2024/02/01 更新

写真a

カサイ ユキ
笠井 由紀
Yuki Kasai
所属
研究開発機構 専任研究員
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外部リンク

学位

  • 博士(理学) ( 神戸大学 )

学歴

  • 1992年3月
     

    神戸大学   理学研究科   生物学専攻   修了

経歴

  • 2012年4月 - 2017年3月

    中央大学

  • 2014年10月 - 2015年3月

    お茶の水女子大学

  • 2013年10月 - 2014年3月

    お茶の水女子大学

  • 2008年4月 - 2012年3月

    北里大学

  • 2010年4月 - 2011年3月

    北里大学

  • 2009年6月 - 2010年3月

    独立行政法人製品評価技術基盤機構バイオテクノロジー本部客員研究員

  • 1997年10月 - 2008年3月

    (株)海洋バイオテクノロジー研究所研究員

  • 1992年4月 - 1997年3月

    兵庫県立中央農業技術センター生物工学研究所研究員

  • 中央大学   助教C

▼全件表示

所属学協会

  • 日本生物工学会

  • 日本微生物生態学会

  • 日本ゲノム微生物学会

研究キーワード

  • 環境浄化

  • 遺伝子工学

  • 有害物質分解

  • バイオれメディエーション

  • 微細藻類

  • 嫌気ベンゼン分解

研究分野

  • ライフサイエンス / 応用微生物学

  • ライフサイエンス / 分子生物学

論文

  • Precise excision of a selectable marker gene in transgenic Coccomyxa strains by the piggyBac transposase 査読

    Yuki Kasai, Kenta Matsuzaki, Fukiko Ikeda, Yuya Yoshimitsu, Shigeaki Harayama

    ALGAL RESEARCH-BIOMASS BIOFUELS AND BIOPRODUCTS   27   152 - 161   2017年11月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ELSEVIER SCIENCE BV  

    The piggyBac transposon isolated from the cabbage looper moth Trichoplusia ni, is integrated into the host genome, and then excised from it without leaving a footprint. The piggyBac transposon system has been used as a genomic engineering tool in a variety of organisms. In this study, we used two improved versions of the piggyBac transposase (PBase) to create marker-free transgenic strains of the unicellular green alga Coccomyxa sp. strain KJ as follows: Uracil-auxotrophic (Ura(-)) mutants of strain KJ defective in the gene for uridine monophosphate synthase (KJUMPS) were isolated on agar plates containing 5-fluoroorotic acid. Subsequently, cDNA of KJUMPS (cKJUMPS) was cloned between the promoter and terminator of the elongation factor 1 alpha gene to construct a cKJUMPS expression cassette. A DNA fragment carrying the cKJUMPS expression cassette flanked with piggyBac transposon terminal repeats was then constructed (TR_cKJUMPS) and introduced into an Ura-mutant, and Ura(+) transformants were isolated. One of the Ura(+) transformants was named strain TR2-7. Hyperactive PBase (hyPBase) is a mutant PBase with increased excision and integration frequencies. Herein, we synthesized a coding sequence for hyPBase (KJhyPBase), which has optimized codons for expression in strain KJ, and its expression cassette was introduced into strain TR2-7. Fourteen transformants stably carried the KJhyPBase expression cassette, and TR_cKJUMPS was excised from seven of these. We also introduced an expression cassette of KJhyPBase_Ex, which encodes the excision-competent/integration-defective R372A/K375A/D450N mutant of KJhyPBase (KJhyPBase_Ex), into strain TR2-7, and found that the excision frequency of TR_cKJUMPS in KJhyPBase_Ex transformants was significantly higher than that in KJhyPBase transformants. In further experiments, we purified His-tagged KJhyPBase_Ex, and transfected it into strain TR2-7 using electroporation. Under these conditions, TR_cKJUMPS was precisely excised at a frequency of 8.8x10(-8) cell(-1) .The present data extend applications of the present piggyBac transposase-catalyzed excision system in green algae.

    DOI: 10.1016/j.algal.2017.09.007

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  • Stimulation of albino regeneration from rice tissue culture by proline under high osmosis: A possible relationship with an endogenous transposable element os-mudr 査読

    S. Yoshida, Y. Kasai, K. Tamaki, K. Watanabe, M. Fujino, C. Nakamura

    Biotechnology and Biotechnological Equipment   12 ( 1 )   3 - 7   1998年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Proline stimulates proliferation of callus and somaclonal variation, particularly albino regeneration, in rice tissue culture under high osmosis. Albino regenerants from scutellum-derived callus exhibited various deletion patterns in chloroplast DNA, similar to those reported in anther-derived albino regenerants. Through differential display one PCR product was selected which showed enhanced expression by proline in scutellum-derived callus. Northern blot analysis revealed a 2.6 kb major transcript. A deduced amino acid sequence of a partial cDNA clone (976 b) selected from a cDNA library constructed from proline-treated callus showed 36% homology with a maize protein (MURA) encoded by an autonomous regulatory transposable element MuDR. A rice homologue to maize MuDR (designated as Os-MuDR) was shown to be present as a single copy gene in Japanese rice cultivars. Our results suggest a possible relationship between Os-MuDR and the high frequency of somatic mutations including albino regeneration in rice tissue culture. © 1998 Taylor and Francis Group, LLC.

    DOI: 10.1080/13102818.1998.10818956

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書籍等出版物

  • Microbial Bioremediation of Non-metals: Current Research

    Horizon Scientific Press  2011年6月 

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  • Microbial biodegradation: Genomics and Molecular Biology

    Caister Academic Press  2008年1月 

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  • 水環境の浄化・改善技術

    シーエムシー出版  2004年11月 

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  • Pseudomonas: Volume 3: Biosynthesis of Macromolecules and Molecular Metabolism

    Springer  2004年6月 

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MISC

  • Construction of Marker-Free Transgenic Strains of Chlamydomonas reinhardtii Using a Cre/loxP-Mediated Recombinase System

    Yuki Kasai, Shigeaki Harayama

    PLOS ONE   11 ( 8 )   p.e0161733   2016年8月

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

    The Escherichia coli bacteriophage P1 encodes a site-specific recombinase called Cre and two 34-bp target sites of Cre recombinase called loxP. The Cre/loxP system has been used to achieve targeted insertion and precise deletion in many animal and plant genomes. The Cre/loxP system has particularly been used for the removal of selectable marker genes to create marker-free transgenic organisms. For the first time, we applied the Cre/loxP-mediated site-specific recombination system to Chlamydomonas reinhardtii to construct markerfree transgenic strains. Specifically, C. reinhardtii strains cc4350 and cc124 carrying an aphVIII expression cassette flanked by two direct repeats of loxP were constructed. Separately, a synthetic Cre recombinase gene (CrCRE), the codons of which were optimized for expression in C. reinhardtii, was synthesized, and a CrCRE expression cassette was introduced into strain cc4350 carrying a single copy of the loxP-flanked aphVIII expression cassette. Among 46 transformants carrying the CrCRE expression cassette stably, the excision of aphVIII by CrCre recombinase was observed only in one transformant. We then constructed an expression cassette of an in-frame fusion of ble to CrCRE via a short linker peptide. The product of ble (Ble) is a bleomycin-binding protein that confers resistance to bleomycin-related antibiotics such as Zeocin and localizes in the nucleus. Therefore, the ble-(linker)-CrCRE fusion protein is expected to localize in the nucleus. When the ble( linker)-CrCRE expression cassette was integrated into the genome of strain cc4350 carrying a single copy of the loxP-flanked aphVIII expression cassette, CrCre recombinase-mediated excision of the aphVIII expression cassette was observed at a frequency higher than that in stable transformants of the CrCRE expression cassette. Similarly, from strain cc124 carrying a single loxP-flanked aphVIII expression cassette, the aphVIII expression cassette was successfully excised after introduction of the ble-(linker)-CrCRE expression cassette. The ble-(linker)-CrCRE expression cassette remained in the genome after excision of the aphVIII expression cassette, and it was subsequently removed by crossing with the wildtype strain. This precise Cre-mediated deletion method applicable to transgenic C. reinhardtii could further increase the potential of this organism for use in basic and applied research.

    DOI: 10.1371/journal.pone.0161733

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  • Construction of a self-cloning system in the unicellular green alga Pseudochoricystis ellipsoidea

    Yuki Kasai, Kohei Oshima, Fukiko Ikeda, Jun Abe, Yuya Yoshimitsu, Shigeaki Harayama

    BIOTECHNOLOGY FOR BIOFUELS   8 ( 94 )   2015年6月

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    記述言語:英語   出版者・発行元:BIOMED CENTRAL LTD  

    Background: Microalgae have received considerable interest as a source of biofuel production. The unicellular green alga Pseudochoricystis ellipsoidea (non-validated scientific name) strain Obi appears to be suitable for large-scale cultivation in outdoor open ponds for biodiesel production because it accumulates lipids to more than 30 % of dry cell weight under nitrogen-depleted conditions. It also grows rapidly under acidic conditions at which most protozoan grazers of microalgae may not be tolerant. The lipid productivity of this alga could be improved using genetic engineering techniques; however, genetically modified organisms are the subject of regulation by specific laws. Therefore, the aim of this study was to develop a self-cloning-based positive selection system for the breeding of P. ellipsoidea.
    Results: In this study, uracil auxotrophic mutants were isolated after the mutagenesis of P. ellipsoidea using either ultraviolet light or a transcription activator-like effector nuclease (TALEN) system. The cDNA of the uridine monophosphate synthase gene (PeUMPS) of P. ellipsoidea was cloned downstream of the promoter of either a beta-tubulin gene (PeTUBULIN1) or the gene for the small subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase (PeRBCS) to construct the pUT1 or pUT2 plasmid, respectively. These constructs were introduced into uracil auxotroph strains, and genetically complementary transformants were isolated successfully on minimal agar plates. Use of Noble agar as the solidifying agent was essential to avoid the development of false-positive colonies. It took more than 6 weeks for the formation of colonies of pUT1 transformants, whereas pUT2 transformants formed colonies in 2 weeks. Real-time PCR revealed that there were more PeUMPS transcripts in pUT2 transformants than in pUT1 transformants. Uracil synthesis (Ura(+)) transformants were also obtained using a gene cassette consisting solely of PeUMPS flanked by the PeRBCS promoter and terminator.
    Conclusions: A self-cloning-based positive selection system for the genetic transformation of P. ellipsoidea was developed. Self-cloned P. ellipsoidea strains will require less-stringent containment measures for large-scale outdoor cultivation.

    DOI: 10.1186/s13068-015-0277-0

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  • Tropicibacter naphthalenivorans gen. nov., sp nov., a polycyclic aromatic hydrocarbon-degrading bacterium isolated from Semarang Port in Indonesia

    Theresia Umi Harwati, Yuki Kasai, Yumiko Kodama, Dwi Susilaningsih, Kazuya Watanabe

    INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY   59   392 - 396   2009年2月

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    記述言語:英語   出版者・発行元:SOC GENERAL MICROBIOLOGY  

    An aerobic, Gram-negative, motile bacterium, strain C02(T) was isolated from seawater obtained from Semarang Port in Indonesia. Cells of strain C02(T) were peritrichously flagellated and rod-shaped. Strain C02(T) was able to degrade naphthalene, alkylnaphthalenes and phenanthrene. 16S rRNA gene sequence analysis revealed that this strain was affiliated with the family Rhodobacteraceae in the class Alphaproteobacteria and was related most closely to Marinovum algicola FF3(T) (95.7% similarity) and Thalassobius aestuarii JC2049(T) (95.2%). The DNA G + C content of strain C02(T) was 64.6 mol%. The major cellular fatty acids were C(18:1)omega 7c (50.9% of the total), C(16:0) (17.9%), 11 methyl C(18:1)omega 7c (14.7%), C(18:1)omega 9c (2.9%) and C(19:0) cyclo omega 8c (2.4%), and the predominant respiratory lipoquinone was ubiquinone-10. Based on physiological, chemotaxonomic and phylogenetic data, strain C02(T) is suggested to represent a novel species of a new genus, for which the name Tropicibacter naphthalenivorans gen. nov., sp. nov. is proposed. The type strain of Tropicibacter naphthalenivorans is C02(T) (=JCM 14838(T) =DSM 19561(T)).

    DOI: 10.1099/ijs.0.65821-0

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  • Tropicimonas isoalkanivorans gen. nov., sp nov., a branched-alkane-degrading bacterium isolated from Semarang Port in Indonesia

    Theresia Umi Harwati, Yuki Kasai, Yumiko Kodama, Dwi Susilaningsih, Kazuya Watanabe

    INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY   59   388 - 391   2009年2月

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    記述言語:英語   出版者・発行元:SOC GENERAL MICROBIOLOGY  

    An aerobic, Gram-negative, motile bacterium, strain B51(T), was isolated from seawater obtained from Semarang Port in Indonesia. Cells of strain B51(T) were peritrichously flagellated and rod-shaped. Strain B51(T) was able to degrade alkanes, branched alkanes and alkyl naphthalenes. 16S rRNA gene sequence analysis revealed that strain B51(T) was affiliated with the family Rhodobacteraceae, and was related most closely to Thioclava pacifica TL 2(T) (94.6% similarity). The DNA G + C content of strain B51(T) was 66.5 mol%. The major cellular fatty acids were C(18:1)omega 7c (84.9%), C(18:1)omega 9c (13.8%), C(16:0) (8.7%), C(18:0) (6.4%) and anteiso-C(15:0) (5.8%) and the major quinone was ubiquinone-10. Based on its phenotypic and phylogenetic characteristics, strain B51(T) is considered to represent a novel species of a new genus, for which the name Tropicimonas isoalcanivorans gen. nov., sp. nov. is proposed. The type strain of the type species is B51(T) (=JCM 14837(T) =DSM 19548(T)).

    DOI: 10.1099/ijs.0.65822-0

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  • Characterizing and screening Oil Degrading Microbes for Land and Beach Reclamation in Indonesia

    Dwi Susilaningsih, Theresia Umi Harwati, Yuki Kasai, Kazuya Watanabe, Fumiyoshi Okazaki, Shigeaki Harayama, Yantyati Widyastuti, Bambang Prasetya

    In proceeding of: International Conference on Urbanisation, Land Use, Land Degradation and Environment, At Turkey   2009年

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  • Tranquillimonas alkanivorans gen. nov., sp nov., an alkane-degrading bacterium isolated from Semarang Port in Indonesia

    Theresia Umi Harwati, Yuki Kasai, Yumiko Kodama, Dwi Susilaningsih, Kazuya Watanabe

    INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY   58   2118 - 2121   2008年9月

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    記述言語:英語   出版者・発行元:SOC GENERAL MICROBIOLOGY  

    Strain A34(T), an obligately halophilic, Gram-negative, non-motile, rod-shaped bacterium, was isolated from seawater obtained from Semarang Port in Indonesia. It possesses a pink pigment and degrades short-chain alkanes. It is positive for catalase and oxidase and reduces nitrate to nitrite. Analyses of 16S rRNA gene sequences revealed a clear affiliation of this strain with the family 'Rhodobacteraceae' in the class Alphaproteobacteria, with its closest relatives being Salipiger mucosus A3(T) (94.9% sequence similarity) and Palleronia marisminoris B33(T) (93.4%). The DNA G + C content was 69.1 mol%. The major cellular fatty acids of strain A34(T) were C(18:1)omega 7c (56.2%), C(19:0) cyclo omega 8c (26.0%) and C(16:0) (9.1 %), while the predominant respiratory lipoquinone was ubiquinone-10. Based on the physiological and phylogenetic data, it is proposed that strain A34(T) should be classified in a new genus and species, for which the name Tranquillimonas alkanivorans gen. nov., sp. nov. is proposed. The type strain of Tranquillimonas alkanivorans is strain A34(T) (JCM 14836(T) =DSM 19547(T)).

    DOI: 10.1099/ijs.0.65817-0

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  • Characterization of diverse hydrocarbon-degrading bacteria isolated from Indonesian seawater

    Teresia Umi Harwati, Yuki Kasai, Yumiko Kodama, Dwi Susilaningsih, Kazuya Watanabe

    MICROBES AND ENVIRONMENTS   22 ( 4 )   412 - 415   2007年12月

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    記述言語:英語   出版者・発行元:JAPANESE SOC MICROBIAL ECOLOGY, DEPT BIORESOURCE SCIENCE  

    Seawater sampled from the Semarang port in Indonesia was streaked onto inorganic-medium plates coated with weathered crude oil, and 153 strains were isolated. Analyses of 16S rRNA gene sequences identified 67 different phylotypes affiliated with Alphaproteobacteria (111 strains/44 phylotypes), Gammaproteobacteria (8/8) and Actinobacteria (34/15). The organisms represented by 36 phylotypes could transform petroleum components in pure cultures. Many of them were affiliated with genera yet unrelated to hydrocarbon degradation. Strains unaffiliated with known genera were also obtained. Results suggest that many as-yet-unknown hydrocarbon-degrading bacteria are present in tropical marine environments.

    DOI: 10.1264/jsme2.22.412

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  • Degradative capacities and bioaugmentation potential of an anaerobic benzene-degrading bacterium strain DN11

    Yuki Kasai, Yumiko Kodama, Yoh Takahata, Toshihiro Hoaki, Kazuya Watanabe

    ENVIRONMENTAL SCIENCE & TECHNOLOGY   41 ( 17 )   6222 - 6227   2007年9月

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    記述言語:英語   出版者・発行元:AMER CHEMICAL SOC  

    Azoarcus sp. strain DN11 is a denitrifying bacterium capable of benzene degradation under anaerobic conditions The present study evaluated strain DN11 for its application to bioaugmentation of benzene-contaminated underground aquifers. Strain DN11 could grow on benzene, toluene, m-xylene, and benzoate as the sole carbon and energy sources under nitrate-reducing conditions, although o- and p-xylenes were transformed in the presence of toluene. Phenol was not utilized under anaerobic conditions. Kinetic analysis of anaerobic benzene degradation estimated its apparent affinity and inhibition constants to be 0.82 and 11 mu M, respectively. Benzene-contaminated groundwater taken from a former coal-distillation plant site was anaerobically incubated in laboratory bottles and supplemented with either inorganic nutrients (nitrogen, phosphorus, and nitrate) alone, or the nutrients plus strain DN11, showing that benzene was significantly degraded only when DN11 was introduced. Denaturing gradient gel electrophoresis of PCR-amplified 16S rRNA gene fragments, and quantitative PCR revealed that DN11 decreased after benzene was degraded. Following the decrease in DN11 16S rRNA gene fragments corresponding to bacteria related to Owenweeksia hongkongensis and Pelotomaculum isophthalicum, appeared as strong bands, suggesting possible metabolic interactions in anaerobic benzene degradation. Results suggest that DN11 is potentially useful for degrading benzene that contaminates underground aquifers at relatively low concentrations.

    DOI: 10.1021/es062842p

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  • Hydroxylations of substituted naphthalenes by Escherichia coli expressing aromatic dihydroxylating dioxygenase genes from polycyclic aromatic hydrocarbon-utilizing marine bacteria

    Kazutoshi Shindo, Ayako Osawa, Yuki Kasai, Nobuko Lba, Ayako Saotome, Norihiko Misawa

    JOURNAL OF MOLECULAR CATALYSIS B-ENZYMATIC   48 ( 3-4 )   77 - 83   2007年9月

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    記述言語:英語   出版者・発行元:ELSEVIER SCIENCE BV  

    Bioconversion experiments of various mono- or di-substituted naphthalenes such as dimethylnaphthalenes were carried out using the cells of Escherichia coli that expressed aromatic dihydroxylating dioxygenase genes (phnA1A2A3A4 and phdABCD) from polycyclic aromatic hydrocarbon-utilizing marine bacteria, Nocardioides sp. KP7 and Cycloclasticus sp. A5, respectively. We found that the former dioxygenase PhnA1A2A3A4 had broad substrate preference for these compounds and often was able to hydroxylate their methyl groups. Specifically, 1,4-dimethylnaphthalene was predominantly bioconverted into 1,4-dihydroxymethyl naphthalene. (c) 2007 Elsevier B.V. All rights reserved.

    DOI: 10.1016/j.molcatb.2007.06.007

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  • 嫌気ベンゼン分解菌DN11株を用いる土壌・地下水の浄化技術

    高畑陽, 笠井由紀, 帆秋利洋, 渡辺一哉

    大成建設技術センター報   ( 40 )   43 - 1   2007年

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  • Rapid intrinsic biodegradation of benzene, toluene, and xylenes at the boundary of a gasoline-contaminated plume under natural attenuation

    Yoh Takahata, Yuki Kasai, Toshihiro Hoaki, Kazuya Watanabe

    APPLIED MICROBIOLOGY AND BIOTECHNOLOGY   73 ( 3 )   713 - 722   2006年12月

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    記述言語:英語   出版者・発行元:SPRINGER  

    A groundwater plume contaminated with gasoline constituents [mainly benzene, toluene, and xylenes (BTX)] had been treated by pumping and aeration for approximately 10 years, and the treatment strategy was recently changed to monitored natural attenuation (MNA). To gain information on the feasibility of using MNA to control the spread of BTX, chemical and microbiological parameters in groundwater samples obtained inside and outside the contaminated plume were measured over the course of 73 weeks. The depletion of electron aceptors (i.e., dissolved oxygen, nitrate, and sulfate) and increase of soluble iron were observed in the contaminated zone. Laboratory incubation tests revealed that groundwater obtained immediately outside the contaminated zone (the boundary zone) exhibited much higher potential for BTX degradation than those in the contaminated zone and in uncontaminated background zones. The boundary zone was a former contaminated area where BTX were no longer detected. Denaturing gradient gel electrophoresis (DGGE) analysis of polymerase chain reaction (PCR)-amplified bacterial 16S rRNA gene fragments revealed that DGGE profiles for groundwater samples obtained from the contaminated zone were clustered together and distinct from those from uncontaminated zones. In addition, unique bacterial rRNA types were observed in the boundary zone. These results indicate that the boundary zone in the contaminant plumes served as a natural barrier for preventing the BTX contamination from spreading out.

    DOI: 10.1007/s00253-006-0522-3

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  • RNA-based stable isotope probing and isolation of anaerobic benzene-degrading bacteria from gasoline-contaminated groundwater

    Y Kasai, Y Takahata, M Manefield, K Watanabe

    APPLIED AND ENVIRONMENTAL MICROBIOLOGY   72 ( 5 )   3586 - 3592   2006年5月

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    記述言語:英語   出版者・発行元:AMER SOC MICROBIOLOGY  

    Stable isotope probing (SIP) of benzene-degrading bacteria in gasoline-contaminated groundwater was coupled to denaturing gradient gel electrophoresis (DGGE) of DNA fragments amplified by reverse transcription-PCR from community 16S rRNA molecules. Supplementation of the groundwater with [C-13(6)]benzene together with an electron acceptor (nitrate, sulfate, or oxygen) showed that a phylotype affiliated with the genus Azoarcus specifically appeared in the C-13-RNA fraction only when nitrate was supplemented. This phylotype was also observed as the major band in DGGE analysis of bacteria] 16S rRNA gene fragments amplified by PCR from the gasoline-contaminated groundwater. In order to isolate the Azoarcus strains, the groundwater sample was streaked on agar plates containing nonselective diluted CGY medium, and the DGGE analysis was used to screen colonies formed on the plates. This procedure identified five bacterial isolates (from 60 colonies) that corresponded to the SIP-identified Azoarcus phylotype, among which two strains (designated DN11 and AN9) degraded benzene under denitrifying conditions. Incubation of these strains with [C-14]benzene showed that the labeled carbon was mostly incorporated into (CO2)-C-14 within 14 days. These results indicate that the Azoarcus population was involved in benzene degradation in the gasoline-contaminated groundwater under denitrifying conditions. We suggest that RNA-based SIP identification coupled to phylogenetic screening of nonselective isolates facilitates the isolation of enrichment/isolation-resistant microorganisms with a specific function.

    DOI: 10.1128/AEM.72.5.3586-3592.2006

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  • Development of a PCR method for the detection and quanti cation of benzoyl-CoA reductase genes and its application to monitored natural attenuation

    A Hosoda, Y Kasai, N Hamamura, Y Takahata, K Watanabe

    BIODEGRADATION   16 ( 6 )   591 - 601   2005年12月

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    記述言語:英語   出版者・発行元:SPRINGER  

    Benzoyl coenzyme A reductase (BCR) catalyzes dearomatization of benzoyl coenzyme A (benzoyl-CoA), which is the central step in the anaerobic degradative pathways for a variety of aromatic compounds. This study developed a PCR method for the detection and quanti cation of BCR genes in bacterial strains and environmental samples. PCR primers were designed by aligning known BCR genes in Thauera, Azoarcus and Rhodopseudomonas species, and their utility was assessed by amplifying BCR fragments from aromatic-hydrocarbon degrading anaerobes and other bacteria. BCR fragments with the expected sizes were obtained from denitrifying and phototrophic aromatics degraders. The positive signals were also obtained from Geobacter metallireducens and xylene-degrading sulfate-reducing bacterium ( strain mXyS1) but not from other aromatics-degrading sulfate-reducing bacteria and aerobic bacteria. When the PCR was used for analyzing a natural attenuation (NA) site, the positive signal was obtained only from gasoline-contaminated groundwater; sequence analysis of these amplicons revealed that most of them exhibited substantial similarities to the known BCRs. Quantitative competitive PCR analysis estimated BCR-gene copies to account for 10-40% of bacterial 16S rRNA gene copies in the contaminated groundwater, indicating that bacteria possessing BCR genes were highly enriched in the contaminated groundwater. In microcosm bioremediation tests using the contaminated groundwater, the copy number of BCR gene was approximately 10-fold increased in the course of aromatics degradation under denitrifying conditions but not under sulfidogenic conditions. These results suggest the utility of the PCR method for assessing the potential of denitrifying bacteria for aromatic-compound degradation in groundwater.

    DOI: 10.1007/s10532-005-0826-5

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  • Physiological and molecular characterization of a microbial community established in unsaturated, petroleum-contaminated soil

    Y Kasai, Y Takahata, T Hoaki, K Watanabe

    ENVIRONMENTAL MICROBIOLOGY   7 ( 6 )   806 - 818   2005年6月

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    記述言語:英語   出版者・発行元:BLACKWELL PUBLISHING LTD  

    The microbial communities established in soil samples from an unsaturated, petroleum-contaminated zone and from an adjacent uncontaminated site were characterized by physiological and molecular approaches. Possible electron acceptors such as sulfate and nitrate had been completely depleted in these soil samples. Slurries of these soil samples were incubated in bottles in the presence of hydrocarbon indicators (benzene, toluene, xylene and decane), and the degradation of these compounds was examined. Supplementation with electron acceptors stimulated hydrocarbon degradation, although the stimulatory effect was small in the contaminated soil. The initial degradation rates in the contaminated soil under fermentative/methanogenic conditions were comparable to those under aerobic conditions. The microbial populations in the original soil samples were analysed by cloning and sequencing of polymerase chain reaction (PCR)-amplified bacterial and archaeal 16S rRNA gene fragments, showing that the sequences retrieved from these soils were substantially different. For instance, Epsilonproteobacteria, Gammaproteobacteria, Crenarchaeota and Methanosarcinales could only be detected at significant levels in the contaminated soil. Denaturing gradient gel electrophoresis (DGGE) analyses of 16S rRNA gene fragments amplified by PCR from the incubated soil-slurry samples showed that supplementation of the electron acceptors resulted in a shift in the major populations, while the DGGE profiles after incubating the contaminated soil under the fermentative/methanogenic conditions were not substantially changed. These results suggest that petroleum contamination of the unsaturated zone resulted in the establishment of a fermentative/methanogenic community with substantial hydrocarbon-degrading potential.

    DOI: 10.1111/j.1462-2920.2005.00754.x

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  • Microbial communities in oil-contaminated seawater

    S Harayama, Y Kasai, A Hara

    CURRENT OPINION IN BIOTECHNOLOGY   15 ( 3 )   205 - 214   2004年6月

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    記述言語:英語   掲載種別:書評論文,書評,文献紹介等   出版者・発行元:CURRENT BIOLOGY LTD  

    Although diverse bacteria capable of degrading petroleum hydrocarbons have been isolated and characterized, the vast majority of hydrocarbon-degrading bacteria, including anaerobes, could remain undiscovered, as a large fraction of bacteria inhabiting marine environments are uncultivable. Using culture-independent rRNA approaches, changes in the structure of microbial communities have been analyzed in marine environments contaminated by a real oil spill and in micro- or mesocosms that mimic such environments. Alcanivorax and Cycloclasticus of the gamma-Proteobacteria were identified as two key organisms with major roles in the degradation of petroleum hydrocarbons. Alcanivorax is responsible for alkane biodegradation, whereas Cycloclasticus degrades various aromatic hydrocarbons. This information will be useful to develop in situ bioremediation strategies for the clean-up of marine oil spills.

    DOI: 10.1016/j.copbio.2004.04.002

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  • ガソリン汚染地下水におけるBTXの自然減衰評価

    高畑陽, 帆秋利洋, 笠井由紀, 渡辺一哉

    大成建設技術センター報   ( 37 )   40 - 1   2004年

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  • Molecular characterization and substrate preference of a polycyclic aromatic hydrocarbon dioxygenase from Cycloclasticus sp strain A5

    Y Kasai, K Shindo, S Harayama, N Misawa

    APPLIED AND ENVIRONMENTAL MICROBIOLOGY   69 ( 11 )   6688 - 6697   2003年11月

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    記述言語:英語   出版者・発行元:AMER SOC MICROBIOLOGY  

    Cycloclasticus sp. strain A5 is able to grow with petroleum polycyclic aromatic hydrocarbons (PAHs), including unsubstituted and substituted naphthalenes, dibenzothiophenes, phenanthrenes, and fluorenes. A set of genes responsible for the degradation of petroleum PAHs was isolated by using the ability of the organism to oxidize indole to indigo. This 10.5-kb DNA fragment was sequenced and found to contain 10 open reading frames (ORFs). Seven ORFs showed homology to previously characterized genes for PAH degradation and were designated phn genes, although the sequence and order of these phn genes were significantly different from the sequence and order of the known PAH-degrading genes. The phnA1, phnA2, phnA3, and phnA4 genes, which encode the alpha and beta subunits of an iron-sulfur protein, a ferredoxin, and a ferredoxin reductase, respectively, were identified as the genes coding for PAH dioxygenase. The phnA4A3 gene cluster was located 3.7 kb downstream of the phnA2 gene. PhnA1 and PhnA2 exhibited moderate (less than 62%) sequence identity to the alpha and beta subunits of other aromatic ring-hydroxylating dioxygenases, but motifs such as the Fe(11)binding site and the [2Fe-2S] cluster ligands were conserved. Escherichia coli cells possessing the phnA1A2A3A4 genes were able to convert phenanthrene, naphthalene, and methylnaphthalene in addition to the tricyclic heterocycles dibenzofuran and dibenzothiophene to their hydroxylated forms. Significantly, the E. coli cells also transformed biphenyl and diphenylmethane, which are ordinarily the substrates of biphenyl dioxygenases.

    DOI: 10.1128/AEM.69.11.6688-6697.2003

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  • 27-B-08 ガソリン汚染地下水中のBTX分解菌の解析(バイオレメディエーション,一般講演)

    笠井 由紀, 高畑 陽, 帆秋 利洋, 渡辺 一哉

    日本微生物生態学会講演要旨集   ( 19 )   2003年

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    記述言語:日本語   出版者・発行元:日本微生物生態学会  

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  • 総説:石油汚染のバイオレメディエーション

    共著

    土壌環境センター技術ニュース   ( 7 )   73 - 77   2003年

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    記述言語:日本語   出版者・発行元:土壌環境センター  

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  • Bacteria belonging to the genus Cycloclasticus play a primary role in the degradation of aromatic hydrocarbons released in a marine environment

    Y Kasai, H Kishira, S Harayama

    APPLIED AND ENVIRONMENTAL MICROBIOLOGY   68 ( 11 )   5625 - 5633   2002年11月

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    記述言語:英語   出版者・発行元:AMER SOC MICROBIOLOGY  

    To identify the bacteria that play a major role in the aerobic degradation of petroleum polynuclear aromatic hydrocarbons (PAHs) in a marine environment, bacteria were enriched from seawater by using 2-methylnaphthalene, phenanthrene, or anthracene as a carbon and energy source. We found that members of the genus Cycloclasticus became predominant in the enrichment cultures. The Cycloclasticus strains isolated in this study could grow on crude oil and degraded PAH components of crude oil, including unsubstituted and substituted naphthalenes, dibenzothiophenes, phenanthrenes, and fluorenes. To deduce the role of Cycloclasticus strains in a coastal zone oil spill, propagation of this bacterial group on oil-coated grains of gravel immersed in seawater was investigated in beach-simulating tanks that were I In wide by 1.5 m long by 1 m high. The tanks were two-thirds filled with gravel, and seawater was continuously introduced into the tanks; the water level was varied between 30 cm above and 30 cm below the surface of the gravel layer to simulate a 12-h tidal cycle. The number of Cycloclasticus cells associated with the grains was on the order of 10(3) cells/g of grains before crude oil was added to the tanks and increased to 3 x 10(6) cells/g of grains after crude oil was added. The number increased further after 14 days to 10(8) cells/g of grains when nitrogen and phosphorus fertilizers were added, while the number remained 3 x 10(6) cells/g of grains when no fertilizers were added. PAH degradation proceeded parallel with the growth of Cycloclasticus cells on the surfaces of the oil-polluted grains of gravel. These observations suggest that bacteria belonging to the genus Cycloclasticus play an important role in the degradation of petroleum PAHs in a marine environment.

    DOI: 10.1128/AEM.68.11.5625-5633.2002

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  • A transcriptionally active maize MuDR-like transposable element in rice and its relatives

    N Asakura, C Nakamura, T Ishii, Y Kasai, S Yoshida

    MOLECULAR GENETICS AND GENOMICS   268 ( 3 )   321 - 330   2002年11月

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    記述言語:英語   出版者・発行元:SPRINGER-VERLAG  

    Two Mu-like transposable elements were cloned from a rice genomic library using a partial cDNA clone that exhibits high homology to the mudrA gene of the maize element MuDR. Database searches led to the identification of six other sequences that carried highly homologous terminal inverted repeats (TIRs). All the rice elements possessed similar to200-by TIRs, and four were flanked by 9-bp target-site duplications (TSDs). The longer of the two cloned elements, OsMu4-2, could potentially encode a protein colinear with a MURA-like transposase, but it had stop codons in the coding region indicating that it is a pseudogene. All the other elements had large internal deletions. Direct dinucleotide repeats were found in two elements at positions flanking the deleted regions, suggesting that the deletions arose via the interrupted-gap-repair mechanism. Sequences related to empty sites of insertion were found in OsMu4-2 and one of the elements identified in the databases. These results provide evidence that the rice OsMu element was active and transposed in the past. Analysis of OsMu4-2 cDNAs revealed two types of transcripts produced by alternative splicing. Genomic Southern analysis suggested that OsMu4-2 was conserved in rice species with the A genome, but a deleted version was unique to japonica subspecies. Some wild rice species harbored paralogous copies of the OsMu element.

    DOI: 10.1007/s00438-002-0737-7

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  • Predominant growth of Alcanivorax strains in oil-contaminated and nutrient-supplemented sea water

    Y Kasai, H Kishira, T Sasaki, K Syutsubo, K Watanabe, S Harayama

    ENVIRONMENTAL MICROBIOLOGY   4 ( 3 )   141 - 147   2002年3月

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    記述言語:英語   出版者・発行元:BLACKWELL PUBLISHING LTD  

    We found that bacteria closely related to Alcanivorax became a dominant bacterial population in petroleum-contaminated sea water when nitrogen and phosphorus nutrients were supplied in adequate quantity. The predominance of Alcanivorax bacteria was demonstrated under three experimental conditions: (i) in batch cultures of sea water containing heavy oil; (ii) in columns packed with oil-coated gravel undergoing a continuous sea water flow; and (iii) in a large-scale tidal flux reactor that mimics a beach undergoing tidal cycles with fresh sea water. These results suggest that bacteria related to Alcanivorax are major players in the bioremediation of oil-contaminated marine environments.

    DOI: 10.1046/j.1462-2920.2002.00275.x

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  • The TOL plasmid pWWO xylN gene product from Pseudomonas putida is involved in m-xylene uptake

    Y Kasai, J Inoue, S Harayama

    JOURNAL OF BACTERIOLOGY   183 ( 22 )   6662 - 6666   2001年11月

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    記述言語:英語   出版者・発行元:AMER SOC MICROBIOLOGY  

    The upper operon of the TOL plasmid pWWO of Pseudomonas putida encodes a set of enzymes involved in the conversion of toluene and xylenes to their carboxylic acid derivatives. The last gene of the upper operon, xylN, encodes a 465-amino-acid polypeptide which exhibits significant sequence similarity to FadL, an outer membrane protein involved in fatty acid transport in Escherichia coli. To analyze the role of the xylN gene product, xylN on TOL plasmid pWW0 was disrupted by inserting a kanamycin resistance gene, and the phenotypes of P. putida harboring the wild-type and xylN mutant TOL plasmids were characterized. The growth of P. putida harboring the wild-type TOL plasmid was inhibited by a high concentration of m-xylene, while that of P. putida harboring the xylN mutant TOL plasmid was not. The apparent K, value for the oxidation of m-xylene in intact cells of the xylN mutant was fourfold higher than that of the wild-type strain, although the TOL catabolic enzyme activities in cell extracts from the two strains were almost identical. We therefore presume that the xylN gene product is a porin involved in the transport of m-xylene and its analogues across the outer membrane. Western blot analysis confirmed the localization of xylN in the outer membrane.

    DOI: 10.1128/JB.183.22.6662-6666.2001

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  • 論説:海を守るバクテリア

    生物工学会誌   2001年9月

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  • Molecular detection of marine bacterial populations on beaches contaminated by the Nakhodka tanker oil-spill accident

    Y Kasai, H Kishira, K Syutsubo, S Harayama

    ENVIRONMENTAL MICROBIOLOGY   3 ( 4 )   246 - 255   2001年4月

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    記述言語:英語   出版者・発行元:BLACKWELL SCIENCE LTD  

    In January 1997, the tanker Nakhodka sank in the Japan Sea, and more than 5000 tons of heavy oil leaked. The released oil contaminated more than 500 km of the coastline, and some still remained even by June 1999, To investigate the long-term influence of the Nakhodka oil spill on marine bacterial populations, sea water and residual oil were sampled from the oil-contaminated zones 10, 18, 22 and 29 months after the accident, and the bacterial populations in these samples were analysed by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rDNA fragments. The dominant DGGE bands were sequenced, and the sequences were compared with those in DNA sequence libraries. Most of the bacteria in the sea water samples were classified as the Cytophaga-Flavobacterium-Bacteroides phylum, a-Proteobacteria or cyanobacteria, The bacteria detected in the oil paste samples were different from those detected in the sea water samples; they were types related to hydrocarbon degraders, exemplified by strains closely related to Sphingomonas subarctica and Alcanivorax borkumensis, The sizes of the major bacterial populations in the oil paste samples ranged from 3.4 x 10(5) to 1.6 x 10(6) bacteria per gram of oil paste, these low numbers explaining the slow rate of natural attenuation.

    DOI: 10.1046/j.1462-2920.2001.00185.x

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  • 論説:流出油のバイオレメディエーション

    笠井 由紀

    ペトロテック   23 ( 5 )   371 - 375   2000年5月

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    記述言語:日本語   出版者・発行元:石油学会  

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  • Petroleum biodegradation in marine environments

    Shigeaki Harayama, Hideo Kishira, Yuki Kasai, Kazuaki Shutsubo

    Journal of Molecular Microbiology and Biotechnology   1 ( 1 )   63 - 70   1999年8月

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    記述言語:英語  

    Petroleum-based products are the major source of energy for industry and daily life. Petroleum is also the raw material for many chemical products such as plastics, paints, and cosmetics. The transport of petroleum across the world is frequent, and the amounts of petroleum stocks in developed countries are enormous. Consequently, the potential for oil spills is significant, and research on the fate of petroleum in a marine environment is important to evaluate the environmental threat of oil spills, and to develop biotechnology to cope with them. Crude oil is constituted from thousands of components which are separated into saturates, aromatics, resins and asphaltenes. Upon discharge into the sea, crude oil is subjected to weathering, the process caused by the combined effects of physical, chemical and biological modification. Saturates, especially those of smaller molecular weight, are readily biodegraded in marine environments. Aromatics with one, two or three aromatic rings are also efficiently biodegraded
    however, those with four or more aromatic ring are quite resistant to biodegradation. The asphaltene and resin fractions contain higher molecular weight compounds whose chemical structures have not yet been resolved. The biodegradability of these compounds is not yet known. It is known that the concentrations of available nitrogen and phosphorus in seawater limit the growth and activities of hydrocarbon-degrading microorganisms in a marine environment. In other words, the addition of nitrogen and phosphorus fertilizers to an oil-contaminated marine environment can stimulate the biodegradation of spilled oil. This notion was confirmed in the large-scale operation for bioremediation after the oil spill from the Exxon Valdez in Alaska. Many microorganisms capable of degrading petroleum components have been isolated. However, few of them seem to be important for petroleum biodegradation in natural environments. One group of bacteria belonging to the genus Alcanivorax does become predominant in an oil-contaminated marine environment, especially when nitrogen and phosphorus fertilizers are added to stimulate the growth of endogenous microorganisms.

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  • Proline stimulates albino regeneration from anther- and seed-derived rice callus under high osmosis

    S Yoshida, Y Kasai, K Watanabe, M Fujino

    JOURNAL OF PLANT PHYSIOLOGY   155 ( 1 )   107 - 109   1999年7月

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    記述言語:英語   出版者・発行元:GUSTAV FISCHER VERLAG  

    Proline added in subculture medium stimulated albino regeneration from anther- and seed-derived callus in rice (Oryza sativa). This proline effect was synergistic with sorbitol. About one half of albino regenerants from the seed-derived callus showed various patterns of plastid DNA deletions. This suggested that the mechanism of proline-induced albinism in seed callus-derived regenerants is similar to that in anther callus-derived regenerants.

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  • 論説:流出油対策としてのバイオテクノロジー

    高圧ガス   1999年2月

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  • Stimulation of albino regeneration from rice tissue culture by proline under high osmosis: A possible relationship with an endogenous transposable element Os-MuDR

    S Yoshida, Y Kasai, K Tamaki, K Watanabe, M Fujino, C Nakamura

    BIOTECHNOLOGY & BIOTECHNOLOGICAL EQUIPMENT   12 ( 1 )   3 - 7   1998年

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    記述言語:英語   出版者・発行元:DIAGNOSIS PRESS LTD  

    Proline stimulates proliferation of callus and somaclonal variation, particularly albino regeneration, in rice tissue culture under high osmosis. Albino regenerants from scutellum-derived callus exhibited various deletion patterns in chloroplast DNA, similar to those reported in anther-derived albino regenerants. Through differential display one PCR product was selected which showed enhanced expression by proline in scutellum-derived callus. Northern blot analysis revealed a 2.6 kb major transcript. A deduced amino acid sequence of a partial cDNA clone (976 b) selected from a cDNA library constructed from proline-treated callus showed 36% homology with a maize protein (MURA) encoded by an autonomous regulatory transposable element MuDR. A rice homologue to maize MuDR (designated as Os-MuDR) was shown to be present as a single copy gene in Japanese rice cultivars. Our results suggest a possible relationship between Os-MuDR and the high frequency of somatic mutations including albino regeneration in rice tissue culture.

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  • アグロバクテリウムによるカーネーションの形質転換

    岩井豊通, 笠井由紀

    兵庫県農業技術センター研究報告(農業編)   44 ( 44 )   p.5 - 8   1996年

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    記述言語:日本語   出版者・発行元:兵庫県立中央農業技術センター  

    CiNii Books

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    その他リンク: http://agriknowledge.affrc.go.jp/RN/2010561506

▼全件表示

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    日本農芸化学会  2017年 

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  • Aerobic and anaerobic benzene degradation pathway of Azoarcus sp. strain DN11

    日本農芸化学会2016年度大会  2016年 

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    会議種別:ポスター発表  

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    日本農芸化学会  2015年 

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    第12回クラミドモナス研究会  2015年 

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    微細藻類バイオマス利用シンポジウム  2015年 

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    微細藻類バイオマス利用シンポジウム  2015年 

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    微細藻類バイオマス利用シンポジウム  2015年 

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    微細藻類バイオマス利用シンポジウム  2015年 

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  • 油脂生産性微細藻類Pseudococcomyxa ellipsoideaにおけるセルフクローニングシステムの開発

    第7回日本ゲノム微生物学会年会  2013年 

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  • ベンゼン分解菌 DN11 株の培養条件によるベンゼン分解能の影響

    土木学会第67回年次学術講演会  2013年 

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    微細藻類バイオマス利用国際シンポジウム  2013年 

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    微細藻類バイオマス利用国際シンポジウム  2013年 

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  • 嫌気ベンゼン分解菌Azoarcus sp. DN11株の特異的検出法

    日本水環境学会講演集/日本水環境学会  2011年 

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  • 嫌気ベンゼン分解菌Azoarcus sp. DN11株を用いる石炭ガス製造工場跡地の浄化実証試験

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    日本生物工学会 北日本支部シンポジウム  2011年 

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  • 嫌気芳香族化合物分解系遺伝子の分子モニタリング技術の開発

    第19回日本微生物生態学会大会講演要旨集/日本微生物生態学会  2003年 

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  • ガソリン汚染地下水中のBTX分解菌の解析

    第19回日本微生物生態学会大会講演要旨集/日本微生物生態学会  2003年 

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  • TGGE法による石油分解微生物群集の解析

    第3回マリンバイオテクノロジー学会大会講演要旨集/マリンバイオテクノロジー学会  1999年 

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  • 日本海重油流出事故現場の微生物ポピュレーションの解析(2)

    日本生物工学会大会講演要旨集/日本生物工学会  1999年 

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  • 日本海重油流出事故現場の石油分解細菌のポピュレーションの解析

    日本生物工学会東日本支部「生物工学フォーラム」要旨集/日本生物工学会東日本支部  1999年 

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  • 日本海重油流出事故現場の微生物ポピュレーションの解析

    日本生物工学会大会講演要旨集/日本生物工学会  1998年 

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  • Bioremediationの現状と可能性

    日本海洋学会大会講演要旨集 特殊号:秋季/日本海洋学会  1998年 

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  • 石油汚染とバイオレメディエーション

    TECNO OCEAN '98国際シンポジウム論文集/社団法人国際海洋科学技術協会  1998年 

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Works(作品等)

  • バイオマスエネルギー技術研究開発/戦略的次世代バイオマスエネルギー利用技術開発事業(次世代技術開発)/高油脂生産微細藻類の大規模培養と回収および燃料化に関する研究開発

    2013年4月 -  

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  • 海洋微生物等を活用した県産素材の高付加価値化

    2011年4月 -  

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  • バイオマスエネルギー技術研究開発/戦略的次世代バイオマスエネルギー利用技術事業(次世代技術開発)/油分生産性の優れた微細藻類の育種・改良技術の研究開発

    2011年4月 -  

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  • 嫌気ベンゼン分解の分子機構の解明

    2009年4月 -  

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  • 生分解・処理メカニズムの解析と制御技術の開発

    2002年4月 -  

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共同研究・競争的資金等の研究課題

  • トランスクリプトームビッグデータ解析を活用した高油脂生産Coccomyxaの創生

    研究課題/領域番号:18K05561  2018年4月 - 2022年3月

    日本学術振興会  科学研究費助成事業  基盤研究(C)  中央大学

    笠井 由紀

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    配分額:4160000円 ( 直接経費:3200000円 、 間接経費:960000円 )

    CoccomyxaのDYRK1変異株は野生株の2倍の速度で油脂を生産する。トランスクリプトーム解析の結果、DYRK1変異株では油脂合成に関与する複数の遺伝子の転写活性が野生株よりも速いタイミングで変動していることが明らかとなった。DYRK1変異株を分子育種の親株にするために、CRISPR/Cas9でDYRK1のみを破壊した株を作製した。また、硝酸塩要求性を導入し生物学的封じ込めが可能な株の作製にも成功した。

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  • 高油脂生産微細藻類の大規模培養と回収および燃料化に関する研究開発

    2015年 -  

    委託研究 

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    資金種別:競争的資金

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  • 油分生産性の優れた微細藻類の育種・改良技術の開発

    2012年 -  

    委託研究 

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    資金種別:競争的資金

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  • 嫌気ベンゼン分解の分子機構の解明

    研究課題/領域番号:21580118  2009年 - 2011年

    日本学術振興会  科学研究費助成事業  基盤研究(C)  北里大学

    笠井 由紀

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    配分額:4940000円 ( 直接経費:3800000円 、 間接経費:1140000円 )

    嫌気ベンゼン分解菌DN11株を使って、嫌気ベンゼン分解経路とそれに係る遺伝子の同定・解析を行った。中間代謝産物の解析からDN11株はベンゼンをトルエンに変換する経路で分解することが示唆された。ベンゼンで発現誘導を受ける遺伝子を解析し、嫌気トルエン分解遺伝子群の近傍にメチル化酵素遺伝子を検出した。この遺伝子を大腸菌で発現させベンゼンと反応させるとトルエンと考えられるピークが検出された。

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  • 深海環境下における油の微生物分解速度およびその支配因子の評価

    研究課題/領域番号:19360400  2007年 - 2009年

    日本学術振興会  科学研究費助成事業  基盤研究(B)  千葉工業大学

    柴田 清, 渡辺 一哉, 笠井 由紀, 滝口 泰之, 渡辺 一哉

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    配分額:14690000円 ( 直接経費:11300000円 、 間接経費:3390000円 )

    本研究では深海における沈船中残存油の微生物分解処理の可能性を検討するために、高圧下におけるフェノールの微生物分解について実験的に調査した。その結果、Pseudomonas stutzeri単離株を用いた場合、海表面条件下ではフェノール分解が進行することが確認されたが、2.0MPa~5.0MPa加圧下では分解の進行が確認できなかった。一方、深海泥試料を添加した場合は常圧よりも加圧下の方が分解の進行が速いことが観察され、深海環境下でも有機物の微生物分解が進行する可能性が示された。

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  • 沈船による油汚染リスク削減を目指した高圧下の深海における油の微生物分解挙動解析

    研究課題/領域番号:16360444  2004年 - 2005年

    日本学術振興会  科学研究費助成事業  基盤研究(B)  独立行政法人海上技術安全研究所

    柴田 清, 原 正一, 渡辺 一哉, 笠井 由紀

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    配分額:14700000円 ( 直接経費:14700000円 )

    1.深海環境下での油分解実験
    相模湾あるいは東京湾で採取した天然海水に、被分解模擬油としてn-ヘキサデカンあるいはn-デカン、および栄養源としてリン酸とアンモニウム塩を加え、深海を模擬した20MPa、5℃あるいは25℃の好気条件で、10日から75日間緩やかに攪拌して分解の進行を調査した。また比較対照のために高圧下で模擬油無添加、および常圧下でも圧力以外同じ条件で実験を行った。所定日数経過後、模擬油と海水の試料に内部標準物質としてn-デカンあるいはn-ヘキサデカンを一定量加え、n-ヘキサンで油分を抽出後、その被分解模擬油と内部標準物質の濃度比をGC/MSで測定し、実験初期条件の場合と比較することにより被分解物質の分解率を求めた。その結果、初期の海水条件に起因すると思われるばらつきはあるものの、高圧下でも約10日間で50%以上の分解率が観測されることがあった。
    2.微生物コンソーシア解析
    油分解実験前後の海水中の微生物コンソーシアの変化をPCR-DGGE法(変性ゲル電気泳動法)によって観察した。その結果、各々の実験条件毎に得られるバンド分布に顕著な変化が観察され、それぞれ個別の微生物コンソーシアが発達したと認められる。しかし、分解率と同様に形成される微生物コンソーシアに統一した傾向を見いだしにくく、油分の分解に寄与した微生物を特定するには至っていない。
    3.沈船情報の収集
    過去約100年の間に座礁や沈没した総トン数100トン以上の船舶を対象に、遭難日時、遭難原因、遭難地点、遭難時搭載物、遭難時の船体損傷、遭難時死傷者数、沈没地点水深、船舶名、船種、総トン数、寸法、主機械、馬力、速力、搭載燃料、満載貨物量、起工・進水・竣工年月日、船主又は所属、建造所、遭難後対処措置の調査を行い、1206件についてデータベースとして整理した。

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