ~ 20 to ~45 bp upstream of the start codon-coding sites (referred to as the start codons) within the 5′-UTR (untranslated region), ~ 25 to ~ 60 bp downstream of the start codons, and promoter regions. For the adjacent regions downstream of the stop codons and on and around the gene ends, most of the IRs with a repeat unit length of ≥ 8 bp and a spacer size of ≤ 8 bp were parts of the intrinsic terminators, regardless of the location, and presumably used for Rho-independent transcription termination. In contrast, fewer IRs were present in the small region preceding the start codons. In E. coli, IRs with cruciform-forming potential are actively placed or excluded in the regulatory regions for the initiation and termination of transcription and translation, indicating their deep involvement or influence in these processes.

    DOI

  • Involvement of the response regulator CtrA in the extracellular DNA production of the marine bacterium Rhodovulum sulfidophilum.

    Komatsu, H, Yamamoto, J, Suzuki, H, Nagao, N, Hirose, Y, Ohyama, T, Umekage, S, Kikuchi, Y

    Gen. Appl. Microbiol.   64   103 - 107  2018年  [査読有り]

    DOI PubMed

  • Magnesium chloride and polyamine can differentiate mouse embryonic stem cells into trophectoderm or endoderm

    Jun-ichi Tanase, Takehiro Yokoo, Yuuki Matsumura, Makoto Kinoshita, Yo Kikuchi, Hirofumi Suemori, Takashi Ohyama

    Biochem. Biophys. Res. Commum.   482 ( 4 ) 764 - 770  2017年01月  [査読有り]

     概要を見る

    Magnesium chloride and polyamines stabilize DNA and chromatin. Furthermore, they can induce nucleosome aggregation and chromatin condensation in vitro. To determine the effects of elevating the cation concentrations in the nucleus of a living cell, we microinjected various concentrations of mono-, di- and polyvalent cation solutions into the nuclei of mouse embryonic stem (ES) cells and traced their fates. Here, we show that an elevation of either MgCl2, spermidine or spermine concentration in the nucleus exerts a significant effect on mouse ES cells, and can differentiate a certain population of the cells into trophectoderm, a lineage that mouse ES cells do not normally generate, or endoderm. It is hypothesized that the cell differentiation was most probably caused by the condensation of chromatin including the Oct3/4 locus, which was induced by the elevated concentrations of these cations. (C) 2016 Elsevier Inc. All rights reserved.

    DOI

  • Cloning and characterization of a human genomic sequence that alleviates repeat-induced gene silencing

    Miki Fukuma, Yuto Ganmyo, Osamu Miura, Takashi Ohyama, Noriaki Shimizu

    Plos One   11 ( 4 ) e0153338  2016年04月  [査読有り]

     概要を見る

    Plasmids bearing a mammalian replication initiation region (IR) and a nuclear matrix attachment region (MAR) are spontaneously amplified in transfected mammalian cells, and such amplification generates chromosomal homogeneously staining regions (HSRs) or extrachromosomal double minutes (DMs). This method provides a novel, efficient, and rapid way to establish cells that stably produce high levels of recombinant proteins. However, because IR/MAR plasmids are amplified as repeats, they are frequently targeted by repeat-induced gene silencing (RIGS), which silences a variety of repeated sequences in transgenes and the genome. To address this problem, we developed a novel screening system using the IR/MAR plasmid to isolate human genome sequences that alleviate RIGS. The screen identified a 3,271 bp sequence (B-3-31) that elevated transgene expression without affecting the amplification process. Neither non-B structure (i.e., the inverted repeats or bending) nor known epigenetic modifier elements such as MARs, insulators, UCOEs, or STARs could explain the anti-silencing activity of B-3-31. Instead, the activity was distributed throughout the entire B-3-31 sequence, which was extremely A/T-rich and CpG-poor. Because B-3-31 effectively and reproducibly alleviated RIGS of repeated genes, it could be used to increase recombinant protein production.

    DOI

  • The gene transfer agent-like particle of the marine phototrophic bacterium Rhodovulum sulfidophilum

    Nobuyoshi Nagao, Junya Yamamoto, Hiroyuki Komatsu, Hiromichi Suzuki, Yuu Hirose, So Umekage, Takashi Ohyama, Yo Kikuchi

    Biochem. Biophys. Rep.   4   369 - 374  2015年12月  [査読有り]

     概要を見る

    Gene transfer agents (GTAs) are shaped like bacteriophage particles but have many properties that distinguish them from bacteriophages. GTAs play a role in horizontal gene transfer in nature and thus affect the evolution of prokaryotic genomes. In the course of studies on the extracellular production of designed RNAs using the marine bacterium Rhodovulum sulfidophilum, we found that this bacterium produces a GTA-like particle. The particle contains DNA fragments of 4.5. kb, which consist of randomly fragmented genomic DNA from the bacterium. This 4.5-kb DNA production was prevented while quorum sensing was inhibited. Direct observation of the particle by transmission electron microscopy revealed that the particle resembles a tailed phage and has a head diameter of about 40. nm and a tail length of about 60. nm. We also identified the structural genes for the GTA in the genome. Translated amino acid sequences and gene positions are closely related to those of the genes that encode the Rhodobacter capsulatus GTA. This is the first report of a GTA-like particle from the genus Rhodovulum. However, gene transfer activity of this particle has not yet been confirmed. The differences between this particle and other GTAs are discussed.

    DOI

  • Functional analyses of carnivorous plant-specific amino acid residues in S-like ribonucleases

    Naoki Arai, Emi Nishimura, Yo Kikuchi, Takashi Ohyama

    Biochem. Biophys. Res. Commum.   465 ( 1 ) 108 - 112  2015年09月  [査読有り]

     概要を見る

    Unlike plants with no carnivory, carnivorous plants seem to use S-like ribonucleases (RNases) as an enzyme for carnivory. Carnivorous plant-specific conserved amino acid residues are present at four positions around the conserved active site (CAS). The roles of these conserved amino acid residues in the enzymatic function were explored in the current study by preparing five recombinant variants of DA-I, the S-like RNase of Drosera adelae. The k(cat) and k(cat)/K-m values of the enzymes revealed that among the four variants with a single mutation, the serine to glycine mutation at position 111 most negatively influenced the enzymatic activity. The change in the bulkiness of the amino acid residue side-chain seemed to be the major cause of the above effect. Modeling of the three dimensional (3D) structures strongly suggested that the S to G mutation at 111 greatly altered the overall enzyme conformation. The conserved four amino acid residues are likely to function in keeping the two histidine residues, which are essential for the cleavage of RNA strands, and the CAS in the most functional enzymatic conformation. (C) 2015 Elsevier Inc. All rights reserved.

    DOI PubMed

  • Structural and functional characteristics of S-like ribonucleases from carnivorous plants

    Emi Nishimura, Shinya Jumyo, Naoki Arai, Kensuke Kanna, Marina Kume, Jun-ichi Nishikawa, Jun-ichi Tanase, Takashi Ohyama

    Planta   240 ( 1 ) 147 - 159  2014年07月  [査読有り]

     概要を見る

    Although the S-like ribonucleases (RNases) share sequence homology with the S-RNases involved in the self-incompatibility mechanism in plants, they are not associated with this mechanism. They usually function in stress responses in non-carnivorous plants and in carnivory in carnivorous plants. In this study, we clarified the structures of the S-like RNases of Aldrovanda vesiculosa, Nepenthes bicalcarata and Sarracenia leucophylla, and compared them with those of other plants. At ten positions, amino acid residues are conserved or almost conserved only for carnivorous plants (six in total). In contrast, two positions are specific to non-carnivorous plants. A phylogenetic analysis revealed that the S-like RNases of the carnivorous plants form a group beyond the phylogenetic relationships of the plants. We also prepared and characterized recombinant S-like RNases of Dionaea muscipula, Cephalotus follicularis, A. vesiculosa, N. bicalcarata and S. leucophylla, and RNS1 of Arabidopsis thaliana. The recombinant carnivorous plant enzymes showed optimum activities at about pH 4.0. Generally, poly(C) was digested less efficiently than poly(A), poly(I) and poly(U). The kinetic parameters of the recombinant D. muscipula enzyme (DM-I) and A. thaliana enzyme RNS1 were similar. The k (cat)/K (m) of recombinant RNS1 was the highest among the enzymes, followed closely by that of recombinant DM-I. On the other hand, the k (cat)/K (m) of the recombinant S. leucophylla enzyme was the lowest, and was similar to 1/30 of that for recombinant RNS1. The magnitudes of the k (cat)/K (m) values or k (cat) values for carnivorous plant S-like RNases seem to correlate negatively with the dependency on symbionts for prey digestion.

    DOI PubMed

  • Positions of pluripotency genes and hepatocyte-specific genes in the nucleus before and after mouse ES cell differentiation

    K. Udagawa, T. Ohyama

    Genet. Mol. Res.   13 ( 1 ) 1979 - 1988  2014年  [査読有り]

     概要を見る

    Spatial positioning of genes in the cell nucleus plays an important role in the regulation of genomic functions. Evidence for changes in gene positioning associated with transcriptional activity has been reported. However, our understanding of this phenomenon is still quite limited. We examined how pluripotency genes and hepatocyte-specific genes behave during the differentiation of mouse embryonic stem (ES) cells into hepatocytes, by targeting the loci of the Klf4, Nanog, Oct4, Sox2, Cyp7a1, Pck1, Tat, and Tdo2 genes, and using three-dimensional fluorescence in situ hybridization analyses. We found that each gene has a distinctly inherent localization profile in the ES cell nucleus. During differentiation, the Klf4, Nanog, Oct4, Cyp7a1, Pck1, and Tat loci shifted toward the nuclear center, while the Sox2 and Tdo2 loci shifted toward the periphery. The Klf4, Nanog, Oct4, and Tdo2 seem to prefer the outer regions, rather than the inner regions, when they are active. We also found that the radial positioning of the focused genes in the hepatocyte cell nucleus was highly correlated with the local GC content and the gene density of the surrounding region, but not with gene activity.

    DOI PubMed

  • S-like ribonuclease gene expression in carnivorous plants

    Emi Nishimura, Minako Kawahara, Reina Kodaira, Marina Kume, Naoki Arai, Jun-ichi Nishikawa, Takashi Ohyama

    Planta   238 ( 5 ) 955 - 967  2013年11月  [査読有り]

     概要を見る

    Functions of S-like ribonucleases (RNases) differ considerably from those of S-RNases that function in self-incompatibility. Expression of S-like RNases is usually induced by low nutrition, vermin damage or senescence. However, interestingly, an Australian carnivorous plant Drosera adelae (a sundew), which traps prey with a sticky digestive liquid, abundantly secretes an S-like RNase DA-I in the digestive liquid even in ordinary states. Here, using D. adelae, Dionaea muscipula (Venus flytrap) and Cephalotus follicularis (Australian pitcher plant), we show that carnivorous plants use S-like RNases for carnivory: the gene da-I encoding DA-I and its ortholog cf-I of C. follicularis are highly expressed and constitutively active in each trap/digestion organ, while the ortholog dm-I of D. muscipula becomes highly active after trapping insects. The da-I promoter is unmethylated only in its trap/digestion organ, glandular tentacles (which comprise a small percentage of the weight of the whole plant), but methylated in other organs, which explains the glandular tentacles-specific expression of the gene and indicates a very rare gene regulation system. In contrast, the promoters of dm-I, which shows induced expression, and cf-I, which has constitutive expression, were not methylated in any organs examined. Thus, it seems that the regulatory mechanisms of the da-I, dm-I and cf-I genes differ from each other and do not correlate with the phylogenetic relationship. The current study suggests that under environmental pressure in specific habitats carnivorous plants have managed to evolve their S-like RNase genes to function in carnivory.

    DOI

  • The genome folding mechanism in yeast

    Hajime Kimura, Yasutoshi Shimooka, Jun-ichi Nishikawa, Osamu Miura, Shigeru Sugiyama, Shuji Yamada, Takashi Ohyama

    J. Biochem.   154 ( 2 ) 137 - 147  2013年08月  [査読有り]

     概要を見る

    The structure of the nucleosome has been solved at atomic resolution, and the genome-wide nucleosome positions have been clarified for the budding yeast Saccharomyces cerevisiae. However, the genome-wide three-dimensional arrangement of nucleosomal arrays in the nucleus remains unclear. Several studies simulated overall interphase chromosome architectures by introducing the putative persistence length of the controversial 30-nm chromatin fibres into the modelling and using data-fitting approaches. However, the genome-folding mechanism still could not be linked with the chromosome shapes, to identify which structures or properties of chromatin fibres or DNA sequences determine the overall interphase chromosome architectures. Here we demonstrate that the paths of nucleosomal arrays and the chromatin architectures themselves are determined principally by the physical properties of genomic DNA and the nucleus size in yeast. We clarified the flexibilities and persistence lengths of all linker DNAs of the organism, deduced their spatial expanses and simulated the architectures of all 16 interphase chromosomes in the nucleus, at a resolution of beads-on-a-string chromatin fibre. For the average spatial distance between two given loci in a chromosome, the model predictions agreed well with all experimental data reported to date. These findings suggest a general mechanism underlying the folding of eukaryotic genomes into interphase chromosomes.

    DOI

  • Regions of unusually high flexibility occur frequently in human genomic DNA

    Hajime Kimura, Dai Kageyama, Mika Furuya, Shigeru Sugiyama, Noboru Murata, Takashi Ohyama

    Biosci. Biotechnol. Biochem.   77 ( 3 ) 612 - 617  2013年03月  [査読有り]

     概要を見る

    Remarkable progress has been made in genome science during the past decade, but understanding of genomes of eukaryotes is far from complete. We have created DNA flexibility maps of the human, mouse, fruit fly, and nematode chromosomes. The maps revealed that all of these chromosomes have markedly flexible DNA regions (We named them SPIKEs). SPIKEs occur more frequently in the human chromosomes than in the mouse, fruit fly, and nematode chromosomes. Markedly rigid DNA regions (rSPIKEs) are also present in these chromosomes. The ratio of the number of SPIKEs to the total number of SPIKEs and rSPIKEs correlated positively with evolutionary stage among the organisms. Repetitive DNA sequences with flexible and rigid properties contribute to the formation of SPIKEs and rSPIKEs respectively. However, non-repetitive flexible and rigid sequences appear to play a major role in SPIKE and rSPIKE formation respectively. They might be involved in the genome-folding mechanism of eukaryotes.

    DOI PubMed

  • Most methylation-susceptible DNA sequences in human embryonic stem cells undergo a change in conformation or flexibility upon methylation

    Yasutoshi Shimooka, Jun-Ichi Nishikawa, Takashi Ohyama

    Biochemistry   52 ( 8 ) 1344 - 1353  2013年02月  [査読有り]

     概要を見る

    DNA methylation in eukaryotes occurs on the cytosine bases in CG, CHG, and CHH (where H indicates non-G nucleotides) contexts and provides an important epigenetic mark in various biological processes. However, the structural and physical properties of methylated DNA are poorly understood. Using nondenaturing polyacrylamide gel electrophoresis, we performed a systematic study of the influence of DNA methylation on the conformation and physical properties of DNA for all CG, CHG, and CHH contexts. In the CG context, methylated multimers of the CG/CG-containing unit fragment migrated in gels slightly faster than their unmethylated counterparts. In the CHG context, both homo- and hemimethylation caused retarded migration of multimers of the CAG/CTG-containing fragment. In the CHH context, methylation caused or enhanced retarded migration of the multimers of CAA/TTG-, CAT/ATG-, CAC/GTG-, CTA/TAG-, or CTT/AAG-containing fragments. These results suggest that methylation increases DNA rigidity in the CG context and introduces distortions into several CHG and CHH sequences. More interestingly, we found that nearly all of the methylation repertoires in the CHG context and 98% of those in the CHH context in human embryonic stem cells were species that undergo conformational changes upon methylation. Similarly, most of the methylation repertoires in the Arabidopsis CHG and CHH contexts were sequences with methylation-induced distortion. We hypothesize that the methylation-induced properties or conformational changes in DNA may facilitate nucleosome formation, which provides the essential mechanism for alterations of chromatin density. © 2013 American Chemical Society.

    DOI PubMed

  • Selective association between nucleosomes with identical DNA sequences

    Jun-ichi Nishikawa, Takashi Ohyama

    Nucl. Acids Res.   41 ( 3 ) 1544 - 1554  2013年02月  [査読有り]

     概要を見る

    Self-assembly is the autonomous organization of constituents into higher order structures or assemblages and is a fundamental mechanism in biological systems. There has been an unfounded idea that self-assembly may be used in the sensing and pairing of homologous chromosomes or chromatin, including meiotic chromosome pairing, polytene chromosome formation in Diptera and transvection. Recent studies proved that double-stranded DNA molecules have a sequence-sensing property and can self-assemble, which may play a role in the above phenomena. However, to explain these processes in terms of self-assembly, it first must be proved that nucleosomes retain a DNA sequence-sensing property and can self-assemble. Here, using atomic force microscopy (AFM)-based analyses and a quantitative interaction assay, we show that nucleosomes with identical DNA sequences preferentially associate with each other in the presence of Mg2+ ions. Using Xenopus borealis 5S rDNA nucleosome-positioning sequence and 601 and 603 sequences, homomeric or heteromeric octa-or tetranucleosomes were reconstituted in vitro and induced to form weak intracondensates by MgCl2. AFM clearly showed that DNA sequence-based selective association occurs between nucleosomes with identical DNA sequences. Selective association was also detected between mononucleosomes. We propose that nucleosome self-assembly and DNA self-assembly constitute the mechanism underlying sensing and pairing of homologous chromosomes or chromatin.

    DOI PubMed

  • A designed curved DNA sequence remarkably enhances transgene expression from plasmid DNA in mouse liver

    S. Fukunaga, G. Kanda, J. Tanase, H. Harashima, T. Ohyama, H. Kamiya

    Gene Ther.   19 ( 8 ) 828 - 835  2012年08月  [査読有り]

     概要を見る

    The intranuclear disposition of plasmid DNA is extremely important for transgene expression. The interactions between the plasmid DNA and the histone proteins are one of the keys for controlling the disposition. In this study, the effects of a left-handedly curved sequence (20-40 repeated A center dot T tracts) on transgene expression from a plasmid were examined in vivo. A naked luciferase plasmid with the curved sequence was delivered into mouse liver by a hydrodynamics-based injection, and the luciferase activities were quantitated at various time points. Interestingly, transgene expression was markedly increased by the addition of the curved sequence. An analysis of the nucleosome positions near the left-handedly curved sequence suggested that the sequence functions as an acceptor of the histone core and allows nucleosome sliding, resulting in transcriptional activation. These results suggested that the designed curved DNA sequences could control transgene expression from plasmid DNAs in vivo.

    DOI

  • Nuclear localization of reporter genes activated by curved DNA

    Koji Udagawa, Hajime Kimura, Hideyuki Tanabe, Takashi Ohyama

    J. Biosci. Bioeng.   113 ( 4 ) 431 - 437  2012年04月  [査読有り]

     概要を見る

    Curved DNA structures with a left-handed superhelical conformation can activate eukaryotic transcription. Mechanistically, these structures favor binding to histone cores and can function as a docking site for sliding nucleosomes. Thus, promoters with this kind of curved DNA can adopt a more open structure, facilitating transcription initiation. However, whether the curved DNA segment can affect localization of a reporter gene is an open question. Localization of a gene in the nucleus often plays an important role in its expression and this phenomenon may also have a curved DNA-dependent mechanism. We examined this issue in transient and stable assay systems using a 180-bp synthetic curved DNA with a left-handed superhelical conformation. The results clearly showed that curved DNA of this kind does not have a property to deliver reporter constructs to nuclear positions that are preferable for transcription. We also identify the spatial location to which electroporation delivers a reporter plasmid in the nucleus. (C) 2011, The Society for Biotechnology, Japan. All rights reserved.

    DOI

  • Competence of an artificial bent DNA as a transcriptional activator in mouse ES cells

    Jun-ichi Tanase, Tasuku Mitani, Koji Udagawa, Jun-ichi Nishikawa, Takashi Ohyama

    Mol. Biol. Rep.   38 ( 1 ) 37 - 47  2011年01月  [査読有り]

     概要を見る

    Curved DNA structures with a left-handed superhelical conformation can activate eukaryotic transcription. However, their potency in transgene activation in embryonic stem (ES) cells has not been examined. T20 is an artificial curved DNA of 180 bp that serves as a transcriptional activator. We investigated the effect of T20 on transcription in mouse ES cell lines or hepatocytes differentiated from them. We established 10 sets of cell lines each harboring a single copy of the reporter construct. Each set comprised a T20-harboring cell line and a T20-less control cell line. Analyses showed that in ES cells and in hepatocytes originating from these cells, T20 both activated and repressed transcription in a manner that was dependent on the locus of reporter. The present and previous studies strongly suggest that in cells that have a strict gene regulation system, transcriptional activation by T20 occurs only in a transcriptionally active locus in the genome.

    DOI

  • Highly Efficient Chromatin Transcription Induced by Superhelically Curved DNA Segments: The Underlying Mechanism Revealed by a Yeast System

    Jun-ichi Tanase, Nobuyuki Morohashi, Masashi Fujita, Jun-ichi Nishikawa, Mitsuhiro Shimizu, Takashi Ohyama

    BIOCHEMISTRY   49 ( 11 ) 2351 - 2358  2010年03月  [査読有り]

     概要を見る

    Superhelically curved DNA structures can strongly activate transcription in mammalian cells. However, the mechanism underlying the activation has not been clarified. We investigated this mechanism in yeast cells, using 108, 180, and 252 bp synthetic curved DNA segments. Even in the presence of nucleosomes, these DNAs activated transcription front it UAS-deleted. CYC1 promoter that is silenced in the presence of nucleosomes. The fold-activations of transcription by these segments, relative to the transcription on the control that lacked Such segments, were 51.4, 63.4, and 56.4, respectively. The superhelically curved DNA structures favored nucleosome formation. However, the translational positions of the nucleosomes were dynamic. The high mobility of the nucleosomes on the superhelically curved DNA Structures seemed to influence the mobility of the nucleosomes formed oil the promoter and eventually enhanced the access to the center region or one TATA sequence. Functioning as a dock for the historic core and allowing nucleosome sliding seem to be the mechanisms underlying the transcriptional activation by superhelically curved DNA Structures in chromatin. The present study provides important clues for designing and constructing artificial chromatin modulators, as a tool for chromatin engineering.

    DOI PubMed CiNii

  • Effects of carriers on transgene expression from plasmids containing a DNA sequence with high histone affinity

    Hiroyuki Kamiya, Satoki Fukunaga, Takashi Ohyama, Hideyoshi Harashima

    INTERNATIONAL JOURNAL OF PHARMACEUTICS   376 ( 1-2 ) 99 - 103  2009年07月  [査読有り]

     概要を見る

    The intranuclear disposition of plasmid DNA is highly important for transgene expression. The effects of a left-handedly curved sequence with high histone affinity on transgene expression were examined in COS-7 cells with two kinds of carriers (Lipofectamine Plus and TransIT-LT1). Three plasmids containing the curved sequence at different positions were transfected. The transgene expression was affected by the position of the left-handedly curved sequence, and the sequence at appropriate locations enhanced the expression from plasmid DNAs. However, the position effects on the expression differed from those obtained by electroporation of the same plasmid DNAs in a naked form. In addition, the degree of expression enhancement seemed to depend on the carriers. These results suggest that the left-handedly curved sequence with high histone affinity could increase the transgene expression from a plasmid delivered with carriers. (C) 2009 Elsevier B.V. All rights reserved.

    DOI

  • The human actin-related protein hArp5: Nucleo-cytoplasmic shuttling and involvement in DNA repair

    Kumiko Kitayama, Mariko Kamo, Yukako Oma, Ryo Matsuda, Takafumi Uchida, Tsuyoshi Ikura, Satoshi Tashiro, Takashi Ohyama, Barbara Winsor, Masahiko Harata

    EXPERIMENTAL CELL RESEARCH   315 ( 2 ) 206 - 217  2009年01月  [査読有り]

     概要を見る

    Certain actin-related proteins (Arps) of budding yeast are localized in the nucleus, and have essential roles as stoichiometric components of histone acetyltransferase (HAT) and chromatin remodeling complexes. On the other hand, identification of vertebrate nuclear Arps and their functional analyses are just beginning. We show that human Arp5 (hArp5) proteins are localized in the nucleus, and that arp5 Delta yeast cells are partially complemented by hArp5. Thus, hArp5 is a novel member of the nuclear Arps of vertebrates, which possess evolutionarily conserved functions from yeast to humans. We show here that hArp5 shuttles between the nucleus and the cytoplasm. Furthermore, after the induction of DNA double strand breaks (DSB), cell growth and the accumulation of phosphorylated histone H2AX (gamma-H2AX) are impaired by hArp5 depletion. Association of hArp5 with the hIno80 chromatin remodeling enzyme and decrease of chromatinbound hIno80 by hArp5-depletion indicate that hArp5 may have a role in the recruitment of the hINO80 complex to chromatin. Overexpression of hArp5 and hIno80 enhanced gamma-H2AX accumulation. These observations suggest that hArp5 is involved in the process of DSB repair through the regulation of the chromatin remodelling machinery. (C) 2008 Elsevier Inc. All rights reserved.

    DOI PubMed CiNii

  • A common mechanical property shared by yeast nucleosomal DNAs.

    Kimura, H, Ohyama, T

    J. Adv. Sci.   20   37 - 40  2008年  [査読有り]

    DOI

  • The location of the left-handedly curved DNA sequence affects exogenous DNA expression in vivo

    Hiroyuki Kamiya, Satoki Fukunaga, Takashi Ohyama, Hideyoshi Harashima

    ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS   461 ( 1 ) 7 - 12  2007年05月  [査読有り]

     概要を見る

    The intranuclear disposition of a plasmid is extremely important for transgene expression. The effects of a left-handedly curved sequence with high histone affinity on plasmid expression were examined in vivo. A naked luciferase-plasmid was delivered into mouse liver by a hydrodynamics-based injection, and the luciferase activities were quantitated at various time points. The location of the left-handedly curved sequence determined the transgene expression, without affecting the amount of intranuclear exogenous DNA. The plasmid containing the curved sequence at the location that results in the exposure of the TATA box out of the nucleosome core showed the highest expression. These results suggest that sequences with high histone affinity could control transgene expression from plasmids in vivo. (c) 2007 Elsevier Inc. All rights reserved.

    DOI PubMed CiNii

  • 180塩基対の左巻き超らせん様ベントDNAにより活性化されたプロモーターのクロマチン構造

    SUMIDA Noriyuki, SONOBE Haruyuki, OHYAMA Takashi

    Journal of advanced science   19 ( 1/2 ) 22 - 28  2007年

    DOI CiNii

  • Regulation of chromatin structure by curved DNA: How activator binding sites become accessible

    Takashi Ohyama

    Nuclear Dynamics: Molecular Biology and Visualization of the Nucleus     227 - 238  2007年  [査読有り]

     概要を見る

    A single somatic cell of humans contains DNA fibers of a total length of approximately 2 m, which are compacted, without entanglement, into the nucleus of approximately 1×10-5 m in diameter. To greater or lesser degrees, all organisms compact their DNA. Biologically important DNA regions, such as the origins of DNA replication, regulatory regions of transcription, and recombination loci, must all be compacted. The tightly constrained DNA, however, presents the appropriate environment for replication, transcription, and recombination to take place.

    DOI

  • Self-assembly of double-stranded DNA molecules at nanomolar concentrations

    Shotaro Inoue, Shigeru Sugiyama, Andrew A. Travers, Takashi Ohyama

    BIOCHEMISTRY   46 ( 1 ) 164 - 171  2007年01月  [査読有り]

     概要を見る

    Some proteins have the property of self-assembly, known to be an important mechanism in constructing supramolecular architectures for cellular functions. However, as yet, the ability of double-stranded (ds) DNA molecules to self-assemble has not been established. Here we report that dsDNA molecules also have a property of self-assembly in aqueous solutions containing physiological concentrations of Mg2+. We show that DNA molecules preferentially interact with molecules with an identical sequence and length even in a solution composed of heterogeneous DNA species. Curved DNA and DNA with an unusual conformation and property also exhibit this phenomenon, indicating that it is not specific to usual B-form DNA. Atomic force microscopy (AFM) directly reveals the assembled DNA molecules formed at concentrations of 10 nM but rarely at 1 nM. The self-assembly is concentration-dependent. We suggest that the attractive force causing DNA self-assembly may function in biological processes such as folding of repetitive DNA, recombination between homologous sequences, and synapsis in meiosis.

    DOI PubMed CiNii

  • A designed curved DNA segment that is a remarkable activator of eukaryotic transcription

    Noriyuki Sumida, Jun-ichi Nishikawa, Haruka Kishi, Miho Amano, Takayo Furuya, Haruyuki Sonobe, Takashi Ohyama

    FEBS JOURNAL   273 ( 24 ) 5691 - 5702  2006年12月  [査読有り]

     概要を見る

    To identify artificial DNA segments that can stably express transgenes in the genome of host cells, we built a series of curved DNA segments that mimic a left-handed superhelical structure. Curved DNA segments of 288 bp (T32) and 180 bp (T20) were able to activate transcription from the herpes simplex virus thymidine kinase (tk) promoter by approximately 150-fold and 70-fold, respectively, compared to a control in a transient transfection assay in COS-7 cells. The T20 segment was also able to activate transcription from the human adenovirus type 2 E1A promoter with an 18-fold increase in the same assay system, and also activated transcription from the tk promoter on episomes in COS-7 cells. We also established five HeLa cell lines with genomes containing T20 upstream of the transgene promoter and control cell lines with T20 deleted from the transgene locus. Interestingly, T20 was found to activate transcription in all the stable transformants, irrespective of the locus. This suggests that the T20 segment may allow stable expression of transgenes, which is of importance in many fields, and may also be useful for the construction of nonviral vectors for gene therapy.

    DOI PubMed CiNii

  • Genetic information carried in DNA conformation and properties

    Takashi Ohyama, Noriyuki Sumida, Jun-ichi Tanase, Shotaro Inoue, Takahiro Okabe

    DNA Structure, Chromatin and Gene Expression, 2006     71 - 84  2006年  [査読有り]

     概要を見る

    The conformation and mechanical properties of DNA provide additional structural and functional dimensions to chromatin organization and gene expression. The reason why transcriptional regulatory regions often contain curved DNA structures has been roughly understood They seem to construct chromatin structures that leave target elements exposed, permitting activators to recognize them and bind. Concerning the mechanical properties of DNA, a surprisingfinding was recently made for eukaryotic class II gene promoters. They have common mechanical properties irrespective of the promoter type. These properties are suggested to function as general markers in promoter recognition by transcription factors. Here, we discuss genetic information carried in DNA conformation and properties.

  • Structural property of DNA that migrates faster in gel electrophoresis, as deduced by CD spectroscopy

    A Matsugami, K Tani, K Ouhashi, S Uesugi, M Morita, T Ohyama, M Katahira

    NUCLEOSIDES NUCLEOTIDES & NUCLEIC ACIDS   25 ( 4-6 ) 417 - 425  2006年  [査読有り]

     概要を見る

    Bent DNAs are known to migrate slower than ordinary DNA in non-denaturing polyacrylamide gel electrophoresis. In contrast, several satellite DNAs have been shown to migrate fast. The structural property that causes the fast migration, however, is not clarified so far on molecular basis. We have investigated the structural property of a satellite DNA, which contains consecutive purine sequences and migrates faster in gel, by CD spectroscopy. Partial formation of an A-form-like structure has been suggested. Reduction in DNA length due to the formation of the A-form-like structure may be responsible for the fast migration. The pronounced rigidity of DNA may also contribute to the behavior.

    DOI

  • An S-like ribonuclease gene is used to generate a trap-leaf enzyme in the carnivorous plant Drosera adelae

    T Okabe, Y Iwakiri, H Mori, T Ogawa, T Ohyama

    FEBS LETTERS   579 ( 25 ) 5729 - 5733  2005年10月  [査読有り]

     概要を見る

    Carnivorous plants usually grow in nutrient-deficient habitats, and thus they partly depend on insects for nitrogen and phosphate needed for amino acid and nucleotide synthesis. We report that a sticky digestive liquid from a sundew, Drosera adelae, contains an abundant amount of an S-like ribonuclease (RNase) that shows high amino acid-sequence similarity to S-like RNases induced by phosphate starvation or wounding in normal plants. By giving leaves an RNase "coat", D. adelae seems to achieve two requirements simultaneously to adapt itself to its specific surroundings: it obtains phosphates from insects, and defends itself against pathogen attack. (c) 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

    DOI PubMed CiNii

  • Structural analysis of the gene encoding Drosera adelae S-like ribonuclease DA-I.

    Okabe, T, Futatsuya, C, Tanaka, O, Ohyama, T

    Journal of Advanced Science   17 ( 3/4 ) 218 - 224  2005年  [査読有り]

    DOI CiNii

  • A highly distinctive mechanical property found in the majority of human promoters and its transcriptional relevance

    Y Fukue, N Sumida, J Tanase, T Ohyama

    NUCLEIC ACIDS RESEARCH   33 ( 12 ) 3821 - 3827  2005年  [査読有り]

     概要を見る

    A recent study revealed that TATA boxes and initiator sequences have a common anomalous mechanical property, i.e. they comprise distinctive flexible and rigid sequences when compared with the other parts of the promoter region. In the present study, using the flexibility parameters from two different models, we calculated the average flexibility profiles of 1004 human promoters that do not contain canonical promoter elements, such as a TATA box, initiator (Inr) sequence, downstream promoter element or a GC box, and those of 382 human promoters that contain the GC box only. Here, we show that they have a common characteristic mechanical property that is strikingly similar to those of the TATA box-containing or Inr-containing promoters. Their most interesting feature is that the TATA- or Inr-corresponding region lies in the several nucleotides around the transcription start site. We have also found that a dinucleotide step from -1 to +1 (transcription start site) has a slight tendency to adopt CA that is known to be flexible. We also demonstrate that certain synthetic DNA fragments designed to mimic the average mechanical property of these 1386 promoters can drive transcription. This distinctive mechanical property may be the hallmark of a promoter.

    DOI PubMed CiNii

  • Improvement of the aluminum borate whisker-mediated method of DNA delivery into rice callus

    K Mizuno, W Takahashi, T Ohyama, T Shimada, O Tanaka

    PLANT PRODUCTION SCIENCE   7 ( 1 ) 45 - 49  2004年03月  [査読有り]

     概要を見る

    We improved the aluminum borate whisker-mediated method of DNA delivery into rice callus (Oryza saliva L., cv. Notohikari). The following factors were examined: amount of whiskers, the kind of apparatus for agitation, the type of whiskers, duration of agitation, and agitation speed. Twenty callused scutellar tissues were agitated in a 1.5 ml microtube containing aluminum borate whiskers (ABW), liquid medium, and the plasmid pAct1-F carrying the beta-glucuronidase (GUS) gene. After the agitation, the scutellar tissues were subjected to transient GUS expression assay, which visualizes GUS-expressing cells as blue spots. Efficiencies of DNA delivery were evaluated as the number of blue spots resulting from the assay. In the present study, we succeeded in improving the efficiency of DNA delivery by changing the apparatus for agitation from rotary (Vortex Genie 2) to multidirectional (MT-360), and the type of ABW from Alborex Y to an aminosilane-coated Alborex (Alborex YS3A).

    DOI CiNii

  • 左向きのねじれを持ったベントDNAはエピソーム内でTATAボックス依存的に転写を活性化する

    NISHIKAWA Jun-ichi, FUKUE Yoshiro, OHYAMA Takashi

    Journal of advanced science   16 ( 3-4 ) 99 - 103  2004年

    DOI CiNii

  • Core promoter elements of eukaryotic genes have a highly distinctive mechanical property

    Y Fukue, N Sumida, J Nishikawa, T Ohyama

    NUCLEIC ACIDS RESEARCH   32 ( 19 ) 5834 - 5840  2004年  [査読有り]

     概要を見る

    In spite of the abundant data on DNA sequence, the mechanical aspects of promoter DNA remain poorly understood. We classified 1871 human and 196 mouse RNA polymerase II promoters and investigated average flexibility profiles of the human promoters containing either a TATA box or an initiator (Inr) sequence only. Here, we show that TATA boxes and Inr sequences have a common anomalous mechanical property: they are comprised of distinctively flexible and rigid sequences, compared with the other parts of the promoter region. The +2 position in the Inr consensus sequence does not favor adenine to keep the high flexibility and thus this position is more accurately represented as 'T, G, C>A'. Additionally, it was also found that DNA region upstream of TATA box or Inr sequence is more rigid than region downstream of each element. These properties may function as a marker for recognition by TATA-binding protein and Inr-binding protein.

    DOI PubMed CiNii

  • Left-handedly curved DNA regulates accessibility to cis-DNA elements in chromatin

    J Nishikawa, M Amano, Y Fukue, S Tanaka, H Kishi, Y Hirota, K Yoda, T Ohyama

    NUCLEIC ACIDS RESEARCH   31 ( 22 ) 6651 - 6662  2003年11月  [査読有り]

     概要を見る

    There is little information on chromatin structure that allows access of trans-acting transcription factors. Logically, the target DNA elements become accessible by either exposing themselves towards the environment on the surface of the nucleosome, or making the regulatory region free of the nucleosome. Here, we demonstrate that curved DNA that mimics a negative supercoil can play both roles in the promoter region. By constructing 35 reporter plasmids and using in vivo assay systems, we scrutinized the relationships between upstream DNA geometry, nucleosome positioning and promoter activity. When the left-handedly curved DNA was linked to the herpes simplex virus thymidine kinase (HSV tk) promoter at a specific rotational phase and distance, the curved DNA attracted the nucleosome and the TATA box was thereby left in the linker DNA with its minor groove facing outwards, which led to the activation of transcription. Neither planar curving, nor right-handedly curved DNA nor straight DNA had this effect. Our results seem to provide a clue for solving the problem of why curved DNA is often located near transcriptional control regions.

    DOI PubMed CiNii

  • The curved DNA structure in the 5 '-upstream region of the light-responsive genes: its universality, binding factor and function for cyanobacterial psbA transcription

    M Asayama, H Kato, J Shibato, M Shirai, T Ohyama

    NUCLEIC ACIDS RESEARCH   30 ( 21 ) 4658 - 4666  2002年11月  [査読有り]

     概要を見る

    A unique DNA curvature, the CIT, has been found in the 5'-upstream region of the psbA2 gene, which exhibits basal, light-responsive and circadian rhythmic transcription, in a unicellular photosynthetic cyanobacterium, Microcystis aeruginosa K-81. In this study, we report the universality of curvatures found in 5'-upstream regions in the psbA family and the function of the curvature in gene expression. Intrinsic curvatures were identified within 1000 bp upstream from the psbA genes in another cyanobacterium, a red alga and in plants (monocot and dicot). Mutagenized curvatures were constructed and confirmed to have disrupted architecture by gel electrophoresis and atomic force microscopy. Relatively small amounts but light-responsive transcripts of psbA2 were observed in cyanobacterial transformants harboring the mutagenized curvature under light/dark and light/high-light conditions. This shows that the curvature is important for basal transcription. In vitro primer extension and DNA mobility shift assay revealed that factors which might bind to the region upstream from the bending center contribute to the effective basal transcription of psbA2.

    DOI PubMed CiNii

  • Multimerization of restriction fragments by magnesium-mediated stable base pairing between overhangs: A cause of electrophoretic mobility shift

    H Tagashira, M Morita, T Ohyama

    BIOCHEMISTRY   41 ( 40 ) 12217 - 12223  2002年10月  [査読有り]

     概要を見る

    The electrophoretic mobility shift assay (EMSA) or simply the "gel shift assay" is one of the most sensitive methods for studying the ability of a protein to bind to DNA. EMSAs are also widely used to investigate protein- or sequence-dependent DNA bending. Here we report that electrophoresis using physiological concentrations of Mg2+ can cause a mobility shift of restriction fragments in nondenaturing polyacrylamide gels as the overhangs form stable base pairs. This phenomenon was observed at even 37 degreesC. The retardation was, however, more pronounced at low temperatures, where a three-nucleotide overhang 5'-GAC also caused a mobility shift. The stability of the pairing was generally high when the overhangs of four nucleotides display high GC content, while the mobility shift caused by 5'-AATT was greater than those caused by 5'-GATC, 5'-TCGA, and 5'-CTAG. This observation should be taken into account to avoid misinterpretation of the data when the EMSA, especially the circular permutation gel mobility shift assay, is performed using a running buffer that contains Mg2+ ions. The stable adhesion between short overhangs may present an important basis for genome stability and many genetic processes occurring in living cells.

    DOI PubMed CiNii

  • Intrinsic DNA bends: an organizer of local chromatin structure for transcription

    T Ohyama

    BIOESSAYS   23 ( 8 ) 708 - 715  2001年08月  [査読有り]

     概要を見る

    DNA with a curved trajectory of its helix axis is called bent DNA, or curved DNA. Interestingly, biologically important DNA regions often contain this structure, irrespective of the origin of DNA. In the last decade, considerable progress has been made in clarifying one role of bent DNA in prokaryotic transcription and its mechanism of action. However, the role of bent DNA in eukaryotic transcription remains unclear. Our recent study raises the possibility that bent DNA is implicated in the "functional packaging" of transcriptional regulatory regions into chromatin. In this article, I review recent progress in bent DNA research in eukaryotic transcription, and summarize the history of bent DNA research and several subjects relevant to this theme. Finally, I propose a hypothesis that bent DNA structures that mimic a negative supercoil, or have a right-handed superhelical writhe, organize local chromatin infrastructure to help the very first interaction between cis-acting DNA elements and activators that trigger transcription. (C) 2001 John Wiley & Sons, Inc.

    DOI

  • Linearization and integration of DNA into cells preferentially occurs at intrinsically curved regions from human LINE-1 repetitive element

    T Kusakabe, Y Sugimoto, T Maeda, Y Nakajima, M Miyano, J Nishikawa, S Tone, Y Kawaguchi, K Koga, T Ohyama

    GENE   274 ( 1-2 ) 271 - 281  2001年08月  [査読有り]

     概要を見る

    A bent DNA library was constructed from human genomic DNA, from which a new clone belonging to the human LINE-1 sequence family was isolated and characterized. This clone, with a length of 378 base pairs and termed HBC-1 (human bent clone-1), contained an intrinsically occurring curved DNA structure. By permutation analysis, the center of curvature of this fragment was mapped onto the nucleotide position 886 from the 5' terminus of the complete LINE-1 sequence. Reporter plasmids, which contain HBC-1, were effectively integrated into human chromosome, indicating that the bent DNA structure provides a preferential donor site for the integration of human LINE-1 sequences. The present finding may provide an explanation as to why some inactivated LINE-1 sequences on human chromosomes carry the deletion at their 5' termini. (C) 2001 Elsevier Science B.V. All rights reserved.

    DOI PubMed CiNii

  • A common feature shared by bent DNA structures locating in the eukaryotic promoter region

    M Miyano, T Kawashima, T Ohyama

    MOLECULAR BIOLOGY REPORTS   28 ( 1 ) 53 - 61  2001年03月  [査読有り]

     概要を見る

    Eukaryotic promoters often contain a bent DNA structure, suggesting that this structure plays some role in transcription. To reveal the role, we need more information on the promoters that contain or flank a bent DNA structure. In this study, we collected such promoters by the following approach: we first isolated human genomic DNA fragments that contained at least one bent DNA structure, then shotgun cloned them into a promoter trap vector, screened DNA fragments that functioned as a promoter, and finally found the promoters of interest by determining the bent DNA locus and the region expressing promoter activity. From 1187 recombinant plasmids, we isolated 51 that showed promoter activity. Structural and functional analyses of randomly selected 10 clones with inserts of 548-913 bp demonstrated 11 sequences that could drive transcription. Unexpectedly, all of these clones met our purpose: i.e., each segment that showed a promoter activity (67-179 bp) was very close to the bent DNA structure (spanning about 150 bp in all clones), and in some cases overlapped it. More interestingly, these bent DNA structures all had a superhelical writhe. We propose a hypothesis that in the bent-DNA-containing eukaryotic promoters, bent DNA organizes local chromatin infrastructure appropriately for transcription initiation.

    DOI

  • Effect of upstream DNA architecture on transcription of a human LINE-1 retrotransposon sequence.

    Miyano, M, Okabe, T, Kusakabe, T, Ohyama, T

    J. Adv. Sci.   13 ( 1/2 ) 1 - 6  2001年  [査読有り]

     概要を見る

    We have cloned a human genomic DNA fragment that contains a complex bent DNA structure and a LINE-1 (L1) sequence. The bent DNA structure spans from 70 to -440 relative to the transcription start site of L1, and contains a left-handed sup erhelical structure from about 70 to -200. When we changed the rotational orientation of the bent DNA relative to the L1 promoter by inserting double-stranded oligonucleotides between positions -3 and -4, L1 promoter activity was profoundly affected Inserts of 5 and 16 by altered the rotational position by about 180°, and stimulated transcription 2- to 3-fold Insertions of 11 and 21 base pairs, which altered the rotational orientati on minimally, had much less effect. We present here the first experimental evidence that upstream DNA architecture influences the machinery employed in L1 transcription.

    DOI CiNii

  • Contamination of Escherichia coli insertion element DNA sequences in human genome databases.

    Tone, S, Hatada, S, Minatogawa, Y, Kurihara, K, Kohara, M, Kubo, T, Kawashima, T, Ohyama, T

    J. Adv. Sci.   13 ( 4 ) 537 - 540  2001年  [査読有り]

     概要を見る

    During investigation of the molecular mechanism of large DNA fragmentation in apoptosis, we found that one of our clones, which were derived from 30kb DNA fragments generated from heat-treated human cells, mached perfectly with many sequences in E. coli genomic DNA, E. coli cDNA, a Neiserria plasmid, and several human genomic DNAs and cDNAs. All had exactly the same 153bp sequence. In fact, 1147bp DNA, containing the 153bp sequence, was apparently shared between human chromosomes 12, 13, and 21 and E. coli. This DNA was identified as E. coli insertion element 5. We need to pay carefull attention to such contaminations in genome data bases.

    DOI CiNii

  • Quantitation of mRNA using in vitro RNA amplification and northern hybridization

    S Tone, T Kubo, T Ohyama, T Kusakabe, Y Minatogawa

    ANALYTICAL BIOCHEMISTRY   284 ( 2 ) 420 - 422  2000年09月  [査読有り]

    DOI PubMed CiNii

  • Conservation of DNA bend sites with identical superhelical twists among the human, mouse, bovine, rabbit and chiken β-globin genes.

    Wanapirak, C, Onishi, Y, Wada-Kiyama, Y, Ohyama, T, Kiyama, R

    DNA Research   7 ( 4 ) 253 - 259  2000年08月  [査読有り]

    DOI

  • Isolation of replicational cue elements from a library of bent DNAs of Aspergillus oryzae

    T Kusakabe, Y Sugimoto, Y Hirota, S Tone, Y Kawaguchi, K Koga, T Ohyama

    MOLECULAR BIOLOGY REPORTS   27 ( 1 ) 13 - 19  2000年03月  [査読有り]

     概要を見る

    Two fragments that could function as replicational cue elements were isolated from a genomic DNA digest of Aspergillus oryzae on the basis of abnormal behavior in polyacrylamide gel electrophoresis. The vector used in this study contained a scaffold-associated region of the Drosophila melanogaster ftz gene to provide nuclear retention. Neither fragment contained a yeast ARS consensus sequence or an eukaryotic topoisomerase II binding sequence. One of the fragments showed sequence homology with the mitochondrial replication origin of Candida utilis and a portion of mitochondrial DNA of Aspergillus nidulans. This plasmid carrying the cue fragment could also replicate in HeLa and NIH3T3 cells.

  • Influence of highly curved DNA segments on in vivo topology of plasmids

    T Ohyama, M Miyano, S Sakuma

    MOLECULAR BIOLOGY REPORTS   26 ( 4 ) 269 - 276  1999年12月  [査読有り]

     概要を見る

    Recombinant plasmids carrying a highly curved DNA structure are sometimes unstable in Escherichia coli. In order to know the underlying mechanism, several plasmids carrying one or two highly bent DNA segment(s) from the human adenovirus type 2 (Ad2) enhancer and/or origin region of phage lambda replication were systematically constructed and propagated in E. coli. The highly bent DNA segments disturbed the action of DNA topoisomerases: i.e. they were shown to be able to produce an anomalously wide spectrum of linking number topoisomers that tails toward lower supercoiling with a little of the DNA actually positively supercoiled. Furthermore, bent DNA caused multimeric plasmid formation. The linking number topoisomers and multimers seemed to be intermediate topological states of the bent DNA-containing plasmids that would lead to the deletion occurring in them. The nucleotide sequence of a deletion product of a bent DNA-containing plasmid showed that the putative source of the severe topological constraint was entirely eliminated from the plasmid.

    DOI PubMed

  • An intrinsic DNA curvature found in the cyanobacterium Microcystis aeruginosa K-81 affects the promoter activity of rpoD1 encoding a principal sigma factor

    M Asayama, Y Hayasaka, M Kabasawa, M Shirai, T Ohyama

    JOURNAL OF BIOCHEMISTRY   125 ( 3 ) 460 - 468  1999年03月  [査読有り]

     概要を見る

    The rpoD1 gene in the unicellular cyanobacterium Microcystis aeruginosa K-81 encodes a principal sigma factor of RNA polymerase and is transcribed under light and dark conditions to produce multiple monocistronic transcripts. In the 5'-upstream region from rpoD1 Promoter 2, which has a sequence of Escherichia coli type, we found a sequence-directed DNA curvature with an AT-rich sequence. Insertions of 2 to 21 base pairs introduced into the curved center changed a gross geometry of the original curved DNA structure. The rpoD1 promoter activities assayed in vivo by using transcriptional lacZ fusions were correlated with the change in the gross geometry in not only a cyanobacterium but also E. coli. In addition, RNA polymerase binding to the rpoD1 promoter region and the efficiency of the mRNA synthesis from the rpoD1 Promoter 2 were also affected in vitro by the change in the geometry. These results suggest that the tertiary structure of the curved DNA is important for the rpoD1 transcription. The deletion of the center region of the curvature resulted in a considerable reduction of the transcription from Promoter 2 in the cyanobacterium. This report demonstrates that a curved DNA plays a significant role in transcription in cyanobacteria, and that this functional curvature is located in the 5'-upstream region from the rpoD gene, which encodes a principal sigma factor in eubacteria.

  • In vivo gene transfer to mouse spermatogenic cells by deoxyribonucleic acid injection into seminiferous tubules and subsequent electroporation

    Y Yamazaki, H Fujimoto, H Ando, T Ohyama, Y Hirota, T Noce

    BIOLOGY OF REPRODUCTION   59 ( 6 ) 1439 - 1444  1998年12月  [査読有り]

     概要を見る

    An in vivo gene transfer technique for living mouse testes was used to develop a novel transient expression assay system for transcriptional regulatory elements of spermatogenic specific genes. The combination of DNA injection into seminiferous tubules and subsequent in vivo electroporation resulted in an efficient and convenient assay system for gene expression during spermatogenesis. The transfer of the firefly luciferase reporting gene driven by the Protamine-1 (Prm-1) enhancer region revealed a significant increase in the activity of the reporter enzyme. Histochemical studies of the transfer of the lacZ gene driven by the Prm-1 enhancer showed specific lacZ expression only in haploid spermatid cells in adult testes, corresponding with the expression pattern of endogenous Prm-1. We were able to detect long-lasting transgene expression in the transfected spermatogenic cells. A group of spermatogenic differentiating cells maintained the transfected lacZ expression after more than 2 mo of transfection, suggesting that spermatogenic stem cells and/or spermatogonia could also incorporate foreign DNA and that the transgene could be transmitted to the progenitor cells derived from a transfected proliferating germ cell.

  • Suppression of electrophoretic anomaly of bent DNA segments by the structural property that causes rapid migration

    T Ohyama, H Tsujibayashi, H Tagashira, K Inano, T Ueda, Y Hirota, K Hashimoto

    NUCLEIC ACIDS RESEARCH   26 ( 21 ) 4811 - 4817  1998年11月  [査読有り]

     概要を見る

    Intrinsic DNA curvature is speculated to be a common feature of all satellite DNA sequences and may aid in the tight winding of DNA in constitutive heterochromatin. Several satellite DNAs, however, show unusually rapid migration in non-denaturing polyacrylamide gels, which is just the opposite behavior of that shown by curved DNA structures. Employing bovine satellite I DNA monomer, we attempted to understand the molecular mechanism of 'rapid migration'. The phenomenon of rapid migration was temperature-dependent and to a small extent polyacrylamide-concentration-dependent. Physiological or near-physiological concentrations of Mg2+ and Ca2+ ions bent the rapid migrating DNA segment, Predominance of purine-purine base stacking over purine-pyrimidine in nucleotide sequence was strongly indicated to be the cause of the rapid migration, Furthermore, they seemed to be implicated in the formation of induced DNA bend. We also found that the satellite I monomer contains an intrinsic DNA curvature as do many other satellites, Heretofore, the rapid migration property has concealed the presence of curvature.

    DOI PubMed CiNii

  • Effect of upstream superhelical writhe on the function of eukaryotic promoters.

    Amamo, M, Nishikawa, J, Hirota, Y, Ohyama, T

    Nucl. Acids Symp. Ser.   ( 39 ) 255 - 266  1998年

  • A method for quantification of gene expression using in vitro transcription and Northern hybridization.

    Tone, S, Ohyama, T, Minatogawa, Y

    Nucl. Acids Symp. Ser.   ( 39 ) 81 - 82  1998年

  • Sense of superhelical writhe of a bent DNA influences eukaryotic promoter function

    T Ohyama, J Nishikawa, M Amano

    FASEB JOURNAL   11 ( 9 ) A1374 - A1374  1997年07月  [査読有り]

  • Deoxyribonuclease secreted from an insectivorous plant Drosera adelae.

    Okabe, T, Mori, H, Ohyama, T

    Nucl. Acids Symp. Ser.   ( 37 ) 127 - 128  1997年

  • Characterization of DNA fragments that show anomalously rapid migration in nondenaturing polyacrylamide gels.

    Tsujibayashi, H, Tagashira, H, Ohyama, T

    Nucl. Acids Symp. Ser.   ( 37 ) 279 - 280  1997年

  • Bent DNA in the human adenovirus type 2 E1A enhancer is an architectural element for transcription stimulation

    T Ohyama

    JOURNAL OF BIOLOGICAL CHEMISTRY   271 ( 44 ) 27823 - 27828  1996年11月  [査読有り]

     概要を見る

    The upstream half of the human adenovirus type 2 enhancer adopts a curved DNA structure. Most of the enhancer elements are within the curvature, suggesting that this unusual structure is linked to enhancer function. To verify this experimentally, I constructed in vitro transcription assay systems which could distinguish any effects generated by conformational changes in a DNA template, The curved DNA conformation in the enhancer clearly affected the extent of the stimulation of the E1A gene transcription: assays using the wildtype DNA template showed that the moderately curved enhancer was superior to the highly curved enhancer in transcriptional stimulation. In additional experiments, the enhancer region was substituted with a curved DNA derived from the bacteriophage lambda origin of replication, Assays using this mutant revealed that this curved segment could also act as an enhancer when it had the proper conformation. Consequently, DNA conformation may play a general role in transcriptional stimulation.

  • Cloning of human promoter sequences that carry a curved DNA structure.

    Miyano, M, Tone, S, Kadokawa, Y, Ohyama, T

    Nucl. Acids Symp. Ser.   ( 35 ) 265 - 266  1996年

  • Cloning of DNA fragments derived from 30kbp DNA occurring in early phase of apoptosis.

    Tone, S, Kurihara, K, Ohyama, T, Shinohara, K

    Nucl. Acids Symp. Ser.   ( 35 ) 215 - 216  1996年

  • ADJACENT UPSTREAM SUPERHELICAL WRITHE INFLUENCES AN ESCHERICHIA-COLI PROMOTER AS MEASURED BY IN-VIVO STRENGTH AND IN-VITRO OPEN COMPLEX-FORMATION

    Y HIROTA, T OHYAMA

    JOURNAL OF MOLECULAR BIOLOGY   254 ( 4 ) 566 - 578  1995年12月  [査読有り]

     概要を見る

    This work investigates the effect on transcription of superhelical writhe located in the region immediately upstream of the -35 consensus sequence of Escherichia coli promoters. A set of double-stranded oligonucleotides, each with an unique DNA configuration, were designed, synthesized and substituted into an area of naturally occurring right-handed superhelical curvature immediately. upstream of the beta-lactamase promoter in plasmid pUC19. All the mutants showed reduced promoter activities in E. coli cells. However, rightward superhelical writhe clearly facilitated transcription when compared with the effect produced by a straight DNA segment. Leftward writhe greatly repressed transcription. A plane curve hewed an intermediate effect. This phenomenon was due not only to the difference in the ability of the segment to drive an open complex formation, but also to the difference in the binding affinity of RNA polymerase to the promoter. The positive effect of rightward writhe was also observed in vivo for the promoter of the tetracycline resistance gene of pBR322. The sense and extent of superhelical writhe of a DNA curvature seem to determine its influence on promoter activity. (C) 1995 Academic Press Limited

    DOI PubMed CiNii

  • A MURINE THY-1.2 REPORTER VECTOR CONTAINING A SV40 ORIGIN FOR RAPID CLONING AND ANALYSIS OF EUKARYOTIC PROMOTERS

    Y KADOKAWA, T KUSAKABE, Y KAMACHI, K ISOBE, H KONDOH, T OHYAMA

    GENE   153 ( 2 ) 277 - 278  1995年02月  [査読有り]

     概要を見る

    A new vector, pATO, was constructed for rapid cloning and analysis of eukaryotic promoters. When a recombinant pATO, carrying a promoter sequence in its multiple cloning site, was introduced into COS cells, Thy-1.2 protein was produced on the cell surface, and was easily identified by an fluorescein-conjugated anti-Thy-1.2 antibody. The intensity of the fluorescence reflected the strength of the inserted promoter. Since pATO could replicate efficiently in COS cells, the recombinant plasmids recovered from a single COS cell were sufficient to transform Escherichia coli cells. This plasmid is applicable for the rapid and labor saving cloning of promoter elements.

    DOI PubMed CiNii

  • Effects of an upstream DNA curvature on the strength of HSV thymidine kinase promoter.

    Ohyama, T, Kadokawa, Y, Hirota, Y

    Nucl. Acids Symp. Ser.   ( 34 ) 67 - 68  1995年

  • High-Efficiency Shotgun Cloning of Curved DNA Segments from Chromosomal DNA

    T. Ohyama, T. Kusakabe

    Analytical Biochemistry   212 ( 1 ) 287 - 289  1993年07月  [査読有り]

    DOI

  • A possible function of DNA curvature in transcription.

    Ohyama, T, Hirota, Y

    Nucl. Acids Symp. Ser.   29   153 - 154  1993年

  • ATP and Mg2+ ions regulate both conformation and aminoacylation capacity of transfer RNA.

    Ohyama, T, Takemura, S, Nishikawa, K

    J. Adv. Sci.   5 ( 2 ) 39 - 42  1993年  [査読有り]

     概要を見る

    It has been known that transfer RNA assumes a functional conformation in the presence of certain metal ions, most commonly magnesium ions. Several Mg2+-binding sites of high affinity have been revealed by X-ray crystallographic analyses of yeast tRNAPhe. On the other hand, ATP, a naturally occurring high-energy compound and which is one of the substrates in aminoacylation of tRNA, is known to have ability to chelate Mg2+. We therefore examined the possibility that the conformation of tRNA is regulated by the quantitative balance between Mg2+ and ATP, and consequently biological activity of tRNA is modulated as well. Structural transition of tRNA was monitored by measuring the melting profiles of yeast tRNATyr in the presence of Mg2+ and/or ATP at various [ATP]/[Mg2+] ratios. Tyrosine-accepting activity of yeast tRNATyr was also measured under the similar solution conditions. These results clearly indicated that conformation of tRNA and its aminoacylation capacity can be easily modulated by changing the relative concentration ratio of Mg2+ and ATP.

    DOI CiNii

  • AN ULTRARAPID METHOD FOR THE RECOVERY OF DNA FROM GELS

    T OHYAMA

    ANALYTICAL BIOCHEMISTRY   208 ( 1 ) 209 - 211  1993年01月  [査読有り]

    DOI PubMed CiNii

  • Alteration of the curved helical structure located in the upstream region of the β-lactamase promoter of plasmid pUC19 and its effect on transcription.

    Ohyama, T, Nagumo, M, Hirota, Y, Sakuma, S

    Nucleic Acids Research   20 ( 7 ) 1617 - 1622  1992年04月  [査読有り]

    DOI PubMed CiNii

  • DNA conformation of the region preceding the β-lactamase promoter of pUC19 which is required for efficient transcription.

    Hirota, Y, Ohyama, T

    Nucl. Acids Symp. Ser.   ( 27 ) 151 - 152  1992年

  • Functional significance of the DNA curvature located near a promoter : an analysis using the β-lactamase promoter of pUC19.

    Hirota, Y, Ohyama, T

    Nucl. Acids Symp. Ser.   ( 25 ) 117 - 118  1991年

  • UPSTREAM HALF OF ADENOVIRUS TYPE-2 ENHANCER ADOPTS A CURVED DNA CONFORMATION

    T OHYAMA, S HASHIMOTO

    NUCLEIC ACIDS RESEARCH   17 ( 10 ) 3845 - 3853  1989年05月  [査読有り]

    DOI PubMed CiNii

  • STUDY ON THE FUNCTION OF THE BENT DNA WITHIN ADENOVIRUS TYPE-2 EIA ENHANCER - ANALYSIS USING AN INVITRO TRANSCRIPTION SYSTEM

    T OHYAMA, M NAGUMO, S SAKUMA

    SIXTEENTH SYMPOSIUM ON NUCLEIC ACIDS CHEMISTRY   21 ( 21 ) 15 - 16  1989年

  • STUDIES ON T-UTILIS TRANSFER RNATYR VARIANTS WITH ENZYMATICALLY ALTERED D-LOOP SEQUENCES .2. RELATIONSHIP BETWEEN THE TERTIARY STRUCTURE AND TYROSINE ACCEPTANCE

    T OHYAMA, K NISHIKAWA, S TAKEMURA

    JOURNAL OF BIOCHEMISTRY   99 ( 3 ) 859 - 866  1986年03月  [査読有り]

  • Effects of a conformational change in tRNA on its aminoacylation capacity : a study using enzymatically reconstructed variants of T. utilis tRNATyr.

    Ohyama, T, Nishikawa, K, Takemura, S

    Nucl. Acids Res. Symp. Ser.   ( 16 ) 213 - 215  1985年

  • STUDIES ON T-UTILIS TRANSFER-RNA TYR VARIANTS WITH ENZYMATICALLY ALTERED D-LOOP SEQUENCES .1. DELETION OF THE CONSERVED SEQUENCE GM-G AND ITS EFFECTS ON AMINOACYLATION AND CONFORMATION

    T OHYAMA, K NISHIKAWA, S TAKEMURA

    JOURNAL OF BIOCHEMISTRY   97 ( 1 ) 29 - 36  1985年  [査読有り]

  • Enzymatic replacement of nucleotide sequences in the D-loop of T. utilis tRNATyr and its effect on aminoacylation.

    Ohyama, T, Nishikawa, K, Takemura, S

    Nucl. Acids Res. Symp. Ser.   ( 12 ) 137 - 140  1983年

  • Peculiar transition observed on the temperature dependence of water permeability of a novel hydrophilic polyamide membrane.

    Sumitomo, H, Hashimoto, K, Ohyama, T

    Polymer Bulletin   1 ( 9 ) 635 - 639  1979年  [査読有り]

  • Water permeation and solute rejection behaviors of a novel hydrophilic polyamide membrane.

    Sumitomo, H, Hashimoto, K, Ohyama, T

    Polymer Bulletin   1 ( 2 ) 133 - 136  1978年  [査読有り]

▼全件表示

書籍等出版物

  • エピジェネティクス

    大山 隆, 東中川 徹( 担当: 共著)

    裳華房  2016年

  • Intrinsic homology-sensing and assembling property of chromatin fiber. In Bernstein, H. and Bernstein, C. (eds.)

    Nishikawa, J, Shimooka, Y, Ohyama, T

    Meiosis. InTech  2013年

  • 大山隆監修、西川一八・清水光弘編集「ベーシックマスター生化学」

    オーム社  2008年

  • Regulation of chromatin structure by curved DNA: how activator binding sites become accessible. In Nagata, K. and Takeyasu, K. (eds.), Nuclear Dynamics-Molecular Biology and Visualization of the Nucleus-.

    Springer-Verlag (Tokyo)  2007年

  • Regulation of chromatin structure by curved DNA: how activator binding sites become accessible. In Nagata, K. and Takeyasu, K. (eds.), Nuclear Dynamics-Molecular Biology and Visualization of the Nucleus-.

    Springer-Verlag (Tokyo)  2007年

  • 東中川徹、大山隆、清水光弘編「ベーシックマスター分子生物学」

    オーム社  2006年

  • Genetic information carried in DNA conformation and properties. In Kiyama, R. and Shimizu, M. (eds), DNA structure, Chromatin and Gene Expression.

    Transworld Research Network (Kerala)  2006年

  • Genetic information carried in DNA conformation and properties. In Kiyama, R. and Shimizu, M. (eds), DNA structure, Chromatin and Gene Expression.

    Transworld Research Network (Kerala)  2006年

  • The role of unusual DNA structures in chromatin organization for transcription. In Ohyama, T. (ed.), DNA Conformation and Transcription.

    Springer (New York)  2005年

  • Curved DNA and Transcription in eukaryotes. In Ohyama, T. (ed.), DNA Conformation and Transcription.

    Springer (New York)  2005年

  • Curved DNA and prokaryotic promoters: a mechanism for activation of transcription. In Ohyama, T. (ed.), DNA Conformation and Transcription.

    Springer (New York)  2005年

  • The role of unusual DNA structures in chromatin organization for transcription. In Ohyama, T. (ed.), DNA Conformation and Transcription.

    Springer (New York)  2005年

  • Curved DNA and Transcription in eukaryotes. In Ohyama, T. (ed.), DNA Conformation and Transcription.

    Springer (New York)  2005年

  • Curved DNA and prokaryotic promoters: a mechanism for activation of transcription. In Ohyama, T. (ed.), DNA Conformation and Transcription.

    Springer (New York)  2005年

  • 実験室の小さな生き物たち.(共著)

    羊土社  1999年

  • 細胞生物学辞典(共著)

    中外医学社  1992年

▼全件表示

Misc

  • 食虫植物の消化酵素の起源

    大山隆

    食虫植物研究会会誌   72   37 - 42  2021年

  • ゲノムDNAはいかにして折り畳まれるか—DNA物性とゲノム収納—

    大山隆, 木村元, 下岡保俊

    実験医学増刊号「細胞核—遺伝情報制御と疾患」   27   2715 - 2722  2009年

    記事・総説・解説・論説等(商業誌、新聞、ウェブメディア)  

  • DNAの高次構造と物理的特性に印された遺伝情報

    大山隆

    蛋白質核酸酵素   53 ( 1 ) 1 - 11  2008年

    記事・総説・解説・論説等(商業誌、新聞、ウェブメディア)  

    CiNii

  • ヒストン高親和性配列が外来DNAの発現に与える影響

    福永 賢輝, 大山 隆, 原島 秀吉, 紙谷 浩之

    薬剤学 = Journal of Pharmaceutical Science and Technology, Japan   67 ( 0 )  2007年05月

    CiNii

  • 脳・神経特異的に発現するアクチン関連タンパク質ArpNαの遺伝子発現制御および神経分化への関与の解析

    尾間由佳子, 大山隆, 西森克彦, 原田昌彦

    日本分子生物学会年会プログラム・講演要旨集   26th   461  2003年11月

    J-GLOBAL

  • Characterization of the nucleases secreted from Drosera Adelae.

    Okabe, T, Ohyama, T

    Proceedings of the 4th international carnivorous plant conference     141 - 143  2002年

    研究発表ペーパー・要旨(国際会議)  

  • 食虫植物Drosera adelaeが分泌するRNaseのcDNAクローニング

    岡部隆宏, 小川智久, 森仁志, 大山隆

    日本分子生物学会年会プログラム・講演要旨集   23rd  2000年

    J-GLOBAL

  • Molecular mechanism underlying bent DNA-mediated transcriptional activation

    T Ohyama, J Nishikawa

    MOLECULAR BIOLOGY OF THE CELL   10   95A - 95A  1999年11月

    研究発表ペーパー・要旨(国際会議)  

  • ベントDNAによる真核細胞プロモーターからの活性化

    天野 美保, 西川 純一, 大山 隆

    日本分子生物学会年会プログラム・講演要旨集   21   395 - 395  1998年12月

    CiNii

  • ベントDNAはどのようなプロモーターに存在するのか -ヒトゲノムを用いた解析-

    宮野 勝, 大山 隆

    日本分子生物学会年会プログラム・講演要旨集   21   395 - 395  1998年12月

    CiNii

  • すぺてのサテライトDNAにベントDNA構造が存在するか?

    田頭 英樹, 大山 隆

    日本分子生物学会年会プログラム・講演要旨集   21   478 - 478  1998年12月

    CiNii

  • ベントDNA構造を介した転写調節のメカニズム

    大山隆

    生化学   70   1339 - 1344  1998年

    記事・総説・解説・論説等(学術雑誌)  

  • モウセンゴケとゆかいな仲間たち

    大山隆

    実験医学   15 ( 6 ) 672  1997年

    記事・総説・解説・論説等(商業誌、新聞、ウェブメディア)  

  • モウセンゴケD.adelaeが分泌するデオキシリボヌクレアーゼ

    前田拓志, 杉元康志, 刀祢重信, 大山隆

    日本分子生物学会年会プログラム・講演要旨集   19th   246  1996年07月

    J-GLOBAL

  • ベントDNA構造の生物学的意義

    大山 隆

    Viva origino   24 ( 1 ) 86 - 87  1996年03月

    CiNii

  • 転写調節におけるベントDNAの役割

    大山隆

    Viva Origino   24   199 - 210  1996年

    記事・総説・解説・論説等(学術雑誌)  

  • Drosera adelaeの腺毛から分泌されるヌクレアーゼの精製とキャラクタリゼーション

    前田拓志, 山本賢, 杉元康志, 大山隆

    日本分子生物学会年会プログラム・講演要旨集   18th   236  1995年11月

    J-GLOBAL

  • ベントDNAの形と機能

    大山隆

    メビオ   10 ( 10 ) 6 - 13  1993年

    記事・総説・解説・論説等(商業誌、新聞、ウェブメディア)  

    CiNii

  • 転写因子が誘起するDNA二重らせんの曲がり.(共著)

    南雲正彦, 大山隆

    実験医学   8 ( 4 ) 364 - 367  1990年

    記事・総説・解説・論説等(商業誌、新聞、ウェブメディア)  

    CiNii

  • 正の超らせん構造をとったプラスミドDNA

    大山隆

    ラジオアイソトープ   39 ( 6 ) 55  1990年

    記事・総説・解説・論説等(学術雑誌)  

  • ベントDNAと転写制御

    大山隆

    蛋白質核酸酵素   35 ( 3 ) 248 - 252  1990年

    記事・総説・解説・論説等(商業誌、新聞、ウェブメディア)  

    CiNii

▼全件表示

受賞

  • JB論文賞 (2014)

    日本生化学会  

共同研究・競争的資金等の研究課題

  • 食虫植物の消化酵素の起源と進化:遺伝子発現様式の変更による新規形質獲得の普遍性

    科学研究費 基盤研究(C)

    研究期間:

    2020年04月
    -
    2023年03月
     

    大山 隆

  • リン酸基結合分子の変化によるリン吸収端構造変化とそのクロマチン可視化応用

    科学研究費  基盤研究(B)

    研究期間:

    2019年
    -
    2021年
     

    江島 丈雄

  • ヒトゲノムDNAの折り畳み原理の解明

    科学研究費  基盤研究(C)

    研究期間:

    2014年
    -
    2016年
     

    大山 隆

  • 機能性RNAの微生物生産およびRNA発現菌の利用

    科学研究費  基盤研究(A)

    研究期間:

    2013年
    -
    2015年
     

    菊池 洋

  • 遺伝情報収納・発現・継承の時空間場

    科学研究費  新学術領域研究(研究領域提案型)

    研究期間:

    2008年
    -
    2013年
     

    平岡 秦

  • ゲノムにおける高頻度組換え部位の形成基盤:新視点からの解析

    科学研究費  挑戦的萌芽研究

    研究期間:

    2011年
    -
    2012年
     

    大山 隆

  • 遺伝情報収納のダイナミクス

    科学研究費  新学術領域研究(研究領域提案型)

    研究期間:

    2008年
    -
    2012年
     

    大山 隆

  • 非B型DNA配列を利用した遺伝子発現制御による高効率・安定的遺伝子改変動物の開発

    科学研究費  新学術領域研究(研究課題提案型)

    研究期間:

    2008年
    -
    2010年
     

    三谷 匡

  • 細胞核ダイナミクスにおけるゲノムの機能的収納機構の解明

    科学研究費  特定領域研究

    研究期間:

    2007年
    -
    2008年
     

    大山 隆

  • DNAの高次構造と物理的特性に印された遺伝情報の解読

    科学研究費  基盤研究C(一般)

    研究期間:

    2006年
    -
    2007年
     

    大山 隆

  • 細胞核ダイナミクスにおけるゲノムの機能的収納機構の解明

    科学研究費  特定領域研究

    研究期間:

    2005年
    -
    2006年
     

    大山 隆

  • 転写開始のためのクロマチン基盤構造の構築機構:ベントDNAが担う役割の解明

    科学研究費  基盤研究(C)

    研究期間:

    2003年
    -
    2004年
     

    大山 隆

  • Elucidation of genetic information carried in DNA conformation and properties

    Grant-in-Aid for Scientific Research

    研究期間:

    2003年
    -
     
     

  • 転写の開始段階に必須なクロマチン構造の実体解明

    科学研究費  基盤研究(C)

    研究期間:

    2001年
    -
    2002年
     

    大山 隆

  • 真核細胞プロモーターにおけるベントDNAの機能発現メカニズムの解析

    科学研究費  基盤研究(C)

    研究期間:

    1997年
    -
    1998年
     

    大山 隆

  • ベントDNA構造を持ったヒト遺伝子プロモーターの選択的単離と構造および機能解析

    科学研究費  基盤研究(C)

    研究期間:

    1995年
    -
    1996年
     

    大山 隆

  • カイコの卵エクジステロイドの生理作用と作用機構の解析

    科学研究費  基盤研究(C)

    研究期間:

    1995年
    -
    1996年
     

    園部 治之

▼全件表示

講演・口頭発表等

  • DNAの物理的特性とゲノムダイナミクス

    大山隆  [招待有り]

    第94回日本生化学会大会シンポジウム  

    発表年月: 2021年11月

  • Novel effects of metal cations on nucleic acids, chromatin and undifferentiated state of ES cells

    大山 隆  [招待有り]

    8th Annual World Congress of Molecular & Cell Biology -Exploring life, Inspiring innovation, Creating feature-  

    発表年月: 2018年

  • 遺伝情報収納の基本原理

    大山 隆  [招待有り]

    第6回都医学研シンポジウム「DNAとゲノム:二重らせん構造を超えて」  

    発表年月: 2016年

  • DNAの物理的特性に隠された情報と機能

    大山 隆  [招待有り]

    日本分析化学会第63年会 特別シンポジウム「異分野との融合」  

    発表年月: 2014年

  • Self-organizing and self-assembling properties of chromatin fiber.

    大山 隆  [招待有り]

    1st Singapore-Japan-India Joint Symposium on Protein-DNA Interactions in Prokaryotic Nucleoid and Eukaryotic Chromatin  

    発表年月: 2013年

  • ゲノム折り畳みの基本原理と間期染色体構造(B)

    大山 隆  [招待有り]

    CREST講演会シンポジウム「光が拓く細胞解析の最前線」  

    発表年月: 2011年

  • ゲノム収納の原理と染色体構造

    大山 隆  [招待有り]

    第62回染色体学会年会 公開シンポジウム1「染色体構造-形態と分子との対話-」  

    発表年月: 2011年

  • ゲノム折り畳みの基本原理と間期染色体構造(A)

    大山 隆  [招待有り]

    平成23年度日本生化学会関東支部例会特別企画  

    発表年月: 2011年

  • ヌクレオソーム間の選択的相互作用

    第28回染色体ワークショップ  

    発表年月: 2011年

  • 出芽酵母間期染色体の三次元構造とゲノム折り畳み原理

    第28回染色体ワークショップ  

    発表年月: 2011年

  • Sequence-dependent selective interaction between nucleosomes

    International Symposium on the Physicochemical Field for Genetic Activities  

    発表年月: 2011年

  • The prime determinants of interphase chromosome structures

    International Symposium on the Physicochemical Field for Genetic Activities  

    発表年月: 2011年

  • Structure of yeast interphase chromosomes and the genome folding principle

    International Symposium on the Physicochemical Field for Genetic Activities  

    発表年月: 2011年

  • ヌクレオソームの自己集合

    第27回染色体ワークショップ  

    発表年月: 2010年

  • 間期クロマチン構造のin silicoモデリング

    第27回染色体ワークショップ  

    発表年月: 2010年

  • イオン環境とDNAの空間的拡がり

    第27回染色体ワークショップ  

    発表年月: 2010年

  • ヒトゲノムの物理的特性とクロマチン構造

    第27回染色体ワークショップ  

    発表年月: 2010年

  • ゲノム収納の原理とクロマチン繊維の基本構造

    第27回染色体ワークショップ  

    発表年月: 2010年

  • DNA物性とクロマチン・染色体構造との関係

     [招待有り]

    細胞システムコロキウム  

    発表年月: 2010年

  • 間期クロマチン三次元構造の構築原理

    第33回日本分子生物学会年会・第83回日本生化学会大会合同大会  

    発表年月: 2010年

  • 食虫植物のS-likeリボヌクレアーゼ:その発現様式と生物学的意義

    第33回日本分子生物学会年会・第83回日本生化学会大会合同大会  

    発表年月: 2010年

  • ゲノムにおける物性の特異領域とその生物学的意義

    第33回日本分子生物学会年会・第83回日本生化学会大会合同大会  

    発表年月: 2010年

  • 人工ベントDNAにより活性化されたトランスジーンの核内局在

    第33回日本分子生物学会年会・第83回日本生化学会大会合同大会  

    発表年月: 2010年

  • クロマチン構造の人為的改変による遺伝子の高効率発現

    第33回日本分子生物学会年会・第83回日本生化学会大会合同大会  

    発表年月: 2010年

  • DNAの機械的特性と遺伝子機能

    第33回日本分子生物学会年会・第83回日本生化学会大会合同大会  

    発表年月: 2010年

  • メチル化DNAの高次構造と物性

    第33回日本分子生物学会年会・第83回日本生化学会大会合同大会  

    発表年月: 2010年

  • ヌクレオソームの自己集合

    第33回日本分子生物学会年会・第83回日本生化学会大会合同大会  

    発表年月: 2010年

  • 正の超らせんを擬態したベントDNA上に形成されるヌクレオソーム

    第33回日本分子生物学会年会・第83回日本生化学会大会合同大会  

    発表年月: 2010年

  • 核内イオン環境の変化が細胞分化に及ぼす影響

    第33回日本分子生物学会年会・第83回日本生化学会大会合同大会  

    発表年月: 2010年

  • 間期クロマチンの三次元構造とゲノム収納の原理

    第33回日本分子生物学会年会・第83回日本生化学会大会合同大会 ワークショップ「クロマチンと染色体の構造:学際的アプローチによる挑戦」  

    発表年月: 2010年

  • S-like RNase gene expression in carnivorous plants

    The American Society for Cell Biology 50th Annual Meeting  

    発表年月: 2010年

  • Self-assembly of nucleosomes

    The American Society for Cell Biology 50th Annual Meeting  

    発表年月: 2010年

  • DNA flexibility maps of eukaryotic genomes

    The American Society for Cell Biology 50th Annual Meeting  

    発表年月: 2010年

  • Three-dimensional architecture of chromatin fibers in the interphase nucleus

    The American Society for Cell Biology 50th Annual Meeting  

    発表年月: 2010年

  • 出芽酵母におけるDNA構造による遺伝子発現とクロマチン構造の制御

    第32回日本分子生物学会年会  

    発表年月: 2009年

  • ハエトリソウにおけるS-likeリボヌクレアーゼ遺伝子の発現制御

    第32回日本分子生物学会年会  

    発表年月: 2009年

  • 食虫植物S-likeリボヌクレアーゼ遺伝子の発現とエピジェネティクス

    第32回日本分子生物学会年会  

    発表年月: 2009年

  • 正の超らせんを擬態したベントDNAのゲノム内分布

    第32回日本分子生物学会年会  

    発表年月: 2009年

  • ヒトゲノムの物理的特性とクロマチン構造

    第32回日本分子生物学会年会  

    発表年月: 2009年

  • ヒトゲノムDNAにおける物性特異領域の生物学的意義:動態解析の試み

    第32回日本分子生物学会年会  

    発表年月: 2009年

  • 人工ベントDNAにより活性化されたトランスジーンの核内局在:マウスES細胞を用いた解析

    第32回日本分子生物学会年会  

    発表年月: 2009年

  • 人工ベントDNAにより活性化されたトランスジーンの核内局在:HeLa細胞を用いた解析

    第32回日本分子生物学会年会  

    発表年月: 2009年

  • 遺伝子発現に有利なクロマチン構造の人為的構築

    第32回日本分子生物学会年会  

    発表年月: 2009年

  • クロマチン工学による外来遺伝子の安定的発現

    第32回日本分子生物学会年会  

    発表年月: 2009年

  • 間期クロマチン構造のin silicoモデリング

    第32回日本分子生物学会年会  

    発表年月: 2009年

  • ヌクレオソームの自己集合

    第32回日本分子生物学会年会  

    発表年月: 2009年

  • イオン環境とDNAの空間的拡がり

    第32回日本分子生物学会年会  

    発表年月: 2009年

  • ゲノム収納の原理とクロマチン繊維の基本構造

    第32回日本分子生物学会年会ワークショップ「クロマチン機能構造の階層性(Hierarchy in chromatin structure and function)」  

    発表年月: 2009年

  • 食虫植物の生存戦略:環境応答遺伝子の食虫機構への転用

    第82回日本生化学会大会  

    発表年月: 2009年

  • 間期細胞核におけるクロマチン繊維の構造

    第82回日本生化学会大会シンポジウム「クロマチン生物学の近未来(Chromatin Biology in Immediate Future)」  

    発表年月: 2009年

  • 遺伝情報収納の基本メカニズム

    文科省科研費新学術領域研究「遺伝情報場」第2回班会議  

    発表年月: 2009年

  • Localization of transgenes activated by an artificial curved DNA

    発表年月: 2009年

  • ツルギバモウセンゴケda-I遺伝子発現のエピジェネティクス

    第8回核ダイナミクス研究会  

    発表年月: 2009年

  • クロマチン繊維の自己組織化

    第26回染色体ワークショップ  

    発表年月: 2009年

  • Stable transgene expression based on chromatin engineering

    The Wilhelm Bernhard Workshop 21st International Workshop on the Cell Nucleus  

    発表年月: 2009年

  • 3D structure of 10 nm chromatin fiber deduced from DNA flexibility

    The Wilhelm Bernhard Workshop 21st International Workshop on the Cell Nucleus  

    発表年月: 2009年

  • How genomic DNA is functionally folded in a nucleus

    The Wilhelm Bernhard Workshop 21st International Workshop on the Cell Nucleus  

    発表年月: 2009年

  • How genomic DNA is functionally folded in a nucleus

    18th Lake Shirakaba Conference  

    発表年月: 2009年

  • Self and non-self discrimination property of nucleosomes

    Irago Conference 2018 (Tokyo) 2018  

  • Function of the IR cluster occurring in the distal enhancer region of the mouse Oct3/4 gene

    Irago Conference 2018 (Tokyo) 2018  

  • DNA methylation as a discrimination factor in the nucleosome self-assembly

    ASCB | EMBO 2018 meeting (San Diego) 2018  

  • Putative functions of cruciform motifs in the Saccharomyces cerevisiae genome

    ASCB | EMBO 2018 meeting (San Diego) 2018  

  • The underlying mechanism of glandular cell-specific gene expression in carnivorous plants

    ASCB | EMBO 2018 meeting (San Diego) 2018  

  • Co-localization of cruciform motifs and the mammalian poly(A) signals

    ASCB | EMBO 2019 meeting (Washington, DC) 2019  

  • Digestive enzyme genes of a sundew, Drosera adelae: Mechanisms underlying the adhesive trap-specific gene expression

    大山 隆

    ASCB | EMBO 2019 meeting (Washington, DC) 2019  

  • Function of the IR cluster located in the upstream of the Oct3/4 gene

    ASCB | EMBO 2019 meeting (Washington, DC) 2019  

  • A role of physical properties of DNA in the transcription of non-coding RNA

    ASCB | EMBO 2019 meeting (Washington, DC) 2019  

▼全件表示

特定課題研究

  • 食虫植物C. follicularisの消化酵素の起源と進化

    2020年  

     概要を見る

     フクロユキノシタ(Cephalotusfollicularis)は南西オーストラリアの1都市アルバニーの周辺にのみ自生する食虫植物であり、袋状の捕虫葉で昆虫などを捕らえ、内部に蓄えた消化液で消化して吸収する。本研究により、消化液に含まれるタンパク質群の種類が一般の植物が生体防御や周辺の有機物の分解のために用いているタンパク質群の種類と高い共通性を示すことが明らかになった。

  • RNAの自己集合能の探索

    2019年   伊藤大直

     概要を見る

     我々は、相同DNA間や相同ヌクレオソーム間で自発的な集合現象(自己集合)が起きることを世界に先駆けて見出し、報告した(Inoue et al., Biochemistry 2007; Ohyama and Nishikawa, Nucl. Acids Res. 2013)。今回、このような集合現象が、RNAの相同分子間でも起きるか否かについて検討した。具体的には、HSVd(hop stunt viroid)の低濃度水溶液を一定温度下でインキュベートして、経時的な変化が起きるか否かを原子間力顕微鏡(AFM)で観察した。興味深いことに、HSVdは、Mg2+イオンなどのカチオンを一切含まない水溶液中で自発的に集合しやすい性質を有していることが明らかになった。

  • 相同クロマチン繊維間に見られる自己集合現象の駆動原理の解明

    2018年  

     概要を見る

     我々は、相同DNA間や相同ヌクレオソーム間で自発的な集合現象(自己集合)が起きることを世界に先駆けて見出し、報告した。今回、このような集合現象が、RNAの相同分子間でも起きるか否かについて検討を始めることした。具体的には、合成DNA(転写鋳型)とT7RNAポリメラーゼを用いてPSTVd(potato spindle tuber viroid:ジャガイモやせいもウイロイド)の塩基配列に相当する数種類のRNA断片を試験管内で合成した。現在、電気泳動における各分子の挙動について、鋭意解析を進めている。

  • non-B DNAならびに特異なDNA物性に隠された機能と情報の解明

    2017年  

     概要を見る

     ゲノム上には、非B型DNAあるいは特殊DNA構造とよばれる様々なDNAが存在する。今回、cruciform DNA(十字架構造とも呼ばれる)の生物学的意義を解明する研究の一環として、大腸菌、出芽酵母ならびにマウスの各ゲノムを対象とした以下の研究を行った。大腸菌と出芽酵母の各ゲノムに関しては、十字架構造形成能を有するIR(inverted repeat)に関する全ゲノムマップを作成し、当該IRの分布に関する偏りを解明した。マウスゲノムに関しては、ES細胞のOct3/4遺伝子の上流に存在するIRをDR(direct repeat: 十字架構造形成不能)に改変した。今後、その影響について解析する。

  • 相同クロマチン繊維間に見られる自己集合現象の基盤原理の解明

    2017年  

     概要を見る

     我々は、相同DNA間や相同ヌクレオソーム間で自発的な集合現象(自己集合)が起きることを世界に先駆けて見出し、報告した。これらの発見から、細胞内の相同クロマチン繊維間や相同染色体間で起きる集合・対合現象は、相同DNA間ならびに相同ヌクレオソーム間の各自己集合現象を基盤としていると推論できる。そこで我々は、各種のクロマチン繊維を人工的に作製し、水溶液中でのそれらの挙動を解析することで、上記の仮説を実証する計画である。しかし、与えられた研究費で可能な研究は限定されるので、今回は、クロマチン繊維を作製する際に用いるDNA鋳型を各種作製した。さらに、特定の領域をメチル化したDNA鋳型も作製した。

  • DNAの特殊な構造や物性に隠された機能と情報の解明

    2016年  

     概要を見る

     近年、DNAの高次構造や物理的特性が重要な機能や情報を担っている場合があることが明らかになってきた。しかし、これらが有する機能や情報について、これまでに解明された点は極めて僅かである。本研究では、バイオインフォマティクスによる機能予測とゲノム編集による実験的検証を用いて、DNAの特殊な構造や物理的特性が有する生物学的な機能や情報の全容を解明することを最終的な目標にしている。その一環として、本研究では逆方向反復配列(IR)に着目して、その一次構造やゲノムワイドな分布について解析を進めた。その結果、出芽酵母ゲノム上でのIRの分布にはある特徴があることが明らかになった。

  • ゲノムワイド物性インフォマティクスから解く高頻度組換え部位出現の分子基盤

    2014年  

     概要を見る

     減数分裂の際、クロマチン内の特定の領域に組換えのホットスポットが形成される。その分子的背景を明らかにするために、我々はこれまで、出芽酵母、分裂酵母およびマウスのゲノムを対象にして、DSB(二本鎖切断)のホットスポットの物理的特性の解析を進めてきた。そして、平均像としては、DSBのホットスポットの二本鎖DNA崩壊エネルギーが周辺領域のそれに比べて特徴的に高いことを解明した。しかし、個別的に見ると必ずしもこの特徴を示さないものも存在する。そこで本研究では、DSBホットスポットの形成を個別的に説明できる分子基盤について追究した。その結果、様々な物性パラメーターの総合的特徴がDSBの位置を指定する情報として機能している可能性が高いことを明らかにした。

  • 相同染色体やクロマチンの対合の背景にある自己集合原理の分子的理解

    2014年  

     概要を見る

     細胞内では、相同DNA間、相同クロマチン繊維間、相同染色体間の対合現象が数多く見られるが、その分子機構は未解明である。我々は、少なくとも相同DNA間、相同ヌクレオソーム間では、生理的濃度のMg2+イオンさえ存在すれば、タンパク質等の介在なしに自己集合現象が起きることを世界に先立って見出し、報告した(Inoue et al., 2007、Nishikawa & Ohyama, 2013)。なお、ヌクレオソームの自己集合現象もDNAの塩基配列の相同性を基盤にしている。本研究では、相同クロマチン繊維間の選択的集合を試験管内で再現する系の構築を試みた。その結果、部分的な集合が起きる系はできたが、まだ不十分であり、継続して条件検討を進めている。

  • ハエトリソウの全ゲノム配列解析から解く食虫植物進化の分子機構

    2013年  

     概要を見る

     食虫植物は世界中に約600種存在し、我が国にも20種ほどが自生している。一般にこれらの植物は、窒素・リンなどが不足した環境を自生地としており、特殊な葉や根を用いて昆虫などの小動物を捕らえて消化・吸収することで、環境に適応している。我々は、生物形態・機能の進化と遺伝子機能の進化の関係に興味をもち、食虫植物の研究を行ってきた。これまでに、Drosera adelae(和名:ツルギバモウセンゴケ)、Dionaea muscipula(ハエトリソウ)、Cephalotus follicularis(フクロユキノシタ)が、S-like リボヌクレアーゼ(RNase)を消化液中に大量に含むことを発見していた(Okabe et al., 2005; Nishimura et al., 2013)。本研究課題では、通常の植物が食虫植物に進化してきた過程で、どのような遺伝子が関与したかを解明することを目的としている。しかし、今回の助成額と期間でこの研究を実施することは無理なので、Aldrovanda vesiculosa(ムジナモ), Nepenthes bicalcarata(ウツボカズラの一種)、Sarracenia leucophylla(アミメヘイシソウ)を新たに加えた6種の食虫植物のS-like RNaseの構造解析、ならびにそれらと非食虫植物のS-like RNaseの比較構造解析を行った。その結果、食虫植物のS-like RNaseだけに保存されたアミノ酸残基と非食虫植物のS-like RNaseだけに保存されたアミノ酸残基が存在すること、さらに、植物における系統関係を超えて、食虫植物のS-like RNaseがひとつの類縁グループを形成することが明らかになった。また、クラスIのS-like RNaseの場合、植物の進化上の近さよりも機能的相同性の方がより強く酵素の構造に反映していることが明らかになった。

  • 高頻度組換え部位形成の分子基盤の解明

    2013年  

     概要を見る

     遺伝情報に関するこれまでの常識は、『遺伝情報は核酸塩基の配列として符号化されている』というものであった。しかし近年、この常識が“改訂”され始めた。DNAの高次構造や物理的特性にも遺伝情報が“印”されていることが明らかになってきたからである。近年、このような“高次”の遺伝情報は、クロマチンの構築や遺伝子発現制御など、様々な現象に関与していることが明らかになってきた。本研究では、減数分裂時において高頻度組換え部位が形成される際に使われていると推察される高次遺伝情報の実体を解明するために行われた。 減数分裂時の高頻度組換え部位に共通したコンセンサス配列は存在しないことが分かっている。従って、組換え部位を指定する情報が塩基配列の並びとしてではなく、それ以外の形をとってゲノムに印されている可能性が高い。しかし、その情報の実体は未だに解明されていない。Chenらは高頻度組換え部位の形成にはDNAの構造特性が関与していると主張しているが、それがどのような特性であるかについては解明できていない(Nucleic Acids Res. 41, e68, 2013)。因みに彼らが開発した、塩基配列から高頻度組換え部位か否かを判定するプログラムは、高頻度組換え部位以外の領域に対しても70%以上の確率で高頻度組換え部位であるという判定をしてしまう。そこで本研究では、出芽酵母とマウスの各ゲノムクロマチン内で組換え部位が決定される情報基盤を解明する目的でDNAの構造特性の解析を進めた。まず、二本鎖DNAの崩壊エネルギー(duplex disruption energy: DDE)を2 bp単位の高分解能で解析できるコンピュータプログラムMIUdde.ver12を開発した。これを用いて、出芽酵母とマウスのゲノムワイドのDDEマップを作成した。その結果、減数分裂時に形成される高頻度組換え部位のDDEが異常に高いことが明らかになった。

  • ゲノムにおける高頻度組換え部位の形成基盤:新視点からの解析

    2010年  

     概要を見る

     前年度に続き、出芽酵母のクロマチンを対象としたコンピュータシミュレーションによる解析を行った。今回はまず、ヌクレオソームの密度に視点を置いて、高密度領域と低密度領域の三次元構造について検討した。高密度領域の解析からは、出芽酵母の間期染色体内には、いわゆる30 nmクロマチン線維が形成される可能性のある領域が数多く存在することが明らかになった。さらに、このような領域は、次のような3つのカテゴリーに分類することができた。“one-start型”の折り畳み構造をとる可能性が高い領域、“two-start型”の折り畳み構造をとる可能性が高い領域、それに規則正しい構造をとらないと予想される領域である。この他、30 nmクロマチン線維が形成されるほど規則的なヌクレオソーム連鎖は形成されないが、領域全体としてみると、極めて“閉じた構造”になっている領域も散見された。いずれにせよ、これらの領域が部位特異的組み換え現象に関与することはないと予測される。一方、低密度領域のクロマチン三次元構造については、共通した特徴は今のところ見出せていない。しかし、特異的組み換え部位はヌクレオソーム密度が低く、しかも“開いた”クロマチン構造を持つ領域内に存在すると予測される。現在、特異的組み換え部位をクロマチン構造内にマップする作業を急いでいる。

  • 高次遺伝情報の解読:エキソンのDNAだけがもつ特異的柔軟性の生物学的意義

    2010年  

     概要を見る

     本研究は、エキソンを構成するDNAの柔軟性がプロモーター領域を構成するDNAの柔軟性よりも高いことを発見したことに端を発している。2009年度は、出芽酵母のCYC1プロモーター(チトクロームcアイソフォーム1遺伝子のプロモーター)を用いた実験的アプローチを行い、その結果から次の仮説を導いた。「エキソン以外の領域にはヌクレオソームのポジショニング情報が含まれている(遺伝子の機能制御のため)。一方、エキソンにはこのような情報は不必要でヌクレオソームが出来さえすれば良い(単にゲノムDNAの凝縮のため)」。もしこの仮説が正しいのであれば、プロモーター領域と第1エキソンの間に存在する5’非翻訳領域も第1エキソンよりも硬いDNAで構成されていることになる。2010年度は、この点について調べた。具体的には、任意の真核生物遺伝子の第1エキソンの上・下流それぞれ50 bpの領域を解析することだけを条件にして、約50名の解析者に自由な解析(解析サンプル数も各自自由)をさせ、二つの領域の間にDNAの柔軟性における差異が有るか否かを検討するという手法をとった。したがって、解析対象は生物種や遺伝子に関して多種多様であった。しかし、得られた結果は、どれも下流の50 bpが上流の50 bpよりも若干柔軟であるというものであった。なお、この解析ではゲノムデータベースを検索する際に転写開始点の情報を無視したので、一部のサンプルにはプロモーターの一部の領域が含まれていた可能性がある。この点を考慮に入れても、今回の解析で5’非翻訳領域は第1エキソンの始めの領域よりも若干硬いDNAでできていることが明らかになった。

  • ゲノムにおける高頻度組換え部位の形成基盤:新視点からの解析

    2009年  

     概要を見る

     研究費の関係から、出芽酵母第3染色体の間期クロマチン構造をコンピュータ計算により予測し、高頻度組換え部位の特徴を解析することにした。構造予測は以下の方法で行った。まず、柔軟性の異なる約500 bpのDNA断片を数種類用意し、原子間力顕微鏡(AFM)で観察した。そして、Moukhtarらの方法(Phys. Rev. Lett., 2007)に従って、AFM像から持続長を決定した。次に、得られたデータをもとにDNAの相対的柔軟性と持続長との関係を明らかにし、第3染色体内のすべてのリンカーDNAの持続長を割り出した。そしてそのデータから、DNAの単位長さを3 bpとした場合の結合角θを計算して各リンカーの拡がりを計算した。また、ヌクレオソームを跨いだリンカーDNA間の角度は90度とし、リンカーDNAの回転的位相に合わせてヌクレオソームを配置した。このようにしてモデリングした第3染色体の空間的拡がりは、この染色体がある時間断面でとりうるひとつの状態と考えることができる。従って、特定の領域間の空間距離を測定するために、このようなモデリングを500回以上繰り返して距離の平均値を求めた。得られた結果は、すでに報告のある実験データと全て良い一致を示すことが判明し、モデリングの手法の正しさを証明した。従って我々は、間期クロマチンの構造モデリングに世界で初めて成功したものと思われる。現在、このモデルを用いて高頻度組換え部位領域の特徴を解析している。

  • 高次遺伝情報の解読:エキソンのDNAだけがもつ特異的柔軟性の生物学的意義

    2009年  

     概要を見る

     第1エキソンの上流には必ずプロモーターが存在する。プロモーターは硬いDNAでできていることが知られている。同時に進行させている他の研究との関連で、本研究ではプロモーターから第1エキソンにかけて形成されるクロマチンの特徴を明らかにすることにした。この解析からも、エキソンが柔らかいDNAでできている理由を解明する手がかりが得られると考えたからである。プロモーターには出芽酵母CYC1プロモーター(チトクロームcアイソフォーム1遺伝子のプロモーター)からUAS配列を除去したものを用いた。なお、このプロモーターには、TATA1~TATA5とよばれる5個のTATA配列があり、転写には上流側に存在するTATA1とTATA2が関与していることが知られている。間接末端標識法、ヌクレオソームリピ-トアッセイ、ChIPアッセイ、およびDNaseIフットプリントアッセイによりクロマチン構造の解析を行った。その結果、このプロモーター上には1つ、さらにその上流には2つのヌクレオソームがポジショニングすることが明らかになった。なお興味深いことに、やはり硬い構造であるベントDNAをUAS欠失領域に挿入すると、これらのヌクレオソームのスライディングが容易になり、TATA3配列の露出度が上がって転写が活性化することが明らかになった。残念ながら、まだエキソン1領域のクロマチン構造に関する情報が得られていないが、今回の解析から大胆な仮説を立てると次のようになる。エキソン以外の領域にはヌクレオソームのポジショニング情報が含まれている(遺伝子の機能制御のため)。一方、エキソンにはこのような情報は不必要でヌクレオソームが出来さえすれば良い(単にゲノムDNAの凝縮のため)。現在、さらに詳細な解析を進めている。

  • クロマチン工学の開拓

    2008年  

     概要を見る

     現在、組換えDNA技術を基盤とした遺伝子操作技術は、生命科学の諸分野に欠くことのできない基礎技術となっている。しかし、真核生物の遺伝子発現を思い通りに制御する技術はまだなく、生命科学における大きな技術的課題となっている。遺伝子発現制御の主要な段階は転写であるが、真核生物では転写はクロマチン内で行われるため、転写制御領域に転写開始に適したクロマチン構造が構築されるかどうかが遺伝子発現を左右する鍵となる。そこで本研究では、クロマチン工学を「遺伝子発現に有利なクロマチン構造を人為的に構築する新しい遺伝子機能制御技術」と定義して、その開拓に先鞭をつけることを目的とした。 我々は、負の超らせんを擬態した180塩基対の合成ベントDNA(T20)をクロマチン構造の“モデュレーター”として用いることで、HeLa細胞やCOS-7細胞中でレポーター遺伝子の発現を安定的に昂進できることを既に見出していた。そこで今回、T20の機能が細胞分化の影響を受けるかどうかについて詳細に検討することにした。まず、プロモーターの上流にT20をもつレポーターをマウスES細胞のゲノムに1コピーだけ導入して10株の細胞株を樹立した。次いでこれらからT20のみを欠失させて同数の対照株を樹立した。各細胞を肝細胞に分化させてT20の効果を調べたところ、未分化状態の細胞内でも、分化後の細胞内でも、T20がプロモーターを活性化している場合が多いことが分かった。現在、細胞分化に伴って構造が変化したクロマチン領域と構造的に不変であったクロマチン領域を明らかにする解析を始めている。この他、一過的遺伝子発現系において転写を活性化できるZ型DNAを作製することができた。以上のように、単年度の研究ながら、多くの研究成果が得られた。

  • ゲノムにおける高頻度組換え部位の形成基盤:新視点からの解析

    2008年  

     概要を見る

     DNAの相同組換えにおいて、組換え部位が決定されるメカニズムはまだ解明されていない。ただし、特定の塩基配列は利用されていないことは明らかになっている。そこで本研究では、出芽酵母第3染色体を対象として、DNAの物理的特性の解析を行い、クロマチン内で組換え部位が決まるメカニズムを明らかにすることを目的とした。出芽酵母第3染色体を解析対象にした理由は、染色体内のすべての高頻度組換え部位が既に解明されているからである。なお、DNAの物理的特性にもさまざまなものがあるが、今回はDNAの柔軟性を解析した。DNAは柔軟性という視点で見ると不均一な分子である。柔らかい領域もあれば、硬い領域もある。さらに、異方的(anisotropic)に柔らかい領域もある。柔らかい領域とは、力を加えた時に曲がりやすい領域のことで、硬い領域とは、その逆の性質を示す領域のことである。そして、異方的に柔らかい領域とは、ある特定の方向には曲がりやすいが、それ以外の方向には曲がりにくい領域を指す。 まず、第3染色体をトリヌクレオチドステップの単位に分け(ウィンドウとよぶ)、1 bpずつずらして(スライディングステップとよぶ)柔軟性の解析を行った。しかし、高頻度組換え部位だけが示す特有の特徴は見出せなかった。そこで次に、ウィンドウを5 bp、10 bp、50 bpと拡げ、スライディングステップもさまざまに変化させて解析を行った。その結果、高頻度組換え部位の多くに共通した興味深い特徴が明らかになった。現在、論文作成中であるのでその詳細には触れないが、DNAの柔軟性におけるこの特徴を認識するすることが組換え現象の最初のステップであると推察される。この認識過程がタンパク質(またはタンパク質複合体)によってなされるのか、あるいはその他のメカニズムが存在するのか、今後、引き続き検討する予定である。

  • プロモーターの物理的特性に印された遺伝情報の解明

    2006年  

     概要を見る

     遺伝子の転写を制御する領域であるプロモーターは、分子生物学においてこれまでに最もよく研究された領域のひとつである。しかし見方を変えると、プロモーターを構成するDNAに関して分子生物学が明らかにした知見は、その塩基配列に関するものだけである。我々は最近、ヒトとマウスのクラスII遺伝子のプロモーターを対象として、DNAの機械的性質の解析を行った。そして、プロモーターのDNAを硬さ・柔らかさという視点から見ると、すべてのプロモーターに共通した、そして他の領域には見られない独特の性質があることを発見した。具体的には、プロモーターの内部に一カ所、極めて柔らかい特性を持つDNAと極めて硬い特性を持つDNAが隣り合わせになった部位があり、この部位を境に上・下流のDNAの平均的硬さが変化することを発見した。しかしこれまでの研究では、解析範囲が転写開始点上流-500から下流+100の範囲に限られており、それ以外の領域にこのような特徴を有する領域があるか否かは不明であった。また、この点を明らかにするためには、ゲノムワイドな解析が行えるコンピュータプログラムが必要であるが、そのようなプログラムはこれまでなかった。そこで本研究課題では、ゲノムワイドな解析を目的としたコンピュータプログラムの開発を試みた。その結果、300Mbの機械的特性をわずか1時間程度で解析できるプログラムを作成することに成功した。このプログラムを用いた解析は緒についたばかりであり、上記の疑問に対する答えはまだ得られていないが、ゲノムワイドの機械的特性という観点からは、興味深い知見が得られ始めている。たとえば、酵母第3染色体を用いた解析では、遺伝子間領域(プロモーターを含む)は、ORF(open reading frame)よりも硬いDNAでできていることが判明した。現在、当初の目的に沿った解析を進めている。

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