Researcher Database
Last Updated :2024/10/02
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Profile and Settings
Profile and Settings
Name (Japanese)
AsanoName (Kana)
ShinichiroName
200901053198142950
Alternate Names
Achievement
Research Interests
- 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
Research Areas
Research Experience
- 2021/04 - Today Hokkaido University Graduate School of Agriculture Research Faculty of Agriculture Professor
- 2010 - 2012 北海道大学 (連合)農学研究科(研究院) 准教授
- 1999/04 - 2007/03 Associate Professor
- 2007 - 農学部 准教授
Education
Awards
Published Papers
- Toshiki Nakanishi, Shin-ichiro Asano, Hisanori Bando, Masanao SatoJournal of Insect Biotechnology and Sericology 93 (1) 1 - 10 2024/05/28 [Refereed]
- Mami Sakai, Satoshi Kakutani, Shin-ichiro Asano, Masanao Sato, Hisanori BandoVirus Research 291 198195 - 198195 0168-1702 2021/01 [Refereed][Not invited]
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 cellsMari Sekiguchi, Sayuri Ishikawa, Shin-ichiro Asano, Hisanori Bando, Masanao SatoJournal of Insect Biotechnology and Sericology 89 (3) 45 - 53 2020/09 [Refereed]
- Akio Kawahara, Kazuhiro Iiyama, Shin-Ichiro Asano, Oumi Nishi, Chisa Yasunaga-AokiJournal of Insect Biotechnology and Sericology 89 (2) 25 - 30 1884-7978 2020Two novel cry-type gene homologues, tentatively named cryPp1 and cryPp2, were detected in the genomic DNA of Paenibacillus popilliae ATCC 14706T, both of which were similar to genes encoding Cry18 proteins, suggesting their similar modes of action. Ours is the first report concerning multiple Cry homologues, detected from a sole strain of P. popilliae. In order to characterize the roles of the two Cry homologues in pathogenicity, a trial for the expression of recombinant proteins in Escherichia coli was conducted, which revealed that the optimization of rare codons, such as supplementation of tRNA genes, was necessary.
- Design and prototyping of a Bombyx mori nucleopolyhedrovirus-based genome assembly from PCR-amplified DNA fragmentsNaoto Harada, Daiki Ishibashi, Shin-ichiro Asano, Masanao Sato, Hisanori BandoInsect Biotechnology and Sericology 87 (3) 97 - 108 2018/10 [Refereed][Not invited]
- 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 1346-986X 2017/12/25 [Not refereed][Not invited]
- 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 1346-986X 2017/12/25 [Not refereed][Not invited]
- 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 1346-986X 2017/12/25 [Not refereed][Not invited]
- 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 1346-986X 2017/12/25 [Not refereed][Not invited]
- 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 1346-986X 2016/12/10 [Not refereed][Not invited]
- 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 1346-986X 2016/12/10 [Not refereed][Not invited]
- KURNIA Yudistira Wahyu, FUJITA Ryosuke, SATO Masanao, ISAWA Haruhiko, ASANO Shin‐ichiro, BINH Ngo Dinh, BANDO HisanoriJournal of Insect Biotechnology and Sericology 85 (2) 39‐47 1346-8073 2016/09/12 [Not refereed][Not invited]
- Kota Kawakami, Yudistira Wahyu Kurnia, Ryosuke Fujita, Toshiaki Ito, Haruhiko Isawa, Shin-ichiro Asano, Ngo Dinh Binh, Hisanori BandoARCHIVES OF VIROLOGY 161 (4) 801 - 809 0304-8608 2016/04 [Refereed][Not invited]
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 1346-986X 2015/12/20 [Not refereed][Not invited]
- 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 2015/09/25 [Not refereed][Not invited]
- Ryosuke Fujita, Chikako Ono, Isamu Ono, Shin-ichiro Asano, Hisanori BandoBIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 464 (4) 1297 - 1301 0006-291X 2015/09 [Refereed][Not invited]
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-AokiMeta Gene 4 29 - 44 2214-5400 2015/06/01 [Refereed][Not invited]
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 BandoPLOS ONE 10 (3) e0119580 1932-6203 2015/03 [Refereed][Not invited]
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 1346-986X 2014/12/10 [Not refereed][Not invited]
- Kazuhiro Iiyama, Masahiro Otao, Kazuki Mori, Hiroaki Mon, Jae Man Lee, Takahiro Kusakabe, Kousuke Tashiro, Shin-Ichiro Asano, Chisa Yasunaga-AokiBIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 78 (5) 891 - 897 0916-8451 2014/05 [Refereed][Not invited]
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 HyakumachiPLANT CELL REPORTS 33 (1) 99 - 110 0721-7714 2014/01 [Refereed][Not invited]
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 1346-986X 2013/12/30 [Not refereed][Not invited]
- Masako Yokoo, Ryosuke Fujita, Yumiko Nakajima, Mamoru Yoshimizu, Hisae Kasai, Shin-ichiro Asano, Hisanori BandoBiochemical and Biophysical Research Communications 439 (1) 18 - 22 0006-291X 2013/09/13 [Refereed][Not invited]
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-ichiroThe Journal of Sericultural Science of Japan The Japanese Society of Sericultural Science 82 (1) 13 - 18 1346-8073 2013/06/28Cry39Aa 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 HisanoriJ Insect Biotechnol Sericology 82 (1) 25 - 32 1346-8073 2013/06/28 [Not refereed][Not invited]
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 AsanoJOURNAL OF INVERTEBRATE PATHOLOGY 113 (2) 123 - 128 0022-2011 2013/06 [Refereed][Not invited]
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 TakahashiMicrobes and Environments 28 (1) 128 - 134 1342-6311 2013 [Refereed][Not invited]
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. - Kazuhiro Iiyama, Oumi Nishi, Hiroaki Mon, Jae Man Lee, Takahiro Kusakabe, Shin-ichiro Asano, Chisa Yasunaga-Aoki, Susumu ShimizuJournal of Insect Biotechnology and Sericology The Japanese Society of Sericultural Science 82 (1) 1 - 11 1346-8073 2013 [Refereed][Not invited]
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 SusumuJournal of Insect Biotechnology and Sericology 82 (2) 45 - 48 2013 [Refereed][Not invited]
- Masataka Ohsawa, Miki Tanaka, Kenta Moriyama, Mitsuaki Shimazu, Shin-ichiro Asano, Kazuhisa Miyamoto, Kohsuke Haginoya, Toshiaki Mitsui, Tomoaki Kouya, Masayuki Taniguchi, Hidetaka HoriAPPLIED AND ENVIRONMENTAL MICROBIOLOGY 78 (13) 4755 - 4757 0099-2240 2012/07 [Refereed][Not invited]
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 BandoARCHIVES OF VIROLOGY 155 (4) 577 - 581 0304-8608 2010/04 [Refereed][Not invited]
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 cellsFujita R, Asano S, Sahara K, Bando H41th Annual meeting of the society for invertebrate pathology and 9th international conference on Bacillus thuringiensis 19 - 19 2008/08 [Refereed][Not invited]
- K. Kojima, K. Kojima, K. Oritani, T. Nakatsukasa, S. Asano, K. Sahara, H. BandoVirus Research Elsevier 130 (1) 202 - 209 0168-1702 2007/12/01 [Not refereed][Not invited]
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 cellsFujita R, Asano S, Sahara K, Bando H40th Annual meeting of the society for invertebrate pathology and 1st international forum on entomopathogenic nematodes and symbiotic bacteria 95 - 95 2007/08 [Refereed][Not invited]
- R Fujita, T Matsuyama, J Yamagishi, K Sahara, S Asano, H BandoJOURNAL OF VIROLOGY 80 (5) 2390 - 2395 0022-538X 2006/03 [Refereed][Not invited]
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 HVirus Research 112 (1-2) 38 - 41 2005/10 [Refereed][Not invited]
- 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 2004/12 [Refereed][Not invited]
- 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Research bulletin of the University Farm, Hokkaido University 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 33, 9-13 (33) 9 - 13 0385-6445 2003/03 [Not refereed][Not invited]
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 BandoVIRUS RESEARCH 90 (1-2) 253 - 261 0168-1702 2002/12 [Not refereed][Not invited]
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. - Antisense and double-strand RNA interference in a silkworm ovarian cell lineRyoko Isobe, Katsura Kojima, Ken Sahara, Shin-ichiro Asano, Hisanori BandoJournal of Insect Biotechnology and Sericology 71 (1) 43 - 47 1346-8073 2002Antisense 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.
- 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Research bulletin of the University Farm,Hokkaido University 北海道大学農学部附属農場 32, 61-69. (32) 61 - 69 0385-6445 2001/03 [Not refereed][Not invited]
- T Hayakawa, K Kojima, K Nonaka, M Nakagaki, K Sahara, S Asano, T Iizuka, H BandoVIRUS RESEARCH 66 (1) 101 - 108 0168-1702 2000/01 [Refereed][Not invited]
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. BandoArchives of Virology 143, 127-143 127 - 143 0304-8608 1998/03/27 [Not refereed][Not invited]
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 IizukaCURRENT MICROBIOLOGY 35 (1) 1 - 8 0343-8651 1997/07 [Refereed][Not invited]
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 YamamotoAPPLIED AND ENVIRONMENTAL MICROBIOLOGY 63 (3) 1054 - 1057 0099-2240 1997/03 [Refereed][Not invited]
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 BandoARCHIVES OF VIROLOGY 142 (2) 393 - 399 0304-8608 1997 [Refereed][Not invited]
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 YamamotoCURRENT MICROBIOLOGY 32 (4) 195 - 200 0343-8651 1996/04 [Refereed][Not invited]
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. IizukaArchives of Virology 140, 1147-1155 (6) 1147 - 1155 0304-8608 1995/06/01 [Not refereed][Not invited]
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 日本蠶絲學會 63 (5) p361 - 366 0037-2455 1994/10 [Refereed][Not invited]
- Ikuhiro Nakatani, Shinichiro Asano, Hisanori Bando, Toshihiko IizukaJournal of Sericultural Science of Japan 63 (2) 140 - 148 0037-2455 1994 [Refereed][Not invited]
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 MuraiJournal of Sericultural Science of Japan 63 (4) 303 - 309 0037-2455 1994 [Refereed][Not invited]
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.
MISC
- 前田麻亜子, 坪田拓也, 門宏明, 増田亮津, 松田拓也, 浅野眞一郎, 伴戸久徳, 瀬筒秀樹, 瀬筒秀樹, 荒川和晴, 荒川和晴, 日下部宜宏, 佐藤昌直, 佐藤昌直 日本分子生物学会年会プログラム・要旨集(Web) 46th- 2023
- 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
- 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
- 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
- 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
- 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
- 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
- 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
- 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
- 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 2016/12 [Not refereed][Not invited]
- 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 2016/06
- 浅野 眞一郎 昆虫と自然 51- (3) 4 -7,図巻頭1p 2016/03
- 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
- 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
- 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
- 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 2014/03/09 [Not refereed][Not invited]
- 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 2014/03/09 [Not refereed][Not invited]
- 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
- 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
- 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 2013/03/18 [Not refereed][Not invited]
- 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 2013/03/15 [Not refereed][Not invited]
- 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 2013/03/15 [Not refereed][Not invited]
- 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 2013/03/15 [Not refereed][Not invited]
- Chikako Ono, Takanori Kamagata, Hitomi Taka, Ken Sahara, Shin-ichiro Asano, Hisanori Bando VIRUS RESEARCH 165- (2) 197 -206 2012/05 [Not refereed][Not invited]
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
- 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 2011/03/20 [Not refereed][Not invited]
- 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
- 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 [Not refereed][Not invited]
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. - Shin-ichiro Asano JOURNAL OF PESTICIDE SCIENCE 36- (1) 91 -93 2011 [Not refereed][Not invited]
- Takuya Yamaguchi, Ken Sahara, Hisanori Bando, Shin-ichiro Asano JOURNAL OF INVERTEBRATE PATHOLOGY 105- (3) 243 -247 2010/11 [Not refereed][Not invited]
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. - ASANO Shin-ichiro 講演要旨集 (35) 34 -34 2010/04/21
- 浅野 眞一郎 蚕糸・昆虫バイオテック = Sanshi-konchu biotec 77- (3) 181 -186 2008/12/01 [Not refereed][Not invited]
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 2008/11 [Not refereed][Not invited]
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 2008/10/31 [Not refereed][Not invited]
- 微生物の事典 Ⅲ朝倉書店 2008 [Not refereed][Not invited]
- ONO Chikako, NAKATSUKASA Tomonori, NISHIJIMA Yasuyuki, ASANO Shin-ichiro, SAHARA Ken, BANDO Hisanori Journal of insect biotechnology and sericology 76- (3) 161 -167 2007/10/30 [Not refereed][Not invited]
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 [Not refereed][Not invited]
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 2006/09 [Not refereed][Not invited]
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 2006/08 [Not refereed][Not invited]
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 2006/03 [Not refereed][Not invited]
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 [Not refereed][Not invited]
- FUJITA Ryousuke, ASANO Shinichiro, SAHARA Ken, BANDO Hisanori Journal of Insect Biotechnology and Sericology 74- (3) 125 -128 2005/10/28 [Not refereed][Not invited]
- T Abe, N Miyake, Y Nishijima, R Fujita, K Sahara, S Asano, H Bando VIRUS RESEARCH 112- (1-2) 38 -41 2005/09 [Not refereed][Not invited]
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
- 浅野 眞一郎, 伊藤 岳 化学と生物 42- (8) 501 -503 2004/08/25 [Not refereed][Not invited]
- 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 2004/07 [Not refereed][Not invited]
We have developed a new procedure for Agrobacterium-mediated transformation of plants in the genus Beta using shoot-base as the material for Agrobacterium infection. The frequency of regeneration from shoot bases was analyzed in seven accessions of sugarbeet (Beta vulgaris) and two accessions of B. maritima to select materials suitable for obtaining transformed plants. The frequency of transformation of the chosen accessions using Agrobacterium strain LBA4404 and selection on 150-mg/l kanamycin was found to be higher than that in previously published methods. Genomic DNA analysis and beta-glucuronidase reporter assays showed that the transgene was inherited and expressed in subsequent generations. In our method, shoot bases are prepared by a simple procedure, and transformation does not involve the callus phase, thus minimizing the occurrence of somaclonal variations. - 佐藤 研二, 浅野 真一郎 Japanese journal of nematology 34- (2) 79 -88 2004 [Not refereed][Not invited]
Bacillus huringiensis serovar roskildiensisから新規なcry遺伝子であるcry21Balをクローニングし、その塩基配列を決定した。本遺伝子のORF（アミノ酸1286残基から成るCry21Balをコードする鎖長3858bpのDNA）の塩基配列は、既知の線虫毒性タンパク質であるCry21Aalをコードする遺伝子（すなわちcry21Aal）のORFの塩基配列と75％一致した。また、Cry21Balの推定アミノ酸配列はCry21Aalのアミノ酸配列と67％一致した。これらの結果は、Cry21Balが線虫毒性タンパク質である可能性を示唆している。 - S Asano, C Yamashita, T Iizuka, K Takeuchi, S Yamanaka, D Cerf, T Yamamoto BIOLOGICAL CONTROL 28- (2) 191 -196 2003/10 [Not refereed][Not invited]
A strain of Bacillus thuringiensis (Bt) subsp. galleriae highly toxic to the cupreous chafer, Anomala cuprea, was isolated from a soil sample collected in Japan. The strain, SDS-502, produces a crystal consisting of a 130-kDa protein. The gene encoding the protein was cloned in Escherichia coli using antiserum directed to the protein. The gene was expressed in E coli, producing a 130kDa protein toxic to A. cuprea. Sequencing of the cloned gene indicated a typical Bt cry gene with substantial homology to cry8 genes. Based on the peptide sequence comparison, the gene found in SDS-502 was designated as cry8Da (AB089299). The cry8Da gene was highly expressed in SDS-502 in a laboratory scale bioreactor. The fermentation product of SDS-502 was formulated for soil application and tested in a peanut field for chafer control along with a chemical insecticide reference, a fenthion organophosphate granular formulation. Judging from the amount of undamaged nuts harvested from plots of different treatments, plots treated with SDS-502 had significantly better insect control than the untreated plots. The chemical insecticide plots showed no significant difference in nut damage-from the Bt-treated or control plots. (C) 2003 Elsevier Science (USA). All rights reserved. - MATSUYAMA Takahiro, ASANO Shinichiro, SAHARA Ken, BANDO Hisanori Journal of Insect Biotechnology and Sericology 72- (2) 87 -94 2003/06/30
- MATSUYAMA Takahiro, ASANO Shinichiro, SAHARA Ken, BANDO Hisanori Journal of Insect Biotechnology and Sericology 72- (2) 87 -94 2003/06/30 [Not refereed][Not invited]
- 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 [Not refereed][Not invited]
- ITO Takeshi, SAHARA Ken, BANDO Hisanori, ASANO Shinichiro Journal of Insect Biotechnology and Sericology 71- (3) 123 -128 2002/10/31 [Not refereed][Not invited]
- ISOBE Ryoko, KOJIMA Katsura, SAHARA Ken, ASANO Shin-ichiro, BANDO Hisanori Journal of Insect Biotechnology and Sericology 71- (1) 43 -47 2002/02/28 [Not refereed][Not invited]
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 [Not refereed][Not invited]
- 2002 [Not refereed][Not invited]
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 2001/03/29 [Not refereed][Not invited]
- 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 2001/03/29 [Not refereed][Not invited]
- 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
- K. Kojima, T. Hayakawa, S. Asano, H. Bando Archives of Virology 146- (7) 1407 -1414 2001 [Not refereed][Not invited]
- K Sahara, H Saito, K Kikuchi, Y Yamada, S Asano, H Bando JAPANESE JOURNAL OF APPLIED ENTOMOLOGY AND ZOOLOGY 45- (2) 94 -98 2001 [Not refereed][Not invited]
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. - Katsura Kojima, Aya Hirano, Shin-Ichiro Asano, Ken Sahara, Hisanori Bando Journal of Sericultural Science of Japan 70- (2) 103 -108 2001 [Not refereed][Not invited]
Recent studies of BmDNV-2 predicted a unique replication mechanism different from the parvoviruses and implied that the common terminal sequences (CTS) of 53 nucleotides and the viral structural protein with the DNA polymerase motif (p128) played important roles in the viral DNA replication. To understand the replication mechanism of BmDNV-2, the function of the CTS and the properties of the viral DNA polymerase were studied. The gel mobility shift assay revealed that at least one of the viral structural proteins bound to the 3'-CTS, emphasizing the propriety of the idea that the CTS was a putative replication origin of the viral DNA. On the other hand, an increase of the DNA polymerase activity with characteristic properties was observed in the midgut of the silkworm infected with BmDNV-2. These observations suggest that the farther characterization of the BmDNV-2 specific DNA polymerase is indispensable to understanding the replication mechanism of BmDNV-2. © 2001, The Japanese Society of Sericultural Science. All rights reserved. - Ken Sahara, Hiroshi Saito, Kunio Kikuchi, Yasuhiro Yamada, Shin-Ichiro Asano, Hisanori Bando Japanese Journal of Applied Entomology and Zoology 45- (2) 94 -98 2001 [Not refereed][Not invited]
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
- Junya Yamagishi, Shinichiro Asano, Ken Sahara, Toshihiko Iizuka, Hisanori Bando Journal of Sericultural Science of Japan 69- (4) 271 -276 2000 [Not refereed][Not invited]
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 [Not refereed][Not invited]
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) - Junya Yamagishi, Shinichiro Asano, Ken Sahara, Toshihiko Iizuka, Hisanori Bando Journal of Sericultural Science of Japan 69- (4) 271 -276 2000 [Not refereed][Not invited]
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
- Yulia Pujiastuti, Shin-ichiro Asano, Ken Sahara, Hlsanori Bando, Toshihiko Ilzuka Journal of Sericultural Science of Japan 68- (3) 195 -199 1999 [Not refereed][Not invited]
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 [Not refereed][Not invited]
- N Matsuki, S Asano, H Bando, T Iizuka APPLIED ENTOMOLOGY AND ZOOLOGY 33- (1) 205 -205 1998/02 [Not refereed][Not invited]
- A Rizali, S Asano, K Sahara, H Bando, BW Lay, S Hastowo, T Iizuka APPLIED ENTOMOLOGY AND ZOOLOGY 33- (1) 111 -114 1998/02 [Not refereed][Not invited]
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
- Shin-ichiro Asano, Yulia Pujiastuti, Ken Sahara, Hisanori Bando, Toshihiko Iizuka, Har unori Kikuta Journal of Sericultural Science of Japan 67- (3) 237 -242 1998 [Not refereed][Not invited]
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. - ASANO SHIN-ICHIRO, PUJIASTUTI YULIA, SAHARA KEN, BANDO HISANORI, KIKUTA HARUNORI, IIZUKA TOSHIHIKO Journal of Insect Biotechnology and Sericology 67- (3) 237 -242 1998 [Not refereed][Not invited]
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, cry1E 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 cry1C and cry1E genes. It seems that they have the novel cry genes. - N Matsuki, S Asano, H Bando, T Iizuka APPLIED ENTOMOLOGY AND ZOOLOGY 32- (4) 583 -588 1997/11 [Not refereed][Not invited]
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 1997/11 [Not refereed][Not invited]
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. - KIKUTA Harunori, KUROIWA Manabu, ASANO Shinichiro, IIZUKA Toshihiko Journal of the College of Dairying. Natural science 22- (1) 81 -97 1997/10 [Not refereed][Not invited]
- SAHARA KEN, FUKUTANI SHOUKO, SAITOH HIROSHI, NAKADA TOHRU, ASANO SHIN-ICHIRO, BANDO HISANORI, KAWAMURA NAOKO, IIZUKA TOSHIHIKO The Journal of Sericultural Science of Japan 66- (2) 141 -144 1997/04/28 [Not refereed][Not invited]
- Ken Sahara, Youko Tanaka, Yasuhiro Yamada, Hiroshi Saitoh, Tohru Nakada, Shin-ichiro Asano, Hisanori Bando, Toshihiko Iizuka, Naoko Kawamura Journal of Sericultural Science of Japan 66- (3) 207 -211 1997 [Not refereed][Not invited]
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. - Ken Sahara, Yasuhiro Yamada, Hiroshi Saitoh, Tohru Nakada, Shinichiro Asano, Hisanori Bando, Toshihiko Iizuka, Naoko Kawamura Journal of Sericultural Science of Japan 66- (5) 341 -345 1997 [Not refereed][Not invited]
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. - Ken Saharan, P. Rama, Yasuhiro Yamada, Hiroshi Saitoh, Shin-Ichiro Asano, Hisanori Bando, Toshihiko Iizuka, Tohru Nakada, Mohana Rao Journal of Sericultural Science of Japan 66- (5) 364 -366 1997 [Not refereed][Not invited]
- KAGEYASU SEIJI, HAYAKAWA TOHORU, ISAWA HARUHIKO, ASANO SHINICHIRO, SAHARA KEN, IIZUKA TOSHIHIKO, BANDO HISANORI The Journal of Sericultural Science of Japan 66- (6) 477 -483 1997 [Not refereed][Not invited]
The five viruses, Bombyx mori densonucleosis virus type 1 (BmDNV-1), -type 2 (BmDNV-2), Bombyx mori nuclear polyhedrosis virus (BmDPV), Bombyx mori cytoplasmic polyhedrosis virus (BmCPV), and Infectious flacherie virus (IFV), are the agents causing fetal disease of the silkworm. The aim of this study is to establish a simple and highly sensitive PCR system for detection of all the viruses. A combination of the RT-PCR and the nested PCR was found to be effective to amplify DNA and/or RNA virus genome sequences in a reaction maintaining high sensitivity and specificity. In this report, the primer design and the condition of the PCR for the detection of all the viruses are described. - 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 [Not refereed][Not invited]
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)＊ - Ken Sahara, Youko Tanaka, Yasuhiro Yamada, Hiroshi Saitoh, Tohru Nakada, Shin-ichiro Asano, Hisanori Bando, Toshihiko Iizuka, Naoko Kawamura Journal of Sericultural Science of Japan 66- (3) 207 -211 1997 [Not refereed][Not invited]
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. - SAHARA KEN, FUKUTANI SHOUKO, SAITOH HIROSHI, NAKADA TOHRU, ASANO SHIN-ICHIRO, BANDO HISANORI, KAWAMURA NAOKO, IIZUKA TOSHIHIKO Journal of Insect Biotechnology and Sericology 66- (2) 141 -144 1997 [Not refereed][Not invited]
- KAGEYASU SEIJI, HAYAKAWA TOHORU, ISAWA HARUHIKO, ASANO SHINICHIRO, SAHARA KEN, IIZUKA TOSHIHIKO, BANDO HISANORI The Journal of Sericultural Science of Japan 66- (6) 477 -483 1997 [Not refereed][Not invited]
The five viruses, Bombyx mori densonucleosis virus type 1 (BmDNV-1), -type 2 (BmDNV-2), Bombyx mori nuclear polyhedrosis virus (BmDPV), Bombyx mori cytoplasmic polyhedrosis virus (BmCPV), and Infectious flacherie virus (IFV), are the agents causing fetal disease of the silkworm. The aim of this study is to establish a simple and highly sensitive PCR system for detection of all the viruses. A combination of the RT-PCR and the nested PCR was found to be effective to amplify DNA and/or RNA virus genome sequences in a reaction maintaining high sensitivity and specificity. In this report, the primer design and the condition of the PCR for the detection of all the viruses are described. - 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 [Not refereed][Not invited]
- Ken Sahara, Yasuhiro Yamada, Hiroshi Saitoh, Tohru Nakada, Shinichiro Asano, Hisanori Bando, Toshihiko Iizuka, Naoko Kawamura Journal of Sericultural Science of Japan 66- (5) 341 -345 1997 [Not refereed][Not invited]
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 [Not refereed][Not invited]
- 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 [Not refereed][Not invited]
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 [Not refereed][Not invited]
- Ken Saharan, P. Rama, Yasuhiro Yamada, Hiroshi Saitoh, Shin-Ichiro Asano, Hisanori Bando, Toshihiko Iizuka, Tohru Nakada, Mohana Rao Journal of Sericultural Science of Japan 66- (5) 364 -366 1997 [Not refereed][Not invited]
- Jun Sasaki, Shinichiro Asano, Toshihiko Iizuka, Hisanori Bando, Bibiana W. Lay, Sugyo Hastowo, Gary K. Powell, Takashi Yamamoto Current Microbiology 32- (4) 195 -200 1996/04 [Not refereed][Not invited]
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) 339 -339 1996/03/05 [Not refereed][Not invited]
- Asano Shin-ichiro Memoirs of the Faculty of Agriculture, Hokkaido University 19- (7) 529 -563 1996 [Not refereed][Not invited]
- Jun Sasaki, Shinichiro Asano, Hisanori Bando, Toshihiko Iizuka, Bibiana W. Lay, Sugyo Hastowo Journal of Sericultural Science of Japan 65- (1) 56 -61 1996 [Not refereed][Not invited]
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. - Naoki Hashimoto, Jun Sasaki, Shinichiro Asano, Hisanori Bando, Toshihiko Iizuka Journal of Sericultural Science of Japan 65- (3) 185 -191 1996 [Not refereed][Not invited]
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. - Naoki Hashimoto, Jun Sasaki, Shinichiro Asano, Hisanori Bando, Toshihiko Iizuka Journal of Sericultural Science of Japan 65- (3) 185 -191 1996 [Not refereed][Not invited]
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. - Jun Sasaki, Shinichiro Asano, Hisanori Bando, Toshihiko Iizuka, Bibiana W. Lay, Sugyo Hastowo Journal of Sericultural Science of Japan 65- (1) 56 -61 1996 [Not refereed][Not invited]
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 [Not refereed][Not invited]
- ASANO Shoji, HORI Hidetaka Applied entomology and zoology 30- (2) 369 -374 1995 [Not refereed][Not invited]
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. - S ASANO, J SASAKI, H BANDO, T IIZUKA JAPANESE JOURNAL OF APPLIED ENTOMOLOGY AND ZOOLOGY 38- (4) 300 -302 1994/11 [Not refereed][Not invited]
- 浅野真一郎 東北蚕糸研究報告 19- 34 -34 1994
- 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) 146 -146 1993/04/03 [Not refereed][Not invited]
- 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) 149 -149 1993/04/03 [Not refereed][Not invited]
- 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) 149 -149 1993/04/03 [Not refereed][Not invited]
- Shinichiro Asano, Hisanori Bando, Toshihiko Iizuka Journal of Sericultural Science of Japan 62- (3) 223 -227 1993 [Not refereed][Not invited]
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. - Shinichirou Asano, Hisanori Bando, Toshihiko Iizuka, Harunori Kikuta Journal of Sericultural Science of Japan 62- (3) 210 -215 1993 [Not refereed][Not invited]
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) 211 -211 1992/09/10 [Not refereed][Not invited]
- 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) 212 -212 1992/09/10 [Not refereed][Not invited]
- ASANO SHINICHIRO, IIZUKA TOSHIHIKO Journal of Insect Biotechnology and Sericology 60- (6) p475 -479 1991/12 [Not refereed][Not invited]
In order to identify the insecticidal activity of 20 Bacillus thuringiensis subspecies which produce bipyramidal crystals, dot blot hybridization of DNA purified from the subspecies was performed with a cloned crystal protein gene (cryl-1 gene) from subsp. kurstaki HD-1. The DNAs from the ten subspecies exhibiting on insecticidal activity against the silkworm, Bombyx mori, strongly hybridized with the probe prepared from the cloned crystal protein gene. However, DNAs from the other ten subspecies lacking insecticidal activity did not hybridize with the probe. Similar results were also obtained in the experiment using a probe prepared from the DNA fragment that is specific for insecticidal activity. Based on these results, we concluded that the intensity of the insecticidal activity against the silkworm can be determined DNA hybridization. - 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) 103 -103 1991/09/15 [Not refereed][Not invited]
- ASANO SHINICHIRO, IIZUKA TOSHIHIKO Journal of Insect Biotechnology and Sericology 59- (5) 375 -380 1990/10 [Not refereed][Not invited]
The amount of protoxin from the diamond-shaped clystals produced by twenty Bacillus thuringiensis subspecies/strains was compared with the peptide level using SDS treatment as a simple and improved method. The insecticidal activities of the spore and crystal samples from these twenty strains were tested against 5th instar larvae of the silkworm by peroral injection. Lepidopteran insecticidal activities were recognized in the strains in which 130 to 140kDa peptides were detected except for the subsp. japonensis in which 120kDa protoxin was not detected. However, insecticidal activity was not revealed in the strains in which 130 to 140kDa peptides were not detected. - Fujio Baba, Shinichiro Asano, Toshihiko Iizuka Journal of Sericultural Science of Japan 59- (6) 487 -489 1990 [Not refereed][Not invited]
- KIKUTA H Memoirs of the Faculty of Agriculture,Hokkaido University 16- (4) p383 -389,図8p 1989 [Not refereed][Not invited]
Books etc
Association Memberships
- 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
Research Projects
- Japan Society for the Promotion of Science：Grants-in-Aid for Scientific ResearchDate (from‐to) : 2021/10 -2025/03Author : 仲井 まどか, 浅野 眞一郎, 坂本 卓磨タイワンカブトムシ(別名サイカブトムシ：Oryctes rhinoceros）は、ヤシ類の害虫である。本種の防除には、1970年代から1980年代にかけて天敵ウイルス（Oryctes rhinoceros nudivirus: OrNV) を用いた防除が実施され、南太平洋のフィジーなどではヤシの被害を抑えることに成功した。しかし、2007年にグアム（米国）にウイルス抵抗性のハプロタイプ（Gタイプ）が侵入したことが報告され、その後もGタイプの侵入が、パプアニューギニア、ソロモン諸島、ハワイなどに拡大していることから太平洋州の各国でヤシ類の植物保護において大きな脅威になっている。また、パラオにはGタイプとそれ以外のハプロタイプ（Sタイプ）が混在しておりヤシの被害も少ない。本研究は、Gタイプの害虫としての特徴と天敵ウイルスを用いた生物的防除の機構を解明することによりその防除の成否を決定する要因の解明を目的とする。本研究において国際共同研究を強化することにより、太平洋州全域のヤシ林の保護を実現させることを目指している。 2021年度は、新型コロナウイルス感染拡大のため、GタイプおよびSタイプの輸入ができなかった。そこで、天敵ウイルスによる本種の防除メカニズムの解明について研究を行った。具体的には、幼虫および成虫に対するウイルス接種法の検討を行った。接種方法が確立できれば、宿主のステージごとのウイルス伝播率を定量化することができる。室内実験においてウイルスの伝播率が、幼虫から幼虫、幼虫から成虫、成虫から幼虫、成虫から成虫のどのステージごとにどれだけ伝播するか比較できれば、 OrNVを用いた生物的防除の成否を決定するメカニズムの解明に繋がる。また、培養細胞を用いたウイルス増殖を検討した。さらに、沖縄県において採集したタイワンカブトムシを大量飼育し、生物検定の基盤確立を行った。
- Japan Society for the Promotion of Science：Grants-in-Aid for Scientific ResearchDate (from‐to) : 2017/04 -2021/03Author : Nakai MadokaCoconut Rhinoceros Beetle (Oryctes rhinoceros) is a pest of coconut palms and oil palms. Natural enemy of this species, nudivirus, was used to control this species in the 1970s and 1980s, and succeeded in reducing damage to palms in the South Pacific including Fiji. However, in 2007, it was reported that a haplotype (a strain of the same species with a different genotype), in which the virus is ineffective, invaded Guam (USA), and since then this G-type has been expanding its distribution to Palau, Hawaii, Solomon Islands, Papua New Guinea, and other countries. In this study, we isolated natural enemy viruses from Palau populations in order to search for virus strains that could be used to control the G-type.
- Japan Society for the Promotion of Science：Grants-in-Aid for Scientific ResearchDate (from‐to) : 2015/04 -2017/03Author : Hisanori Bando, ASANO SHINICHIROBombyx mori nucleopolyhedrovirus (BmNPV) H4 that had genome sharing high sequence similarity (approx. 97%) with that of the type strain of BmNPV T3 showed marked growth advantage in silkworm larvae. Studies using chimeric recombinant BmNPV between H4 and T3 demonstrated that a viral membrane protein gene gp64 as a determinant in the in vivo fitness of BmNPV H4. Further analyses identified amino acid positions involved in the BmNPV H4 phenotype of efficient growth in silkworm larvae. These results suggested the possibility to control growth ability of BmNPV in silkworm larvae by gp64-targetted genome manipulation.
- Japan Society for the Promotion of Science：Grants-in-Aid for Scientific ResearchDate (from‐to) : 2014 -2016Author : ASANO SHINICHIROI have been conducting research aiming at elucidating the insecticidal activity mechanism of Cry44Aa toxin produced by Bacillus thuringiensis entomocidus INA 288, which is known to have highly insecticidal activity against Aedes aegypti and Culex pippins which intermediate tropical infectious diseases. As a result of research for Cry44Aa toxin receptor molecules in A. aegypti which play an important role in insecticidal activity mechanism, alkaline phosphatase and ABC transferase were identified as candidate molecules.
- Japan Society for the Promotion of Science：Grants-in-Aid for Scientific ResearchDate (from‐to) : 2012/04 -2015/03Author : MASNAORI Koike, AIUCHI Daigo, ASANO Shin-ichiro, MASUDA Toshio, SEKINE MuneyukiFungal entomopathogens have been widely investigated as biological control agents of pest insects in attempts to improve the sustainability of crop protection. Simultaneous biological control of both insect pests and plant pathogens (dual control) has been reported for the hypocrealean fungal entomopathogens, Beauveria bassiana, Metharizium spp. and Lecanicillium spp. And accumulating evidence shows that Beauveria spp. can colonize a wide array of plant species endophytically. Furthermore, traits that are important for insect pathogenicity are also involved in pathogenicity to plant pathogen and plant pathogenic nematode.
- Japan Society for the Promotion of Science：Grants-in-Aid for Scientific ResearchDate (from‐to) : 2010 -2012Author : ASANO ShinichiroTo find the novel Cry toxin with specific insecticidal activity in adult Coleoptera pests, we were screening by PCR and Bioassay from our B. thuringiensis strains’ library. As microbial control agent against Coleoptera adults to conduct for the scarab beetle adult and leaf beetles adult of Cry8D toxin, and to elucidate the insecticidal activity mechanism adult-specific, is a flame pest control, I develop a new BT agents.
- 病原微生物を用いた害虫防除科学研究費補助金Date (from‐to) : 2008 -2012
- Microbiol Pest ManagementGrant-in-Aid for Scientific ResearchDate (from‐to) : 2008 -2012
- 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Date (from‐to) : 2008
- Research for molecilar biology of Bacillus thuringiensis insecticidal crystal proteinCooperative ResearchDate (from‐to) : 2008
- Japan Society for the Promotion of Science：Grants-in-Aid for Scientific ResearchDate (from‐to) : 2003 -2006Author : BANDO Hisanori, ASANO Shinichiro, SAHARA KenBaculovirus and Bt (Bacillus thuringiensis) are used as microbial pesticides all over the world and the former also provide a most efficient foreign gene expression system in eukaryotes (baculovector system). We studied the molecular basis in the safety of these entomopathogenic microorganisms. Recent studies demonstrated that a baculovirus (AcMNPV) can enter the nucleus of mammalian cells independently of the cell cycle without multiplication and effectively express foreign genes without expressing its own genes, suggesting that the baculovirus can be used as a low risk viral vector in the field of gene therapy. Contrary to the general perception, we elucidated that AcMNPV expressed viral genes at least partly in mammalian cells through the usual infection pathway. In addition, the expression of cellular gene (at least β-actin) was upregulated in the mammalian cells inoculated with AcMNPV. However, the expression of viral genes was inhibited to low level in mammalian cells partly by histone deacetylation mechanism and further the transcription of some viral genes started at incorrect site(s). As for the B. thuringiensis (Bt) strains, we investigated mainly the mechanisms of Vero cell toxicity by non-hemolytic enterotoxin C (NheC) that was thought to be the most important enterotoxin causing food-poisoning. It was elucidated that NheC secreted from a clinical isolate of B. cereus (Bc) strain possessing Vero cell toxicity could attach to the surface of Vero cells through the N-terminal region of 30 amino acids. On the other hand, the corresponding N-terminal region was truncated possibly by post-translational cleavage in the NheC secreted from Bt strains used in the commercial products. In addition, the N-terminal truncated from of NheC didn't show Vero cell toxicity any more.
- A New approach to the practical use of insect-tolerant lines of sugar beet via foreign gene transferJapan Society for the Promotion of Science：Grants-in-Aid for Scientific ResearchDate (from‐to) : 1999 -2002Author : KANAZAWA Akira, IKEGUCHI Shojiro, ASANO Shin-ichiroThe larvae of cabbage armyworm (Mamestra brassicae L.) bring heavy damage on the final yield of beet-roots and subsequently, sugar production, by eating stems and leaves of sugar beet plants. In order to establish sugar beet cultivars that are tolerant to insects including cabbage armyworm, we have been developing transgenic sugar beet lines harboring cryIA(b), a gene encoding insecticide protein from Bacillus thuringiensis. In the present study, based on the results so far obtained, we tried to make transgenic sugar beet lines that show increased insecticide activity aiming at practical use of the transgenic lines. First, we introduced cryIC into sugar beet plants and analyzed the effect of its expression. We chose cryIC from various candidates of insecticide protein genes, since its gene product showed highest insecticide activity against cabbage armyworm among various insecticide proteins that were obtained by expressing cloned genes in E. coli. Meanwhile, reports were published that described that the introduction of insecticide protein genes into chloroplast gave increased accumulation of the protein and consequently, gave increased insecticide activity of plants. We, therefore, developed vectors available for chloroplast transformation and selection system of transformed chloroplast in order to introduce insecticide protein genes into sugar beet chloroplasts. We also succeeded in introducing several genes practically useful for sugar beet production and evaluated the effect of gene expression. In addition, we analyzed the stability of the expression of foreign genes in plant cells in order to make sure their experssion.
- Ministry of Education, Culture, Sports, Science and Technology：Grants-in-Aid for Scientific Research(基盤研究(B))Date (from‐to) : 1999 -2000Author : 飯塚 敏彦, Shin-ichiro ASANO, 浅野 眞一郎, Ken SAHARA1. Screening of a scarab-cidal B.thuringiensis (1999)In order to find of a scarab-cidal B.thuringiensis, we were screening our B.thuringiensis stocks.2. Analysis of the scarab-cidal B.thuringiensis strain (gallariae BBT2-8)(2000)We found the scarab-cidal Bt.BBT2-8, then we cloned the crystal protein genes (cryB28 gene) have a scarab-cidal activity. This cryB28 gene was expressed in E.coli and acrystariferous B.thuringiensis strain (BT51). The expression products had a highly activity against scarabe.3. Screening of a nematoda-cidal B.thuringiensis (1999-2000)In order to find of a nematoda-cidal B.thuringiensis, we were screening our B.thuringiensis stocks by using PCR.We could not find nematodacidal B.thuringiensis strains.4. Screening of novel lepidopteran-cidal B.thuringiensis (1999)In order to find of novel lepidopteran-cidal B.thuringiensis, we were screening our B.thuringiensis stocks. We picked up the novel lepidopteran acitive Bt strain (wuhanensis), then we cloned the novel cry1 gene (cry1Db1) from wuhanensis have insecticidal activities against Spodoptera litura, Plutella xylostella and Bombyx mori.
- 文部科学省：科学研究費補助金(萌芽的研究)Date (from‐to) : 1998 -1999Author : 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。
- Japan Society for the Promotion of Science：Grants-in-Aid for Scientific ResearchDate (from‐to) : 1997 -1999Author : SHIMAMOTO Yoshiya, KANAZAWA Akira, ASANO Shinichiro, SANO Yoshio1)Development of chloroplast transgenic system in sugarbeet We have progressed to develop a chloroplast transgenic system in sugarbeet in which is introduced cry genes to chloroplast genome and produce efficiently insecticidal crystal protein (ICP) in chloroplast. Plasmid carrying selective factor (aadA), marker factor (GUS), promoter (rrn) and terminator (rps 16) was incorporated into disc tissue of shoot of sugarbeet by microprojectile bombardment. In the tissue culture regeneration plant has not achieved. Repeatedly plasmid was constructed and incorporated into the sugarbeet. 2)Development of concentration system of protein upon organelle in sugarbeet We have progressed to develop a concentration system of toxic protein upon organelle in sugarbeet. Base sequence coding transit peptide to chloroplast was cloned from tobacco plant and was linked with position of N terminal in GUS gene. Thus the plasmid was constructed and introduced into sugarbeet by Agrobacterium method. 3)Expression of cry gene in transformant sugarbeet Several transformants carrying cryIA(b) were detected by PCR southern, ICP analysis and PCR southern blotting in half of plants regenerating after procedure of Agrobacterium method. Several leaves of these transformants were supplied for cabbage armyworm with the third larva age. Larva of cabbage armyworm was able to grow normally on leaves with non-cry gene and not to grow on several leaves of transformant. 4)Screening for efficient cry gene to produce insecticidal protein Screening for efficient cry gene to produce insecticidal protein was conducted for cabbage armyworm. CryIC gene has more insecticidal ability than cryIA(b) in larva of cabbage armyworm.
- 文部科学省：科学研究費補助金(基盤研究(C))Date (from‐to) : 1996 -1996Author : 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。
- Japan Society for the Promotion of Science：Grants-in-Aid for Scientific ResearchDate (from‐to) : 1995 -1996Author : BANDO Hisanori, SAHARA Ken, ASANO ShinichiroBombyx mori denso nucleosis virus (Yamanashi isolate ; BmDNV-2) is a small spherical virus with a bi-partite genome. In this study, to elucidate the nature of BmDNV-2, the viral DNA replication mechanism, genome organization and function of viral proteins were investigated. Analysis of the replicative intermediates (RI) of the viral DNA suggested the self-priming and hairpin-transfer model for parvovirus replication could not be applied to the replication of BmDNV-2. Nucleotide sequencing studies of the genomic DNAs showed that, unlike BmDNV-1 and the other parvoviruses the BmDNV-2 terminal sequences did not contained the large palindromic sequence which could form a stable hairpin structure. This was confirmed by the secondary structure prediction using a computer analyzes program. These results also suggested that the BmDNV-2 had a unique replication mechanisms other than the self-priming and hairpin-transfer mechanism. Furthermore, the sequence analyzes elucidated that the BmDNV-2 genome contained 6 major open reading frames (ORFs), 4 ORFs in VD1 and 2in VD2. Immunochemical studies demonstrated that the major four viral structural proteins (VP1,2,3 and 4) were encoded in the ORF2 of VD1. Followingly, the functions of the nonstructural proteins (NSPs) were investigated by transient expression assay using the luciferase as a reporter. The NSPs, p37 and p14 which were encoded in ORF3 and4 of VD1, respectively, transactivated the tentative promoter sequence for the major structural protein gene, suggesting that these proteins were concerned in the regulation of the expression fo structural proteins. In addition, p27 which was an NSP encoded in VD2 could transactivate all of the viral promoters implying that p27 is a key regulatory protein in the BmDNV-2 replication. In conclusion, this study elucidated the genome structure and the genome organization of the BmDNV-2. The results revealed that the BmDNV-2 is a new type of single-stranded DNA virus with a unique replication mechanism which was different from those of parvoviruses. Furthermore, the functional analyzes of the viral proteins suggested that the viral gene expression was regulated in the complicated manner by the proteins encoded in viral DNAs. And, p27 will be a key regulatory protein in the replication of the BmDNV-2 and may be one of the factor concerning in the determination of the host specificity.
- Japan Society for the Promotion of Science：Grants-in-Aid for Scientific ResearchDate (from‐to) : 1994 -1996Author : IIZUKA Toshihiko, SAHARA Ken, ASANO Shinichiro, BANDO HisanoriBecillus thuringiensis produces parasporal crystal protein which has insecticidal activity. This protein has been expressed by cry genes from B.thuringiensis. In the research. origonucleotide primers were synthesized depending on each cry gene specific region and DNA probes were prepared by PCR method. According to apply this method. cry genes from B.thuringiensis were easily and quickly isolated and identified. We have also isolated B.thuringiensis strains from soil samples in Hokkaido and Indonesia. Cry genes from these isolates were identified by using PCR method and a couple of novel cry genes were analyzed for nucleotide sequencing. By the result. cryV gene which has both of Lepidopteracidal and Coleopteracidal activities were found from serovar kurstaki INA-02. These results should be applied for microbial control agents in the near future.
- Japan Society for the Promotion of Science：Grants-in-Aid for Scientific ResearchDate (from‐to) : 1995 -1995Author : IIZUKA Toshihiko, HASTOWO Sugyo, LAY Bibiana W., SAHARA Ken, ASANO Shinichiro, BANDO HisanoriBacillus thuringiensis(Bt)is a gram-positive soil bacterium distinguished from other bacilli by its production of insecticidal protein. Because of their strong insecticidal activity and highly restricted host specificity, Bt has been used for microbiological control of plant-destroying insect. Recently, new Bts with unique characteristic traits have been isolated from various area in the world. Aim of our project is to isolate Bts with unique characteristics from Indonesia where is the habitant of huge number of insect species. In the last two years, we isolated many Bts from the soil collected from more than 50 areas in Indonesia. This year, we tried to obtain further Bts with unique properties from the areas where we have been able to obtain Bts before and characterized them. We newly obtained 17 Bt strains from 6 areas including forests and sericultural farms, resulting in obtaining more than 80 Bt isolates in the three years-collaboration. In the H-serotyping, the isolates were classified into three subspecies, alesti, kenyae and indiana. It was demonstrated by the following researches such as analysis of ICP by SDS-PAGE,ICP gene-typing by PCR method established in our Lab, and bio-assay of the host specificity that our Bt collection contained three unique isolates which had never been reported. The results from the studies on the properties of these three unique Bt isolates lead us to the tentative conclusions that the minor mutation in the ICP gene results in the change of host specificity and that there is new ICP gene which has quite different nucleotide sequence in nature.
- Japan Society for the Promotion of Science：Grants-in-Aid for Scientific ResearchDate (from‐to) : 1993 -1994Author : IIZUKA Toshihiko, LAY Bibiana w., SAHARA Ken, ASANO Shinichiro, BANDO HisanoriBacillus thuringiensis (BT) is known as the bacteria group which produces the insecticidal crystal proteins (ICP) and has been used for microbiological control of plant-destroying insect because of its highly restricted host specificity. Nowadays, new Bts with unique characteristic traits have been isolated from various areas in the world. Aim of our project is to isolate Bts from some areas in Indonesia where is a habitant of huge numbers of insect species. In the last year, we isolated many Bts from the soil under big trees as reported. This year, we tried to obtain further Bts from several areas and studied its biological characteristics. As a result, we obtained more than 30 BT isolates form about 50 areas containing forest and sericultural field. In the H-serotyping, the isolates were classified into subspp. thuringiensis, sotto, darmstadiensis, aizawai but some were untypable. In the two years-collaboration, we obtained more than 60 BT isolates already and analyzed gene construction especially on crystal protein (cry) gene by PCR method established in our laboratory. Furthermore, the host specificity of the isolates were studied. The results of the experiments showed that the classification by H-serotype is not always reflected in the grouping of BT by its host spectrum. For example, some isolates obtained from Indonesia had the insecticidal activity against Spodoptera litura but it was classified into subsp. kurstaki which was a group of BT without insecticidal activity against the insect. One of those isolates, INA02, had the cryV gene which seemed to have an activity against S.litura. Then we cloned and determined the nucleotide sequence of the cryV gene of INA02. On the other hand, we found another new type of BT which had the insecticidal activity against Aedes japonicus. We identified a cryII gene in this isolate (SKW isolate) which showed the sequence homology to the cryIIA gene. The function of this gene is under investigation. Another isolate (INA67) which was classified into subspp. tenebrionis showed the insecticidal activity against a coleoptera. However, this isolate contained no cryIII or V gene which were reported as the coleopteracidal crystal protein genes.
- 文部科学省：科学研究費補助金(奨励研究(A))Date (from‐to) : 1993 -1993Author : 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。
- 日本学術振興会：科学研究費助成事業Date (from‐to) : 1992 -1992Author : 伴戸 久徳, 浅野 真一郎家蚕濃核病ウイルスには現在、伊那株(BmDNV-1)と山梨株(BmDNV-2)に代表される2種類のウイルスが存在し、これらは品種特異性などの点で著しく異なっている。本研究ではこれらのウイルスの違いを分子生物学的な基盤に於いて明らかにするとともに、濃核病発生予防のための基礎的情報を得る目的で以下の実験を行った。 BmDNV-1はすでにその遺伝子構造及び複製様式からパルボウイルスに属する事が判明している。ところが、BmDNV-2に於いてはゲノム末端にヘアピン構造は認められず複製様式に於いてパルボウイルスとは異なっている事が推定された。本ウイルスの分類上の位置づけを明瞭にするためにはウイルスDNAの遺伝情報に関して明らかにする必要がある。そこで2種類のゲノムDNA(VD1,VD2)の塩基配列の解析を行った。まず、VD2に関してその全塩基配列を決定した。VD2は約6000塩基からなりその塩基組成には片寄りがみられた。すなわち末端領域は非常にG-Crichでありそれ以外の領域は逆に非常にA-Trichであった。また、本配列には少なくとも2個の大きなオープン・リーディング・フレーム(ORF)が認められ、これらは別々のDNA鎖に存在した。これらのORFにコードされるタンパク質に関して解析を進めるためそれらに対するペプチド抗体を作成中である。VD1に於いてもVD2同様塩基配列の片寄りが認められた。現在、遺伝情報の解析中である。以上の結果は、BmDNV-2がこれまで知られているDNVとは全く異なるタイプのウイルスである事を示している。一方、BmDNV-1とBmDNV-2に於いて明かとなった塩基配列を基に合成したオリゴヌクレオチドを用いたPCR法の応用を検討したところ、極めてて高感度でそれぞれの塩基配列が検出可能であり、この方法の診断及び疫学への応用が可能であると考えられた。
- Japan Society for the Promotion of Science：Grants-in-Aid for Scientific ResearchDate (from‐to) : 1991 -1992Author : IIZUKA Toshihiko, KIKUTA Harunori, ASANO Shinichiro, BANDO HisanoriA insecticidal crystal protein (ICP) produced by Bacillus thuringiensis (BT), which shows highly activity against the Lepidopteran insect, should be studied from the following two ways: 1. Newly isolation and identification of BT. : Newly BT isolates from soil samples which were collected in the natural conserved area, were identified by routine technique of H-serotype and by newly developed technique depending on agarose gel electrophoresis of cryl genes. After oligo-primers designed by a specific domain were synthesized, these cryI genes were amplified by PCR method with template DNA from BT subspecies. The newly identification technique was very effective in order to clear a high active strain comparing with already known BT strains. 2. Nucleotide sequence analysis of ICP genes. : BT subsp. wuhanensis has been known as high active strain against the silkworm and the cabbage moth. According to a report of Iizuka et al. (1993), subsp. wuhanensis has included cryIA(b), cryIA(c) and cryID genes. In this study, the nucleotide sequence of cryIA(b) was analyzed and compared with another cryIA(b) genes from subspp. thuringiensis berliner 1715 and kurstaki HD-2. The differentiation of amino acid composition between wuhanensis and HD-2 was only one within a active domain. A regulatory mechanisms of each protein expression encoded by three genes was in progressed.
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