2018年12月, 平成30年度茨城大学学長表彰, 茨城大学

2017年11月, 平成29年度(第16回)日本農学進歩賞

A novel monocarboxylate transporter involved in 3-hydroxykynurenine transport for ommochrome coloration
Hirosumi Uchiyama; Yoko Takasu; Minoru Moriyama; Kazutoshi Yoshitake; Hironobu Uchiyama; Tetsuya Iizuka; Keiro Uchino; Genta Okude; Yutaka Banno; Seigo Kuwazaki; Kimiko Yamamoto; Shunsuke Yajima; Hideki Sezutsu; Toshiki Tamura; Ryo Futahashi; Mizuko Osanai-Futahashi, ラスト(シニア)オーサー
2023年06月

昆虫の変異体から読み解くオモクローム色素による着色機構とその応用
二橋 美瑞子, 筆頭著者
蚕糸・昆虫バイオテック, 2022年08月, ［招待有り］

Diversity of melanin synthesis genes in insects
Ryo Futahashi; Shigeyuki Koshikawa; Genta Okude; Mizuko Osanai-Futahashi, Elsevier
Advances in Insect Physiology, 2022年04月18日, ［査読有り］

〔主要な業績〕The red egg gene as a novel effective egg color marker for silkworm transgenesis
Mizuko Osanai-Futahashi; Keiro Uchino; Toshiki Tamura and Hideki Sezutsu, 筆頭著者, Ommochromes are major pigments involved in coloration of eggs, eyes, and epidermis of arthropods. The recessive homozygous of egg and eye color mutant of Bombyx mori, red egg (re), exhibits red eggs and dark red eyes instead of normal purplish-brown eggs and black eyes, due to a defect in ommochrome pigment synthesis. The gene responsible for the re mutant is a major facilitator superfamily transporter gene, Bm-re. Here, we demonstrate that the re phenotype can be effectively rescued by an intact Bm-re gene driven by the Bombyx Actin A3 promoter or the baculovirus Immediate Early 1 promoter, indicating that the Bm-re gene can be used as a marker gene for visual screening of transgenic silkworms. The coloration of eggs rescued by the Bm-re transgene could be distinguished from that of host mutant eggs from diapausing period through head pigmentation stage. This allows transgenic screening at earlier embryonic stages and over a longer time period compared to conventional 3xP3 fluorescent markers, without requiring the skill and equipment to detect stemmata fluorescence., Elsevier
Insect Biochemistry and Molecular Biology, 2022年01月25日, ［査読有り］

〔主要な業績〕Mutations in a β-group of solute carrier gene are responsible for egg and eye coloration of the brown egg 4 (b-4) mutant in the silkworm, Bombyx mori
Tomihara K; Satta K; Matsuzaki S; Yoshitake K; Yamamoto K; Uchiyama H; Yajima S; Futahashi R; Katsuma S; Osanai-Futahashi M; Kiuchi T., 責任著者, Elsevier
Insect Biochemistry and Molecular Biology, 2021年07月29日, ［査読有り］

A novel partial agonist of GPBA reduces blood glucose level in a murine glucose tolerance test
Rina Enomoto; Aya Kurosawa; Yoshiaki Nikaido; Misaki Mashiko; Toshihiko Saheki; Nozomi Nakajima; Satoshi Kuroiwa; Michinari Otobe; Maki Ohsaki; Kazuya Tooyama; Yusuke Inoue; Nobuo Kuwabara; Osamu Kikuchi; Tadahiro Kitamura; Itaru Kojima; Yuko Nakagawa; Tamio Saito; Hiroyuki Osada; Mizuko Futahashi; Hideki Sezutsu; Shigeki Takeda, GPBA is a G protein-coupled receptor that is activated by bile acids. Because activation of GPBA leads to increased cAMP levels and secretion of incretins and insulin, GPBA has been proposed as a promising drug target for the treatment of metabolic syndrome. Previously, we have developed a ligand-screening system to identify novel agonists of GPBA by means of a fusion protein of GPBA with G protein stimulatory a subunit (Gs alpha) and by a [S-35] GTP gamma S-binding assay. To express the GPBA-Gs alpha fusion protein, transgenic silkworms were employed in this study, and cell membrane fractions were prepared from their fat body or pupae. We applied them to the screening of a chemical library that contains 10,625 compounds from the RIKEN Natural Products Depository (NPDepo). Eventually, a unique partial agonist, GUM2, was successfully identified. Our results indicated that the GPCR-G alpha fusion proteins were beneficial for ligand identification and that the transgenic silkworms were useful for large-scale production of GPCRs. In HEK293 cells transiently expressing GPBA, GUM2 showed 50% effective concentration (EC50) of 3.5 +/- 2.4 mu M and induced GPBA internalization as effectively as did an endogenous agonist, TLC. We also confirmed that GUM2 stimulates insulin secretion in MIN6 cells. Moreover, a single 2 mg/kg dose of GUM2 significantly reduced blood glucose levels in mice during an intraperitoneal glucose tolerance test even though GUM2 is only a partial agonist with a low intrinsic activity. We concluded that GUM2 is a good candidate for research on GPBA signaling under physiological conditions and for the development of GPBA-targeting therapeutic compounds., ELSEVIER SCIENCE BV
EUROPEAN JOURNAL OF PHARMACOLOGY, 2017年11月, ［査読有り］

RNAseqから明らかになった昆虫の色素合成と色覚の新知見
二橋美瑞子; 二橋亮, 筆頭著者, 日本農芸化学会 ; 1962-
化学と生物, 2016年05月20日, ［査読有り］, ［招待有り］

Positional cloning of a Bombyx pink-eyed white egg locus reveals the major role of cardinal in ommochrome synthesis
M. Osanai-Futahashi; K-i Tatematsu; R. Futahashi; J. Narukawa; Y. Takasu; T. Kayukawa; T. Shinoda; T. Ishige; S. Yajima; T. Tamura; K. Yamamoto; H. Sezutsu, 筆頭著者, Ommochromes are major insect pigments involved in coloration of compound eyes, eggs, epidermis and wings. In the silkworm Bombyx mori, adult compound eyes and eggs contain a mixture of the ommochrome pigments such as ommin and xanthommatin. Here, we identified the gene involved in ommochrome biosynthesis by positional cloning of B. mori egg and eye color mutant pink-eyed white egg (pe). The recessive homozygote of pe has bright red eyes and white or pale pink eggs instead of a normal dark coloration due to the decrease of dark ommochrome pigments. By genetic linkage analysis, we narrowed down the pe-linked region to similar to 258 kb, containing 17 predicted genes. RNA sequencing analyses showed that the expression of one candidate gene, the ortholog of Drosophila haem peroxidase cardinal, coincided with egg pigmentation timing, similar to other ommochrome-related genes such as Bm-scarlet and Bm-re. In two pe strains, a common missense mutation was found within a conserved motif of B. mori cardinal homolog (Bm-cardinal). RNA interference-mediated knockdown and transcription activator-like effector nuclease (TALEN)-mediated knockout of the Bm-cardinal gene produced the same phenotype as pe in terms of egg, adult eye and larval epidermis coloration. A complementation test of the pe mutant with the TALEN-mediated Bm-cardinal-deficient strain showed that the mutant phenotype could not be rescued, indicating that Bm-cardinal is responsible for pe. Moreover, knockdown of the cardinal homolog in Tribolium castaneum also induced red compound eyes. Our results indicate that cardinal plays a major role in ommochrome synthesis of holometabolous insects., NATURE PUBLISHING GROUP
HEREDITY, 2016年02月, ［査読有り］

A Major Facilitator Superfamily protein encoded by TcMucK gene is not required for cuticle pigmentation, growth and development in Tribolium castaneum.
Seulgi Mun; Mi Young Noh; Mizuko Osanai-Futahashi; Subbaratnam Muthukrishnan; Karl J. Kramer; Yasuyuki Arakane, Insect cuticle pigmentation and sclerotization (tanning) are vital physiological processes for insect growth, development and survival. We have previously identified several colorless precursor molecules as well as enzymes involved in their biosynthesis and processing to yield the mature intensely colored body cuticle pigments. A recent study indicated that the Bombyx mori (silkmoth) gene, BmMucK, which encodes a protein orthologous to a Culex pipiens quiquefasciatus (Southern house mosquito) cis,cis, muconate transporter, is a member of the "Major Facilitator Superfamily" (MFS) of transporter proteins and is associated with the appearance of pigmented body segments of naturally occurring body color mutants of B. mori. While RNA interference of the BmMucK gene failed to result in any observable phenotype, RNAi using a dsRNA for an orthologous gene from the red flour beetle, Tribolium castaneum, was reported to result in molting defects and darkening of the cuticle and some body parts, leading to the suggestion that orthologs of MucK genes may differ in their functions among insects. To verify the role and essentiality of the ortholog of this gene in development and body pigmentation function in T castaneum we obtained cDNAs for the orthologous gene (TcMucK) from RNA isolated from the GA-1 wild-type strain of T. castaneum. The sequence of a 1524 nucleotides-long cDNA for TcMucK which encodes the putatively full-length protein, was assembled from two overlapping RT-PCR fragments and the expression profile of this gene airing development was analyzed by real-time PCR. This cDNA encodes a 55.8 kDa protein consisting of 507 amino acid residues and includes 11 putative transmembrane segments. Transcripts of TcMucK were detected throughout all of the developmental stages analyzed. The function of this gene was explored by injection of two different double-stranded RNAs targeting different regions of the TcMucK gene (dsTcMucKs) into young larvae to down-regulate transcripts during subsequent stages of insect development until the adult stage. RNA interference of TcMucK had no observable effects on larval, pupal or adult pigmentation. In addition, it did not affect larval-larval, larval-pupal and pupal-adult molting or survival. Thus, in contrast to the results of Zhao et al. (2012), our study demonstrates that TcMucK is not essential for growth, development or cuticle pigmentation of T. castaneum. (C) 2014 Elsevier Ltd. All rights reserved., PERGAMON-ELSEVIER SCIENCE LTD
Insect Biochemistry and Molecular Biology, 2014年, ［査読有り］

Large Scale Full-Length cDNA Sequencing Reveals a Unique Genomic Landscape in a Lepidopteran Model Insect, Bombyx mori
Yoshitaka Suetsugu; Ryo Futahashi; Hiroyuki Kanamori; Keiko Kadono-Okuda; Shun-ichi Sasanuma; Junko Narukawa; Masahiro Ajimura; Akiya Jouraku; Nobukazu Namiki; Michihiko Shimomura; Hideki Sezutsu; Mizuko Osanai-Futahashi; Masataka G Suzuki; Takaaki Daimon; Tetsuro Shinoda; Kiyoko Taniai; Kiyoshi Asaoka; Ryusuke Niwa; Shinpei Kawaoka; Susumu Katsuma; Toshiki Tamura; Hiroaki Noda; Masahiro Kasahara; Sumio Sugano; Yutaka Suzuki; Haruhiko Fujiwara; Hiroshi Kataoka; Kallare P Arunkumar; Archana Tomar; Javaregowda Nagaraju; Marian R Goldsmith; Qili Feng; Qingyou Xia; Kimiko Yamamoto; Toru Shimada; Kazuei Mita, Abstract

The establishment of a complete genomic sequence of silkworm, the model species of Lepidoptera, laid a foundation for its functional genomics. A more complete annotation of the genome will benefit functional and comparative studies and accelerate extensive industrial applications for this insect. To realize these goals, we embarked upon a large-scale full-length cDNA collection from 21 full-length cDNA libraries derived from 14 tissues of the domesticated silkworm and performed full sequencing by primer walking for 11,104 full-length cDNAs. The large average intron size was 1904 bp, resulting from a high accumulation of transposons. Using gene models predicted by GLEAN and published mRNAs, we identified 16,823 gene loci on the silkworm genome assembly. Orthology analysis of 153 species, including 11 insects, revealed that among three Lepidoptera including Monarch and Heliconius butterflies, the 403 largest silkworm-specific genes were composed mainly of protective immunity, hormone-related, and characteristic structural proteins. Analysis of testis-/ovary-specific genes revealed distinctive features of sexual dimorphism, including depletion of ovary-specific genes on the Z chromosome in contrast to an enrichment of testis-specific genes. More than 40% of genes expressed in specific tissues mapped in tissue-specific chromosomal clusters. The newly obtained FL-cDNA sequences enabled us to annotate the genome of this lepidopteran model insect more accurately, enhancing genomic and functional studies of Lepidoptera and comparative analyses with other insect orders, and yielding new insights into the evolution and organization of lepidopteran-specific genes., Oxford University Press (OUP)
G3: Genes, Genomes, Genetics, 2013年09月01日, ［査読有り］

Efficient TALEN construction for Bombyx gene targeting.
Yoko Takasu; Suresh Sajwan; Takaaki Daimon; Mizuko Osanai-Futahashi; Keiro Uchino; Hideki Sezutsu; Toshiki Tamura; Michal Zurovec, Engineered nucleases are artificial enzymes able to introduce double stranded breaks at desired genomic locations. The double stranded breaks start the error-prone repair process of non-homologous end-joining (NHEJ), which eventually leads to the induction of mutations at target sites. We showed earlier that ZFNs and TALENs are able to induce NHEJ mutations in the B. mori genome. In order to optimize our mutagenesis protocol, we modified one of the reported truncated TALEN scaffolds and optimized it for use in the B. mori embryo. We also established a novel B. mori somatic cell assay suitable for the preselection of highly efficient TALENs directly in the B. mori model system. We compared the efficiency of several TALEN pairs based on three different frameworks using the BmBLOS2 gene. The new active TALENs show one order of magnitude higher efficiency than those we used previously. We confirmed the utility of our improved protocol by mutagenesis of the autosomal gene, red egg (Bm-re) and showed that it allows obtaining homozygous mutants in G1. Our procedure minimizes the chance of failure in B. mori gene targeting experiments. © 2013 Takasu et al.
PLoS ONE, 2013年, ［査読有り］

オモクローム色素合成の最新の知見―ショウジョウバエのパラダイムを超えて―　　　　　　　　　　　　　　　
二橋美瑞子, 筆頭著者
蚕糸・昆虫バイオテック, 2013年, ［招待有り］

Identification of the Bombyx red egg gene reveals the involvement of a novel transporter family gene in the late steps of the insect ommochrome biosynthesis pathway.
Mizuko Osanai-Futahashi; Ken-ichiro Tatematsu; Kimiko Yamamoto; Junko Narukawa; Keiro Uchino; Takumi Kayukawa; Tetsuro Shinoda; Yutaka Banno; Toshiki Tamura; Hideki Sezutsu, 筆頭著者, Ommochromes are one of the major pigments involved in coloration of eggs, eyes, and body surface of insects. However, the molecular mechanisms of the final steps of ommochrome pigment synthesis have been largely unknown. The eggs of the silkworm Bombyx mori contain a mixture of ommochrome pigments, and exhibit a brownish lilac color. The recessive homozygous of egg and eye color mutant, red egg (re), whose eggs display a pale orange color instead of normal dark coloration, has been long suggested to have a defect in the biosynthesis of the final ommochrome pigments. Here, we identify the gene responsible for the re locus by positional cloning, mutant analysis, and RNAi experiments. In the re mutants, we found that a 541-bp transposable element is inserted into the ORF of BGIBMGA003497-1 (Bm-re) encoding a novel member of a major facilitator super-family transporter, causing disruption of the splicing of exon 9, resulting in two aberrant transcripts with frameshifts yielding nonfunctional proteins lacking the C-terminal transmembrane domains. Bm-re function in pigmentation was confirmed by embryonic RNAi experiments. Homologs of the Bm-re gene were found in all insect genomes sequenced at present, except for 12 sequenced Drosophila genomes, which seemed to correlate with the previous studies that have demonstrated that eye ommochrome composition is different from other insects in several Dipterans. Knockdown of the Bm-re homolog by RNAi in the red flour beetle Tribolium castaneum caused adult compound eye coloration defects, indicating a conserved role in ommochrome pigment biosynthesis at least among holometabolous insects., AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
Journal of Biological Chemistry, 2012年, ［査読有り］

A visible dominant marker for insect transgenesis.
Mizuko Osanai-Futahashi; Takahiro Ohde; Junya Hirata; Keiro Uchino; Ryo Futahashi; Toshiki Tamura; Teruyuki Niimi; Hideki Sezutsu, 筆頭著者, Transgenesis of most insects currently relies on fluorescence markers. Here we establish a transformation marker system causing phenotypes visible to the naked eye due to changes in the color of melanin pigments, which are widespread in animals. Ubiquitous overexpression of arylalkylamine-N-acetyl transferase in the silkworm, Bombyx mori, changes the color of newly hatched first-instar larvae from black to a distinctive light brown color, and can be used as a molecular marker by directly connecting to baculovirus immediate early 1 gene promoter. Suppression of black pigmentation by Bm-arylalkylamine-N-acetyl transferase can be observed throughout the larval stages and in adult animals. Alternatively, overexpression in another gene, B. mori beta-alanyl-dopamine synthetase (Bm-ebony), changes the larval body color of older instars, although first-instar larvae had normal dark coloration. We further show that ectopic Bm-arylalkylamine-N-acetyl transferase expression lightens coloration in ladybird beetle Harmonia axyridis and fruit fly Drosophila melanogaster, highlighting the potential usefulness of this marker for transgenesis in diverse insect taxa., NATURE PUBLISHING GROUP
Nature Communications, 2012年, ［査読有り］

Coevolution of telomeric repeats and telomeric repeat-specific non-LTR retrotransposons in insects.
Mizuko Osanai-Futahashi; Haruhiko Fujiwara, 筆頭著者, In the telomeres of the silkworm Bombyx mori, telomeric repeat-specific non-long terminal repeat (LTR) retrotransposon SARTBm1 is accumulated in the TTAGG telomeric repeats. Here, we identify novel telomeric repeat-specific non-LTR retrotransposons, SARTTc family, from the red flour beetle Tribolium castaneum in the unconventional TCAGG telomeric repeats. To compare the sequence specificity of SARTBm1 and SARTTc1, we developed a comparable ex vivo retrotransposition assay. Both SARTBm1 and SARTTc1 preferred the telomeric sequence of their hosts, suggesting that the target specificity of these retrotransposons coevolved with their host's telomeric repeats. Swapping experiment indicated that the endonuclease domain is involved in recognizing the target sequence. Moreover, SARTBm1 proteins could retrotranspose 3'untranslated region (UTR) sequence of SARTTc1 as well as their own 3'UTR, whereas SARTTc1 proteins could only retrotranspose their own 3'UTRs. These results provide insights to the mechanism and divergence of sequence specificity and 3'UTR recognition in non-LTR retrotransposons., OXFORD UNIV PRESS
Molecular Biology and Evolution, 2011年, ［査読有り］

Structural basis for telomerase catalytic subunit TERT binding to RNA template and telomeric DNA
Meghan Mitchell; Andrew Gillis; Mizuko Futahashi; Haruhiko Fujiwara; Emmanuel Skordalakes, Telomerase is a specialized DNA polymerase that extends the 3′ ends of eukaryotic linear chromosomes, a process required for genomic stability and cell viability. Here we present the crystal structure of the active Tribolium castaneum telomerase catalytic subunit, TERT, bound to an RNA-DNA hairpin designed to resemble the putative RNA-templating region and telomeric DNA. The RNA-DNA hybrid adopts a helical structure, docked in the interior cavity of the TERT ring. Contacts between the RNA template and motifs 2 and B′ position the solvent-accessible RNA bases close to the enzyme active site for nucleotide binding and selectivity. Nucleic acid binding induces rigid TERT conformational changes to form a tight catalytic complex. Overall, TERT-RNA template and TERT-telomeric DNA associations are remarkably similar to those observed for retroviral reverse transcriptases, suggesting common mechanistic aspects of DNA replication between the two families of enzymes. © 2010 Nature America, Inc. All rights reserved.
Nature Structural and Molecular Biology, 2010年04月, ［査読有り］

Unique functions of repetitive transcriptomes.
Gerald G. Schumann; Elena V. Gogvadze; Mizuko Osanai-Futahashi; Azusa Kuroki; Carsten Münk; Haruhiko Fujiwara; Zoltan Ivics; Anton A. Buzdin, Repetitive sequences occupy a huge fraction of essentially every eukaryotic genome Repetitive sequences cover more than 50% of mammalian genomic DNAs, whereas gene exons and protein coding sequences occupy only similar to 3% and 1%, respectively Numerous genomic repeats include genes themselves They generally encode "selfish" proteins necessary for the proliferation of transposable elements (TEs) in the host genome The major part of evolutionary "older" TEs accumulated mutations over time and fails to encode functional proteins However, repeats have important functions also on the RNA level Repetitive transcripts may serve as multifunctional RNAs by participating in the antisense regulation of gene activity and by competing with the host encoded transcripts for cellular factors In addition, genomic repeats include regulatory sequences like promoters, enhancers, splice sites, polyadenylation signals, and insulators, which actively reshape cellular transcriptomes TE expression is tightly controlled by the host cells, and some mechanisms of this regulation were recently decoded Finally, capacity of TEs to proliferate in the host genome led to the development of multiple biotechnological applications, ELSEVIER ACADEMIC PRESS INC
International Review of Cell and Molecular Biology, 2010年

The genome of the model beetle and pest Tribolium castaneum
Springer Science and Business Media LLC
Nature, 2008年03月23日

The genome of a lepidopteran model insect, the silkworm Bombyx mori.
International Silkworm Genome Consortium, Bombyx mori, the domesticated silkworm, is a major insect model for research, and the first lepidopteran for which draft genome sequences became available in 2004. Two independent data sets from whole-genome shotgun sequencing were merged and assembled together with newly obtained fosmid- and BAC-end sequences. The remarkably improved new assembly is presented here. The 8.5-fold sequence coverage of an estimated 432 Mb genome was assembled into scaffolds with an N50 size of similar to 3.7 Mb; the largest scaffold was 14.5 million base pairs. With help of a high-density SNP linkage map, we anchored 87% of the scaffold sequences to all 28 chromosomes. A particular feature was the high repetitive sequence content estimated to be 43.6% and that consisted mainly of transposable elements. We predicted 14,623 gene models based on a CLEAN-based algorithm, a more accurate prediction than the previous gene models for this species. Over three thousand silkworm genes have no homologs in other insect or vertebrate genomes. Some insights into gene evolution and into characteristic biological processes are presented here and in other papers in this issue. The massive silk production correlates with the existence of specific IRNA clusters, and of several sericin genes assembled in a cluster. The silkworm's adaptation to feeding on mulberry leaves, which contain toxic alkaloids, is likely linked to the presence of new-type sucrase genes, apparently acquired from bacteria. The silkworm genome also revealed the cascade of genes involved in the juvenile hormone biosynthesis pathway, and a large number of cuticular protein genes. (C) 2008 Elsevier Ltd. All rights reserved., PERGAMON-ELSEVIER SCIENCE LTD
Insect Biochemistry and Molecular Biology, 2008年, ［査読有り］

Genome-wide screening and characterization of transposable elements and their distribution analysis in the silkworm, Bombyx mori.
Mizuko Osanai-Futahashi; Yoshitaka Suetsugu; Kazuei Mita; Haruhiko Fujiwara, 筆頭著者, To elucidate the contribution of transposable elements (TEs) to the silkworm genome structure and evolution, we have conducted genome-wide analysis of TEs using the newly released genome assembly. The TEs made up 35% of the genome and contributed greatly to the genome size. Non-long terminal repeat retrotransposons (non-LTRs) and short interspersed nuclear elements (SINEs) were the predominant TE classes. From characterization of the TE distribution in the genome, it was revealed that non-LTRs, especially R1 clade elements, are frequently inserted into GC-rich regions. The GC content of non-LTRs themselves was over 40%, which indicate their contribution to the GC content of the insertion region. TEs accumulated in regions with low gene density, and there were relatively strong positive correlations between TE density and chromosomal recombination rate, We also characterized the clade distribution of the non-LTRs. The silkworm non-LTRs represented 10 of the 16 previously defined clades, which had the most variety than that reported for other genomes. Two partial CRE clade elements were found, which is one of the most ancient lineages of non-LTRs, and have been only found in Trypanosoma and fungi before. This analysis suggests that Bombyx genome is influenced by numerous amounts and variety of TEs. (C) 2008 Elsevier Ltd. All rights reserved., PERGAMON-ELSEVIER SCIENCE LTD
Insect Biochemistry and Molecular Biology, 2008年, ［査読有り］

転移因子によるゲノム言語の構築と進化　　　　　　　　　　　　　　　
二橋美瑞子、藤原晴彦
実験医学, 2008年

A novel target-specific gene delivery system combining baculovirus and sequence-specific long interspersed nuclear elements.
Tomoko Kawashima; Mizuko Osanai; Ryo Futahashi; Tetsuya Kojima; Haruhiko Fujiwara, Transposable elements are valuable for somatic and germ-line transformation. However, long interspersed nuclear elements (LINEs) have not been used because of poor information on the transposition mechanism. We have developed a novel gene delivery system combining baculovirus AcNPV and two silkworm LINEs, SART1 and R1, which integrate into specific sequences of telomeric repeats and 28S ribosomal DNA, respectively. When two LINEs containing the enhanced green fluorescent protein gene recombined into AcNPV were infected into fifth instar larvae of the silkworm, we observed target-specific retrotransposition of LINEs at 72 h post-infection, using polymerase chain reaction amplification and sequencing. Telomere- and 28S rDNA-specific transposition occurred in all nine tissues tested, including the ovary and testis. This is the first demonstration of site-specific gene delivery in living larvae. Insertion efficiencies were dependent on the virus titer for injection and the host strains of Bombyx mori. Using this system, we successfully detected the intergeneration transmission of retrotransposed sequences. In addition, AcNPV-mediated SART1 also transposed into telomere of another lepidopteran, Orgyia recens, suggesting that this system is useful for a wide variety of AcNPV-infectious insects. Site-specific gene delivery by virus-mediated LINE will be a potential gene therapy tool to avoid harmful unexpected insertions. (C) 2007 Elsevier B.V. All rights reserved., ELSEVIER SCIENCE BV
Virus Research, 2007年, ［査読有り］

Characterization of the sequence specificity of the R1Bm endonuclease domain by structural and biochemical studies.
Nobuo Maita; Hideyuki Aoyagi; Mizuko Osanai; Masahiro Shirakawa; Haruhiko Fujiwara, R1Bm is a long interspersed element (LINE) inserted into a specific sequence within 28S rDNA of the silkworm genome. Of two open reading frames (ORFs) of R1Bm, ORF2 encodes a reverse transcriptase (RT) and an endonuclease (EN) domain which digests specifically both top and bottom strand of the target sequence in 28S rDNA. To elucidate the sequence specificity of EN domain of R1Bm (R1Bm EN), we examined the cleavage tendency for the target sequences, and found that 50-A(G/C)(A/T)!(A/G) T-30 is the consensus sequence (!= cleavage site). We also determined the crystal structure of R1Bm EN at 2.0 angstrom resolution. Its structure was basically similar to AP endonuclease family, but had a special beta-hairpin at the edge of the DNA binding surface, which is a common feature among EN of LINEs. Point-mutations on the DNA binding surface of R1Bm EN significantly decreased the cleavage activities, but did not affect the sequence recognition in most residues. However, two mutants Y98A and N180A had altered cleavage patterns, suggesting an important role of these residues (Y98 and N180) for the sequence recognition of R1Bm EN. In addition, Y98A mutant showed another cleavage pattern, that implies de novo design of novel sequence-specific EN., OXFORD UNIV PRESS
Nucleic Acids Research, 2007年, ［査読有り］

Identification and characterization of the telomerase reverse transcriptase of Bombyx mori (silkworm) and Tribolium castaneum (flour beetle).
Mizuko Osanai; Kenji K Kojima; Ryo Futahashi; Satoshi Yaguchi; Haruhiko Fujiwara, 筆頭著者, Chromosomal ends of most eukaryotes are composed of simple telomeric repeats. Arthropod telomeres are generally constituted by TTAGG pentanucleotide repeats; however, some insect species including Drosophila melanogaster do not have telomeric repeats. In contrast, the domestic silkworm Bombyx mori contains TTAGG-type telomeric repeats, but the telomerase activity has not been detected in all investigated tissues. To search for a cause of unusual telomere structure in insects, we here identified telomerase reverse transcriptase (TERT) subunit from the domestic silkworm B. mori and the flour beetle Tribolium castaneum. This is the first report of telomerase genes from arthropods. The domestic silkworm TERT gene (BnzoTERT) and the flour beetle TERT gene (TcasTERT) both did not have the N-terminal GQ motif Comparison between cDNA and genomic DNA of BmoTERT revealed that it includes no introns. BmoTERT contains five ATG codons in its 5'UTR, which could reduce the translation of ElmoTERT proteins. Also, Northern hybridization indicated that BmoTERT is transcribed at a very low level. These unique features of BmoTERT possibly explain the undetectable Bombyx telomerase activity. (c) 2006 Elsevier B.V. All rights reserved., ELSEVIER SCIENCE BV
Gene, 2006年, ［査読有り］

Functional roles of 3'-terminal structures of template RNA during in vivo retrotransposition of non-LTR retrotransposon, R1Bm
Tomohiro Anzai; Mizuko Osanai; Mitsuhiro Hamada; Haruhiko Fujiwara, R1Bm is a non-LTR retrotransposon found specifically within 28S rRNA genes of the silkworm. Different from other non-LTR retrotransposons encoding two open reading frames (ORFs), R1Bm structurally lacks a poly (A) tract at its 3' end. To study how R1Bm initiates reverse transcription from the poly (A)-less template RNA, we established an in vivo retrotransposition system using recombinant baculovirus, and characterized retrotransposition activities of R1Bm. Target-primed reverse transcription (TPRT) of R1Bm occurred from the cleavage site generated by endonuclease (EN). The 147 bp of 3'-untranslated region (3'UTR) was essential for efficient retrotransposition of R1Bm. Even using the complete R1Bm element, however, reverse transcription started from various sites of the template RNA mostly with 5'-UG-3' or 5'-UGU-3' at their 3' ends, which are presumably base-paired with 3' end of the EN-digested 28S rDNA target sequence, 5'-AGTAGATAGGGACA-3'. When the downstream sequence of 28S rDNA target was added to the 3' end of R1 unit, reverse transcription started exactly from the 3' end of 3'UTR and retrotransposition efficiency increased. These results indicate that 3'-terminal structure of template RNA including read-through region interacts with its target rDNA sequences of R1Bm, which plays important roles in initial process of TPRT in vivo., OXFORD UNIV PRESS
Nucleic Acids Research, 2005年, ［査読有り］

Essential domains for ribonucleoprotein complex formation required for retrotransposition of telomere-specific non-long terminal repeat retrotransposon SART1.
Takumi Matsumoto; Mitsuhiro Hamada; Mizuko Osanai; Haruhiko Fujiwara
Molecular and Cellular Biology, 2005年, ［査読有り］

Telomere-specific non-LTR retrotransposons and telomere maintenance in the silkworm, Bombyx mori.
Haruhiko Fujiwara; Mizuko Osanai; Takumi Matsumoto; Kenji K. Kojima, Most insects have telomeres that consist of pentanucleotide (TTAGG) telomeric repeats, which are synthesized by telomerase. However, all species in Diptera so far examined and several species in other orders of insect have lost the (TTAGG)(n) repeats, suggesting that some of them recruit telomerase-independent telomere maintenance. The silkworm, Bombyx mori, retains the TTAGG motifs in the chromosomal ends but expresses quite a low level of telomerase activity in all stages of various tissues. Just proximal to a 6-8-kb stretch of the TTAGG repeats in B. mori, more than 1000 copies of non-LTR retrotransposons, designated TRAS and SART families, occur among the telomeric repeats and accumulate. TRAS and SART are abundantly transcribed and actively retrotransposed into TTAGG telomeric repeats in a highly sequence-specific manner. They have three possible mechanisms to ensure specific integration into the telomeric repeats. This article focuses on the telomere structure and telomere-specific non-LTR retrotransposons in B. mori and discusses the mechanisms for telomere maintenance in this insect., SPRINGER
Chromosome Research, 2005年

Essential motifs in the 3' untranslated region required for retrotransposition and the precise start of reverse transcription in non-long-terminal-repeat retrotransposon SART1.
Mizuko Osanai; Hidekazu Takahashi; Kenji K Kojima; Mitsuhiro Hamada; Haruhiko Fujiwara, 筆頭著者, Non-long-terminal-repeat (non-LTR) retrotransposons amplify their copies by reverse transcribing mRNA from the 3' end, but the initial processes of reverse transcription are still unclear. We have shown that a telomere-specific non-LTR retrotransposon of the silkworm, SART1, requires the 3' untranslated region (3' UTR) for retrotransposition. With an in vivo retrotransposition assay, we identified several novel motifs within the 3' UTR involved in precise and efficient reverse transcription. Of 461 nucleotides (nt) of the 3' UTR, the central region, from nt 163 to nt 295, was essential for SART1 retrotransposition. Of five putative stem-loops formed in RNA for the SART1 3' UTR, the second stem-loop (nt 159 to 221) is included in this region. Loss of the 3' region (nt 296 to 461) in the 3' UTR and the poly(A) tract resulted in decreased and inaccurate reverse transcription, which starts mostly from several telomeric repeat-like GGUU sequences just downstream of the second stem-loop. These results suggest that short telomeric repeat-like sequences in the 3' UTR anneal to the bottom strand of (TTAGG)(n) repeats. We also demonstrated that the mRNA for green fluorescent protein (GFP) could be retrotransposed into telomeric repeats when the GFP coding region is fused with the SART1 3' UTR and SART1 open reading frame proteins are supplied in trans., AMER SOC MICROBIOLOGY
Molecular and Cellular Biology, 2004年, ［査読有り］

チョウ目鱗粉の表面ナノ構造形成制御の分子基盤
北村勇斗; 大平健太; 高須陽子; 内野恵郎; 飯塚哲也; 瀬筒秀樹; 二橋亮; 二橋美瑞子; 二橋美瑞子
日本分子生物学会年会プログラム・要旨集(Web), 2024年

カイコ辻田褐卵(b-t)変異体の原因遺伝子として新規オモクローム色素関連遺伝子を同定
内山広純; 八木橋泰仁; 千葉龍之介; 吉武和敏; 伴野豊; 山本公子; 高須陽子; 飯塚哲也; 内野恵郎; 瀬筒秀樹; 内山博允; 矢嶋俊介; 二橋亮; 二橋美瑞子
日本分子生物学会年会プログラム・要旨集(Web), 2021年

カイコ蛹期翅高発現遺伝子のRNA-seq解析およびノックインによる遺伝子の空間的発現の解析
廣田遥菜; 二橋亮; 高須陽子; 飯塚哲也; 内野恵郎; 田村俊樹; 瀬筒秀樹; 二橋美瑞子
日本蚕糸学会大会・蚕糸・昆虫機能学術講演会講演要旨集, 2020年

カイコ変異体優性赤蟻Iaにおける濃色メラニンの着色抑制メカニズム
坂寄和哉; 二橋亮; 坪田拓也; 飯塚哲也; 山本公子; 瀬筒秀樹; 二橋美瑞子
日本蚕糸学会大会・蚕糸・昆虫機能学術講演会講演要旨集, 2018年03月19日

カイコのオモクローム変異体淡赤眼白卵(pe)と第四褐卵(b‐4)の解析
二橋美瑞子; 松崎将平; 立松謙一郎; 二橋亮; 高須陽子; 粥川琢巳; 石毛太一郎; 内山博允; 矢嶋俊介; 内野恵郎; 田村俊樹; 篠田徹郎; 山本公子; 瀬筒秀樹
日本蚕糸学会大会・蚕糸・昆虫機能学術講演会講演要旨集, 2017年03月17日

胆汁酸受容体TGR5に対する新規リガンドの解析
榎本理奈; 二階堂良亮; 黒沢綾; 黒岩聡; 井上裕介; 乙部理也; 斎藤臣雄; 桑原伸夫; 瀬筒秀樹; 二橋美瑞子; 武田茂樹
日本分子生物学会年会プログラム・要旨集(Web), 2016年

カイコ卵色変異体の解析と遺伝子組換えマーカーへの応用
二橋美瑞子; 冨田秀一郎; 立松謙一郎; 内野恵朗; 田村俊樹; 瀬筒秀樹
日本蚕糸学会大会・蚕糸・昆虫機能学術講演会講演要旨集, 2014年03月09日

肉眼で判別できるカイコの遺伝子組換えマーカーの開発
二橋美瑞子; 立松謙一郎; 山本公子; 生川潤子; 内野恵郎; 粥川琢巳; 篠田徹郎; 田村俊樹; 伴野豊; 大出高弘; 平田隼也; 二橋亮; 新美輝幸; 瀬筒秀樹
農業生物資源研究所主な研究成果, 2013年06月

カイコの体色変異を引き起こす新規遺伝子の同定とマーカーへの応用
二橋美瑞子; 立松謙一郎; 山本公子; 内野恵朗; 伴野豊; 二橋亮; 田村俊樹; 瀬筒秀樹
日本蚕糸学会大会・蚕糸・昆虫機能学術講演会講演要旨集, 2012年03月18日

昆虫のオモクローム系色素合成に関わる新規遺伝子の同定
二橋美瑞子; 立松謙一郎; 山本公子; 内野恵郎; 伴野豊; 田村俊樹; 瀬筒秀樹
日本動物学会大会予稿集, 2011年08月20日

カイコ卵色変異体赤卵(red egg)は,新規遺伝子に欠損が存在する
二橋美瑞子; 立松謙一郎; 山本公子; 内野恵郎; 伴野豊; 田村俊樹; 瀬筒秀樹
日本蚕糸学会大会・蚕糸・昆虫機能学術講演会講演要旨集, 2011年03月20日

Development of sequence-specific gene delivery system using LINE
Haruhiko Fujiwara; Azusa Kuroki; Mizuko Futahashi; Haruka Yatabe; Tomoko Kawashima
GENES & GENETIC SYSTEMS, 2009年12月

テロメラーゼの制御
長内美瑞子
日本医事新報, 2007年, ［招待有り］
筆頭著者

動物の体色がわかる図鑑　　　　　　　　　　　　　　　
秋山 豊子、池田 譲、伊藤 祥輔、合田 真、近藤 滋、四宮 愛、高橋 明義、 橋本 寿史、廣部 知久、福澤 利彦、二橋 美瑞子、二橋 亮、持田 浩治、森本 元、吉岡 伸也、若松 一雅, 分担執筆
動物の体色がわかる図鑑, 2022年07月08日
9784766136272

〔主要な業績〕Pigments, Pigment Cells and Pigment Patterns
Ryo Futahashi and Mizuko Osanai-Futahashi (Editors: Hashimoto; H.; Goda; M.; Futahashi; R.; Kelsh; R.; Akiyama; T. ), 分担執筆
Springer, 2021年07月, ［査読有り］
9789811614903

〔主要な業績〕カイコの科学　　　　　　　　　　　　　　　
二橋美瑞子(45-46),二橋亮・二橋美瑞子(51-53); 日本蚕糸学会編, 分担執筆
朝倉書店, 2020年06月26日
9784254420432

色素細胞 第2版　　　　　　　　　　　　　　　
二橋美瑞子・二橋 亮, 共著
慶応義塾大学出版会, 2015年08月31日
9784766422528

A novel monocarboxylate transporter involved in 3-hydroxykynurenine transport for lepidopteran ommochrome coloration　　　　　　　　　　　　　　　
Mizuko Osanai-Futahashi
XXVII International Congress of Entomology (ICE2024), 2024年08月27日, ［招待有り］

カイコの卵とチョウの翅のオモクローム着色を担う3OHKトランスポーターの解明　　　　　　　　　　　　　　　
内山広純、二橋亮、二橋美瑞子
日本蚕糸学会第94回大会, 2024年03月15日

コロナ放電を利用したカイコのゲノム編集　　　　　　　　　　　　　　　
内野恵郎、北村勇斗、大平健太、高須陽子、飯塚哲也、二橋美瑞子、瀬筒秀樹
日本蚕糸学会第94回大会, 2024年03月15日

鱗翅目の鱗粉(微細構造)形成に関与する遺伝子のRNAiを用いた機能解析　　　　　　　　　　　　　　　
北村勇斗、廣田遥菜、大平健太、二橋亮、二橋美瑞子
第８回蚕糸・昆虫機能利用関東地区学術講演会, 2023年12月09日
20231209

トンボ目の新規テロメア配列の同定　　　　　　　　　　　　　　　
後藤竜弘、鈴木遥、二橋亮、二橋美瑞子
第８回蚕糸・昆虫機能利用関東地区学術講演会, 2023年12月09日
20231209

カメムシ幼虫における動原体構成因子の新たな機能と新規機能解析系の構築　　　　　　　　　　　　　　　
長尾百華、吉川匠、佐藤良賢、峰岸桃子、二橋美瑞子
第８回蚕糸・昆虫機能利用関東地区学術講演会, 2023年12月09日
20231209

チョウ目昆虫のオモクローム色素合成に関与する新規トランスポーター遺伝子　　　　　　　　　　　　　　　
第46回日本分子生物学会年会, 2023年12月07日

Genetic engineering of pigmentation and cuticle-related genes in the silkworm, Bombyx mori.　　　　　　　　　　　　　　　
Mizuko Osanai-Futahashi; Yuto Kitamura; Hirosumi Uchiyama; Ryo Futahashi; Keiro Uchino; Hideki Sezutsu
Entomology 2023, 2023年11月07日, ［招待有り］

分散型動原体を持つカイコにおける動原体タンパク質遺伝子の機能解析　　　　　　　　　　　　　　　
後藤竜弘、鈴木遥、二橋美瑞子
令和 5度蚕糸・昆虫機能利用学術講演会日本蚕糸学会第93回大会, 2023年03月06日

分散型動原体をもつホソヘリカメムシ個体における動原体遺伝子の機能解析　　　　　　　　　　　　　　　
長尾百華、吉川匠、二橋美瑞子
令和 5度蚕糸・昆虫機能利用学術講演会日本蚕糸学会第93回大会, 2023年03月06日

分散型動原体を持つホソヘリカメムシの個体における動原体遺伝子ノックダウンの影響　　　　　　　　　　　　　　　
吉川 匠、佐藤 良賢、 二橋 美瑞子
第45回日本分子生物学会年会, 2022年11月30日

分散型動原体を持つカイコにおける動原体候補遺伝子の機能解析　　　　　　　　　　　　　　　
後藤竜弘、鈴木遥、二橋美瑞子
第7回 蚕糸・昆虫機能利用関東学術講演会, 2022年11月05日

分散型動原体をもつホソヘリカメムシ個体組織における動原体遺伝子の機能解析　　　　　　　　　　　　　　　
長尾百華、吉川匠、佐藤良賢、峰岸桃子、二橋美瑞子
第7回 蚕糸・昆虫機能利用関東学術講演会, 2022年11月05日

カイコの成虫微細構造形成に関与する遺伝子のゲノム編集を用いた機能解析　　　　　　　　　　　　　　　
北村勇斗、廣田遥菜、安藤 俊哉、高須陽子、飯塚哲也、内野恵郎、田村俊樹、瀬筒秀樹、二橋亮、二橋美瑞子
第7回 蚕糸・昆虫機能利用関東学術講演会, 2022年11月05日

カイコうすばね変異体の表現型の解析　　　　　　　　　　　　　　　
工藤慎由、伴野豊、二橋美瑞子
第7回 蚕糸・昆虫機能利用関東学術講演会, 2022年11月05日

分散型動原体を持つホソヘリカメムシの胚と幼虫における動原体遺伝子の機能解析　　　　　　　　　　　　　　　
吉川匠・二橋美瑞子
令和4年度蚕糸・昆虫機能利用学術講演会日本蚕糸学会第92回大会, 2022年03月15日

カイコ蛹期翅におけるトランスクリプトーム解析及び高発現遺伝子のゲノム編集による機能解明　　　　　　　　　　　　　　　
北村勇斗・廣田遥菜・安藤 俊哉・高須陽子・飯塚哲也・内野恵郎・田村俊樹・瀬筒秀樹・二橋亮・二橋美瑞子
令和4年度蚕糸・昆虫機能利用学術講演会日本蚕糸学会第92回大会, 2022年03月15日

カイコ辻田褐卵(b-t)変異体の原因遺伝子として新規オモクローム色素関連遺伝子を同定　　　　　　　　　　　　　　　
内山 広純・八木橋 泰仁・千葉 龍之介・吉武 和敏・伴野 豊・山本 公子・高須 陽子・飯塚 哲也・内野 恵郎・瀬筒 秀樹・内山 博允・矢嶋 俊介・二橋 亮・二橋 美瑞子
第44回日本分子生物学会年会, 2021年12月02日

動原体進化の解明に向けた昆虫の分散型動原体複合体の機能解析　　　　　　　　　　　　　　　
吉川 匠・鈴木 遥・佐藤 良賢・峯岸 桃子・八木橋 泰仁・二橋 美瑞子
第44回日本分子生物学会年会, 2021年12月02日

分散型動原体を持つ4種のトンボにおける胚の免疫染色法の確立およびカイコの個体組織・培養細胞における動原体タンパク質の局在解析　　　　　　　　　　　　　　　
鈴木 遥・八木橋 泰仁・二橋 亮・二橋 美瑞子
第44回日本分子生物学会年会, 2021年12月01日

Pigmentation and cuticular formation in lepidopteran insects.　　　　　　　　　　　　　　　
Mizuko Osanai-Futahashi
MBSJ2020, 2020年12月02日, ［招待有り］

カイコ変異体を用いた昆虫の新規体色関連遺伝子の探索　　　　　　　　　　　　　　　
二橋美瑞子
第62回日本応用動物昆虫学会鹿児島大会, 2018年03月27日, ［招待有り］

カイコ変異体優性赤蟻Iaにおける濃色メラニンの着色抑制メカニズム　　　　　　　　　　　　　　　
坂寄和哉・二橋亮・坪田拓也・飯塚哲也・山本公子・瀬筒秀樹・二橋美瑞子
平成30年度蚕糸・昆虫機能利用学術講演会, 2018年03月20日

カイコの優性変異体Iaにおける濃色メラニンの着色抑制メカニズム　　　　　　　　　　　　　　　
第88回日本動物学会富山大会, 2017年09月21日

カイコのオモクローム変異体淡赤眼白卵(pe)と第四褐卵(b-4)の解析　　　　　　　　　　　　　　　
二橋美瑞子・松崎将平・立松謙一郎・二橋亮・高須陽子・粥川琢巳・石毛太一郎・内山博允・矢嶋俊介・内野恵郎・田村俊樹・篠田徹郎・山本公子・瀬筒秀樹
平成29年度蚕糸・昆虫機能利用学術講演会日本蚕糸学会, 2017年03月21日

Coevolution of telomerase gene and telomere specific retrotransposons in insects.　　　　　　　　　　　　　　　
Mizuko Osanai-Futahashi
Pusan National University Lee Bok Luel Laboratory seminar, 2011年08月30日, ［招待有り］

昆虫のテロメアとそれを標的とするレトロトランスポゾンの共進化　　　　　　　　　　　　　　　
二橋美瑞子
東京大学大学院理学系研究科生物科学専攻 第341回Zoological Conference, 2010年06月23日, ［招待有り］

標的特異的なレトロトランスポゾンのエンドヌクレアーゼの機能と構造　　　　　　　　　　　　　　　
長内美瑞子; 青柳 秀幸; 真板 宣夫; 藤原 晴彦
日本分子生物学会2006フォーラム（シンポジウム：動く遺伝子：ゲノム多様化と新規機能獲得へのインパクト）, 2006年, ［招待有り］

テロメア特異的レトロトランスポゾンの転移における3’UTRの役割　　　　　　　　　　　　　　　
長内美瑞子
東京大学大学院理学系研究科生物科学専攻院生会動植人セミナー, 2003年, ［招待有り］

2001年, 日本分子生物学会

日本動物学会

日本蚕糸学会

昆虫の複眼と鱗粉の微細構造形成メカニズムの分子生物学的解析
基盤研究（C）
茨城大学
2022年04月 - 2025年03月

自然免疫と内部共生の境界線を探る -非自己の許容がもたらす細胞内共生の進化-
基盤研究(B)
琉球大学
2019年04月 - 2023年03月

RNAiが簡便なカメムシ培養細胞株の樹立と分散型動原体の研究　　　　　　　　　　　　　　　
2017年06月 - 2020年03月

カイコ卵色変異系体の解析による昆虫の新規色素合成経路の解明
若手研究(B)
2014年04月 - 2018年03月

昆虫における分散型動原体・局在型動原体の比較解析
特別研究員奨励費
独立行政法人農業生物資源研究所
2012年04月 - 2015年03月

特許第5794620号, 特開2013-005740, 特願2011-139217, アリールアルキルアミン－Ｎ－アセチルトランスフェラーゼ遺伝子とその利用
瀬筒秀樹、内野恵郎、二橋美瑞子

特許第5780631号, 特開2012-205525, 特願2011-072777, カイコの卵および眼の着色に関与する遺伝子およびその利用
瀬筒秀樹・立松謙一郎・二橋美瑞子・山本公子