研究者データベース

浅野 眞一郎(アサノ シンイチロウ)
農学研究院 基盤研究部門 応用生命科学分野
教授

基本情報

所属

  • 農学研究院 基盤研究部門 応用生命科学分野

職名

  • 教授

学位

  • 博士(農学)(北海道大学)

ホームページURL

J-Global ID

研究キーワード

  • cry遺伝子   Bacillus thuringiensis   結晶タンパク質遺伝子   コガネムシ   殺虫性結晶タンパク質   殺線虫活性cry遺伝子   BBMV   cry gene   cryBPl遺伝子   cry 1遺伝子   B.popilliae   cryB28遺伝子   delta-エンドトキシン   promoter   殺鱗翅目活性   分離株   vector   殺コガネムシ活性   B.thuringiensis   殺線虫活性   cry2遺伝子   バインディングアッセイ   N末端解析   ウエスタンブロット   バチルスチューリンゲンシス   ICP遺伝子   バチルス・チューリンゲンシス(BT)   殺虫性タンパク   昆虫ウイルス   デンソウイルス   昆虫病理学   Insect Pathology Applied Entomology   

研究分野

  • 環境・農学 / 昆虫科学 / 昆虫病理学

職歴

  • 2021年04月 - 現在 北海道大学 大学院農学研究院 教授
  • 2010年 - 2012年 北海道大学 (連合)農学研究科(研究院) 准教授
  • 1999年04月 - 2007年03月 - 農学部 助教授
  • 2007年 - 農学部 准教授

学歴

  •         - 1989年   北海道大学   農学部   農業生物学科
  •         - 1989年   北海道大学

所属学協会

  • より   より2000年4月まで   Society Invertebrate Pathology   日本応用動物昆虫学会   日本蚕糸学会   Society Invertebrate Pathology   The Japanease Society of Sericultural Science   

研究活動情報

論文

  • Mami Sakai, Satoshi Kakutani, Shin-ichiro Asano, Masanao Sato, Hisanori Bando
    Virus Research 291 198195 - 198195 2021年01月 [査読有り][通常論文]
     
    The Bombyx mori nucleopolyhedrovirus (BmNPV)-based baculoviral expression vector system is among the most efficient expression vector systems for eukaryotic proteins especially when used in combination with silkworms as a host. We newly isolated a novel BmNPV strain (BmNPV H4) in Hokkaido, Japan that outperforms the type strain T3 in terms of both proliferation and expression of polyhedrin protein in silkworm larvae; however, it proliferates poorly in the BmN cell line. We inferred the gene responsible for the differences in proliferation between viral strains by quantifying amino acid similarity distances in protein functional domains and identifying highly divergent alleles between the H4 and T3 strains. Among proteins that differ markedly in functional domain sequence between H4 and T3, we identified the F gene, which encodes the F protein, as a putative cause of proliferative differences between the two strains. Using recombinant viruses with the F protein-coding sequence exchanged between H4 and T3, we determined that the T3 F protein increases H4 proliferation in BmN while the H4 F protein does not improve T3 proliferation in silkworm larvae. Our results suggest that the BmNPV F protein can strongly affect viral proliferation in a genetic background-specific manner and may be an important target for manipulating the proliferation characteristics of BmNPV-based expression vectors.
  • A single amino acid replacement of tyrosine with histidine at position 172 in BmNPV T3 GP64 decreases viral proliferation in silkworm larvae despite increasing membrane fusion activity with BmN cells
    Mari Sekiguchi, Sayuri Ishikawa, Shin-ichiro Asano, Hisanori Bando, Masanao Sato
    Journal of Insect Biotechnology and Sericology 89 3 45 - 53 2020年09月 [査読有り]
  • Design and prototyping of a Bombyx mori nucleopolyhedrovirus-based genome assembly from PCR-amplified DNA fragments
    Naoto Harada, Daiki Ishibashi, Shin-ichiro Asano, Masanao Sato, Hisanori Bando
    Insect Biotechnology and Sericology 87 3 97 - 108 2018年10月 [査読有り][通常論文]
  • 工藤滉己, 佐藤昌直, 浅野眞一郎, 伴戸久徳
    東北蚕糸・昆虫利用研究報告 42 8‐11  2017年12月25日 [査読無し][通常論文]
  • 原田直人, 佐藤昌直, 浅野眞一郎, 伴戸久徳
    東北蚕糸・昆虫利用研究報告 42 12‐16  2017年12月25日 [査読無し][通常論文]
  • 山口真, 佐藤昌直, 伴戸久徳, 浅野眞一郎
    東北蚕糸・昆虫利用研究報告 42 1‐3  2017年12月25日 [査読無し][通常論文]
  • 齋藤諒, 武田ひとみ, 佐藤昌直, 浅野眞一郎, 伴戸久徳
    東北蚕糸・昆虫利用研究報告 42 4‐7  2017年12月25日 [査読無し][通常論文]
  • 池田優里恵, 佐藤昌直, 伴戸久徳, 浅野眞一郎
    東北蚕糸・昆虫利用研究報告 41 12‐13  2016年12月10日 [査読無し][通常論文]
  • 佐藤文美, 佐藤昌直, 伴戸久徳, 浅野眞一郎
    東北蚕糸・昆虫利用研究報告 41 7‐8  2016年12月10日 [査読無し][通常論文]
  • KURNIA Yudistira Wahyu, FUJITA Ryosuke, SATO Masanao, ISAWA Haruhiko, ASANO Shin‐ichiro, BINH Ngo Dinh, BANDO Hisanori
    Journal of Insect Biotechnology and Sericology 85 2 39‐47  2016年09月12日 [査読無し][通常論文]
  • Kota Kawakami, Yudistira Wahyu Kurnia, Ryosuke Fujita, Toshiaki Ito, Haruhiko Isawa, Shin-ichiro Asano, Ngo Dinh Binh, Hisanori Bando
    ARCHIVES OF VIROLOGY 161 4 801 - 809 2016年04月 [査読有り][通常論文]
     
    We isolated and characterized a novel positive-sense, single-stranded RNA virus from Aedes larvae collected on Okushiri Island, Hokkaido, Japan. This virus, designated Okushiri virus (OKV), replicated in the Aedes albopictus cell line C6/36 with severe cytopathic effects and produced a large number of spherical viral particles that were 50-70 nm in diameter and released into the cell culture medium. The OKV genome consisted of 9,704 nucleotides, excluding the poly(A) tail at the 3'-terminus, and contained three major open reading frames (ORF1, ORF2, and ORF3). ORF1 encoded a putative protein of approximately 268 kDa that included a methyltransferase domain, FtsJ-like methyltransferase domain, helicase domain, and RNA-dependent RNA polymerase domain. The genome organization and results of a phylogenetic analysis based on the amino acid sequence predicted from the nucleotide sequence indicated that OKV is a member of a new insect virus group of negeviruses with a possible evolutionary relationship to some plant viruses. ORF2 and ORF3 were suggested to encode hypothetical membrane-associated proteins of approximately 45 kDa and 22 kDa, respectively. This is the first study on a novel negevirus isolated from mosquito larvae in Japan.
  • 宇多桃香, 佐藤昌直, 浅野眞一郎, 伴戸久徳
    東北蚕糸・昆虫利用研究報告 40 12 - 14 2015年12月20日 [査読無し][通常論文]
  • 石橋大樹, 佐藤昌直, 高ひとみ, 武内潤一, 浅野眞一郎, 伴戸久徳
    日本蚕糸学会大会・蚕糸・昆虫機能学術講演会講演要旨集 85th 58  2015年09月25日 [査読無し][通常論文]
  • Ryosuke Fujita, Chikako Ono, Isamu Ono, Shin-ichiro Asano, Hisanori Bando
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 464 4 1297 - 1301 2015年09月 [査読有り][通常論文]
     
    The Bombyx mori nucleopolyhedrovirus (BmNPV) ie-1 promoter exhibits strong transcriptional activity and is used in transient foreign gene expression systems in insect cells. In a reporter assay experiment using the BmNPV ie-1 promoter, we found that it exhibited activity even in non-host mammalian BHK cells, plant BY-2 cells, and also bacterial Escherichia coli cells. An analysis using a deletion series of the BmNPV ie-1 promoter demonstrated that the core promoter region of this promoter was sufficient to display promoter activity in BHK cells, BY-2 cells, and E. coli cells, whereas upstream elements were required for higher activity in insect cells. Furthermore, we found that the BmNPV ie-1 promoter exhibited sufficient activity for a beta-galactosidase assay in E. coil cells. The results obtained here suggest that the BmNPV ie-1 promoter has potential as a universal promoter for transient expression systems in insect, mammalian, plant, and bacterial cells. (C) 2015 Elsevier Inc. All rights reserved.
  • Kazuhiro Iiyama, Hiroaki Mon, Kazuki Mori, Takumi Mitsudome, Jae Man Lee, Takahiro Kusakabe, Kousuke Tashiro, Shin-ichiro Asano, Chisa Yasunaga-Aoki
    Meta Gene 4 29 - 44 2015年06月01日 [査読有り][通常論文]
     
    A scaffold obtained from whole-genome shotgun sequencing of Paenibacillus popilliae ATCC 14706< sup> T< /sup> shares partial homology with plasmids found in other strains of P. popilliae. PCR and sequencing for gap enclosure indicated that the scaffold originated from a 15,929-bp circular DNA. The restriction patterns of a plasmid isolated from P. popilliae ATCC 14706< sup> T< /sup> were identical to those expected from the sequence thus, this circular DNA was identified as a plasmid of ATCC 14706< sup> T< /sup> and designated pPOP15.9. The plasmid encodes 17 putative open reading frames. Orfs 1, 5, 7, 8, and 9 are homologous to Orfs 11, 12, 15, 16, and 17, respectively. Orf1 and Orf11 are annotated as replication initiation proteins. Orf8 and Orf16 are homologs of KfrA, a plasmid-stabilizing protein in Gram-negative bacteria. Recombinant Orf8 and Orf16 proteins were assessed for the properties of KfrA. Indeed, they formed multimers and bound to inverted repeat sequences in upstream regions of both orf8 and orf16. A phylogenetic tree based on amino acid sequences of Orf8, Orf16 and Kfr proteins did not correlate with species lineage.
  • Chikako Ono, Masanao Sato, Hitomi Taka, Shin-ichiro Asano, Yoshiharu Matsuura, Hisanori Bando
    PLOS ONE 10 3 e0119580  2015年03月 [査読有り][通常論文]
     
    To infect their hosts, DNA viruses must successfully initiate the expression of viral genes that control subsequent viral gene expression and manipulate the host environment. Viral genes that are immediately expressed upon infection play critical roles in the early infection process. In this study, we investigated the expression and regulation of five canonical regulatory immediate-early (IE) genes of Autographa californica multicapsid nucleopolyhedrovirus: ie0, ie1, ie2, me53, and pe38. A systematic transient gene-expression analysis revealed that these IE genes are generally transactivators, suggesting the existence of a highly interactive regulatory network. A genetic analysis using gene knockout viruses demonstrated that the expression of these IE genes was tolerant to the single deletions of activator IE genes in the early stage of infection. A network graph analysis on the regulatory relationships observed in the transient expression analysis suggested that the robustness of IE gene expression is due to the organization of the IE gene regulatory network and how each IE gene is activated. However, some regulatory relationships detected by the genetic analysis were contradictory to those observed in the transient expression analysis, especially for IE0-mediated regulation. Statistical modeling, combined with genetic analysis using knockout alleles for ie0 and ie1, showed that the repressor function of ie0 was due to the interaction between ie0 and ie1, not ie0 itself. Taken together, these systematic approaches provided insight into the topology and nature of the IE gene regulatory network.
  • 石橋大樹, 佐藤昌直, 高ひとみ, 武内潤一, 浅野眞一郎, 伴戸久徳
    東北蚕糸・昆虫利用研究報告 39 5 - 7 2014年12月10日 [査読無し][通常論文]
  • Kazuhiro Iiyama, Masahiro Otao, Kazuki Mori, Hiroaki Mon, Jae Man Lee, Takahiro Kusakabe, Kousuke Tashiro, Shin-Ichiro Asano, Chisa Yasunaga-Aoki
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 78 5 891 - 897 2014年05月 [査読有り][通常論文]
     
    To determine the phylogenetic relationship among Paenibacillus species, putative replication origin regions were compared. In the rsmG-gyrA region, gene arrangements in Paenibacillus species were identical to those of Bacillus species, with the exception of an open reading frame (orf14) positioned between gyrB and gyrA, which was observed only in Paenibacillus species. The orf14 product was homologous to the endospore-associated proteins YheC and YheD of Bacillus subtilis. Phylogenetic analysis based on the YheCD proteins suggested that Orf14 could be categorized into the YheC group. In the Paenibacillus genome, DnaA box clusters were found in rpmH-dnaA and dnaA-dnaN intergenic regions, known as box regions C and R, respectively; this localization was similar to that observed in B. halodurans. A phylogenetic tree based on the nucleotide sequences of the whole replication origin regions suggested that P. popilliae, P. thiaminolyticus, and P. dendritiformis are closely related species.
  • Hideki Takahashi, Kazuhiro Nakaho, Takeaki Ishihara, Sugihiro Ando, Takumi Wada, Yoshinori Kanayama, Shinichiro Asano, Shigenobu Yoshida, Seiya Tsushima, Mitsuro Hyakumachi
    PLANT CELL REPORTS 33 1 99 - 110 2014年01月 [査読有り][通常論文]
     
    Key message Activation of SA-dependent signaling pathway and suppression of JA-dependent signaling pathway seem to play key roles in B. thuringiensis -induced resistance to R. solanacearum in tomato plants. Bacillus thuringiensis, a well-known and effective bio-insecticide, has attracted considerable attention as a potential biological control agent for the suppression of plant diseases. Treatment of tomato roots with a filter-sterilized cell-free filtrate (CF) of B. thuringiensis systemically suppresses bacterial wilt caused by Ralstonia solanacearum through systemic activation of the plant defense system. Comparative analysis of the expression of the Pathogenesis-Related 1(P6) gene, a marker for induced resistance to pathogens, in various tissues of tomato plants treated with CF on their roots suggested that the B. thuringiensis-induced defense system was activated in the leaf, stem, and main root tissues, but not in the lateral root tissue. At the same time, the growth of R. solanacearum was significantly suppressed in the CF-treated main roots but not in the CF-treated lateral roots. This distinct activation of the defense reaction and suppression of R. solanacearum were reflected by the differences in the transcriptional profiles of the main and lateral tissues in response to the CF. In CF-treated main roots, but not CF-treated lateral roots, the expression of several salicylic acid (SA)-responsive defense-related genes was specifically induced, whereas jasmonic acid (JA)-related gene expression was either down-regulated or not induced in response to the CF. On the other hand, genes encoding ethylene (ET)-related proteins were induced equally in both the main and lateral root tissues. Taken together, the co-activation of SA-dependent signaling pathway with ET-dependent signaling pathway and suppression of JA-dependent signaling pathway may play key roles in B. thuringiensis-induced resistance to R. solanacearum in tomato.
  • 高ひとみ, 小野慎子, 佐藤昌直, 浅野眞一郎, 伴戸久徳
    東北蚕糸・昆虫利用研究報告 38 1 - 3 2013年12月30日 [査読無し][通常論文]
  • Masako Yokoo, Ryosuke Fujita, Yumiko Nakajima, Mamoru Yoshimizu, Hisae Kasai, Shin-ichiro Asano, Hisanori Bando
    Biochemical and Biophysical Research Communications 439 1 18 - 22 2013年09月13日 [査読有り][通常論文]
     
    Drosophila Mos1 belongs to the mariner family of transposons, which are one of the most ubiquitous transposons among eukaryotes. We first determined nuclear transportation of the Drosophila Mos1-EGFP fusion protein in fish cell lines because it is required for a function of transposons. We next constructed recombinant baculoviral vectors harboring the Drosophila Mos1 transposon or marker genes located between Mos1 inverted repeats. The infectivity of the recombinant virus to fish cells was assessed by monitoring the expression of a fluorescent protein encoded in the viral genome. We detected transgene expression in CHSE-214, HINAE, and EPC cells, but not in GF or RTG-2 cells. In the co-infection assay of the Mos1-expressing virus and reporter gene-expressing virus, we successfully transformed CHSE-214 and HINAE cells. These results suggest that the combination of a baculovirus and Mos1 transposable element may be a tool for transgenesis in fish cells. © 2013 Elsevier Inc.
  • Ishigaki Shun-ichiro, Bando Hisanori, Asano Shin-ichiro
    Journal of insect biotechnology and sericology 82 1 13 - 18 The Japanese Society of Sericultural Science 2013年06月28日 
    Cry39Aa from Bacillus thuringiensis serovar aizawai BUN1-14 is highly toxic to the larvae of Anopheles stephensi mosquitoes, which transmit malarial parasites. We constructed a homology model of Cry39Aa toxin using the reported structure of Cry4Ba toxin (PDB file 1W99) as a template, and we identified 349KYAYWR354 as the putative loop 1 in domain II. Many studies of various Cry toxins have shown that surface-exposed loops of domain II are critical for toxicity and receptor binding. To investigate the functional role of the putative loop 1 of Cry39Aa toxin, we performed site-directed mutagenesis in the loop. The results of alanine substitutions revealed that the entire structure of loop 1, and aromatic amino acids Y350 and Y352 in particular, are essential for larvicidal activity. In contrast, the toxicities of Cry39Aa mutants with phenylalanine substitutions at these two tyrosine residues did not differ markedly from the toxicity of wild-type Cry39Aa. A competitive binding assay involving A. stephensi brush border membrane vesicles revealed that the alanine mutants showed reduced competition with wild-type Cry39Aa toxin. Our results suggest that the molecular structure of loop 1 in domain II of Cry39Aa toxin is important for toxicity and receptor binding in the midgut of A. stephensi larvae.
  • TAKA Hitomi, ONO Chikako, SATO Masanao, ASANO Shin‐ichiro, BANDO Hisanori
    J Insect Biotechnol Sericology 82 1 25 - 32 2013年06月28日 [査読無し][通常論文]
     
    About two thirds of open reading frames (orfs) predicted on Bombyx mori nucleopolyhedrovirus (BmNPV) genome were dispensable for expression of the reporter gene derived from the polyhedrin promoter in BmN cells when knocked out one at a time (Ono et al., 2012). Effects of removal of multiple genes of BmNPV and genetic interactions of the viral genes have been poorly investigated. In this study, we constructed BmNPV lacking multiple non-essential genes at the orf11-12-13-14 gene cluster and analyzed the expression of EGFP under the control of the polyhedrin gene promoter. Viruses lacking more than two genes except for the one lacking orf13 and orf14 showed distinct phenotypes compared to the single gene knockout viruses. Synergistic, compensatory, and additive relationships were observed in the genetic interaction analyses between pairs of adjacent genes in the gene cluster. In addition, the virus lacking both orf12 and orf13 but carrying the orf12 genomic region at downstream of the polyhedrin locus expressed EGFP at higher levels than the control virus BmGFP (Ono et al., 2012) although the transcription of orf12 was not changed by insertion at the different locus. This increased EGFP expression was not observed with the virus in which the defect of orf12 and orf13 was rescued with orf13 at the same position. These observations suggested that inserting orf12 at the position specifically affected expression of the reporter gene at the polyhedrin locus.
  • Takuya Yamaguchi, Hisanori Bando, Shin-ichiro Asano
    JOURNAL OF INVERTEBRATE PATHOLOGY 113 2 123 - 128 2013年06月 [査読有り][通常論文]
     
    Cry8Da from Bacillus thuringiensis galleriae SDS-502 has insecticidal activity against both the larvae and adult Japanese beetle (Popillia japonica Newman). The receptor determines the specificity of the insecticidal activity of Cry proteins and hence, in order to reveal the mode of action of Cry toxin, receptor identification is a necessary step. However, a receptor for Cry8-type toxin has not been identified in the Scarabaeidae family of insects. Therefore, we aimed to identify the receptor of Cry8Da toxin in adult P. japonica BBMV. A ligand blot showed the Cry8Da toxin only bound to a 150 kDa protein in the BBMV of adult P. japonica. In order to identify the Cry8Da toxin binding protein, it was purified by column chromatography and three internal amino acid sequences were determined. Two of the three internal amino acid sequences shared homology with Coleopteran beta-glucosidases. In addition, the fraction containing the Cry8Da toxin binding protein had beta-glucosidase activity but no aminopeptidase N and alkaline phosphatase activity, both of which are commonly reported as receptors for Cry toxins in Lepidopteran and Dipteran insects. The beta-glucosidase homologous genes could be amplified by PCR using degenerate oligonucleotide primers designed from a conserved sequence of Coleopteran beta-glucosidases and an internal amino acid sequence of the Cry8Da toxin binding protein. Taken together, the beta-glucosidase in adult P. japonica BBMV is the receptor for B. thuringiensis Cry8Da toxin. (C) 2013 Elsevier Inc. All rights reserved.
  • Mitsuro Hyakumachi, Mitsuyoshi Nishimura, Tatsuyuki Arakawa, Shinichiro Asano, Shigenobu Yoshida, Seiya Tsushima, Hideki Takahashi
    Microbes and Environments 28 1 128 - 134 2013年 [査読有り][通常論文]
     
    Bacillus thuringiensis is a naturally abundant Gram-positive bacterium and a well-known, effective bio-insecticide. Recently, B. thuringiensis has attracted considerable attention as a potential biological control agent for the suppression of plant diseases. In this study, the bacterial wilt disease-suppressing activity of B. thuringiensis was examined in tomato plants. Treatment of tomato roots with B. thuringiensis culture followed by challenge inoculation with Ralstonia solanacearum suppressed the development of wilt symptoms to less than one third of the control. This disease suppression in tomato plants was reproduced by pretreating their roots with a cell-free filtrate (CF) that had been fractionated from B. thuringiensis culture by centrifugation and filtration. In tomato plants challenge-inoculated with R. solanacearum after pretreatment with CF, the growth of R. solanacearum in stem tissues clearly decreased, and expression of defense-related genes such as PR-1, acidic chitinase, and β-1,3-glucanase was induced in stem and leaf tissues. Furthermore, the stem tissues of tomato plants with their roots were pretreated with CF exhibited resistance against direct inoculation with R. solanacearum. Taken together, these results suggest that treatment of tomato roots with the CF of B. thuringiensis systemically suppresses bacterial wilt through systemic activation of the plant defense system.
  • 飯山和弘, 西大海, 門宏明, Jae Man Lee, 日下部宜宏, 浅野眞一郎, 青木智佐, 清水進
    Journal of Insect Biotechnology and Sericology 82 1 1 - 11 The Japanese Society of Sericultural Science 2013年 [査読有り][通常論文]
     
    Four housekeeping genes including gapA, groEL, gyrA and pgi were used in order to reveal a phylogenetic relationship among Paenibacillus species. Phylogenetic analysis and end-to-end pairwise analysis were carried out using nucleotide and amino acid sequences of the housekeeping genes. A monophylogenetic clade including P. popilliae, P. thiaminolyticus and P. dendritiformis formed in all phylogenetic trees. The results in both analyses indicated that the relationship between P. larvae subsp. larvae and P. popilliae was comparatively far. The result of the end-to-end pairwise analysis also showed that, among the Paenibacillus species used in this study, the closest species to P. popilliae was P. dendritiformis.
  • liyama Kazuhiro, Mori Kazuki, Mon Hiroaki, Chieda Yuuka, Lee Jae Man, Kusakabe Takahiro, Tashiro Kousuke, Asano Shin-Ichiro, Yasunaga-Aoki Chisa, Shimizu Susumu
    Journal of Insect Biotechnology and Sericology 82 2 45 - 48 2013年 [査読有り][通常論文]
  • Masataka Ohsawa, Miki Tanaka, Kenta Moriyama, Mitsuaki Shimazu, Shin-ichiro Asano, Kazuhisa Miyamoto, Kohsuke Haginoya, Toshiaki Mitsui, Tomoaki Kouya, Masayuki Taniguchi, Hidetaka Hori
    APPLIED AND ENVIRONMENTAL MICROBIOLOGY 78 13 4755 - 4757 2012年07月 [査読有り][通常論文]
     
    The Cry2Aa3 gene was introduced into asporogenic Bacillus thuringiensis, and the synthesized protoxin killed Bombyx mori and Lymantria dispar larvae. Chymotrypsin hydrolyzed the linkages between 49Tyr/Va150 and 145Lys/Ser146 in the protoxin, and 50- and 58-kDa fragments were generated, respectively. Both peptides killed the larvae of both insects.
  • Ryosuke Fujita, Daisuke Ohtsuka, Ken Sahara, Shinichiro Asano, Hisanori Bando
    ARCHIVES OF VIROLOGY 155 4 577 - 581 2010年04月 [査読有り][通常論文]
     
    The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is used as a safer viral vector in mammalian cells with potential applications in gene therapy. However, the mechanism for the insusceptibility of mammalian cells to proliferative infection by entomopathogenic viruses is not well understood. Here, we studied the significance of epigenetic modifications such as histone acetylation, histone methylation and HP1 accumulation for AcMNPV gene expression in mammalian BHK cells. Real-time PCR and chromatin immunoprecipitation with sodium butyrate revealed an important relationship between viral gene expression and histone acetylation, with implications for a mechanism of suppression of AcMNPV gene expression in BHK cells.
  • Supression of AcMNPV gene expression in mammalian cells
    Fujita R, Asano S, Sahara K, Bando H
    41th Annual meeting of the society for invertebrate pathology and 9th international conference on Bacillus thuringiensis 19 - 19 2008年08月 [査読有り][通常論文]
  • K. Kojima, K. Kojima, K. Oritani, T. Nakatsukasa, S. Asano, K. Sahara, H. Bando
    Virus Research 130 1 202 - 209 Elsevier 2007年12月01日 [査読無し][通常論文]
     
    A computer-assisted analysis identified tentative target sequences for regulatory proteins including ecdysone-inducible factors such as BmFTZ-F1 and Broad-Complex Z4 (BR-C Z4) in the ie1 promoter of BmNPV. A transient expression experiment using BmN cells and a series of truncated ie1 promoter constructs demonstrated that the activity of the ie1 promoter responded to alpha-ecdysone and 20-hydroxyecdysone, which required a tridecameric nucleotide stretch (ie1EcRE, 5′-GTGTTATCGACCT-3′) homologous to the ecdysone response element reported for Drosophila (DmEcRE). RT-PCR demonstrated the expression of BmEcR and BmUSP, which are required as ecdysone-specific activators for EcRE-mediated activation, in BmN cells. Furthermore, the ie1 EcRE-mediated response was confirmed by using a recombinant BmNPV possessing a luciferase gene under the control of the ie1 promoter with or without ie1 EcRE. This is the first report of an ecdysone response element in a baculoviral gene promoter. These results also suggested that the regulation of the ie1 by ecdysone may militate viral replication at least under certain conditions during natural infections in vivo. © 2007 Elsevier B.V. All rights reserved.
  • Restricted gene transcription of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) in mammalian cells
    Fujita R, Asano S, Sahara K, Bando H
    40th Annual meeting of the society for invertebrate pathology and 1st international forum on entomopathogenic nematodes and symbiotic bacteria 95 - 95 2007年08月 [査読有り][通常論文]
  • R Fujita, T Matsuyama, J Yamagishi, K Sahara, S Asano, H Bando
    JOURNAL OF VIROLOGY 80 5 2390 - 2395 2006年03月 [査読有り][通常論文]
     
    The gene expression of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) was examined in two types of mammalian cells, human HeLa14 and hamster BHK cells. DNA microarray analysis followed by reverse transcription-PCR identified at least 12 viral genes transcribed in both HeLa14 cells and BHK cells inoculated with AcMNPV. 5' rapid amplification of cDNA ends was carried out to examine the transcriptional fidelity of these genes in HeLa14 cells. The transcription of ie-1, ie-0 and gp64 was initiated at a baculovirus early gene motif, CAGT, accompanied by a TATA motif. In addition, the same splicing observed for ie-0 mRNA in Sf9 cells occurred in HeLa14 cells. While the transcription initiation sites for pe38 and p6.9 were not located in the CAGT motif, most of them were in a typical eukaryotic RNA polymerase II promoter structure (a conventional TATA motif and/or an initiator). Interestingly, the expression of beta-actin was upregulated in the mammalian cells inoculated with AcMNPV. Subsequent experiments using UV-inactivated virus confirmed the upregulation, suggesting that de novo synthesis of viral products is not required for the event. These results indicated that the AcMNPV genome acts as a template for transcription in mammalian cells through the usual infection pathway, though there is no evidence for the functional expression of viral genes at present.
  • Abe T, Miyake N, Nishijima Y, Fujita R, Sahara K, Asano S, Bando H
    Virus Research 112 1-2 38 - 41 2005年10月 [査読有り][通常論文]
  • 昆虫病原ウイルスAcNPVの哺乳動物細胞における遺伝子発現とその影響
    藤田龍介, 浅野眞一郎, 佐原健, 伴戸久徳
    東北蚕糸・昆虫利用研究報告 29 10 - 10 2004年12月 [査読有り][通常論文]
  • 小島 桂, 斎藤 寛, 山田 恭裕, 田村 俊樹, 磯部 良子, 浅野 眞一郎, 佐原 健, 伴戸 久徳
    北大農場研究報告 33, 9-13 33 9 - 13 北海道大学北方生物圏フィールド科学センター耕地圏ステーション生物生産研究農場 2003年03月 [査読無し][通常論文]
     
    Transgenic silkworms possessing artificial anti-viral genes were constructed using a transposon, piggyBac, system and subjected to test for their resistance to Bombyx mori nucleopolyhedrovirus (BmNPV). Silkworms inoculated with BmNPV at the 1st day of the 2nd instar larvae survived till 4th instar larvae, and all of them were found to be transgenic silkworms. Though apparent BmNPV resistance increased in the transgenic silkworms, further improvement of the resistance was required for practical use.
  • Y Yamada, T Matsuyama, GX Quan, T Kanda, T Tamura, K Sahara, S Asano, H Bando
    VIRUS RESEARCH 90 1-2 253 - 261 2002年12月 [査読無し][通常論文]
     
    An immediate-early gene product of baculovirus, IE1, is essential for viral gene expression and for viral DNA replication. It has been demonstrated for Autographa californica nuclear polyhedrosis virus (AcNPV) that the G terminal region of IE1 is required for dimerization. And the acidic N-terminal region of IE1 has been identified as the activation domain. We constructed an N-terminal 267 amino acid (a.a.) truncated mutant of Bombyx mori nuclear polyhedrosis virus (BmNPV) IE1, which was defective as a transactivator of a viral early gene (p35) promoter. We then examined possible IE1 antagonistic functions of this defective IE1, IE1TN, in BmNPV-infected cells. A transient expression experiment demonstrated that IE1TN strongly repressed the activation of the hr 5-dependent p35 promoter derived from BmNPV infection. In addition, Dpn I assay elucidated an inhibitory effect of IE1TN on the hr 5-dependent replication of plasmid in BmN cells induced by NPV infection. A marked reduction in the production of virus was observed when the BmN cells were infected with BmNPV after transfection with IE1TN-expression plasmids. These results suggested that IE1TN could act as an IE1 antagonist in silkworm cells infected with BmNPV. We then analyzed the ability of IE1TN to inhibit the multiplication of BmNPV using transgenic silkworms. The BmNPV-resistance of the transgenic silkworms was very weak, suggesting insufficient expression of the transgene product, IE1TN. (C) 2002 Elsevier Science B.V. All rights reserved.
  • 斎藤 寛, 菊池 邦夫, 山田 恭裕, 飯塚 敏彦, 浅野 眞一郎, 伴戸 久徳, 佐原 健
    北大農学部農場研究報告 32, 61-69. 32 61 - 69 北海道大学農学部附属農場 2001年03月 [査読無し][通常論文]
  • T Hayakawa, K Kojima, K Nonaka, M Nakagaki, K Sahara, S Asano, T Iizuka, H Bando
    VIRUS RESEARCH 66 1 101 - 108 2000年01月 [査読有り][通常論文]
     
    Bombyx mori densonucleosis virus type 2 (BmDNV-2) is a small, spherical virus containing two complementary single-stranded linear DNA molecules (VD1, VD2). BmDNV-2 is a new type of virus with a unique, yet unspecified replication mechanism which is different from that of parvoviruses (Bando, H., Choi, H., Ito, Y., Nakagaki, M., Kawase, S., 1992: Structural analysis on the single-stranded genomic DNAs of the virus newly isolated from silkworm: the DNA molecules share a common terminal sequence, Arch. Virol. 124, 187-193; Bando, H., Hayakawa, T., Asano, S., Sahara, K., Nakagaki, M., Iizuka, T., 1995. Analysis of the genetic information of a DNA segment of a new virus from silkworm, Arch. Virol., 140, 1147-1155; Hayakawa, T., Asano, S., Sahara, K., Iizuka, T., Bando, H., 1997. Detection of replicative intermediate with closed terminus of Bombyx densonucleosis virus. Arch. Virol. 142, 1-7). Recent analyses on the genomic information of BmDNV-2 identified open reading frames which code for three tentative nonstructural proteins and four (VP1 to 4) of the six known structural proteins (Bando, H., Hayakawa, T., Asano, S., Sahara, K., Nakagaki, M., Iizuka, T., 1995. Analysis of the genetic information of a DNA segment of a new virus from silkworm, Arch. Virol., 140, 1147-1155; Nakagaki et al., in preparation). In this report we demonstrate that the two largest ORFs, VD1-ORF1 and VD2-ORF1, code for the two remaining structural proteins. In addition, computer-assisted analysis revealed that the structural protein encoded in VD1-ORF1 contains sequences conserved among various DNA polymerases, and showed an evolutionary relationship with the DNA polymerases involved in protein-primed replication. (C) 2000 Elsevier Science B.V. All rights reserved.
  • H. Isawa, S. Asano, K. Sahara, T. Iizuka, H. Bando, H. Bando
    Archives of Virology 143, 127-143 127 - 143 1998年03月27日 [査読無し][通常論文]
     
    We synthesized the cDNAs of an insect picomavirus, infectious flacherie virus of silkworm (IFV), genomic RNA and inserted it into a bacterial plasmid (pUC119). The 9650 nucleotides (nts) sequence except for the poly(A) tail was obtained from the cloned cDNAs, and the sequence integrity was confirmed by primer extension and direct RNA sequencing. The sequence has a large open reading frame (ORF) of 9255 nts (3085 codons) flanked by the short 5' non-coding region (156 nts) and by the rather long 3' non-coding (239 nts). The structural proteins VP3, 4, 1 and 2 were located at the N-terminus of the polyprotein in this order and were preceded by a tentative small peptide. Computer analysis identified the sequences similar to the consensus sequences of 2C (helicase?), 3C (protease), and 3D (RNA polymerase) conserved among mammalian and plant picorna(-like) viruses. In addition, the predicted genome organization of IFV was quite similar to those of picornaviruses. Further analyses of the characteristics of the genome structure and a tentative phylogenetic tree constructed on the basis of the amino acid sequence similarity emphasized the evolutionary relationships among the insect and plant viruses.
  • J Sasaki, S Asano, N Hashimoto, BW Lay, S Hastowo, H Bando, T Iizuka
    CURRENT MICROBIOLOGY 35 1 1 - 8 1997年07月 [査読有り][通常論文]
     
    A cry2A-type gene, designated as cry2(SKW), was cloned from Bacillus thuringiensis serovar sotto SKW01-10.2-06, and some unique features of the gene were revealed. The cry2(SKW) gene encoded a polypeptide of 635 residues with a predicted molecular mass of 71,137 Da. Cry2(KW) had 95.4% identity with Cry2Aa in amino acid sequence and was two residues longer than Cry2Aa. Two open reading frames (ORFs), designated as orf1 and orf2, were present upstream of the cry2(SKW) and showed high homology with the corresponding ORFs in the cry2Aa operon. The Orf2 from SKW01-10.2-06 contained a region of repeated sequences. However, unlike Cry2Aa, Cry2(SKW) formed the cuboidal crystalline inclusions when the cry2(SKW) gene was expressed in an acrystalliferous B. thuringiensis strain in the absence of the upstream ORFs. Furthermore, Cry2(SKW) was less toxic to a lepidopteran species, Bombyx mori, than Cry2Aa in spite of high homology between the two proteins.
  • S Asano, Y Nukumizu, H Bando, T Iizuka, T Yamamoto
    APPLIED AND ENVIRONMENTAL MICROBIOLOGY 63 3 1054 - 1057 1997年03月 [査読有り][通常論文]
     
    A novel enterotoxin gene was cloned from Bacillus cereus FM1, and its nucleotide sequence was determined, Previously, a 45-kDa protein causing characteristic enterotoxin symptoms in higher animals had been isolated (K. Shinagawa, p. 181-193, in A. E. Pohland et al., ed., Microbial Toxins in Foods and Feeds, 1990) from the same B. cereus strain, but no report of cloning of the enterotoxin gene has been published, In the present study, a specific antibody to the purified enterotoxin was produced and used to screen the genomic library of B. cereus FM1 made with the lambda gt11 vector, An immunologically positive clone was found to contain the full protein-coding region and some 5' and 3' flanking regions, The deduced amino acid sequence of the cloned gene indicated that the protein is rich in beta structures and contains some unusual sequences, such as consecutive Asn residues, In order to clone enterotoxin genes from Bacillus thuringiensis, two PCR primers were synthesized based on the nucleotide sequence of the B. cereus gene. These primers were designed to amplify the full protein-coding region, PCR conducted with DNA preparations from the B. thuringiensis subsp. sotto and B. thuringiensis subsp, israelensis strains successfully amplified a segment of DNA with a size almost identical to that of the protein-coding region of the B. cereus enterotoxin, Nucleotide sequences of the amplified DNA segments showed that these B. thuringiensis strains contain an enterotoxin gene very similar to that of B. cereus. Further PCR screening of additional B. thuringiensis strains with four primer pairs in one reaction revealed that some additional B. thuringiensis strains contain enterotoxin-like genes.
  • T Hayakawa, S Asano, K Sahara, T Iizuka, H Bando
    ARCHIVES OF VIROLOGY 142 2 393 - 399 1997年 [査読有り][通常論文]
     
    Two kinds of Bombyx densonucleosis virus (BmDNV), BmDNV-1 and 2, have been isolated from sericultural farms in Japan or China. These viruses are classified into the family Parvoviridae because of the small spherical virus particle containing a single-stranded linear DNA genome. Recent studies on the genome structure of these viruses suggested that BmDNV-2 was a new type of virus with unique replication mechanism, though that of BmDNV-1 was similar to parvoviruses. However, details about the replication mechanism of BmDNVs have not been reported so far. Here, in order to elucidate the difference on replication mechanism between BmDNVs and parvoviruses, we analyzed the structure of the replicative intermediate (RI) of BmDNV DNAs by PCR using specific primers designed for detection of RI with closed terminal structure (RI-CT) which is expected to be formed by replication with self-priming mechanism. PCR using the DNA from the cells infected with BmDNV-1 could detect the expected DNA fragment, showing the existence of RI-CT. On the other hand, no fragment could be amplified from the virion DNA of the BmDNVs and the DNA extracted from BmDNV-2-infected cells, respectively. These observations strongly suggested that the BmDNV-1 replicates with the ''self-priming and hairpin-transfer'' mechanism similar to the human parvoviruses, while BmDNV-2 does not.
  • J Sasaki, S Asano, T Iizuka, H Bando, BW Lay, S Hastowo, GK Powell, T Yamamoto
    CURRENT MICROBIOLOGY 32 4 195 - 200 1996年04月 [査読有り][通常論文]
     
    A new host specificity was discovered with the insecticidal protein encoded by the cryV gene. The cryV gene was cloned from the Bacillus thuringiensis kurstaki INA-02 strain, which was selected among a number of B. thuringiensis isolates because of its high activity against Spodoptera litura. Analyses by polymerase chain reaction (PCR) revealed that INA-02 contained the cryIA(a) and clyV genes. Since no Spodoptera activity was observed with B. thuringiensis sotto, which contained only cryIA(a), insecticidal activity of the protein encoded by the cryV gene was investigated with several insect species including S. litura. For bioassay, the cryV gene was highly expressed in an acrystalliferous B. thuringiensis strain, BT51. The CryV protein from BT51 was assayed against larvae of three lepidopteran species, Bombyx mori, S. litura, and Plutella xylostella. The protein was highly active against S. litura and P. xylostella, suggestive that the protein contributes to the unique activity of INA-02.
  • H. Bando, T. Hayakawa, S. Asano, K. Sahara, M. Nakagaki, T. Iizuka
    Archives of Virology 140, 1147-1155 6 1147 - 1155 1995年06月01日 [査読無し][通常論文]
     
    In 1983, a parvo-like virus (Yamanashi isolate) was newly isolated from silkworm. However, unlike parvovirus, two DNA molecules (VD1 and 2) were always extracted from purified virions. To investigate the structure and organization of the virus genomes, we determined the complete nucleotide sequence of VD2. The sequence consisted of 6031 nucleotides (nts) and contained a large open reading frame (ORF1) with 3513 nts. A smaller open reading frame (ORF2) with 702 nts was found in the complementary sequence. Computer analysis revealed that both ORFs did not code for the major structural proteins (VP1, 2, 3, and 4). These results suggest that VD2 has not enough information to produce progeny virions by itself. Further, the structural importance of the terminal sequence (CTS) common to both VD1 and VD2 was also predicted by a computer analysis. © 1995 Springer-Verlag.
  • 佐々木 潤, 浅野 真一郎, 伴戸 久徳
    日本蚕糸学雑誌 63 5 p361 - 366 日本蠶絲學會 1994年10月 [査読有り][通常論文]
  • Ikuhiro Nakatani, Shinichiro Asano, Hisanori Bando, Toshihiko Iizuka
    Journal of Sericultural Science of Japan 63 2 140 - 148 1994年 [査読有り][通常論文]
     
    Nucleotide sequence of cryIA (b) gene from B. thuringiensis subsp. wuhanensis (cry IA(b)-W) was determined and compared with other two cryIA (b) genes, one from subsp. kurstaki RD-1(cryIA(b)-H) and other from subsp. thuringiensis berliner 1715 (cryIA (b)-T). The deduced 564 amino acid sequence of cryIA (b) -w was extremely similar to that of cryIA(b)-T, with an exception of amino acid number 207. The entire coding sequence of cryIA(b)-W and crylA(b)-H were cloned separately into pKK233 — 2 and insecticidal proteins (ICP) were expressed in Escherichia coli JM109. After sonication of E, coli cells, solubilized ICPs were directly injected to the mouth of 5th-instar larvae of the silkworm, Bombyx mori. The bioassay results indicated that the ICPs had no toxicity against the silkworm. Iizuka et al. (1992) reported that subsp. wuhanensis contained additional two cryl genes, cryIA (c) and cryID. It should be of interest to determine the nucleotide sequence of these two genes and the insecticidal activity of the gene products. © 1994, The Japanese Society of Sericultural Science. All rights reserved.
  • Toshihiko Iizuka, Seiichiro Ishikawa, Shinichiro Asano, Hisanori Bando, Zhenwei Zheng, Norimoto Murai
    Journal of Sericultural Science of Japan 63 4 303 - 309 1994年 [査読有り][通常論文]
     
    The cryIA (a) gene of Bacillus thuringiensis subsp. sotto encodes delta-endotoxin or insecticidal protein consisting of 1, 180 amino acids. The active toxin was reported to be confined to amino acid residue 29 to 618. To test this a truncated cryIA (a) gene corresponding to the active toxin was isolated using site-directed mutagenesis and expressed in Escherichia coli. The truncated cryIA (a) gene product exhibited insecticidal activity against the silkworm with LD50 at 0.25 to 0.35 μg total E. coli protein per larva. Similarly, a truncated cryIB gene corresponding to the reported active toxin region was isolated using polymerase chain reaction from B. thuringiensis subsp. thuringiensis HD-2. When expressed in E. coli, the truncated cryIB gene product gave toxicity against cabbage butterfly with LD50 at < 0.1μg total E. coli protein per larva. These results demonstrated that the truncated cryIA (a) and IB genes can be used to generate transgenic plants with insecticidal activity. © 1994, The Japanese Society of Sericultural Science. All rights reserved.

書籍

  • バイオロジカル・コントロール-害虫管理と天敵の生物学-
    朝倉書店 2009年

その他活動・業績

  • 中西登志紀, 関口真理, 浅野眞一郎, 伴戸久徳, 佐藤昌直 日本蚕糸学会大会・蚕糸・昆虫機能学術講演会講演要旨集 90th 2020年
  • 橋本健太郎, 佐藤昌直, 伴戸久徳, 浅野眞一郎 東北蚕糸・昆虫利用研究報告 (45) 2020年
  • 関口真理, 佐藤昌直, 浅野眞一郎, 伴戸久徳 日本蚕糸学会大会・蚕糸・昆虫機能学術講演会講演要旨集 90th 2020年
  • 関口真理, 佐藤昌直, 浅野眞一郎, 伴戸久徳 日本蚕糸学会大会・蚕糸・昆虫機能学術講演会講演要旨集 89th 2019年
  • 中西登志紀, 佐藤昌直, 浅野眞一郎, 伴戸久徳 日本蚕糸学会大会・蚕糸・昆虫機能学術講演会講演要旨集 89th 2019年
  • 浅野眞一郎, 小野山雄亮, 中神あゆみ, 中尾悠太, 佐藤昌直, 伴戸久徳 日本蚕糸学会大会・蚕糸・昆虫機能学術講演会講演要旨集 88th 2018年
  • 高ひとみ, 佐藤昌直, 浅野眞一郎, 伴戸久徳 日本蚕糸学会大会・蚕糸・昆虫機能学術講演会講演要旨集 87th 2017年
  • 浅野眞一郎, 中神あゆみ, 中尾悠太, 佐藤昌直, 伴戸久徳 日本蚕糸学会大会・蚕糸・昆虫機能学術講演会講演要旨集 87th 2017年
  • 小池 正徳, 高橋 尚樹, 笹原 勇太, 斉佳 鶴怜, 石井 嶺広, 相内 大吾, 浅野 眞一郎 昆虫と自然 51 (13) 39 -41 2016年12月 [査読無し][通常論文]
  • 小池 正徳, 斉 佳鶴玲, 相内 大吾, 石井 嶺広, 浅野 眞一郎 昆虫と自然 51 (7) 45 -48 2016年06月
  • 高ひとみ, 佐藤昌直, 浅野眞一郎, 伴戸久徳 日本分子生物学会年会プログラム・要旨集(Web) 39th 2016年
  • 高ひとみ, 佐藤昌直, 浅野眞一郎, 伴戸久徳 日本蚕糸学会大会・蚕糸・昆虫機能学術講演会講演要旨集 85th 2015年
  • 宇多桃香, 佐藤昌直, 浅野眞一郎, 伴戸久徳 日本蚕糸学会大会・蚕糸・昆虫機能学術講演会講演要旨集 85th 2015年
  • 小野慎子, 高ひとみ, 佐藤昌直, 浅野眞一郎, 伴戸久徳 日本蚕糸学会大会・蚕糸・昆虫機能学術講演会講演要旨集 84th 70 2014年03月09日 [査読無し][通常論文]
  • 高野恵美子, 高橋こう, 浅野眞一郎, 伴戸久徳 日本蚕糸学会大会・蚕糸・昆虫機能学術講演会講演要旨集 84th 70 2014年03月09日 [査読無し][通常論文]
  • 高ひとみ, 小野慎子, 佐藤昌直, 浅野眞一郎, 伴戸久徳 日本蚕糸学会大会・蚕糸・昆虫機能学術講演会講演要旨集 84th 2014年
  • 高ひとみ, 小野慎子, 佐藤昌直, 浅野眞一郎, 伴戸久徳 日本分子生物学会年会プログラム・要旨集(Web) 37th 2014年
  • 半谷大輝, 伴戸久徳, 浅野眞一郎 日本蚕糸学会大会・蚕糸・昆虫機能学術講演会講演要旨集 83rd 64 2013年03月18日 [査読無し][通常論文]
  • 石垣俊一郎, 伴戸久徳, 浅野眞一郎 日本応用動物昆虫学会大会講演要旨 57th 190 2013年03月15日 [査読無し][通常論文]
  • 小林大介, 伊澤晴彦, 鍬田龍星, 星野啓太, BINH Ngo Dinh, 浅野眞一郎, 伴戸久徳, 糸山享, 沢辺京子 日本応用動物昆虫学会大会講演要旨 57th 5 2013年03月15日 [査読無し][通常論文]
  • 高橋瑛, 伴戸久徳, 浅野眞一郎 日本応用動物昆虫学会大会講演要旨 57th 190 2013年03月15日 [査読無し][通常論文]
  • Chikako Ono, Takanori Kamagata, Hitomi Taka, Ken Sahara, Shin-ichiro Asano, Hisanori Bando VIRUS RESEARCH 165 (2) 197 -206 2012年05月 [査読無し][通常論文]
     
    We constructed a series of gene knockout BmNPVs (KOVs) for each of 141 genes (Gomi et al., 1999; Katsuma et al., 2011)using the BmNPVT3 bacmid system (Ono et al., 2007) and lambda red recombination system (Datsenko and Wanner, 2000). In a subsequent analysis of the properties needed for infection using a marker gene, egfp (enhanced green fluorescent protein gene), inserted into the polyhedrin locus, the knockout viruses (KOVs) were subdivided into four phenotypic types, A to D. Type-A (86 KOVs) showed the ability to expand infections equivalent to the control while type-B (8 KOVs) spread infections more slowly. Type-C (37 KOVs) expressed egfp in transfected-BmN cells but the production of infectious viruses was not observed. Type-D (10 KOVs) showed no ability to express egfp even in the transfection experiments. KOVs lacking genes (pkip (Bm15), gp41 (Bm66), bro-d (Bm131), Bm20, 48, 65, 91, 93, or 101) previously identified as being essential, were placed in the viable type-A and B categories. (C) 2012 Elsevier B.V. All rights reserved.
  • 川上広太, WAHYU Kurnia Yudistira, 伊澤晴彦, BINH Ngo Dinh, 浅野眞一郎, 伴戸久徳 東北蚕糸・昆虫利用研究報告 (37) 2012年
  • 上森翔太, 浅野眞一郎, 佐原健, 伴戸久徳 日本蚕糸学会大会・蚕糸・昆虫機能学術講演会講演要旨集 81st 72 2011年03月20日 [査読無し][通常論文]
  • 川上広太, 伊澤晴彦, 伊藤利章, 浅野眞一郎, BINH Ngo Dinh, 佐原健, 伴戸久徳 東北蚕糸・昆虫利用研究報告 (35/36) 2011年
  • Maria Sugiharti, Chikako Ono, Shin-Ichiro Asano, Ken Sahara, Hisanori Bando, Toshiaki Ito, Yulia Pujiastuti Journal of Biotechnology and Sericology 79 (3) 117 -124 2011年 [査読無し][通常論文]
     
    In 2005, natural epizootics were observed during an outbreak of Setothosea asigna (Lepidoptera: Limacodidae) larvae in an oil palm plantation in South Sumatra, Indonesia. The causative agent was flterable, which implied it was a virus. Since preliminary testing using reverse PCR gave a positive result for the Thosea asigna virus (TaV), we experimented with the purifcation of the viral agent from the diseased larvae. Electron microscopy revealed nonenveloped virus-like particles that were spherical in shape and about 40 nm in diameter. cDNA cloning followed by sequencing demonstrated that RNA purifed from the particles contained two large open-reading-frames (ORFs) with a partly shared sequence and extensive homology (> 98% identity at the nucleotide level) with ORFs of TaV encoding an RNA-dependent RNA polymerase and the capsid protein, respectively. 3´ RACE suggested that there is, like in TaV genomic RNA, no poly(A) tract at the 3´-terminus of the RNA. The pathogenicity of the purifed particles against Limacodidae larvae in Japan was demonstrated to be very strong for Monema favescens and Austrapoda dentata. These results indicated that the agent causing the epizootic disease among S. asigna larvae in the oil palm plantations was TaV which also has potential as a biological control agent for Limacodidae pests in Japan. © 2011, The Japanese Society of Sericultural Science. All rights reserved.
  • 浅野 眞一郎 Journal of pesticide science 36 (1) 91 -93 2011年 [査読無し][通常論文]
  • 浅野眞一郎 日本農薬学会誌 36 (1) 91 -93 2011年 [査読無し][通常論文]
  • Takuya Yamaguchi, Ken Sahara, Hisanori Bando, Shin-ichiro Asano JOURNAL OF INVERTEBRATE PATHOLOGY 105 (3) 243 -247 2010年11月 [査読無し][通常論文]
     
    Bacillus thuringiensis (Bt) Cry8D insecticidal proteins are unique among Cry8 family proteins in terms of its insecticidal activity against adult Scarab beetles, such as Japanese beetle (Popillia japonica Newman). From the sequence homology with other Bt Cry proteins especially those active against beetles, such as Cry3Aa whose 3D structure is available, the structure of the Cry8D protein has been predicted to be a typical three-domain Cry protein type. In addition, the activation process of Cry8D in gut juice of susceptible insects is presumed to be similar to that of Cry3A (Yamaguchi et al., 2008). In this study, the activation process of Cry8Da in insect gut juice was closely examined. Japanese beetle gut juice proteases digested the 130 kDa Cry8Da protein to produce a 64 kDa protein. This 64 kDa protein was active against both adult and larval Japanese beetle and considered to be an activated toxin. N-terminal sequencing of this 64 kDa protein revealed that the Cry8Da leader sequence consisting of 63 amino acid residues from M(1) to F(63) was removed. As in the case of Cry3Aa, the proteases further digested the 64 kDa protein to two 8 kDa and 54 kDa fragments. N-terminal amino acid analysis of these smaller fragments indicated that the proteases digested the loop between Alpha Helix (Alpha for short) 3 and Alpha 4. This means that the 8 kDa fragment consists of Alpha 1-3 of Domain land that the 54 kDa fragment contains the remaining Domain I and full Domain II and Domain III. Size exclusion chromatography and anion exchange chromatography could not separate these 64, 54 and 8 kDa proteins suggesting that the 54 kDa and 8 kDa fragments are still forming the toxin complex equivalent to the 64 kDa protein by size and ionic charge. The sequencing and chromatography results suggest that the gut juice proteases merely nicked the loop between Alpha 3 and Alpha 4. This nicking process appeared to be essential for receptor binding of the Cry8Da toxin. BBMV binding assay revealed that the Cry8Da toxin bound to BBMV preparations from both adult and larval Japanese beetle only after the loop was nicked. Only the 54 kDa fragment bound to the BBMV preparations but not the 64 kDa protein. Ligand blot showed that the protease activated Cry8Da toxin, presumably the 54 kDa fragment, bound to specific BBMV proteins, one or more of those would be receptor(s). The sizes and binding affinities of these Cry8Da-bound proteins of Japanese beetle BBMV differed between larvae and adults. (C) 2010 Elsevier Inc. All rights reserved.
  • 浅野 眞一郎 蚕糸・昆虫バイオテック 77 (3) 181 -186 2009年 [査読無し][通常論文]
  • 浅野 眞一郎 蚕糸・昆虫バイオテック = Sanshi-konchu biotec 77 (3) 181 -186 2008年12月01日 [査読無し][通常論文]
     
    Bacillus thuringiensis(Bt)は1901年にわが国の石渡繁胤によって、カイコに卒倒病を引き起こす病原細菌として、世界に先駆けて報告された。しかしながら、論文が日本語で書かれていたこと、新種の微生物として記載を行わなかったことから、注目を集めなかった。1911年にはBerlinerがドイツのThuringiaの地で、ノシメマダラメイガの斃死幼虫から分離した昆虫病原細菌を、発見地にちなみBacillus thuringiensisをいう学名を与えた。日本国内とりわけ蚕糸昆虫研究者の間では、B. thuringiensisがわが国の石渡によって発見されたsotto菌であることは知られていたが、世界においてこの菌の発見者が石渡であると認識されるようになったのは、その後約半世紀近く経ってからだった。Bt研究については、石渡が本菌の発見者であることからも分かるように、これまでの最先端の研究がわが国の蚕糸昆虫研究者によってなされてきた。ここでは、蚕糸昆虫研究におけるB. thuringiensis研究の歴史と今後の研究について、本特集でとりあげた最近の研究を含め紹介したい。
  • Takuya Yamaguchi, Ken Sahara, Hisanori Bando, Shin-ichiro Asano JOURNAL OF INVERTEBRATE PATHOLOGY 99 (3) 257 -262 2008年11月 [査読無し][通常論文]
     
    A novel cry gene, cry8Db, highly toxic to scarab beetles such as the Japanese beetle, Popillia japonica Newman, was cloned from an isolate of Bacillus thuringiensis (Bt), BBT2-5. The cry8Db gene has 3525 bp nucleotides and codes for a protein of 1174 amino acid residues. The protein, Cry8Db, has typical Bt characteristics such as the 8-block, conserved sequences and the three-domain 3 D toxin structure as defined with Cry3Aa. When the amino acid sequence of Cry8Db was compared with that of Cry8Da whose gene was cloned and characterized in our laboratory earlier, substantial sequence diversities were found in their Domain III. The cry8Db gene was expressed in an acrystalliferous B. thuringiensis strain, BT51. BT51 expressing cry8Db formed a spherical crystal like the natural crystal of BBT2-5. The Cry8Db protein was assayed along with the other scarab active Cry8Da and Cry8Ca against the Japanese beetle. While Cry8Da and Cry8Db had toxicity against both adults and larvae of the Japanese beetle, Cry8Ca was toxic to only larvae. Cry8Ca showed no toxicity against the adult beetle Lip to 30 mu g per 1 cm(2) of leaf discs on which the protein was applied. The activation process of Cry8Db by adult and larval gut juice was compared in vitro with the processes of Cry8Da and Cry8Ca. All three proteins, Cry8Db, Cry8Da and Cry8Ca, produced a toxic core of approximately 70 kDa equally indicating that the activation process does not inactivate the adult activity of Cry8Ca. We concluded that the adult activity of Cry8D proteins is encoded in Domain II. Further tests against other beetle species showed a significant difference between Cry8D's and Cry8Ca but no difference between Cry8Da and Cry8Db. Comparison of 3D structural models of Cry8Ca, Cry8Da and Cry8Db, which were constructed by using Cry3Bb as the structural template, indicated significant structural differences, especially between Cry8Ca and Cry8D proteins, in three major surface-exposed loops of Domain 11 that may be involved in determining the adult beetle activity. (C) 2008 Elsevier Inc. All rights reserved.
  • OHTSUKA Daisuke, NAKATSUKASA Tomonori, FUJITA Ryosuke, ASANO Shin-ichiro, SAHARA Ken, BANDO Hisanori Journal of insect biotechnology and sericology 77 (3) 125 -131 2008年10月31日 [査読無し][通常論文]
  • 微生物の事典 Ⅲ
    朝倉書店 2008年 [査読無し][通常論文]
  • ONO Chikako, NAKATSUKASA Tomonori, NISHIJIMA Yasuyuki, ASANO Shin-ichiro, SAHARA Ken, BANDO Hisanori Journal of insect biotechnology and sericology 76 (3) 161 -167 2007年10月30日 [査読無し][通常論文]
     
    The bacmid system first established for Autographa californica nucleopolyhedrovirus (AcNPV) is a powerful tool for generating recombinant viral DNA in E. coli, which allows one to produce knockout viruses (even viruses missing essential genes) independent of viral replication in host cells. We constructed a bacmid system for the type strain (T3) of Bombyx mori NPV (BT3Bac) and analyzed the functional necessity of a delayed-early gene he65 using the polyhedrin-gene-rescued BmNPV/T3 bacmid (BT3Bac ). An apparent difference in the viral replication profile judging from numbers of polyhedra-forming cells and amounts of viral DNA in the culture medium was not observed between BmNPV/T3 and BT3Bac . On the other hand, the accumulation of viral DNA was delayed by about 12 hrs in he65-knockout BT3Bac (BT3Bac -he65KO) compared to those in BT3Bac or BmNPV/T3, indicating that he65 is not essential for BmNPV replication but critical for an optimal replication in BmN cells. The BmNPV/T3 bacmid system constructed here is useful for the functional analysis of BmNPV genes. +polh +polh +polh +polh
  • Chikako Ono, Tomonori Nakatsukasa, Yasuyuki Nishijima, Shin Ichiro Asano, Ken Sahara, Hisanori Bando Journal of Insect Biotechnology and Sericology 76 (3) 161 -167 2007年 [査読無し][通常論文]
     
    The bacmid system first established for Autographa californica nucleopolyhedrovirus (AcNPV) is a powerful tool for generating recombinant viral DNA in E. coli, which allows one to produce knockout viruses (even viruses missing essential genes) independent of viral replication in host cells. We constructed a bacmid system for the type strain (T3) of Bombyx mori NPV (BT3Bac) and analyzed the functional necessity of a delayed-early gene he65 using the polyhedrin-gene-rescued BmNPV/T3 bacmid (BT3Bac ). An apparent difference in the viral replication profile judging from numbers of polyhedra-forming cells and amounts of viral DNA in the culture medium was not observed between BmNPV/T3 and BT3Bac . On the other hand, the accumulation of viral DNA was delayed by about 12 hrs in he65-knockout BT3Bac (BT3Bac -he65KO) compared to those in BT3Bac or BmNPV/T3, indicating that he65 is not essential for BmNPV replication but critical for an optimal replication in BmN cells. The BmNPV/T3 bacmid system constructed here is useful for the functional analysis of BmNPV genes. +polh +polh +polh +polh
  • Takeshi Ito, Hisanori Bando, Shin-ichiro Asano JOURNAL OF INVERTEBRATE PATHOLOGY 93 (1) 29 -35 2006年09月 [査読無し][通常論文]
     
    Most delta-endotoxins produced by Bacillus thuringiensis require proteolytic processing in order to become active. The in vitro and in vivo activation processes of Cry39A, a delta-endotoxin that is highly toxic to Anopheles stephensi, were investigated. Cry39A with a molecular mass of 72 kDa was processed in vitro into a 60 kDa fragment by trypsin and gut extract from A. stephensi larvae. N-terminal amino acid sequencing of the 60 kDa fragment revealed that trypsin and the protease(s) in the gut extract cleaved Cry39A between Arg(61) and Gly(62). In contrast, 40 and 25 kDa polypeptides were generated in vivo by intramolecular cleavage of the 60 kDa fragment in A. stephensi larvae. Further, a co-precipitation assay was used to investigate the binding property of the activated Cry39A to A. stephensi BBMV. Cry39A bound to A. stephensi BBMV specifically and did not compete with the Cry4Aa toxin. This indicated that the binding molecule(s) for Cry39A might differ from those for Cry4A. In addition, Cry39A preferentially bound to the Triton X-100-insoluble membrane fraction. (c) 2006 Elsevier Inc. All rights reserved.
  • Takeshi Ito, Tomonori Ikeya, Ken Sahara, Hisanori Bando, Shin-ichiro Asano APPLIED AND ENVIRONMENTAL MICROBIOLOGY 72 (8) 5673 -5676 2006年08月 [査読無し][通常論文]
     
    Two novel crystal protein genes, cry30Ba and cry44Aa, were cloned from Bacillus thuringiensis subsp. entomocidus INA288 and expressed in an acrystalliferous strain. Cry44Aa crystals were highly toxic to second-instar Culex pipiens pallens (50% mortality concentration [LC50] = 6 ng/ml) and Aedes aegypti (LC50 = 12 ng/ml); however, Cry30Ba crystals were not toxic.
  • Fujita Ryosuke, Matsuyama Takahiro, Yamagishi Junya, Sahara Ken, Asano Shinichiro, Bando Hisanori Journal of Virology 80 (5) 2390 -2395 2006年03月 [査読無し][通常論文]
     
    The gene expression of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) was examined in two types of mammalian cells, human HeLa14 and hamster BHK cells. DNA microarray analysis followed by RT-PCR identified at least 12 viral genes transcribed in both HeLa14 cells and BHK cells inoculated with AcMNPV. 5'A RACE was carried out to examine the transcriptional fidelity of these genes in HeLa14 cells. The transcription of ie-1, ie-0 and gp64 was initiated at a baculovirus early gene motif, CAGT, accompanied by a TATA motif. In addition, the same splicing observed for ie-0 mRNA in Sf...
  • Ihara Jun-ichiro, Yoshido Atsuo, Asano Shin-ichiro, Bando Hisanori, Sahara Ken Chromosome science 9 (1) 12 -12 2006年 [査読無し][通常論文]
  • FUJITA Ryousuke, ASANO Shinichiro, SAHARA Ken, BANDO Hisanori Journal of insect biotechnology and sericology 74 (3) 125 -128 2005年10月28日 [査読無し][通常論文]
  • T Abe, N Miyake, Y Nishijima, R Fujita, K Sahara, S Asano, H Bando VIRUS RESEARCH 112 (1-2) 38 -41 2005年09月 [査読無し][通常論文]
     
    We happened to discover that the cauliflower mosaic virus (CaMV) 35S promoter inserted into a recombinant Autographa californica multicapsid nucleopolyhedrovirus (rAcMNPV) was strongly activated during the replication of the recombinant virus in Spodoptera frugiperda (Sf9) cells. The expression of the luciferase gene from the 35S promoter in rAcMNPV was remarkably increased late in infection and was resistant to alpha-amanitin treatment. Primer extension indicated that transcriptional initiation from the 35S promoter in Sf9 cells occurred within one of the two baculoviral late promoter TAAG motifs located in the vicinity of the transcription start site in plant cells. These observations suggested that the CaMV 35S promoter served as a transcription start site for AcMNPV-induced RNA polymerase. (c) 2005 Elsevier B.V. All rights reserved.
  • 中務智紀, 小島桂, 田村俊樹, 佐原健, 浅野真一郎, 伴戸久徳 日本蚕糸学会大会・蚕糸・昆虫機能学術講演会講演要旨集 75th 2005年
  • 浅野 眞一郎, 伊藤 岳 化学と生物 42 (8) 501 -503 2004年08月25日 [査読無し][通常論文]
  • H Hisano, Y Kimoto, H Hayakawa, J Takeichi, T Domae, R Hashimoto, J Abe, S Asano, A Kanazawa, Y Shimamoto PLANT CELL REPORTS 22 (12) 910 -918 2004年07月 [査読無し][通常論文]
     
    H. Hisano, Y. Kimoto, H. Hayakawa, J. Takeichi, T. Domae, R. Hashimoto, J. Abe, S. Asano, A. Kanazawa, and Y. Shimamoto :"High frequency Agrobacterium-mediated transformation and plant regeneration via direct shoot formation from leaf explants in Beta vulgaris and B maritima", Plant Cell Reports, 22、910-918*
  • S Asano, C Yamashita, T Iizuka, K Takeuchi, S Yamanaka, D Cerf, T Yamamoto BIOLOGICAL CONTROL 28 (2) 191 -196 2003年10月 [査読無し][通常論文]
     
    Shin-ichiro ASANO, Chikage YAMASHITA, Toshihiko IIZUKA, Katsuyoshi TAKEUCHI, Satoshi YAMANAKA,
    David CERF, and Takashi YAMAMOTO (2003): A strain of Bacillus thuringiensis subsp. galleriae containing a novel cry8 gene highly toxic to Anomala cuprea (Coleoptera: Scarabaeidae), Biological Control, 28: 191-196*
  • Matsuyama Takahiro, Asano Shinichiro, Sahara Ken, BANDO Hisanori Journal of insect biotechnology and sericology 72 (2) 87 -94 2003年06月30日
  • MATSUYAMA Takahiro, ASANO Shinichiro, SAHARA Ken, BANDO Hisanori Journal of insect biotechnology and sericology 72 (2) 87 -94 2003年06月30日 [査読無し][通常論文]
  • Sahara, K., Yoshido, A., Kawamura, N., Ohnuma, A., Abe, H., Mita, K., Oshiki, T., Shimada, T., Asano, S.-I., Bando, H., Yasukochi, Y. Chromosoma 112 (1) 48 -55 2003年 [査読無し][通常論文]
  • ITO Takeshi, SAHARA Ken, BANDO Hisanori, ASANO Shinichiro Journal of insect biotechnology and sericology 71 (3) 123 -128 2002年10月31日 [査読無し][通常論文]
  • ISOBE Ryoko, KOJIMA Katsura, SAHARA Ken, ASANO Shin-ichiro, BANDO Hisanori Journal of insect biotechnology and sericology 71 (1) 43 -47 2002年02月28日 [査読無し][通常論文]
     
    Antisense and double-stranded RNA (dsRNA) interference (RNAi) was investigated in a silkworm ovarian cell line, BmN, using the firefly luciferase as a reporter. The expression of the luciferase from the reporter plasmids was suppressed in the transfected cells when the amount of antisense RNA was in excess of the mRNA amount. The size of the antisense RNA was also found to be an important factor in the interfering efficiency. On the other hand, a small amount of dsRNA induced an efficient RNAi. The sequence-dependency of the dsRNA-derived RNAi was verified in transient expression experiments using the Renillia luciferase, emphasizing the usefulness of dsRNA as a template for gene silencing in the biotechnology of the silkworm.
  • Isobe, R., Kojima, K., Sahara, K., Asano, S. and Bando, H.: "Antisense and double-strand RNA interference in a Silkworm Ovarian Cell Line", J. Insect Biotechnology Sericology, 71:43-47(2002)*
    2002年 [査読無し][通常論文]
  • 2002年 [査読無し][通常論文]
     
    Ito, T., Sahara, K., Bando, H. and Asano, S.: "Cloning and expression of novel crystal protein genes cry39A and 39orf2 from Bacillus thuringiensis subsp. aizawai Bun1-14 encoding mosquitocidal proteins", J. Insect Biotechnology Sericology, 71: 123-128 (2002)*
  • 齋藤 寛, 山田 恭裕, 浅野 眞一郎, 伴戸 久徳, 佐原 健 北海道大学農学部農場研究報告 32 (0) 71 -74 2001年03月29日 [査読無し][通常論文]
  • 山田 恭裕, 齋藤 寛, 浅野 眞一郎, 伴戸 久徳, 佐原 健 北海道大学農学部農場研究報告 32 (0) 75 -79 2001年03月29日 [査読無し][通常論文]
  • 小島桂, 浅野真一郎, 佐原健, 伴戸久徳 東北蚕糸・昆虫利用研究報告 (26) 2001年
  • Kojima K, Hayakawa T, Asano S, Bando H Archives of Virology 146 (7) 1407 -1414 2001年 [査読無し][通常論文]
  • 佐原 健, 斎藤 寛, 菊池 邦夫, 山田 恭裕, 浅野 眞一郎, 伴戸 久徳 日本応用動物昆虫学会誌 45 (2) 94 -98 2001年 [査読無し][通常論文]
     
    Dried-leaf powder of six host plants, Quercus acuta, Q.dentata, Q.acutissima, Q.serrata, Q.mongolica var.grosseserrata and Moulus pumila were evaluated as components of an artificial diet for rearing the wild silkworm Antheraea yamamai. Small-scale rearing of 5 larvae and large-scale rearing of 20 larvae were conducted to compare the viability, growth speed and cocoon quality. With respects to the cocoon qualities, Q.m.var.grosseserrata and M.pumila were preferable components of the artificial diet. For rearing of A.yamamai as an experimental animal, leaf powder of Q.m.var.grosseserrata was...
  • 小島 桂, 平野 文, 浅野 真一郎, 佐原 健, 伴戸 久徳 日本蠶絲學雜誌 70 (2) 103 -108 2001年 [査読無し][通常論文]
     
    2種類の単鎖DNAをゲノムにもつBmDNV-2は、他のパルボウイルスとは異なるゲノムDNAの複製機構を有していると考えられる。本ウイルスの2種類のゲノムは両末端に53塩基からなる共通末端配列CTSをもち、この配列がウイルス複製に重要な機能を持つと考えられていたが、ゲルシフトアッセイの結果、5'-CTSおよび3'-CTSにはウイルス感染時に特異的に結合するタンパク質が存在し、3'-CTSに結合するタンパク質の少なくとも1つはウイルスの構成タンパク質であることが推定された。また、本ウイルスが感染したカイコの中腸には感染特異的DNAポリメラーゼ活性が検出されたが、この活性とウイルス構成タンパク質であるDNAポリメラーゼ様タンパク質(P128)との関連性は、現時点では明らかでない。
  • Ken Sahara, Hiroshi Saito, Kunio Kikuchi, Yasuhiro Yamada, Shin-Ichiro Asano, Hisanori Bando 日本応用動物昆虫学会誌 45 (2) 94 -98 2001年 [査読無し][通常論文]
     
    Dried-leaf powder of six host plants, Quercus acuta, Q. dentata, Q. acutissima, Q. serrata, Q. mongolica var. grosseserrata and Moulus pumila were evaluated as components of an artificial diet for rearing the wild silkworm Antheraea yamamai. Small-scale rearing of 5 larvae and large-scale rearing of 20 larvae were conducted to compare the viability, growth speed and cocoon quality. With respects to the cocoon qualities, Q. m. var. grosseserrata and M. pumila were preferable components of the artificial diet. For rearing of A. yamamai as an experimental animal, leaf powder of Q. m. var. grosseserrata was a suitable component because it resulted in high survival rate, earlier maturation and decrease of abnormal pupae.
  • 小島桂, 浅野真一郎, 佐原健, 伴戸久徳 日本分子生物学会年会プログラム・講演要旨集 23rd 2000年
  • 山岸 潤也, 浅野 真一郎, 佐原 健, 飯塚 敏彦, 伴戸 久徳 日本蠶絲學雜誌 69 (4) 271 -276 2000年 [査読無し][通常論文]
     
    Alternative splicing observed in the mRNAs for the structural proteins of PfDNV seems to be necessary to generate five structural proteins from two separated ORFs. Involvement of several splicing donor sites and acceptor sites in the alternative splicing complicates understanding of the splicing mechanism of the PfDNV. First we developed a method for detection of spliced RNA molecules using the combination of the different two techniques, RT-PCR and primer extension. This splicing-detection method demonstrated that the sites of alternative splicing occurred within the PfDNV RNA was also recognized in S2 cells (a Drosophila cell line). Interestingly, a drastic suppression of the splicing and the accumulation of the unspliced RNA molecules were observed in S2 cells transfected with the PfDNV non-structural proteins (γ and β)-expression plasmids. These results suggested that the cellular factors play an important role on the selection of the specific splicing sites and that the viral nonstruc ural proteins may regulate it in a suppressive manner. © 2000, The Japanese Society of Sericultural Science. All rights reserved.
  • Hayakawa,T., Kojima, K., Nonaka, K., Nakagaki, M., Sahara, K., Asano, S., Iizuka, T. and Bando, H. : Analysis of protein encoded in the bipartite genome of a new type of parvo-like virus isolated from silkworm -structural protein with DNA polymerase mo・・・
    2000年 [査読無し][通常論文]
     
    Hayakawa,T., Kojima, K., Nonaka, K., Nakagaki, M., Sahara, K., Asano, S., Iizuka, T. and Bando, H. :
    Analysis of protein encoded in the bipartite genome of a new type of parvo-like virus isolated from silkworm -structural protein with DNA polymerase motif. Virus Research 66, 101-108 (2000)
  • 山岸 潤也, 浅野 真一郎, 佐原 健, 飯塚 敏彦, 伴戸 久徳 日蚕雑 69 (4) 271 -276 2000年 [査読無し][通常論文]
     
    Alternative splicing observed in the mRNAs for the structural proteins of PfDNV seems to be necessary to generate five structural proteins from two separated ORFs. Involvement of several splicing donor sites and acceptor sites in the alternative splicing complicates understanding of the splicing mechanism of the PfDNV. First we developed a method for detection of spliced RNA molecules using the combination of the different two techniques, RT-PCR and primer extension. This splicing-detection method demonstrated that the sites of alternative splicing occurred within the PfDNV RNA was also recognized in S2 cells (a Drosophila cell line). Interestingly, a drastic suppression of the splicing and the accumulation of the unspliced RNA molecules were observed in S2 cells transfected with the PfDNV non-structural proteins (γ and β)-expression plasmids. These results suggested that the cellular factors play an important role on the selection of the specific splicing sites and that the viral nonstruc ural proteins may regulate it in a suppressive manner. © 2000, The Japanese Society of Sericultural Science. All rights reserved.
  • 小島桂, 浅野真一郎, 佐原健, 飯塚敏彦, 伴戸久徳 東北蚕糸研究報告 (24) 1999年
  • PUJIASTUTI YULIA, 浅野 真一郎, 佐原 健, 伴戸 久徳, 飯塚 敏彦 日本蠶絲學雜誌 68 (3) 195 -199 1999年 [査読無し][通常論文]
     
    B. thuringiensis subsp.wuhanensis contains three types of cryl genes: crylAb, cry 1 Ac and cry ID. Specific cry1Db primers were designed to clone the cry1Dbw gene. The DNA of the cry1Dbw gene sequence exactly matched the cry1Db gene sequence proposed by LAMBERT (1993). We isolated three types of crystal proteins, Cry1Ab, Cry1Dbw and wuhanensis from this strain. The CrylDbw and wuhanensis proteins showed highly toxicities against the silkworm larva, Bombyx mori and the common cutworm, Spodoptera litura. On the other hand, the CrylAb protein was not toxic to B. mori and showed low toxicity to S. litura. We reveal that their toxicities are caused by the cry1Dbw gene, and propose that this gene is very important for controlling the pest insects. © 1999, The Japanese Society of Sericultural Science. All rights reserved.
  • Yulia Pujiastuti, Shin-ichiro Asano, Ken Sahara, Hisanori Bando and Toshihiko Iizuka : "Toxicity of Bacillus thuringiensis subsp. wuhanensis crystal protein to Bombyx mori and Spodoptera litura", J. Seric. Sci. Jpn 68(3), 195-200, (1999)
    1999年 [査読無し][通常論文]
  • N Matsuki, S Asano, H Bando, T Iizuka APPLIED ENTOMOLOGY AND ZOOLOGY 33 (1) 205 -205 1998年02月 [査読無し][通常論文]
  • A Rizali, S Asano, K Sahara, H Bando, BW Lay, S Hastowo, T Iizuka APPLIED ENTOMOLOGY AND ZOOLOGY 33 (1) 111 -114 1998年02月 [査読無し][通常論文]
     
    Bacillus thuringiensis isolates have been recovered from numerous sources, including soil, grain dust, plant leaves, diseased insect larvae from insectaries, and sericulture environments. During a study of B. thuringiensis isolated from mulberry leaves from Indonesia, we found two serovar aizawai isolates. One of the serovar aizawai isolates (Bun 1-14), which was a crystal consisting mainly of 69 kDa peptides, exhibited mosquitocidal activity, while another isolate (Bun 2-1) did not. Both isolates were analyzed by PCR. Although these isolates produced proteinaceous crystals, no cry genes, known as cryI, cryII, cryIII and cryIV, were detected. It appears these strains contain novel cry genes that are responsible for the unique insecticidal activity.
  • 小島桂, 浅野真一郎, 佐原健, 飯塚敏彦, 伴戸久徳 東北蚕糸研究報告 (23) 1998年
  • 浅野 真一郎, プジャスツティ ユリア, 佐原 健, 伴戸 久徳, 菊田 治典, 飯塚 敏彦 日本蠶絲學雜誌 67 (3) 237 -242 1998年 [査読無し][通常論文]
     
    Bacillus thuringiensis reference strains and isolates used in this experiment were stored in our laboratory and originally isolated from soil and dead insects in Hokkaido. In order to find B. thuringiensis strains which have a high toxic activity against Spodoptera litura larvae, these B. thuringiensis strains were bioassayed to the 3rd-instar larvae of S. litura. The identification of cry1 genes from active strains against S. litura larvae were demonstrated by using PCR. Cry1C cloned from serovar entomocidus and serovar kenyae revealed a toxic activity against S. litura larvae. On the contrary, crylE gene cloned from serovar tolworthi and serovar darmstadiensis did not have a toxic activity. On the other hand, even though serovar galleriae Acp10-8 and wuhanensis have a highly toxic activity against S. litura larvae, both of strains did not involve crylC and crylE genes. It seems that they have the novel cry genes. © 1998, The Japanese Society of Sericultural Science. All rights reserved.
  • S. Asano,Y. Pujiastuti, K. Sahara, H. Bando and T. Iizuka:Identification of cry1 genes from Bacillus thuringiensis strains whichhave activity against Spodoptera litura. J. Seric. Sci.Jpn, 67(3),237-242
    1998年 [査読無し][通常論文]
  • N Matsuki, S Asano, H Bando, T Iizuka APPLIED ENTOMOLOGY AND ZOOLOGY 32 (4) 583 -588 1997年11月 [査読無し][通常論文]
     
    Three new pathogenic bacteria were isolated from milky diseased larvae of Popillia japonica NEWMANN, Anomala rufocuprea MOTSCHULSKY and Anomala daimiana HAROLD (Coleoptera: Scarabaeidae) reared in the laboratory after field collection from golf courses in Sapporo in 1993. The morphological features regarding bacterial spores, infectivity host range and plasmid DNA profiles in these isolates showed different types of strains of Bacillus popilliae. Milky diseased larvae of P. japonica and other beetles have not yet been reported in Japan. This report is therefore the first of its kind in Japan.
  • N Matsuki, S Asano, H Bando, T Iizuka APPLIED ENTOMOLOGY AND ZOOLOGY 32 (4) 583 -588 1997年11月 [査読無し][通常論文]
     
    Matsuki, N., Asano, S. -i., Bando, H. and Iizuka, T. : "New Japanese Isolates of Bacillus popilliae Isolated from Milky Diseased Larvae of Popillia japonica, Anomala rufocuprea and Anomala daimiana (Coleoptera : Scarabaeidae)", Appl. Entomol. Zool., 32(4) : 583-588 (1997)*
  • 菊田 治典, 黒岩 学, 浅野 真一郎, 飯塚 敏彦 酪農学園大学紀要. 自然科学編 22 (1) 81 -97 1997年10月 [査読無し][通常論文]
  • 佐原 健, 福谷 晶子, 斉藤 寛, 中田 徹, 浅野 真一郎, 伴戸 久徳, 川村 直子, 飯塚 敏彦 日本蠶絲學雜誌 66 (2) 141 -144 1997年04月28日 [査読無し][通常論文]
  • 佐原 健, 田中 陽子, 山田 恭裕, 斉藤 寛, 中田 徹, 浅野 真一郎, 伴戸 久徳, 川村 直子, 飯塚 敏彦 日本蠶絲學雜誌 66 (3) 207 -211 1997年 [査読無し][通常論文]
     
    Tetraploid male silkworms induced by low temperature treatment are sterile in nature. The previous study showed that the starve-shock application at 60h of the 5th larval instar was effective to induce fertile tetraploid males. In order to determine the most effective length of starvation period, the larvae at 60h of the 5th instar were starved for various length of time (lh to 72h). Prolonged starve-shock application did not always cause high mortality. The increase of fertility was shown in the individuals starved more than 12h. A right-side-up parabolic curve with three peaks at the 15h, 30h and 51h was obtained. The fertility had no relation with both the length of the 5th larval instars and that of pupae. When we consider totally the advantages regarding to high fertility as well as high survival rate, the starve-shock application either for 27h to 30h or for 48h to 54h at 60h of the 5th instar of the tetraploid larvae was preferable method for producing fertile tetraploid males. © 1997, The Japanese Society of Sericultural Science. All rights reserved.
  • 佐原 健, 山田 恭裕, 斉藤 寛, 中田 徹, 浅野 真一郎, 伴戸 久徳, 川村 直子, 飯塚 敏彦 日本蠶絲學雜誌 66 (5) 341 -345 1997年 [査読無し][通常論文]
     
    The autotetraploid male silkworms carrying the sch genes were subjected to the starve-shock in order to increase their fertility. Survival rate of the F1 tetraploids between the cross of tetraploid females and males thus acquired was examined. After 4th ecdysis, mortality of the ZZZW females was quite higher than those of the ZZZZ males or ZZWW females. In the day of prespinning stage and just before the day of pupation, the significant increase in the dead individuals of the ZZZW females was observed. As the genetic background of F1 tetraploids was considered to be similar except four sex chromosomes, Z or W, the sex chromosome constitution may play an important role in the survival of F1 tetraploids. The comparison of the egg size among the F1 tetraploid females and the diploid showed the following order, ZW=ZZZW < ZZWW.On the other hand, the increase in the polygonal pattern size depended on the number of the Z chromosomes, ZW< ZZWW < ZZZW. These results confirmed that the Esd on the W and the Pgd on the Z are independent genes as to the expression of the egg characters. 1) Faculty of Agriculture, Hokkaido University, N9, W9, Kita-ku, Sapporo, 060, Japan. 2) Biological. © 1997, The Japanese Society of Sericultural Science. All rights reserved.
  • 佐原 健, MOHANA RAO RAMA P, 山田 恭裕, 斉藤 寛, 浅野 真一郎, 伴戸 久徳, 飯塚 敏彦, 中田 徹 日本蠶絲學雜誌 66 (5) 364 -366 1997年 [査読無し][通常論文]
  • 景安 聖士, 早川 徹, 伊澤 晴彦, 浅野 真一郎, 佐原 健, 飯塚 敏彦, 伴戸 久徳 日本蚕糸学雑誌 66 (6) 477 -483 1997年 [査読無し][通常論文]
     
    カイコ病原性ウイルスとしては, 核多角体病ウイルス (BmNPV), 濃核病1型ウイルス (BmDNV-1), 濃核病2型ウイルス (BmDNV-2), 細胞質多角体病ウイルス (BmCPV), 伝染性軟化病ウイルス (IFV) が知られている。本研究ではこれら5種類のウイルスゲノムを簡素化したPCR法により高感度で安定して検出するための条件の検討を行った。その結果, それぞれのウイルスに対する特異的プライマーを混合させた反応液中でPCRを行うことで全てのウイルスゲノムが検出可能となった。しかし, 検出感度を高く維持し, かつその特異性を保つためには, ウイルスゲノムRNA検出のためのRT-PCRを独立させることと, nested PCRを行うことが必要であった。ここでは, それぞれのウイルス検出用のプライマーの塩基配列およびPCRの反応条件について報告する。
  • Iizuka, T., Yamaya, K., Rizali, A., Asano, S. -i., Bando, H., Hastowo, S. and Lay, B. W. : "Isolation and identification of a new mosquitocidal isolate, Bacillus thuringiensis subsp. entomocidus INA288", (T. Attathom et al. : Bacillus thuringiensis Bio・・・
    1997年 [査読無し][通常論文]
     
    Iizuka, T., Yamaya, K., Rizali, A., Asano, S. -i., Bando, H., Hastowo, S. and Lay, B. W. : "Isolation and identification of a new mosquitocidal isolate, Bacillus thuringiensis subsp. entomocidus INA288", (T. Attathom et al. : Bacillus thuringiensis Biotechnology and Environmental Benefits, 2 : 152-160 (1997)*
  • 佐原 健, 田中 陽子, 山田 恭裕, 斉藤 寛, 中田 徹, 浅野 真一郎, 伴戸 久徳, 川村 直子, 飯塚 敏彦 『日蚕雑』 66 (3) 207 -211 1997年 [査読無し][通常論文]
     
    Tetraploid male silkworms induced by low temperature treatment are sterile in nature. The previous study showed that the starve-shock application at 60h of the 5th larval instar was effective to induce fertile tetraploid males. In order to determine the most effective length of starvation period, the larvae at 60h of the 5th instar were starved for various length of time (lh to 72h). Prolonged starve-shock application did not always cause high mortality. The increase of fertility was shown in the individuals starved more than 12h. A right-side-up parabolic curve with three peaks at the 15h, 30h and 51h was obtained. The fertility had no relation with both the length of the 5th larval instars and that of pupae. When we consider totally the advantages regarding to high fertility as well as high survival rate, the starve-shock application either for 27h to 30h or for 48h to 54h at 60h of the 5th instar of the tetraploid larvae was preferable method for producing fertile tetraploid males. © 1997, The Japanese Society of Sericultural Science. All rights reserved.
  • 佐原 健, 福谷 晶子, 斉藤 寛, 中田 徹, 浅野 真一郎, 伴戸 久徳, 川村 直子, 飯塚 敏彦 『日蚕雑』 66 (2) 141 -144 1997年 [査読無し][通常論文]
  • 景安 聖士, 早川 徹, 伊澤 晴彦, 浅野 真一郎, 佐原 健, 飯塚 敏彦, 伴戸 久徳 日本蚕糸学雑誌 66 (6) 477 -483 1997年 [査読無し][通常論文]
     
    カイコ病原性ウイルスとしては, 核多角体病ウイルス (BmNPV), 濃核病1型ウイルス (BmDNV-1), 濃核病2型ウイルス (BmDNV-2), 細胞質多角体病ウイルス (BmCPV), 伝染性軟化病ウイルス (IFV) が知られている。本研究ではこれら5種類のウイルスゲノムを簡素化したPCR法により高感度で安定して検出するための条件の検討を行った。その結果, それぞれのウイルスに対する特異的プライマーを混合させた反応液中でPCRを行うことで全てのウイルスゲノムが検出可能となった。しかし, 検出感度を高く維持し, かつその特異性を保つためには, ウイルスゲノムRNA検出のためのRT-PCRを独立させることと, nested PCRを行うことが必要であった。ここでは, それぞれのウイルス検出用のプライマーの塩基配列およびPCRの反応条件について報告する。
  • Hayakawa, T., Asano, S. -i., Sahara, K., Iizuka, T. and Bando H. : "Detection of replicative intermediate with closed terminus of Bombyx densonucleosis virus", Arch. Virol., 142 : 1-7 (1997)*
    1997年 [査読無し][通常論文]
  • 佐原 健, 山田 恭裕, 斉藤 寛, 中田 徹, 浅野 真一郎, 伴戸 久徳, 川村 直子, 飯塚 敏彦 『日蚕雑』 66 (5) 341 -345 1997年 [査読無し][通常論文]
     
    The autotetraploid male silkworms carrying the sch genes were subjected to the starve-shock in order to increase their fertility. Survival rate of the F1 tetraploids between the cross of tetraploid females and males thus acquired was examined. After 4th ecdysis, mortality of the ZZZW females was quite higher than those of the ZZZZ males or ZZWW females. In the day of prespinning stage and just before the day of pupation, the significant increase in the dead individuals of the ZZZW females was observed. As the genetic background of F1 tetraploids was considered to be similar except four sex chromosomes, Z or W, the sex chromosome constitution may play an important role in the survival of F1 tetraploids. The comparison of the egg size among the F1 tetraploid females and the diploid showed the following order, ZW=ZZZW < ZZWW.On the other hand, the increase in the polygonal pattern size depended on the number of the Z chromosomes, ZW< ZZWW < ZZZW. These results confirmed that the Esd on the W and the Pgd on the Z are independent genes as to the expression of the egg characters. 1) Faculty of Agriculture, Hokkaido University, N9, W9, Kita-ku, Sapporo, 060, Japan. 2) Biological. © 1997, The Japanese Society of Sericultural Science. All rights reserved.
  • Asano, S. -i., Nukumizu, Y., Bando, H., Iizuka, T. and Yamamoto, T. : "Cloning of novel enterotoxin genes from Bacillus cereus and Bacillus thuringiensis", Appl. Environ. Microbiol., 63(3) : 1054-1057 (1997)*
    1997年 [査読無し][通常論文]
  • Asano, S. -i., Okumura, E., Kamagata, Y., Sahara, K., Bando, H., Iizuka, T. and Yamamoto, T. : "Mutagenesis of loop3 region of the Bacillus thuringiensis Cry1Ac delta-endotoxin affect DBM's receptor binding", (T. Attathom et al. : Bacillus thuringiensi・・・
    1997年 [査読無し][通常論文]
     
    Asano, S. -i., Okumura, E., Kamagata, Y., Sahara, K., Bando, H., Iizuka, T. and Yamamoto, T. : "Mutagenesis of loop3 region of the Bacillus thuringiensis Cry1Ac delta-endotoxin affect DBM's receptor binding", (T. Attathom et al. : Bacillus thuringiensis Biotechnology and Environmental Benefits, 2 : 49-60 (1997)*
  • Sasaki, J., Asano, S. -i., Hashimoto, N., Lay, B. W., Hastowo, S., Bando, H. and Iizuka, T. : "Characterization of a cry2A gene cloned from an isolate of Bacillus thuringiensis serovar sotto", Curr. Microbiol., 35 : 1-8 (1997)*
    1997年 [査読無し][通常論文]
  • 佐原 健, 山田 恭裕, 斉藤 寛, 浅野 真一郎, 伴戸 久徳, 飯塚 敏彦, 中田 徹 『日蚕雑』 66 (5) 364 -366 1997年 [査読無し][通常論文]
  • Jun Sasaki, Shinichiro Asano, Toshihiko Iizuka, Hisanori Bando, Bibiana W. Lay, Sugyo Hastowo, Gary K. Powell, Takashi Yamamoto Current Microbiology 32 (4) 195 -200 1996年04月 [査読無し][通常論文]
     
    A new host specificity was discovered with the insecticidal protein encoded by the cryV gene. The cryV gene was cloned from the Bacillus thuringiensis kurstaki INA-02 strain, which was selected among a number of B. thuringiensis isolates because of its high activity against Spodoptera litura. Analyses by polymerase chain reaction (PCR) revealed that INA-02 contained the cryIA(a) and cryV genes. Since no Spodoptera activity was observed with B. thuringiensis sotto, which contained only cryIA(a), insecticidal activity of the protein encoded by the cryV gene was investigated with several insect species including S. litura. For bioassay, the cryV gene was highly expressed in an acrystalliferous B. thuringiensis strain, BT51. The CryV protein from BT51 was assayed against larvae of three lepidopteran species, Bombyx mori, S. litura, and Plutella xylostella. The protein was highly active against S. litura and P. xylostella, suggestive that the protein contributes to the unique activity of INA-02.
  • 飯塚 敏彦, 山谷 公美子, Rizali Akhmad, 浅野 真一郎, 伴戸 久徳, Hastowo Sugyo, Lay Bibiana W 日本農藝化學會誌 70 (0) 1996年03月05日 [査読無し][通常論文]
  • 浅野 眞一郎 北海道大学農学部邦文紀要 19 (7) 529 -563 1996年 [査読無し][通常論文]
  • 佐々木 潤, 浅野 真一郎, 伴戸 久徳, LAY BIBIANA W, HASTOWO SUGYO, 飯塚 敏彦 日本蠶絲學雜誌 65 (1) 56 -61 1996年 [査読無し][通常論文]
     
    Polymerase chain reaction (PCR) technique and subsequent agarose gel electrophoresis of restriction enzyme-digested PCR products enabled us to identify the cry V and cry V 465 genes included in Bacillus thuringiensis isolates. At first, PCR using the set of primers which amplified both cry V and cry V 465 genes indicated the presence of at least one of the two cry V-type genes in B. thuringiensis isolates. Next, agarose gel electrophoresis of EcoRI-digested PCR products revealed which of cry V and cry V 465 genes the B. thuringiensis isolates contained. Then, B. thuringiensis isolates of various serovars were examined and the isolates of serovars kurstaki, sotto and aizawai were found to contain the cry V gene. This method was available for searching novel type of cry V gene, because many B. thuringiensis isolates can be examined rapidly and the variants of cry V gene can be detected based on the electrophoretic patterns of the restriction enzyme-digested PCR products. © 1996, The Japanese Society of Sericultural Science. All rights reserved.
  • 橋本 直樹, 佐々木 潤, 浅野 真一郎, 伴戸 久徳, 飯塚 敏彦 日本蠶絲學雜誌 65 (3) 185 -191 1996年 [査読無し][通常論文]
     
    A novel expression vector (pHY/IAaP) for Bacillus thuringiensis insecticidal crystal protein (ICP) genes was constructed by inserting the crylA(a) gene promoter region into a shuttle vector pHY300PLK, and transduced into Bt51, an acrystalliferous strain. In polyacrylamide gel electrophoresis in the presence of detergent, each of the Bt51 transformants, except for the one with cryllA gene, expressed a peptide of the similar size to that produced in the native strain. Furthermore, electron microscopic observations revealed the similarity in the form of ICP between the transformants and the native strains. Since pHY/IAaP seems to have a single ICP gene expressed in Bt51 under the control of crylA(a) gene promoter, the vector is considered to be useful in the studies of the characteristics of the products of an introduced ICP gene. © 1996, The Japanese Society of Sericultural Science. All rights reserved.
  • 橋本 直樹, 佐々木 潤, 浅野 真一郎, 伴戸 久徳, 飯塚 敏彦 『日蚕雑』 65 (3) 185 -191 1996年 [査読無し][通常論文]
     
    A novel expression vector (pHY/IAaP) for Bacillus thuringiensis insecticidal crystal protein (ICP) genes was constructed by inserting the crylA(a) gene promoter region into a shuttle vector pHY300PLK, and transduced into Bt51, an acrystalliferous strain. In polyacrylamide gel electrophoresis in the presence of detergent, each of the Bt51 transformants, except for the one with cryllA gene, expressed a peptide of the similar size to that produced in the native strain. Furthermore, electron microscopic observations revealed the similarity in the form of ICP between the transformants and the native strains. Since pHY/IAaP seems to have a single ICP gene expressed in Bt51 under the control of crylA(a) gene promoter, the vector is considered to be useful in the studies of the characteristics of the products of an introduced ICP gene. © 1996, The Japanese Society of Sericultural Science. All rights reserved.
  • 佐々木 潤, 浅野 真一郎, 伴戸 久徳, 飯塚 敏彦 『日蚕雑』 65 (1) 56 -61 1996年 [査読無し][通常論文]
     
    Polymerase chain reaction (PCR) technique and subsequent agarose gel electrophoresis of restriction enzyme-digested PCR products enabled us to identify the cry V and cry V 465 genes included in Bacillus thuringiensis isolates. At first, PCR using the set of primers which amplified both cry V and cry V 465 genes indicated the presence of at least one of the two cry V-type genes in B. thuringiensis isolates. Next, agarose gel electrophoresis of EcoRI-digested PCR products revealed which of cry V and cry V 465 genes the B. thuringiensis isolates contained. Then, B. thuringiensis isolates of various serovars were examined and the isolates of serovars kurstaki, sotto and aizawai were found to contain the cry V gene. This method was available for searching novel type of cry V gene, because many B. thuringiensis isolates can be examined rapidly and the variants of cry V gene can be detected based on the electrophoretic patterns of the restriction enzyme-digested PCR products. © 1996, The Japanese Society of Sericultural Science. All rights reserved.
  • 「PCR法による Bacillus thuringiensis cry 遺伝子同定法の確立と新規 B. thuringiensis 菌株の発見」
    『北大農邦文紀要』 19 (7) 529 -563 1996年 [査読無し][通常論文]
  • ASANO S, HORI H Appl. Entomol. Zool. 30 (2) 369 -374 1995年 [査読無し][通常論文]
     
    The synergistic effects of supernatants from cultures of nine strains of Bacillus thuringiensis toward the insecticidal activity of δ-endotoxin were investigated using neonates of the common cutworm, Spodoptera litura, and late 3rd instar larvae of the diamondback moth, Plutella xylostella by the diet incorporation method. Synergistic effects between supernatants and δ-endotoxins were detected against S. litura but not toward P. xylostella. Synergistic activity in supernatants against S. litura but not toward P. xylostella. Synergistic activity in supernatants against S. litura was observed in four strains and the occurrence was shown to be dependent on culture media. Interestingly, B. thuringiensis serovar japonensis strain Buibui and B. thuringinensis serovar morrisoni strain san diego, which produce insecticidal crystal proteins specific only to coleopteran larvae, were shown to produce synergistic factor(s) in culture supernatants. The synergistic substance(s) released into supernatants from various strains of B. thuringiensis were apparently the same.
  • 浅野 真一郎, 佐々木 潤, 伴戸 久徳, 飯塚 敏彦 日本応用動物昆虫学会誌 38 (4) 300 -302 1994年11月 [査読無し][通常論文]
  • 浅野 真一郎, 佐々木 潤, 伴戸 久徳, 飯塚 敏彦, 菊田 治典 日本応用動物昆虫学会大会講演要旨 0 (37) 1993年04月03日 [査読無し][通常論文]
  • 温水 友紀, 浅野 真一郎, 伴戸 久徳, 飯塚 敏彦 日本応用動物昆虫学会大会講演要旨 0 (37) 1993年04月03日 [査読無し][通常論文]
  • 松木 伸浩, 浅野 真一郎, 伴戸 久徳, 飯塚 敏彦 日本応用動物昆虫学会大会講演要旨 0 (37) 1993年04月03日 [査読無し][通常論文]
  • 浅野 真一郎, 伴戸 久徳, 飯塚 敏彦 日本蚕糸学雑誌 62 (3) 223 -227 1993年 [査読無し][通常論文]
     
    Oligonucleotide primers including a specific domain of cry II gene sequences were synthesized in order to identify cry II genes which encode the expression of the P2 protein in Bacillus thuringiensis (Bt) strains. The cry II genes of the Bt isolates were amplified by polymerase chain reaction (PCR) and the amplified DNA was used as DNA probe for identification. Cry II B was distinguished from cry II A by Hinc II digestion. The cry II genes in the Bt isolates were identified by agarose gel electrophoresis. Based on the identification of the Bt subspecies, kurstaki HD-1, aizawai IPL and galleriae harboured both cry II A and cry II B genes, which kenyae harboured only the cry II A gene. This procedure was effective for the identification of Bt isolates including both lepidopteracidal and dipteracidal proteins. © 1993, The Japanese Society of Sericultural Science. All rights reserved.
  • 浅野 真一郎 日本蚕糸学雑誌 62 (3) 210 -215 1993年 [査読無し][通常論文]
     
    Experiments were conducted on eight Bacillus thuringiensis isolates identified by KIKUTA (1990). Observation by SEM showed the presence of a larger number of cuboidal crystals compared to the bipyramidal ones. Identification by serotyping was performed and the structure of the cry I and cry II genes in these isolates was determined by the application of the method of oligonucleotide primer synthesis and PCR amplification. Characteristic profiles of Acp 10-4 and GSK 1-1 belonging to Bacillus thuringiensis subsp. kenyae revealed only the presence of the cry II A gene. The other six strains belonging to the subspp, kurstaki and galleriae harboured the cry II A and cry II B genes. The mosquitocidal activity against Aedes japonicus of Acp 10-4 and GSK 1-1 was much higher than that of the other strains. The amount of 135 and 65kDa protoxins consisting of bipyramidal and cuboidal crystals, respectively was determined by SDS-PAGE with a densitometer. The effect of a larger amount of 135kDa protoxins than of 65 kDa protoxins did not affect the expression in these strains. © 1993, The Japanese Society of Sericultural Science. All rights reserved.
  • 浅野 真一郎, 佐々木 潤, 伴戸 久徳, 飯塚 敏彦 日本応用動物昆虫学会大会講演要旨 0 (36) 1992年09月10日 [査読無し][通常論文]
  • 菊田 治典, 浅野 眞一郎, 伴戸 久徳, 飯塚 敏彦 日本応用動物昆虫学会大会講演要旨 0 (36) 1992年09月10日 [査読無し][通常論文]
  • 浅野 真一郎, 飯塚 敏彦 日本蚕糸学雑誌 60 (6) p475 -479 1991年12月 [査読無し][通常論文]
     
    菱形の結晶タンパク質を産生する Bacillus thuringensis の20亜種を材料として, これらの菌株から抽出されたDNAと subsp. kurstaki HD-1からクローニングされた結晶タンパク質遺伝子 (cry 1-1 gene) とのドットブロットハイブリダイゼーションを行った。この結果, 家蚕殺虫活性のある10亜種のDNAにおいては, この結晶タンパク質遺伝子とのハイブリダイズが認められ, 殺虫活性のないほかの亜種ではこれが認められなかった。またこのクローンの家蚕殺虫活性に特異的な領域をプローブとしたドットブロットハイブリダイゼーションによっても同様な結果が得られた。このことから各亜種の家蚕に対する殺虫活性の強さをDNAハイブリダイゼーションにより判別することが可能であると推定された。
  • 飯塚 敏彦, 浅野 真一郎 日本応用動物昆虫学会大会講演要旨 0 (35) 1991年09月15日 [査読無し][通常論文]
  • 浅野 真一郎, 飯塚 敏彦 日本蚕糸学雑誌 59 (5) 375 -380 1990年10月 [査読無し][通常論文]
     
    菱形の結晶タンパク質を産生する20亜種の Bacillus thuringiensis を材料として, 従来のアルカリ処理法ではなく, 約5分間のSDS加熱処理といったより簡単な方法を用いて, 結晶タンパク質のプロトキシンレベルにおける分子量の比較を行った。この結果, 菱形結晶タンパク質を産生する20亜種の中で, 130~145kDaのペプチドが検出された株では, subsp. japonensis を除き全ての亜種でカイコ幼虫に対する殺虫活性が認められた。しかし, 130~140kDaのペプチドが検出されなかった株では, 殺虫活性が認められなかった。このことから, このより簡便なSDS処理を用いて, 結晶タンパク質の鱗翅目昆虫に対する殺虫活性が判別可能と考えられた。
  • 馬場 富二夫, 浅野 真一郎, 飯塚 敏彦 日本蚕糸学雑誌 59 (6) 487 -489 1990年 [査読無し][通常論文]
  • 菊田 治典, 浅野 真一郎, 飯塚 敏彦 北海道大学農学部邦文紀要 16 (4) p383 -389,図8p 1989年 [査読無し][通常論文]

特許

受賞

  • 2016年03月 日本蚕糸学会 日本蚕糸学会賞
     
    受賞者: 浅野 眞一郎

共同研究・競争的資金等の研究課題

  • 文部科学省:科学研究費補助金(基盤研究(C))
    研究期間 : 2014年 -2016年 
    代表者 : 浅野 眞一郎
  • 文部科学省:科学研究費補助金(基盤研究(C))
    研究期間 : 2010年 -2012年 
    代表者 : 浅野 眞一郎
     
    農作物に甚大な被害をおよぼす鞘翅目害虫に対するBT菌株の殺虫活性試験を行ってきた。(マメコガネ成虫、コクヌストモドキ成虫、コガタルリハムシ成虫、ウリハムシ成虫、ウリハムシモドキ成虫、イネドロオイムシ成虫、イネミズゾウウムシ成虫)。バイオアッセィの結果で殺虫活性が認められたのはマメコガネ、コクヌストモドキ、イネドロオイムシ成虫に対してであり、定法に従いCry遺伝子のクローニング、そのCry遺伝子の大量発現と殺虫活性試験を行うとともに、それらのCryタンパク質の各昆虫成虫の消化管におけるトキシンプロセッシング様式の検証を行ったところ、トキシン断片は最終的には54kDa断片であり、α3-α4ヘリックスの間に存在するループ領域が切断されることがトキシンの殺虫活性に深く関与していることを明らかにした。また、特異的殺虫活性を決める要因の一端であるトキシンレセプターの同定を行った結果、マメコガネ成虫のCry8Daトキシンレセプター分子としてβ-グルコシダーゼやアミノペプチデースN、アルカリフォスファターゼが同定された。この中でも、β-グルコシダーゼが成虫におけるレセプター分子として重要であることが明らかとなった。穀物害虫であるコクヌストモドキ成虫からは、トキシンプロセッシングに関わると考えられている消化管のメタロプロテアーゼ遺伝子がクローニングし、Cry8トキシンのプロセッシングの様式の検討を行った。またコクヌストモドキ成虫のCry8トキシンレセプター候補分子として、カドヘリン様タンパク質やグルコシダーゼの遺伝子をクローニングし、これらの分子とCry8Daトキシンとの結合試験を行い、レセプターとしての機能解析を行った。
  • 病原微生物を用いた害虫防除
    科学研究費補助金
    研究期間 : 2008年 -2012年
  • Microbiol Pest Management
    Grant-in-Aid for Scientific Research
    研究期間 : 2008年 -2012年
  • 殺虫性結晶タンパク質産生菌の分子生物学的研究
    共同研究
    研究期間 : 2008年
  • Research for molecilar biology of Bacillus thuringiensis insecticidal crystal protein
    Cooperative Research
    研究期間 : 2008年
  • 文部科学省:科学研究費補助金(基盤研究(B))
    研究期間 : 1999年 -2000年 
    代表者 : 飯塚 敏彦, 浅野 眞一郎, 浅野 眞一郎, 佐原 健
     
    殺コガネムシ活性を有するB.thuringiensis菌株のスクリーニング(1999年)本講座保有のB.thuringiensis菌株の系統保存株の中から、殺コガネムシ活性を有するB.thuringiensisのスクリーニングを行った。殺コガネムシ活性を有するgalleriae BBT2-8株の性状調査(2000年)上記において選抜された、galleriae BBT2-8株の殺コガネムシ活性を有するcry遺伝子(cryB28遺伝子)のクローニングと大腸菌ならびに非結晶産生Bt菌株(Bt51)株での発現を試みた。そしてその遺伝子産物はコガネムシに対して殺虫活性を示した。殺線虫活性を有するB.thuringiensis菌株のスクリーニング(1999-2000年)本講座保有のB.thuringiensis菌株の系統保存株の中から、PCR法を用いて、殺線虫活性を有するB.thuringiensisのスクリーニングを行った。しかしながら、殺線虫活性を有するB.thuringiensisは選抜できなかった。殺鱗翅目活性を有するB.thuringiensis菌株のスクリーニング(1999年)本講座保有のB.thuringiensis菌株の系統保存株の中から、新規の鱗翅目活性を有するBt菌株として、wuhanensis株を選抜し、その株からcrylDb遺伝子をクローニングした。そのcrylDb遺伝子は、ハスモンヨトウ・コナガ・カイコに対して殺虫活性を示した。
  • 文部科学省:科学研究費補助金(萌芽的研究)
    研究期間 : 1998年 -1999年 
    代表者 : 浅野 眞一郎
     
    当該研究者は既に、北海道のゴルフ場にて数種のコガネムシを採取して、そこからB.popilliae菌株(3菌株、var.Mame,var.Hime,var.Sakura)を分離し、それらのB.popilliae菌株の結晶タンバク質を解析した。各菌株の結晶タンパク質のSDS-PAGEによる解析の結果、それぞれの菌株が約80kDaのペプチドからなる結晶タンパク質を産生していることが明らかとなった。そこで、var.Mame株の80kDaのペプチドのN末端アミノ酸配列の解析(10残基)を行った。その結果、報告されている、B.popilliae由来のタンパク質との相同性は認められなかった。既に報告されている、B.popilliae由来の結晶タンパク質遺伝子(cryBP1:cry18A)に特異なオリゴヌクレオチドプライマーを合成し、B.popilliae菌株のcry遺伝子の検索をPCR法を用いて行った。各種プライマーを用いてcry遺伝子検索を行った結果、これらのB.popilliae菌株が、報告されているcryBP1遺伝子を有しないことが明らかとなった。これらのB.popilliae菌株の有するcry遺伝子をクローニングするため、cryBP1遺伝子の上流域に存在するorf1に注目し、このorf1の塩基配列を基にDNAプライマーを作成した。そのプライマーを用いて、B.popilliae var popilliae株のゲノムDNAをテンプレートとして、PCR法によって約300bpの断片が増幅された。この増幅された断片を解析したところ、orf1と高い相同性が認められた。現在は、この増幅されたDNA断片の上・下流域のクローニングを進めている。
  • 文部科学省:科学研究費補助金(基盤研究(C))
    研究期間 : 1996年 -1996年 
    代表者 : 浅野 真一郎
     
    B.thuringiensisにおけるinsecticidal crystal protein(ICP)の発現制御機構が近年、いろいろなICP遺伝子について行われてきた。研究代表者は、B.thuringiensisのICP遺伝子発現ベクターを構築し、各種ICPを発現させることが可能となった。また、B.thuringiensis菌株から各ICP遺伝子のプロモーターをクローニングし(cry1A遺伝子プロモーター、cry2A遺伝子プロモーター、cry3A遺伝子プロモーター、cry4A遺伝子プロモーター等)各種プロモーター制御下でのそれぞれのICP遺伝子の発現制御機構について解析した。cry1Aとcry4A遺伝子に関しては、そのプロモーターでの発現制御機構が、詳細に調べられているがcry2A遺伝子に関してはこの遺伝子のプロモーター制御下でのICP遺伝子発現制御機構は調べられていなかった。cry2A遺伝子プロモーター発現制御機構を調査した結果、cry2A遺伝子の発現は、その上流域に存在する2つのorfの上流に存在するB.thuringiensisの他のcry遺伝子にも共通して見られる、BtIプロモーター制御下で発現されることがプライマーエクステンション法を用いた調査で明かとなった。crylA遺伝子プロモーターの発現制御機構と併せて、新たにcry2A遺伝子プロモーター発現制御機構について本実験で明らかにし、B.thuringiensis遺伝子導入ベクターを2種類構築することができた。
  • 文部科学省:科学研究費補助金(奨励研究(A))
    研究期間 : 1993年 -1993年 
    代表者 : 浅野 眞一郎, 浅野 真一郎
     
    家蚕中腸上皮細胞上に存在する、Bacillus thuringiensis subsp.sottoのdelta-エンドトキシンにたいする受容体についてまず解析を進めた。まず家蚕中腸の組織切片を作成し、切片上にてdelta-エンドトキシンとのバインディングアッセイを行ったところ、家蚕中腸上皮細胞にdelta-エンドトキシンに強い親和性を持つ受容体が存在していることが明らかとなった。Wolfersbergerらの方法に従い、Mg/EDTA沈殿と分画遠心法によって調整した家蚕中腸からBrush border membrane vesicle(BBMV)を調整し、SDS-P AGEによる分画の後、ウエスタンブロットしsotto由来のdelta-エンドトキシンとのバインディングアッセイによる解析によって、BBMV由来の幾つかの分画のバンドとバインディングが認められた。その中でも役130kdalのバンドに強いバインディングが認められたので、そのバンドの分画をSDS-ポリカクリルアミドゲルから切り出して電気泳動的にその分画のペプチドを回収した。その回収した分画のペプチドとdelta-エンドトキシンとをco-incubateし十分にそのペプチドとdelta-エンドトキシンとバインディングさせた後、ウェスタンブロットしたBBMV分画とのバインディングアッセイを行ったところ、バインディングが押さえられていることが明らかとなった。よって、家蚕中腸BBMVをSDS-PAGEによって分画して、ゲルから回収し、その回収した分画をマウスに皮下接種して現在免疫化している。それによって得られた抗体を用いて、家蚕BBMVとsotto由来のdelta-エンドトキシンとのバインディングを阻害することができるかを確かめるつもりである。

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  • 応用分子生物学特論演習
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  • 昆虫病理学
    開講年度 : 2021年
    課程区分 : 学士課程
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    キーワード : 昆虫病理学、昆虫病原、生体防御、細菌、ウイルス、糸状菌、原生動物、昆虫寄生性線虫、微生物防除
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    キーワード : 昆虫機能利用 情報伝達(コミュニケーション)生体防御機構 遺伝子工学
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    キーワード : 応用生命科学,分子生物学,応用分子昆虫学,分子酵素学,遺伝子制御学,生態化学生物学,植物育種学,分子環境生物科学,生物情報分子解析学,ゲノム生化学
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