| 1. |
Akihiro Morio, Jae Man Lee, Tsuguru Fujii, Hiroaki Mon, Akitsu Masuda, Kohei Kakino, Jian Xu, Yutaka Banno, Takahiro Kusakabe, The biological role of core 1β1-3galactosyltransferase (T-synthase) in mucin-type O-glycosylation in Silkworm, Bombyx mori, Insect Biochemistry and Molecular Biology, 10.1016/j.ibmb.2023.103936, 156, 103936-103936, 2023.05. |
| 2. |
Tsuguru Fujii, Maki Kubo, Seigou Kuwazaki, Kimiko Yamamoto, Akio Ohnuma, Yutaka Banno, Toru Shimada, DNA sequence analysis of a chromosomal aberration in irradiation induced burnt (Bu) mutant of Bombyx mori, Journal of Insect Biotechnology and Sericology, 10.11416/jibs.91.3_041, 91, 3, 41-50, 2022.10. |
| 3. |
Miyu Tanaka, Tsuguru Fujii, Hiroaki Mon, Jae Man Lee, Kohei Kakino, Hisayoshi Fukumori, Takeru Ebihara, Takumi Nagasato, Masato Hino, Yoshino Tonooka, Takato Moriyama, Ryosuke Fujita, Yutaka Banno, Takahiro Kusakabe, Silkworm FoxL21 plays important roles as a regulator of ovarian development in both oogenesis and ovariole development., Insect biochemistry and molecular biology, 10.1016/j.ibmb.2022.103737, 143, 103737-103737, 2022.04, The ovary is an important organ in reproduction. In insects, especially lepidopteran insects, the oocytes and reproductive organs develop rapidly during the pupal stage. Despite their drastic morphological changes, the molecular mechanisms of ovary development are not fully understood. In this study, it is found that forkhead box transcription factor L2, member 1 (FoxL21), which is known to be involved in ovarian differentiation and maintenance in vertebrates, is required for the development of the ovary in the silkworm, Bombyx mori. FoxL21 was expressed in the ovary and ovariole during the larval and pupal stage, respectively. In silkworms in which FoxL21 was knocked out by genome editing, multiple ovarian dysfunctions, such as, abnormal egg formation, thinning of the ovariole sheaths, and defective connection of the oviductus geminus with the ovariole were observed. Finally, ovarian transplantation experiments using the knockout silkworms revealed that FoxL21 functions in the ovariole, but not in the oviductus geminus.. |
| 4. |
Hisayoshi Fukumori, Tsuguru Fujii, Daisuke Tanaka, Yutaka Banno, Differences between strains in the tolerance to testis cryopreservation in the silkworm, Bombyx mori, Journal of Insect Biotechnology and Sericology, 10.11416/jibs.91.1_013, 91, 1, 1-19, 2022.03. |
| 5. |
Tanaka, Miyu; Fujii, Tsuguru; Mon, Hiroaki; Lee, Jae Man; Kakino, Kohei; Fukumori, Hisayoshi; Ebihara, Takeru; Nagasato, Takumi; Hino, Masato; Tonooka, Yoshino; Moriyama, Takato; Fujita, Ryosuke; Banno, Yutaka; Kusakabe, Takahiro 2022.04, Silkworm FoxL21 plays important roles as a regulator of ovarian development in both oogenesis and ovariole development, INSECT BIOCHEMISTRY AND MOLECULAR BIOLOGY, 10.1016/j.ibmb.2022.103737, 143, 2022.01. |
| 6. |
Ebihara, Takeru; Masuda, Akitsu; Takahashi, Daisuke; Hino, Masato; Mon, Hiroaki; Kakino, Kohei; Fujii, Tsuguru; Fujita, Ryosuke; Ueda, Tadashi; Lee, Jae Man; Kusakabe, Takahiro, Production of scFv, Fab, and IgG of CR3022 Antibodies Against SARS-CoV-2 Using Silkworm-Baculovirus Expression System, MOLECULAR BIOTECHNOLOGY, 10.1007/s12033-021-00373-0, 63, 12, 1223-1234, 2021.12. |
| 7. |
Tsuguru Fujii, Kohei Kakino, Hisayoshi Fukumori, Masato Hino, Jae Man Lee, Takahiro Kusakabe, Yutaka Banno, Non-molting dwarf (nm-d) as a mutant of Bombyx mori with a defect in purine synthesis., Insect biochemistry and molecular biology, 10.1016/j.ibmb.2021.103636, 103636-103636, 2021.08, There are several known non-molting mutations of the silkworm, Bombyx mori, including non-molting dwarf (nm-d). Larvae with this mutation hatch normally and start eating leaves, but die before the completion of the first ecdysis. Genetic analysis of the nm-d mutation would contribute to the isolation of essential genes for the larval development of lepidopteran insects. To identify the causative gene of the nm-d locus, we conducted RNA-seq based rough mapping. Using two sets of RNA-seq data, one from a pooled sample of normal larvae, and one from a pooled sample of nm-d larvae, the nm-d locus was narrowed to a 500 kb region. Among the genes located in this region, an nm-d-specific exon loss was identified in the Bombyx homolog of the ATIC (5-aminoimidazole-4-carboxamide ribonucleotide transformylase/Inosine 5'-monophosphate cyclohydrolase) (BmATIC) gene, which catalyzes the final two steps of the de novo purine biosynthetic pathway in mammals. PCR and subsequent sequencing analysis revealed that a region containing exon 9 of the BmATIC gene is deleted in the nm-d larvae. A knockout allele of the BmATIC gene (BmATICKO), that was generated using the CRISPR/Cas9 system, revealed that first instar knockout larvae died while exhibiting the dark brown larval body that is a typical feature of mutants that lack uric acid in the integument. Lethal larvae resulted from crosses between +/BmATICKO moths. The uric acid content in the whole-body of the first instar was drastically reduced in the nm-d larvae compared to normal larvae. These results indicated that the BmATIC gene is responsible for the nm-d phenotype, and that nm-d larvae have a defect in purine biosynthesis, including uric acid. We also discuss the possibility that the BmATIC mRNA is maternally transmitted to eggs. Our results indicated that RNA-seq based mapping using pooled samples is a practical method for the identification of the causative genes of lethal mutations.. |
| 8. |
Ryo Nagai, Takeru Ebihara, Kohei Kakino, Akitsu Masuda, Jian Xu, Kosuke Minamihata, Noriho Kamiya, Tatphon Kongkrongtong, Masahiro Kawahara, Hiroaki Mon, Tsuguru Fujii, Takahiro Kusakabe, Jae Man Lee, Production of an active Mus musculus IL-3 using updated silkworm-based baculovirus expression vector system, Journal of Asia-Pacific Entomology, 10.1016/j.aspen.2021.04.011, 24, 3, 544-549, 2021.08, Due to the biological significance and therapeutic potential of Interleukin-3 (IL-3) secreted mainly by activated T cells, various protein expression systems have been challenged to produce recombinant IL3 to meet the increasing demands worldwide. Recently, we established an updated silkworm-based baculovirus expression vector system (silkworm-BEVS), which in most cases, produces eukaryotic proteins in biological or enzymatical active forms with considerable amounts. We attempted to reconstruct and express a recombinant mouse IL-3 (rMmIL-3) with C-terminal His8-Strep tags in silkworm-BEVS in the current study. From our results, we gained an active glycosylated rMmIL-3 protein in a substantial amount and quality. As compared with the E. coli expression system, silkworm-BEVS is a better choice regarding the glycosylations attached in rMmIL-3 and up-scalable system in case that a commercial amount is required in the future. Collectively, our method shares an excellent model to produce interleukin molecular for approaching pharmaceutical applications.. |
| 9. |
Takeru Ebihara, Jian Xu, Yoshino Tonooka, Takumi Nagasato, Kohei Kakino, Akitsu Masuda, Kosuke Minamihata, Noriho Kamiya, Hirokazu Nakatake, Yuuka Chieda, Hiroaki Mon, Tsuguru Fujii, Takahiro Kusakabe, Jae Man Lee, Active human and murine tumor necrosis factor α cytokines produced from silkworm baculovirus expression system, Insects, 10.3390/insects12060517, 12, 6, 2021.06, The tumor necrosis factor α (TNFα) has been employed as a promising reagent in treating autoimmunity and cancer diseases. To meet the substantial requirement of TNFα proteins, we report in this study that mature types of recombinant human and murine TNFα proteins are successfully expressed in the baculovirus expression system using silkworm larvae as hosts. The biological activities of purified products were verified in culture murine L929 cells, showing better performance over a commercial Escherichia coli-derived murine TNFα. By comparing the activity of purified TNFα with or without the tag removal, it is also concluded that the overall activity of purified TNFα cytokines could be further improved by the complete removal of C-terminal fusion tags. Collectively, our current attempt demonstrates an alternative platform for supplying high-quality TNFα products with excellent activities for further pharmaceutical and clinical trials.. |
| 10. |
山本 和典, 藤井 告, 西川 和弘, 田村 圭, 木村 友祐, 福森 寿善, 伴野 豊, イチジクカサンTrilocha variansの繭色の性差と温度が繭色と成虫体色に及ぼす影響
, 蚕糸昆虫バイオテック
, 10.11416/konchubiotec.90.1_041, 1, 90, 41-44, 巻 90 号 1 開始ページ 1_041 終了ページ 1_044 , 2021.01. |
| 11. |
Tsuguru Fujii, Takashi Kiuchi, Takaaki Daimon, Katsuhiko Ito, Susumu Katsuma, Toru Shimada, Kimiko Yamamoto, Yutaka Banno, Development of interspecific semiconsomic strains between the domesticated silkworm, Bombyx mori and the wild silkworm, B. Mandarina, Journal of Insect Biotechnology and Sericology, DOI 10.11416/jibs.90.2_0, 2021.01. |
| 12. |
Tsuguru Fujii, Kohei Kakino, Miyu Tanaka, Jae Man Lee, Takahiro Kusakabe, Yutaka Banno, A defect in purine nucleotide metabolism in the silkworm, Bombyx mori, causes a translucent larval integument and male infertility, Insect Biochemistry and Molecular Biology, 10.1016/j.ibmb.2020.103458, 126, 2020.11, p-oily (op) is a novel mutant of Bombyx mori exhibiting translucent larval integument and male infertility. Elucidation of the causative gene of the op mutant will help understand the genetic mechanism underlying larval integument coloration and male fertility. Using polymorphisms between B. mori and B. mandarina, the op locus was narrowed down to a 375-kb region. Using RNA-seq analysis, we found that op mutants have a frameshift mutation in the KWMTBOMO13770 gene located in the 375-kb region. A database search indicated that this gene is the human cytosolic 5ʹ-nucleotidase II gene (cN-II) homolog in Bombyx, which mediates the conversion of inosine monophosphate (IMP) to inosine, a precursor of uric acid. CRISPR/Cas9-mediated knockout mutants of the Bm-cN-II gene showed translucent integuments, and there appeared translucent larvae in the crosses between knockout moths and +/op moths. Moreover, the translucent phenotype of, and decreased uric acid content in the larval integument caused by the mutations in the Bm-cN-II gene were rescued by oral administration of inosine. These results indicated that the Bm-cN-II gene is responsible for the op phenotype and that the molecular function of the Bm-cN-II gene is the conversion of IMP to inosine. We also discuss the genetic relationship between the Bm-cN-II gene and male fertility.. |
| 13. |
Ryosuke Fujita, Masato Hino, Takeru Ebihara, Takumi Nagasato, Akitsu Masuda, Jae Man Lee, Tsuguru Fujii, Hiroaki Mon, Kohei Kakino, Ryo Nagai, Miyu Tanaka, Yoshino Tonooka, Takato Moriyama, Takahiro Kusakabe, Efficient production of recombinant SARS-CoV-2 spike protein using the baculovirus-silkworm system, Biochemical and Biophysical Research Communications, 10.1016/j.bbrc.2020.06.020, 529, 2, 257-262, 2020.08, In the case of a new viral disease outbreak, an immediate development of virus detection kits and vaccines is required. For COVID-19, we established a rapid production procedure for SARS-CoV-2 spike protein (S protein) by using the baculovirus-silkworm expression system. The baculovirus vector-derived S proteins were successfully secreted to silkworm serum, whereas those formed insoluble structure in the larval fat body and the pupal cells. The ectodomain of S protein with the native sequence was cleaved by the host furin-protease, resulting in less recombinant protein production. The S protein modified in furin protease-target site was efficiently secreted to silkworm serum and was purified as oligomers, which showed immunoreactivity for anti-SARS-CoV-2 S2 antibody. By using the direct transfection of recombinant bacmid to silkworms, we achieved the efficient production of SARS-CoV-2 S protein as fetal bovine serum (FBS)-free system. The resultant purified S protein would be useful tools for the development of immunodetection kits, antigen for immunization for immunoglobulin production, and vaccines.. |
| 14. |
Kohei Kakino, Akitsu Masuda, Masato Hino, Takeru Ebihara, Jian Xu, Hiroaki Mon, Ryosuke Fujita, Tsuguru Fujii, Takahiro Kusakabe, Jae Man Lee, Efficient production of recombinant T7 endonuclease I using silkworm-baculovirus expression vector system, Journal of Asia-Pacific Entomology, 10.1016/j.aspen.2020.05.001, 23, 3, 694-700, 2020.08, Recently, T7 Endonuclease I (T7E1) cleavage assay has been widely employed as an efficient approach for detecting mutations from CRISPR/Cas9 targeted samples. This enzyme is sufficient to detect single- and multiple-base mismatches from various heteroduplex DNA samples. However, T7E1 is quite expensive for researchers to use it only for screening mutations, especially in the condition of a large number of test samples. Regarding the production of this enzyme, to data, only the E. coli system has been reported and the highly overexpressed T7E1 seems toxic to the E. coli host cells. Thus, in this study, we tested whether the silkworm-baculovirus expression vector system (BEVS) is suitable to produce recombinant T7 Endonuclease I (rT7E1). The rT7E1 with N- or C-tags in cultured silkworm cells and silkworm pupae were successfully expressed. Our results demonstrated that the rT7E1-Ntag was highly expressed in silkworm pupae and we obtained rT7E1 proteins in high purity. Moreover, rT7E1 from silkworm-BEVS sufficiently recognized and cleaved the mismatches of designed and CRISPR/Cas9-mediated DNA substrates, which was equivalent to the commercial rT7E1 of the E. coli system. Taken together, our study would greatly support the genome-editing research by providing a cost-effective and active rT7E1 enzyme.. |
| 15. |
Fujii T, Banno Y, Identification of a novel function of the silkworm integument in nitrogen metabolism: Uric acid is synthesized within the epidermal cells in B. mori., Insect biochemistry and molecular biology, 10.1016/j.ibmb.2018.12.014, 105, 43-50, 2019.02. |
| 16. |
Katsumi Togashi, Masato Sasaki, Tsuguru Fujii, Relationship between body size and ovariole number in three cerambycids inhabiting mulberry orchards, Applied Entomology and Zoology, 10.1007/s13355-019-00646-w, 2019.01, © 2019, The Japanese Society of Applied Entomology and Zoology. Natural selection operates on a suite of life-history traits. Those traits are under constraints and trade-offs which often differ among species. Thus, there are an array of suites found in insects. Studies conducted to date predict two reproductive strategies in the family Cerambycidae: body-size dependent ovariole number and pro-ovigeny versus body-size independent ovariole number and synovigeny. To investigate these relationships, we examined the elytral length, the body mass, the number and development of ovarioles, and the content of alimentary canals for adult females of three cerambycid species captured in mulberry orchards. The results confirmed synovigeny in two lamiines Apriona japonica Thomson (Coleoptera: Cerambycidae) and Psacothea hilaris (Pascoe) (Coleoptera: Cerambycidae) and no adult feeding in the cerambycine Xylotrechus chinensis (Chevrolat) (Coleoptera: Cerambycidae). We also found the dependence of ovariole number on body size and pro-ovigeny in X. chinensis, whereas invariable numbers of ovarioles irrespective of body size and synovigeny were found in A. japonica and Ps. hilaris. Two likely different suites of reproductive traits including size dependence of ovariole number and ovigeny are discussed by comparing life-history traits of nine cerambycids reported by the present and previous studies.. |
| 17. |
Tsuguru Fujii, Kazunori Yamamoto, Yutaka Banno, Translucent larval integument and flaccid paralysis caused by genome editing in a gene governing molybdenum cofactor biosynthesis in Bombyx mori, Insect Biochemistry and Molecular Biology, 10.1016/j.ibmb.2018.04.008, 99, 11-16, 2018.08, Translucency of the larval integument in Bombyx mori is caused by a lack of uric acid in the epidermis. Hime'nichi translucent (ohi) is a unique mutation causing intermediate translucency of the larval integument and male-specific flaccid paralysis. To determine the gene associated with the ohi mutation, the ohi locus was mapped to a 400-kb region containing 29 predicted genes. Among the genes in this region, we focused on Bombyx homolog of mammalian Gephyrin (BmGphn), which regulates molybdenum cofactor (MoCo) biosynthesis, because MoCo is indispensable for the activity of xanthine dehydrogenase (XDH), a key enzyme in uric acid biosynthesis. The translucent integument of ohi larvae turned opaque after injection of bovine xanthine oxidase, which is a mammalian equivalent to XDH, indicating that XDH activity is defective in ohi larvae. RT-PCR and sequencing analysis showed that (i) in ohi larvae, expression of the BmGphn gene was repressed in the fat body where uric acid is synthesized, and (ii) there was no amino acid substitution in the ohi mutant allele. Finally, we obtained BmGphn knockout alleles (hereafter denoted as BmGphnΔ) by using CRISPR/Cas9. The resulting ohi/BmGphnΔ larvae had translucent integuments, demonstrating that BmGphn is the gene responsible for the ohi phenotype. Our results show that repressed expression of BmGphn is a causative factor for the defective MoCo biosynthesis and XDH activity observed in ohi larvae. Interestingly, all male BmGphnΔ homozygotes died before pupation and showed a flaccid paralysis phenotype. The genetic and physiological mechanisms underlying this flaccid paralysis phenotype are also discussed.. |
| 18. |
Tsuguru Fujii, Yutaka Banno, Enlargement of egg size by crispr/cas9-mediated knockout of a sex-linked gene in the silkworm, bombyx mori, Journal of Insect Biotechnology and Sericology, 10.11416/jibs.87.3_071, 87, 71-78, 2018.01, © 2018, Japanese Society of Sericultural Sciences. All rights reserved. Ge (giant egg) is a sex-linked mutation in Bombyx mori which leads to the production of large-sized eggs caused by an increase in the number of follicular epithelial cells, each with decreased cell size. We previously determined that a gene orthologous to human Phytanoyl-CoA dioxygenase domain-containing 1 (PHYHD1) is specifically disrupted in Ge and Ge 2 mutants through positional cloning of the Ge locus. However, little is known about the function of the BmPHYHD1 gene in determination of the egg size. In this study, we knocked out BmPHYHD1 using CRISPR/Cas9. We employed a novel knockout protocol developed in other lepidopteran species in which a commercialized Cas9 mixed with two sgRNAs designed for a target gene is injected into newly-laid eggs. First, we confirmed that the protocol was applicable to B. mori with high efficiency. Second, we targeted the BmPHYHD1 gene and obtained G 0 moths laid large size eggs. CRISPR/Cas9-mediated mutations were confirmed in G 1 moths that laid large-sized eggs, demonstrating that the BmPHYHD1 gene controls egg size in B. mori. Finally, the dominant/recessive relationship between Ge and + Ge is discussed and we propose the use of gi (giant egg) and gi 2 (giant egg 2) to represent giant egg mutations, Ge and Ge 2 , respectively. We believe that Ge mutants are an appropriate model for the study of genetic mechanisms mediating the size and number of cells in animals.. |
| 19. |
BANNO Yutaka, FUJII Tsuguru, FUKUMORI Hisayoshi, YAMAMOTO Kimiko, KOBAYASHI Jun, Genetic studies on two egg mutants, "small size egg" and "lethal non-diapausing egg" in the silkworm, Bombyx more, Journal of Insect Biotechnology and Seriology, 10.11416/jibs.86.3_123, 86, 3, 123-126, 2017.10. |
| 20. |
Shigehiro Namiki, Tsuguru Fujii, Toru Shimada, Ryohei Kanzaki, The morphology of antennal lobe projection neurons is controlled by a POU-domain transcription factor Bmacj6 in the silkmoth Bombyx mori, SCIENTIFIC REPORTS, 10.1038/s41598-017-14578-4, 7, 1, 14050, 2017.10, How to wire a neural circuit is crucial for the functioning of the nervous system. Here, we describe the neuroanatomy of the olfactory neurons in the spli mutant strain of silkmoth (Bombyx mori) to investigate the function of a transcription factor involved in neuronal wiring in the central olfactory circuit. The genomic structure of the gene Bmacj6, which encodes a class IV POU domain transcription factor, is disrupted in the spli mutant. We report the neuroanatomical abnormality in the morphology of the antennal lobe projection neurons (PNs) that process the sex pheromone. In addition to the mistargeting of dendrites and axons, we found axonal bifurcation within the PNs. These results indicate that the morphology of neurons in the pheromone processing pathway is modified by Bmacj6.. |
| 21. |
Hisayoshi Fukumori, Jung Lee, Tsuguru Fujii, Zenta Kajiura, Yutaka Banno, Long-term preservation of eri and ailanthus silkworms using frozen gonads, CRYOBIOLOGY, 10.1016/j.cryobiol.2017.05.003, 77, 71-74, 2017.08, Cryopreservation of eri and ailanthus silkworms using frozen gonads was investigated. First, we evaluated the freeze tolerance of ovary and testis in the eri silkworm, which showed high tolerance. Mating between frozen ovary-transplanted females and frozen testis-transplanted males produced 163.0 eggs, yielding 105.7 larvae per moth. In a second experiment, we tested the use of the eri silkworm as a host insect for gonad transplantation from ailanthus silkworm donors. A high success ratio for laid and hatched eggs was demonstrated for ovary transplantation (97.8 and 51.3 eggs per moth, respectively). For testis transplantation, however, the average number of hatched larvae was low (12.0). Mating between host eri females and males in which both frozen ovary and testis of the ailanthus silkworm had been transplanted produced 6.4 fertilized eggs per host moth. Our success in using cross subspecies cryo-preservation between these wild silkworms could lead to the alternative use of hosts between species in other insects. (C) 2017 Elsevier Inc. All rights reserved.. |
| 22. |
Fujii, T, Ohnuma, A, Banno, Y, Abe, H, Molecular characterization of aberrant W chromosomes arising from spontaneous translocations between the Z and W chromosomes in the silkworm, Bombyx mori., Journal of Insect Biotechnology and Sericology., 85, 79-85, 2016.12. |
| 23. |
Tsuguru Fujii, Kimiko Yamamoto, Yutaka Banno, Molybdenum cofactor deficiency causes translucent integument, male-biased lethality, and flaccid paralysis in the silkworm Bombyx mori, INSECT BIOCHEMISTRY AND MOLECULAR BIOLOGY, 10.1016/j.ibmb.2016.03.008, 73, 20-26, 2016.06, Uric acid accumulates in the epidermis of Bombyx mori larvae and renders the larval integument opaque and white. Yamamoto translucent (oya) is a novel spontaneous mutant with a translucent larval integument and unique phenotypic characteristics, such as male-biased lethality and flaccid larval paralysis. Xanthine dehydrogenase (XDH) that requires a molybdenum cofactor (MoCo) for its activity is a key enzyme for uric acid synthesis. It has been observed that injection of a bovine xanthine oxidase, which corresponds functionally to XDH and contains its own MoCo activity, changes the integuments of oya mutants from translucent to opaque and white. This finding suggests that XDH/MoCo activity might be defective in oya mutants. Our linkage analysis identified an association between the oya locus and chromosome 23. Because XDH is not linked to chromosome 23 in B. mori, MoCo appears to be defective in oya mutants. In eukaryotes, MoCo is synthesized by a conserved biosynthesis pathway governed by four loci (MOCS1, MOCS2, MOCS3, and GEPH). Through a candidate gene approach followed by sequence analysis, a 6-bp deletion was detected in an exon of the B. mori molybdenum cofactor synthesis-step 1 gene (BmMOCS1) in the oya strain. Moreover, recombination was not observed between the oya and BmMOCS1 loci. These results indicate that the BmMOCS1 locus is responsible for the oya locus. Finally, we discuss the potential cause of male-biased lethality and flaccid paralysis observed in the oya mutants. (C) 2016 Elsevier Ltd. All rights reserved.. |
| 24. |
Tsuguru Fujii, Akio Ohnuma, Yutaka Banno, Hiroaki Abe, Structural analysis of spontaneous Z-W translocations in the silkworm, Bombyx mori, Journal of Insect Biotechnology and Sericology, 85, 79-85, 2016.01. |
| 25. |
T. Fujii, H. Abe, M. Kawamoto, Y. Banno, T. Shimada, Positional cloning of the sex-linked giant egg (Ge) locus in the silkworm, Bombyx mori, INSECT MOLECULAR BIOLOGY, 10.1111/imb.12150, 24, 2, 213-221, 2015.04, The giant egg (Ge) locus is a Z-linked mutation that leads to the production of large eggs. Cytological observations suggest that an unusual translocation of a large fragment of the W chromosome bearing a putative egg size-determining gene, Esd, gave rise to giant egg mutants. However, there is currently no molecular evidence confirming either a W-Z translocation or the presence of Esd on the W chromosome. To elucidate the origin of giant egg mutants, we performed positional cloning. We observed that the Bombyx mori. orthologue of the human Phytanoyl-CoA dioxygenase domain containing 1 gene (PHYHD1) is disrupted in giant egg mutants. PHYHD1 is highly conserved in eukaryotes and is predicted to be a Fe(II) and 2-oxoglutarate-dependent oxygenase. Exon skipping in one of the two available Ge mutants is probably caused by the insertion of a non-long terminal repeat transposon into intron 4 in the vicinity of the 5 splice site. Segmental duplication in Ge-2, an independent allele, was caused by unequal recombination between short interspersed elements inserted into introns 3 and 5. Our results indicate that (1) Bombyx PHYHD1 is responsible for the Ge mutants and that (2) the Ge locus is unrelated to the W-linked putative Esd. To our knowledge, this is the first report describing the phenotypic defects caused by mutations in PHYHD1 orthologues.. |
| 26. |
Yuichi Egi, Shion Akitomo, Tsuguru Fujii, Yutaka Banno, Katsuhiko Sakamoto, Silkworm strains that can be clearly destined towards either embryonic diapause or direct development by adjusting a single ambient parameter during the preceding generation, ENTOMOLOGICAL SCIENCE, 10.1111/ens.12073, 17, 4, 396-399, 2014.10, In the silkworm, Bombyx mori (Lepidoptera: Bombycidae), embryonic diapause is under maternal control and the decision between diapause and direct development in bivoltine strains depends on environmental factors such as temperature and photoperiod experienced by the preceding generation. We reared ten bivoltine silkworm strains (c10, g32, k06, n25, p21, p22, p24, p44, p50 and p63) under various thermal and light conditions and examined the incidence of embryonic diapause in the next generation to identify strains in which the incidence of diapause can be controlled within the range of 0 to 100% by adjusting a single ambient parameter. Some strains were clearly destined towards either diapause or direct development. The diapause incidence in the c10, p22 and p50 strains was controlled by temperature during the egg stage (0% at 18 degrees C and 100% at 25 degrees C), that in the p50 strain was also determined to be dependent on illumination during the egg stage (0% under continuous darkness and 100% under continuous illumination), and photoperiod during the larval stage regulated diapause in p44 and p50 (0% and 100% under long-day and short-day photoperiod, respectively), when all other external parameters remained constant under each experimental condition. These diapause-controllable silkworm strains might serve as model systems for studies of insect diapause, especially for the differential screening of related factors.. |
| 27. |
Tsuguru Fujii, Hiroaki Abe, Munetaka Kawamoto, Susumu Katsuma, Yutaka Banno, Toni Shimada, Albino (al) is a tetrahydrobiopterin (BH4)-deficient mutant of the silkworm Bombyx mori, INSECT BIOCHEMISTRY AND MOLECULAR BIOLOGY, 10.1016/j.ibmb.2013.03.009, 43, 7, 594-600, 2013.07, Albino (al) is a lethal mutant of Bombyx mori that exhibits a colourless cuticle after the first ecdysis and dies without feeding on mulberry. Previous studies have indicated that sclerotisation was insufficient because of defective phenylalanine and tyrosine metabolism in albino larvae. However, the genetic mechanism underlying the albino phenotype has not been determined. Dopamine plays a central role in insect cuticle colouration and sclerotisation. The pathway for dopamine biosynthesis from phenylalanine involves phenylalanine hydroxylase (PAH; EC 1.14.16.1) and tyrosine hydroxylase (TH; EC 1.14.16.2). Tetrahydrobiopterin (BH4) is an essential cofactor of aromatic amino acid hydroxylases, including PAH and TH. Thus, BH4 is indispensable for cuticle colouration and sclerotisation. Here we report on identifying mutations in the gene that encodes for the Bombyx homolog of 6-pyruvoyl-tetrahydropterin synthase (PTS) which is involved in the biosynthesis of BH4, in 2 strains with different at alleles. In strain a60 (al), a transposable element was inserted in exon 2 of BmPTS. In strain a61 (al(2)), an 11-bp deletion was identified in the exon 2 region of BmPTS. After oral administration of BH4 to the al(2) larvae, the survival rate was effectively increased and the larval integument was pigmented. These results indicated that BmPTS was responsible for the albino mutants of B. mori. We conclude that (i) a mutation in BmPTS leads to an insufficient supply of BH4 and results in defective dopamine biosynthesis and (ii) lack of dopamine results in cuticle colouration and sclerotisation failure. Lemon (tern) is a BH4-deficient mutant. It has been reported that de novo synthesis of zygotic BH4 was indispensable for viability of the embryo in eggs laid by tern (lemllem(l)) females. We found that lem/lem, al(2)/al(2) larvae produced by tern (lem/lem) females were viable during the first instar stage, suggesting that al(2)/al(2) embryo could synthesis BH4 by using maternally transmitted BmPTS. (C) 2013 Elsevier Ltd. All rights reserved.. |
| 28. |
Lingyan Wang, Takashi Kiuchi, Tsuguru Fujii, Takaaki Daimon, Muwang Li, Yutaka Banno, Shingo Kikuta, Takahiro Kikawada, Susumu Katsuma, Toru Shimada, Mutation of a novel ABC transporter gene is responsible for the failure to incorporate uric acid in the epidermis of ok mutants of the silkworm, bombyx mori, Insect Biochemistry and Molecular Biology, 10.1016/j.ibmb.2013.03.011, 43, 7, 562-571, 2013.07, ok mutants of the silkworm, Bombyx mori, exhibit highly translucent larval skin resulting from the inability to incorporate uric acid into the epidermal cells. Here we report the identification of a gene responsible for the ok mutation using positional cloning and RNAi experiments. In two independent ok mutant strains, we found a 49-bp deletion and a 233-bp duplication, respectively, in mRNAs of a novel gene, Bm-. ok, which encodes a half-type ABC transporter, each of which results in translation of a truncated protein in each mutant. Although the Bm-. ok sequence was homologous to well-known transporter genes, white, scarlet, and brown in Drosophila, the discovery of novel orthologs in the genomes of lepidopteran, hymenopteran, and hemipteran insects identifies it as a member of a new distinct subfamily of transporters. Embryonic RNAi of Bm-. ok demonstrated that repression of Bm-. ok causes a translucent phenotype in the first-instar silkworm larva. We discuss the possibility that Bm-ok forms a heterodimer with another half-type ABC transporter, Bmwh3, and acts as a uric acid transporter in the silkworm epidermis. © 2013 Elsevier Ltd.. |
| 29. |
Yutaka Banno, Kiyomi Nagasaki, Marino Tsukada, Yuko Minohara, Junko Banno, Kazuhiro Nishikawa, Kazunori Yamamoto, Kei Tamura, Tsuguru Fujii, Development of a method for long-term preservation of Bombyx mori silkworm strains using frozen ovaries, CRYOBIOLOGY, 10.1016/j.cryobiol.2013.03.004, 66, 3, 283-287, 2013.06, Development of long-term preservation is essential for conservation of stocks of silkworm genetic resources. Thus far, a few methods have been reported, but more improvement is required for practical use. We have developed two effective modifications of a method for long-term preservation using frozen ovaries. One was slow cooling (1 degrees C per min) until -80 degrees C of the donor ovaries made possible by use of a BICELL freezing vessel. Using donor ovaries of 4th instar larvae, the average number of eggs laid per moth increased significantly from 110.7 +/- 53.4 eggs per moth by slow cooling with the BICELL vessel vs 12.3 +/- 10.3 eggs per moth by direct cooling in liquid nitrogen. A second improvement was connecting the thread bodies of the donor ovaries with those of the host in the transplantation step. Females operated on with the new method yielded a significantly higher percentage of moths that laid fertilized eggs than those transplanted with the standard procedure (70.4 +/- 21.6% vs 22.9 +/- 9.3%). (C) 2013 Elsevier Inc. All rights reserved.. |
| 30. |
Lingyan Wang, Takashi Kiuchi, Tsuguru Fujii, Takaaki Daimon, Muwang Li, Yutaka Banno, Susumu Katsuma, Toru Shimada, Reduced expression of the dysbindin-like gene in the Bombyx mori ov mutant exhibiting mottled translucency of the larval skin, GENOME, 10.1139/gen-2012-0127, 56, 2, 101-108, 2013.02, The ov (mottled translucent of Var) mutant, an oily mutant of Bombyx mori, exhibits mottled translucent skin with a varying degree of transparency among individuals. By linkage analysis of 2112 backcross individuals using polymorphic DNA markers, we successfully mapped a 179-kb region of chromosome 20 responsible for the ov phenotype. This region contains nine predicted genes. We compared the mRNA expression of these nine genes between the wild type and mutants and found that the expression of one of them, Bmdysb, was strikingly decreased in the epidermis of ov as well as its allelomorph, ov(p). Moreover, its expression level was well correlated with the degree of transparency among individuals. Bmdysb was homologous to DTNBP1 encoding human dysbindin, a subunit of the biogenesis of lysosome-related organelles complex-1. Our results suggest that the translucent skin may be due to repression of Bmdysb in the ov mutants and that Bmdysb plays an important role in the formation and accumulation of urate granules in the silkworm epidermis.. |
| 31. |
T. Fujii, Y. Banno, H. Abe, S. Katsuma, T. Shimada, A homolog of the human Hermansky-Pudluck syndrome-5 (HPS5) gene is responsible for the oa larval translucent mutants in the silkworm, Bombyx mori, GENETICA, 10.1007/s10709-012-9694-1, 140, 10-12, 463-468, 2012.12, Normally, many granules containing uric acid accumulate in the larval integument of the silkworm, Bombyx mori. These uric acid granules cause the wild-type larval integument to be white or opaque, and the absence of these granules results in a translucent integument. Although about 30 B. mori loci governing larval translucency have been mapped, most have not been molecularly identified yet. Here, based on a structural analysis of a deletion of chromosome 14 that included the oa (aojyuku translucent) locus, we concluded that the BmHPS5 encoding a Bombyx homolog of the HPS5 subunit of biogenesis of lysosome-related organelles complex-2 is the candidate for the oa locus. Nucleotide sequence analyses of cDNAs and genomic DNAs in three mutant strains, each of which were homozygous for the respective allele of the oa locus (oa, oa (2) , and oa (v) ), revealed that each mutant strain has a frame shift or a premature stop codon (caused by deletion or nonsense mutation, respectively) in the BmHPS5 gene. Our findings indicate that some genes that cause the translucent phenotype in Bombyx, some HPS-associated genes in humans, and some genes that cause mutant eye color phenotypes in Drosophila are homologous and participate in an evolutionarily conserved mechanism that leads to biogenesis of lysosome-related organelles.. |
| 32. |
Yutaka Banno, Tsuguru Fujii, The silkworm - An attractive bioresource supported by Japan, GENES & GENETIC SYSTEMS, 87, 6, 396-396, 2012.12. |
| 33. |
Takaaki Daimon, Takeshi Fujii, Tsuguru Fujii, Takeshi Yokoyama, Susumu Katsuma, Tetsuro Shinoda, Toru Shimada, Yukio Ishikawa, Reinvestigation of the Sex Pheromone of the Wild Silkmoth Bombyx mandarina: The Effects of Bombykal and Bombykyl Acetate, JOURNAL OF CHEMICAL ECOLOGY, 10.1007/s10886-012-0164-0, 38, 8, 1031-1035, 2012.08, Sex pheromone investigations of the domesticated silkmoth, Bombyx mori (Lepidoptera: Bombycidae), helped elucidate the molecular and physiological fundamentals of chemical communication in moths, yet little is known about pheromone evolution in bombycid species. Therefore, we reexamined the sex pheromone communication in the wild silkmoth, Bombyx mandarina, which is considered ancestral to B. mori. Our investigations revealed that (a) B. mandarina females produce (E,Z)-10,12-hexadecadienol (bombykol), but not (E,Z)-10,12-hexadecadienal (bombykal) or (E,Z)-10,12-hexadecadienyl acetate (bombykyl acetate), which are pheromone components in other bombycid moths; (b) antennae of male B. mandarina respond strongly to bombykol as well as to bombykal and bombykyl acetate; and (c) bombykal and bombykyl acetate strongly inhibit attraction of B. mandarina males to bombykol in the field. The present study clarifies the evolution of pheromone communication in bombycid moths.. |
| 34. |
Takaaki Daimon, Masaya Yago, Yu-Feng Hsu, Tsuguru Fujii, Yumiko Nakajima, Ryuhei Kokusho, Hiroaki Abe, Susumu Katsuma, Toru Shimada, Molecular phylogeny, laboratory rearing, and karyotype of the bombycid moth, Trilocha varians, JOURNAL OF INSECT SCIENCE, 10.1673/031.012.4901, 12, 49, 2012.04, This study describes the molecular phylogeny, laboratory rearing, and karyotype of a bombycid moth, Trilocha varians (F. Walker) (Lepidoptera: Bombycidae), which feeds on leaves of Ficus spp. (Rosales: Moraceae). The larvae of this species were collected in Taipei city, Taiwan, and the Ryukyu Archipelago (Ishigaki and Okinawa Islands, Japan). Molecular phylogenetic analyses revealed that T. varians belongs to the subfamily Bombycinae, thus showing a close relationship to the domesticated silkworm Bombyx mori (L.), a lepidopteran model insect. A laboratory method was developed for rearing T. varians and the time required for development from the embryo to adult was determined. From oviposition to adult emergence, the developmental zero was 10.47 degrees C and total effective temperature was 531.2 day-degrees, i.e., approximately 30 days for one generation when reared at 28 degrees C. The haploid of T. varians consisted of n = 26 chromosomes. In highly polyploid somatic nuclei, females showed a large heterochromatin body, indicating that the sex chromosome system in T. varians is WZ/ZZ (female/male). The results of the present study should facilitate the utilization of T. varians as a reference species for B. mori, thereby leading to a greater understanding of the ecology and evolution of bombycid moths.. |
| 35. |
Kahori Hara, Tsuguru Fujii, Yutaka Suzuki, Sumio Sugano, Toru Shimada, Susumu Katsuma, Shinpei Kawaoka, Altered expression of testis-specific genes, piRNAs, and transposons in the silkworm ovary masculinized by a W chromosome mutation, BMC GENOMICS, 10.1186/1471-2164-13-119, 13, 119, 2012.03, Background: In the silkworm, Bombyx mori, femaleness is strongly controlled by the female-specific W chromosome. Originally, it was presumed that the W chromosome encodes female-determining gene(s), accordingly called Fem. However, to date, neither Fem nor any protein-coding gene has been identified from the W chromosome. Instead, the W chromosome is occupied with numerous transposon-related sequences. Interestingly, the silkworm W chromosome is a source of female-enriched PIWI-interacting RNAs (piRNAs). piRNAs are small RNAs of 23-30 nucleotides in length, which are required for controlling transposon activity in animal gonads. A recent study has identified a novel mutant silkworm line called KG, whose mutation in the W chromosome causes severe female masculinization. However, the molecular nature of KG line has not been well characterized yet.
Results: Here we molecularly characterize the KG line. Genomic PCR analyses using currently available W chromosome-specific PCR markers indicated that no large deletion existed in the KG W chromosome. Genetic analyses demonstrated that sib-crosses within the KG line suppressed masculinization. Masculinization reactivated when crossing KG females with wild type males. Importantly, the KG ovaries exhibited a significantly abnormal transcriptome. First, the KG ovaries misexpressed testis-specific genes. Second, a set of female-enriched piRNAs was downregulated in the KG ovaries. Third, several transposons were overexpressed in the KG ovaries.
Conclusions: Collectively, the mutation in the KG W chromosome causes broadly altered expression of testis-specific genes, piRNAs, and transposons. To our knowledge, this is the first study that describes a W chromosome mutant with such an intriguing phenotype.. |
| 36. |
Takaaki Daimon, Takeshi Fujii, Masaya Yago, Yu-Feng Hsu, Yumiko Nakajima, Tsuguru Fujii, Susumu Katsuma, Yukio Ishikawa, Toru Shimada, Female sex pheromone and male behavioral responses of the bombycid moth Trilocha varians: comparison with those of the domesticated silkmoth Bombyx mori, NATURWISSENSCHAFTEN, 10.1007/s00114-012-0887-3, 99, 3, 207-215, 2012.03, Analysis of female sex pheromone components and subsequent field trap experiments demonstrated that the bombycid moth Trilocha varians uses a mixture of (E,Z)-10,12-hexadecadienal (bombykal) and (E,Z)-10,12-hexadecadienyl acetate (bombykyl acetate) as a sex pheromone. Both of these components are derivatives of (E,Z)-10,12-hexadecadienol (bombykol), the sex pheromone of the domesticated silkmoth Bombyx mori. This finding prompted us to compare the antennal and behavioral responses of T. varians and B. mori to bombykol, bombykal, and bombykyl acetate in detail. The antennae of T. varians males responded to bombykal and bombykyl acetate but not to bombykol, and males were attracted only when lures contained both bombykal and bombykyl acetate. In contrast, the antennae of B. mori males responded to all the three components. Behavioral analysis showed that B. mori males responded to neither bombykal nor bombykyl acetate. Meanwhile, the wing fluttering response of B. mori males to bombykol was strongly inhibited by bombykal and bombykyl acetate, thereby indicating that bombykal and bombykyl acetate act as behavioral antagonists for B. mori males. T. varians would serve as a reference species for B. mori in future investigations into the molecular mechanisms underlying the evolution of sex pheromone communication systems in bombycid moths.. |
| 37. |
Shinpei Kawaoka, Koji Kadota, Yuji Arai, Yutaka Suzuki, Tsuguru Fujii, Hiroaki Abe, Yuji Yasukochi, Kazuei Mita, Sumio Sugano, Kentaro Shimizu, Yukihide Tomari, Toru Shimada, Susumu Katsuma, The silkworm W chromosome is a source of female-enriched piRNAs, RNA-A PUBLICATION OF THE RNA SOCIETY, 10.1261/rna.027565.111, 17, 12, 2144-2151, 2011.12, In the silkworm, Bombyx mori, the W chromosome plays a dominant role in female determination. However, neither protein-coding genes nor transcripts have so far been isolated from the W chromosome. Instead, a large amount of functional transposable elements and their remnants are accumulated on the W chromosome. PIWI-interacting RNAs (piRNAs) are 23-30-nt-long small RNAs that potentially act as sequence-specific guides for PIWI proteins to silence transposon activity in animal gonads. In this study, by comparing ovary-and testis-derived piRNAs, we identified numerous female-enriched piRNAs. Our data indicated that female-enriched piRNAs are derived from the W chromosome. Moreover, comparative analyses on piRNA profiles from a series of W chromosome mutant strains revealed a striking enrichment of a specific set of transposon-derived piRNAs in the putative sex-determining region. Collectively, we revealed the nature of the silkworm W chromosome as a source of piRNAs.. |
| 38. |
Tsuguru Fujii, Takeshi Fujii, Shigehiro Namiki, Hiroaki Abe, Takeshi Sakurai, Akio Ohnuma, Ryohei Kanzaki, Susumu Katsuma, Yukio Ishikawa, Toru Shimada, Sex-linked transcription factor involved in a shift of sex-pheromone preference in the silkmoth Bombyx mori, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 10.1073/pnas.1107282108, 108, 44, 18038-18043, 2011.11, In the sex-pheromone communication systems of moths, odorant receptor (Or) specificity as well as higher olfactory information processing in males should be finely tuned to the pheromone of conspecific females. Accordingly, male sex-pheromone preference should have diversified along with the diversification of female sex pheromones; however, the genetic mechanisms that facilitated the diversification of male preference are not well understood. Here, we explored the mechanisms involved in a drastic shift in sex-pheromone preference in the silkmoth Bombyx mori using spli mutants in which the genomic structure of the gene Bmacj6, which encodes a class IV POU domain transcription factor, is disrupted or its expression is repressed. B. mori females secrete an similar to 11:1 mixture of bombykol and bombykal. Bombykol alone elicits full male courtship behavior, whereas bombykal alone shows no apparent activity. In the spli mutants, the behavioral responsiveness of males to bombykol was markedly reduced, whereas bombykal alone evoked full courtship behavior. The reduced response of spli males to bombykol was explained by the paucity of bombykol receptors on the male antennae. It was also found that, in the spli males, neurons projecting into the toroid, a compartment in the brain where bombykol receptor neurons normally project, responded strongly to bombykal. The present study highlights a POU domain transcription factor, Bmacj6, which may have caused a shift of sex-pheromone preference in B. mori through Or gene choice and/or axon targeting.. |
| 39. |
T. Fujii, H. Abe, K. Yamamoto, S. Katsuma, T. Shimada, Interspecies linkage analysis of mo, a Bombyx mori locus associated with mosaicism and gynandromorphism, GENETICA, 10.1007/s10709-012-9634-0, 139, 10, 1323-1329, 2011.10, The mo (hereditary mosaic) mutation is one of the most famous and interesting mutations of the silkworm, Bombyx mori. Females homozygous for mo generate mosaic and gynandromorphic offspring due to non-elimination of polar bodies and subsequent double fertilization events, irrespective of the genotype of the mated males. Although mo was first reported in 1927, the locus has not been mapped to linkage groups, as the mutation is unstable and appears to be sensitive to genetic background. In this study, linkage analysis of mo was performed using PCR-based markers on single nucleotide polymorphism linkage maps. Bombyx mandarina was used as the mating partner for the B. mori mo strain, as it is easier to identify polymorphic markers between B. mori and B. mandarina than within B. mori strains. Surprisingly, we identified two homozygous linkage groups (LGs) in all of the 12 B-1 (first backcross generation) moths that had deposited mosaic eggs. It was revealed that + (mo) is located on the M chromosome of B. mandarina, which corresponds to two linkage groups of B. mori, LG 14 and 27. Based on further linkage analysis using B. mori as a mating partner, mo was mapped to LG 14. Additionally, we found that mo activity could be modified by a gene(s) on LG 17.. |
| 40. |
T. Fujii, H. Abe, S. Katsuma, T. Shimada, Identification and characterization of the fusion transcript, composed of the apterous homolog and a putative protein phosphatase gene, generated by 1.5-Mb interstitial deletion in the vestigial (Vg) mutant of Bombyx mori, INSECT BIOCHEMISTRY AND MOLECULAR BIOLOGY, 10.1016/j.ibmb.2011.01.007, 41, 5, 306-312, 2011.05, The vestigial (Vg) mutant is a Z-linked mutant that causes vestigial wings in the silkworm. Bombyx mori. We have previously reported a 1.5-Mb interstitial deletion on the Z chromosome bearing the Vg mutation (Z(Vg) chromosome). In this study, we found that exons 3-8 of a gene named Bmptp-Z encoding a putative tyrosine-specific protein phosphatase are deleted by the 1.5-Mb interstitial deletion. We found that a gene encoding the Bombyx homolog of Drosophila Apterous (BmAp-A) protein is located 4.5 kb downstream of the distal breakpoint of the 1.5-Mb interstitial deletion. Moreover, an in-frame fusion transcript composed of the 5' part of Bmptp-Z and the 3' part of Bmap-A is generated specific to the Z(Vg) chromosome. Effects of the in-frame fusion transcript on the vestigial phenotype are discussed. (C) 2011 Elsevier Ltd. All rights reserved.. |
| 41. |
T. Fujii, T. Daimon, K. Uchino, Y. Banno, S. Katsuma, H. Sezutsu, T. Tamura, T. Shimada, Transgenic analysis of the BmBLOS2 gene that governs the translucency of the larval integument of the silkworm, Bombyx mori, INSECT MOLECULAR BIOLOGY, 10.1111/j.1365-2583.2010.01020.x, 19, 5, 659-667, 2010.10, The larval integument of the silkworm, Bombyx mori, is opaque because urate granules accumulate in the epidermis. Although the biosynthetic pathway of uric acid is well studied, little is known about how uric acid accumulates as urate granules in epidermal cells. In the distinct oily (od) mutant silkworm, the larval integument is translucent because of the inability to construct urate granules. Recently, we have found that the od mutant has a genomic deletion in the B. mori homologue of the human biogenesis of lysosome-related organelles complex1, subunit 2 (BLOS2) gene (BmBLOS2). Here, we performed a molecular and functional characterization of BmBLOS2. Northern blot analysis showed that BmBLOS2 was ubiquitously expressed in various tissues. We analysed the structure of a newly isolated mutant (odB) allelic to od and found a premature stop codon in the coding sequence of BmBLOS2 in this new mutation. Moreover, the translucent phenotype was rescued by the germ-line transformation of the wild-type BmBLOS2 allele into the od mutant. Our results suggest that BmBLOS2 is responsible for the od mutant phenotype and plays a crucial role in biogenesis of urate granules in the larval epidermis of the silkworm. The relationships amongst Hermansky-Pudlak syndrome (HPS) genes in mammals, granule group genes in Drosophila and translucent mutant genes in B. mori are discussed.. |
| 42. |
Tsuguru Fujii, Hroaki Abe, Toru Shimada, Molecular analysis of sex chromosome-linked mutants in the silkworm Bombyx mori, JOURNAL OF GENETICS, 10.1007/s12041-010-0048-4, 89, 3, 365-374, 2010.09, In Bombyx mori, the W chromosome determines the female sex. A few W chromosome-linked mutations that cause masculinization of the female genitalia have been found. In female antennae of a recently isolated mutant, both female-type and male-type Bmdsx mRNAs were expressed, and BmOr1 (bombykol receptor) and BmOr3 (bombykal receptor), which are predominantly expressed in the antennae of male moths, were expressed about 50 times more abundantly in the antennae of mutant females than in those of normal females. These mutants are valuable resources for the molecular analysis of the sex-determination system. Besides the Fern gene, the quantitative egg size-determining gene Esd is thought to be present on the W chromosome, based on the observation that ZWW triploid moths produce larger eggs than ZZW triploids. The most recently updated B. mori genome assembly comprises 20.5 Mb of Z chromosome sequence. Using these sequence data, responsible genes or candidate genes for four Z-linked mutants have been reported. The od (distinct oily) and spli (soft and pliable) are caused by mutation in BmBLOS2 and Bmacj6, respectively. Bmap is a candidate gene for Vg (vestigial). Similarly, Bmprm is a candidate gene for Md (muscle dystrophy), causing abnormal development of indirect flight muscle.. |
| 43. |
Hiroaki Abe, Tsuguru Fujii, Toru Shimada, Kazuei Mita, Novel non-autonomous transposable elements on W chromosome of the silkworm, Bombyx mori, JOURNAL OF GENETICS, 89, 3, 375-387, 2010.09, The sex chromosomes of the silkworm Bombyx mori are designated ZW (XY) for females and ZZ (XX) for males. Numerous long terminal repeat (LTR) and non-LTR retrotransposons, retroposons and DNA transposons have accumulated as strata on the W chromosome. However, there are nucleotide sequences that do not show the characteristics of typical transposable elements on the W chromosome. To analyse these uncharacterized nucleotide sequences on the W chromosome, we used whole-genome shotgun (WGS) data and assembled data that was obtained using male genome DNA. Through these analyses, we found that almost all of these uncharacterized sequences were non-autonomous transposable elements that do not fit into the conventional classification. It is notable that some of these transposable elements contained the Bombyx short interspersed element (Bm1) sequences in the elements. We designated them as secondary-Bm1 transposable elements (SBTEs). Because putative ancestral SBTE nucleotide sequences without Brill do not occur in the WGS data, we suggest that the Brill sequences of SBTEs are not carried on each element merely as a package but are components of each element. Therefore, we confirmed that SBTEs should be classified as a new group of transposable elements.. |
| 44. |
Tsuguru Fujii, Seigo Kuwazaki, Kimiko Yamamoto, Hiroaki Abe, Akio Ohnuma, Susumu Katsuma, Kazuei Mita, Toru Shimada, Identification and molecular characterization of a sex chromosome rearrangement causing a soft and pliable (spli) larval body phenotype in the silkworm, Bombyx mori, GENOME, 10.1139/G09-083, 53, 1, 45-54, 2010.01, We carried out genetic and cytogenetic analyses of X-ray-induced deleterious Z chromosomes that result in a soft and pliable (spli) phenotype in the silkworm, Bombyx mori. In a B. mori strain with a spli phenotype, we found the Z chromosome broken between the sch (1-21.5) and od (1-49.6) loci. We also found a chromosomal fragment bearing a fifth-chromosome locus for egg and eye pigmentation fused to a Z chromosome fragment. By means of fluorescence in situ hybridization using bacterial artificial chromosome clones as probes, we confirmed that the fused chromosome is composed of a fragment of chromosome 5 and a fragment of the Z chromosome. Moreover, a predicted gene, GA002017, the Bombyx ortholog of the Drosophila gene acj6 (Bmacj6), was completely deleted by the Z chromosome breakage event. The relationship between Bmacj6 and the spli phenotype is discussed.. |
| 45. |
Tsuguru Fujii, Masataka Ozaki, Takaaki Masamoto, Susumu Katsuma, Hiroaki Abe, Toru Shimada, A Bombyx mandarina mutant exhibiting translucent larval skin is controlled by the molybdenum cofactor sulfurase gene, GENES & GENETIC SYSTEMS, 10.1266/ggs.84.147, 84, 2, 147-152, 2009.04, During the maintenance of the wild silkworm, Bombyx mandarina, a mutant phenotype exhibiting translucent skin was identified. Based on the crossing experiments with the domesticated silkworm, Bombyx mori, we found that the mutant was controlled by molybdenum cofactor sulfurase (MoCoS) gene. We designated the mutant "Ozaki's translucent" (og(Z)). We found a 2.1-kb deletion containing the transcription initiation site, exons 1 and 2, and the 5' end of exon 3 of the MoCoS gene. The transcript of the MoCoS gene was not detected in the og(Z) homozygote. We concluded that og(Z) is a complete loss-of-function allele generated by a disruption of the MoCoS gene.. |
| 46. |
Susumu Katsuma, Tsuguru Fujii, Shinpei Kawaoka, Toru Shimada, Bombyx mori nucleopolyhedrovirus SNF2 global transactivator homologue (Bm33) enhances viral pathogenicity in B. mori larvae, JOURNAL OF GENERAL VIROLOGY, 10.1099/vir.0.2008/004887-0, 89, Pt 12, 3039-3046, 2008.12, The SNF2 global transactivator gene homologue (Bm33) of Bombyx mori nucleopolyhedrovirus (BmNPV) is one of the genes exclusive to group I NPVs, but its function remains unknown. This study describes the characterization of Bm33. Transcriptional analysis suggested that Bm33 is an early gene, as its transcript was observed at 4 h post-infection in BmNPV-infected BmN cells. To examine the role of Bm33 during BmNPV infection, a Bm33 deletion mutant (BmORF33D) was constructed and its infectivity was characterized in BmN cells and B. mori larvae. BmORF33D did not have any obvious defects in the production of budded viruses (BVs) or occlusion bodies (OBs) in BmN cells compared with wild-type BmNPV. Larval bioassays revealed that deletion of Bm33 did not reduce virus infectivity. However, BmORF33D took approximately 10-15 h longer than wild-type BmNPV to kill B. mori larvae when tested by either BV injection or OB ingestion. These results suggest that Bm33 is not essential for virus growth in vitro or in vivo, but that it accelerates the time of death of B. mori larvae.. |
| 47. |
Tsuguru Fujii, Hiroaki Abe, Susumu Katsuma, Kazuei Mita, Toru Shimada, Mapping of sex-linked genes onto the genome sequence using various aberrations of the Z chromosome in Bombyx mori, INSECT BIOCHEMISTRY AND MOLECULAR BIOLOGY, 10.1016/j.ibmb.2008.03.004, 38, 12, 1072-1079, 2008.12, Many strains of Bombyx mori carry chromosomal aberrations, and they are useful resources for integration between phenotypes and genomic sequences. We compared the molecular structures of three kinds of Z chromosomes, i.e., two strains with chromosome deletions and one strain with translocation involving the Z chromosome. Using polymerase chain reaction markers, we showed that: (1) the Z, chromosome lacks more than 6 Mb, including the proximal end; (2) the Z(Vg) chromosome lacks 1.5 Mb in the interstitial portion; and (3) the +(od)p(Sa)+(p)W carries a 0.6-Mb Z-derived fragment surrounding the +(od) gene. The breakpoint junctions of these deletions and a translocation were precisely determined. Through deletion mapping, we narrowed down the regions where distinct oily (od), vestigial (Vg), and muscle dystrophy (Md) are located and identified a candidate gene for ad. A retroposon-mediated deletion in BmBLOS2-the Bombyx gene homologous to human "biogenesis of lysosome-related organelles complex-1, subunit 2"-was detected in the ad mutant. Although the genes responsible for Vg and Md were not definitively identified, we propose the candidate genes on the basis of their locations and phenotypes. (C) 2008 Elsevier Ltd. All rights reserved.. |
| 48. |
Qingyou Xia, Jun Wang, Zeyang Zhou, Ruiqiang Li, Wei Fan, Daojun Cheng, Tingcai Cheng, Junjie Qin, Jun Duan, Hanfu Xu, Qibin Li, Ning Li, Mingwei Wang, Fangyin Dai, Chun Liu, Ying Lin, Ping Zhao, Huijie Zhang, Shiping Liu, Xingfu Zha, Chunfeng Li, Aichun Zhao, Minhui Pan, Guoqing Pan, Yihong Shen, Zhihong Gao, Zilong Wang, Genhong Wang, Zhengli Wu, Yong Hou, Chunli Chai, Quanyou Yu, Ningjia He, Ze Zhang, Songgang Li, Huanming Yang, Cheng Lu, Jian Wang, Zhonghuai Xiang, Kazuei Mita, Masahiro Kasahara, Yoichiro Nakatani, Kimiko Yamamoto, Hiroaki Abe, Brudrul Ahsan, Takaaki Dai-mon, Koichiro Doi, Tsuguru Fujii, Haruhiko Fujiwara, Asao Fujiyama, Ryo Futahashi, Shin-ichi Hashimoto, Jun Ishibashi, Masafumi Iwami, Keiko Kadono-Okuda, Hiroyuki Kanamori, Hiroshi Kataoka, Susumu Katsuma, Shinpei Kawaoka, Hideki Kawasaki, Yuji Kohara, Toshinori Kozaki, Reginaldo M. Kuroshu, Seigo Kuwazaki, Kouji Matsushima, Hiroshi Minami, Yukinobu Nagayasu, Tatsuro Nakagawa, Junko Narukawa, Junko Nohata, Kazuko Ohishi, Yukiteru Ono, Mizuko Osanai-Futahashi, Katsu-hisa Ozaki, Wei Qu, Ladislav Roller, Shin Sasaki, Takuji Sasaki, Atsushi Seino, Masaru Shimomura, Michihiko Shimomura, Tadasu Shin-I, Tetsuro Shinoda, Takahiro Shiotsuki, Yoshitaka Suetsugu, Sumio Sugano, Makiko Suwa, Yutaka Suzuki, Shige-Haru Takiya, Toshiki Tamura, Hiromitsu Tanaka, Yoshiaki Tanaka, Kazushige Touhara, Tomoyuki Yamada, Minoru Yamakawa, Naoki Yamanaka, Hiroshi Yoshikawa, Yang-Sheng Zhong, Toru Shima-da, Shinichi Morishita, The genome of a lepidopteran model insect, the silkworm Bombyx mori, INSECT BIOCHEMISTRY AND MOLECULAR BIOLOGY, 10.1016/j.ibmb.2008.11.004, 38, 12, 1036-1045, 2008.12, 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.. |
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T. Fujii, T. Yokoyama, O. Ninagi, K. Kakehashi, Y. Obara, M. Nenoi, T. Ishikawa, K. Mita, T. Shimada, H. Abe, Isolation and characterization of sex chromosome rearrangements generating male muscle dystrophy and female abnormal oogenesis in the silkworm, Bombyx mori, GENETICA, 10.1007/s10709-006-9104-7, 130, 3, 267-280, 2007.07, In deletion-mapping of W-specific RAPD (W-RAPD) markers and putative female determinant gene (Fem), we used X-ray irradiation to break the translocation-carrying W chromosome (W-Ze). We succeeded in obtaining a fragment of the W-Ze chromosome designated as Ze(W), having 3 of 12 W-RAPD markers (W-Bonsai, W-Yukemuri-S, W-Yukemuri-L). Inheritance of the Ze(W) fragment by males indicates that it does not include the Fem gene. On the basis of these results, we determined the relative positions of W-Yukemuri-S and W-Yukemuri-L, and we narrowed down the region where Fem gene is located. In addition to the Ze(W) fragment, the Z chromosome was also broken into a large fragment (Z(1)) having the +(sch) (1-21.5) and a small fragment (Z(2)) having the +(od) (1-49.6). Moreover, a new chromosomal fragment (Ze(W)Z(2)) was generated by a fusion event between the Ze(W) and the Z(2) fragments. We analyzed the genetic behavior of the Z(1) fragment and the Ze(W)Z(2) fragment during male (Z/Z(1) Ze(W)Z(2)) and female (Z(1) Ze(W)Z(2)/W) meiosis using phenotypic markers. It was observed that the Z(1) fragment and the Z or the W chromosomes separate without fail. On the other hand, non-disjunction between the Ze(W)Z(2) fragment and the Z chromosome and also between the Ze(W)Z(2) fragment and the W chromosome occurred. Furthermore, the females (2A: Z/Ze(W)Z(2)/W) and males (2A: Z/Z(1)) resulting from non-disjunction between the Ze(W)Z(2) fragment and the W chromosome had phenotypic defects: namely, females exhibited abnormal oogenesis and males were flapless due to abnormal indirect flight muscle structure. These results suggest that Z(2) region of the Z chromosome contains dose-sensitive gene(s), which are involved in oogenesis and indirect flight muscle development.. |
| 50. |
Tsuguru Fujii, Toru Shimada, Sex determination in the silkworm, Bombyx mori: A female determinant on the W chromosome and the sex-determining gene cascade, SEMINARS IN CELL & DEVELOPMENTAL BIOLOGY, 10.1016/j.semcdb.2007.02.008, 18, 3, 379-388, 2007.06, In insects, the sex is determined completely by genetic mechanisms, which at least in somatic tissues, are cell autonomous. The sex of the silkworm, Bombyx mori, is strongly controlled by the presence of the W chromosome. Genetic studies using translocations and deletions of W suggested that a presumptive feminizing gene (Fem) is located in a limited region of the W chromosome. Recent genomic studies revealed a small number of potential candidates for the Fen? gene in this region. In addition, a Bombyx homologue of the Drosophila sex determining gene doublesex has been identified on an autosome and analyzed. Whereas the Drosophila doublesex gene is regulated by activation of splicing in females, the Bombyx doublesex gene (Bmdsx) encodes female- and male-specific mRNAs regulated via male-specific repression of splicing. The vitellogenin gene (Vg) is a target of the BmDSX protein, which directly binds to the Vg promoter. Furthermore, as ectopic expression of the rnale-type Bmdsx induces male-like transformation of the sexual organs, BmDSX may control sex-specific morphological characteristics in Bombyx. This suggests that although upstream events in Drosophila and Bombyx sex determination differ, similarities between the two species do exist in downstream genetic control of sex determination. (c) 2007 Elsevier Ltd. All rights reserved.. |
| 51. |
T. Fujii, N. Tanaka, T. Yokoyama, O. Ninaki, T. Oshiki, A. Ohnuma, Y. Tazima, Y. Banno, M. Ajimura, K. Mita, M. Seki, F. Ohbayashi, T. Shimada, H. Abe, The female-killing chromosome of the silkworm, Bombyx mori, was generated by translocation between the Z and W chromosomes, GENETICA, 10.1007/s10709-005-4147-8, 127, 1-3, 253-265, 2006.05, Bombyx mori is a female-heterogametic organism (female, ZW; male, ZZ) that appears to have a putative feminizing gene (Fem) on the W chromosome. The paternally transmitted mutant W chromosome, Df(p(Sa) + W-p + (od))Fem, derived from the translocation-carrying W chromosome (p (Sa) + W-p + (od)), is inert as femaleness determinant. Moreover, this Df(p (Sa) + (p) W + (od))Fem chromosome has been thought to have a female-killing factor because no female larvae having the Df(p(Sa) + W-p + (od))Fem chromosome are produced. Initially, to investigate whether the Df(p(Sa) + W-p + (od))Fem chromosome contains any region of the W chromosome or not, we analyzed the presence or absence of 12 W-specific RAPD markers. The Df(p(Sa) + W-p + (od))Fem chromosome contained 3 of 12 W-specific RAPD markers. These results strongly indicate that the Df(p(Sa) + W-p + (od))Fem chromosome contains the region of the W chromosome. Moreover, by using phenotypic and molecular markers, we confirmed that the Df(p(Sa) + W-p + (od))Fem chromosome is connected with a partially deleted Z chromosome and that this fused chromosome behaves as a Z chromosome during male meiosis. Furthermore, we demonstrated that the ZZW-type triploid female having the Df(p(Sa) supercript stop + (p) W + (od))Fem chromosome is viable. Therefore, we concluded that the Df(p(Sa) + W-p + (od))Fem chromosome does not have a female-killing factor but that partial deletion of the Z chromosome causes the death of the ZW-type diploid female having the Df(p(Sa) + W-p + (od))Fem chromosome. Additionally, our results of detailed genetic analyses strongly indicate that the female-killing chromosome composed of the Df(p(Sa) + W-p + (od))Fem chromosome and deleted Z chromosome was generated by translocation between the Z chromosome and the translocation-carrying W chromosome, p(Sa) + W-p + (od).. |
| 52. |
H Abe, M Seki, F Ohbayashi, N Tanaka, J Yamashita, T Fujii, T Yokoyama, M Takahashi, Y Banno, K Sahara, A Yoshido, J Ihara, Y Yasukochi, K Mita, M Ajimura, MG Suzuki, T Oshiki, T Shimada, Partial deletions of the W chromosome due to reciprocal translocation in the silkworm Bombyx mori, INSECT MOLECULAR BIOLOGY, 10.1111/j.1365-2583.2005.00565.x, 14, 4, 339-352, 2005.08, In the silkworm, Bombyx mori (female, ZW; male, ZZ), femaleness is determined by the presence of a single W chromosome, irrespective of the number of autosomes or Z chromosomes. The W chromosome is devoid of functional genes, except the putative female-determining gene (Fem). However, there are strains in which chromosomal fragments containing autosomal markers have been translocated on to W. In this study, we analysed the W chromosomal regions of the Zebra-W strain (T(W;3)Ze chromosome) and the Black-egg-W strain (T(W;10)+(w-2) chromosome) at the molecular level. Initially, we undertook a project to identify W-specific RAPD markers, in addition to the three already established W-specific RAPD markers (W-Kabuki, W-Samurai and W-Kamikaze). Following the screening of 3648 arbitrary 10-mer primers, we obtained nine W-specific RAPD marker sequences (W-Bonsai, W-Mikan, W-Musashi, W-Rikishi, W-Sakura, W-Sasuke, W-Yukemuri-L, W-Yukemuri-S and BMC1-Kabuki), almost all of which contained the border regions of retrotransposons, namely portions of nested retrotransposons. We confirmed the presence of eleven out of twelve W-specific RAPD markers in the normal W chromosomes of twenty-five silkworm strains maintained in Japan. These results indicate that the W chromosomes of the strains in Japan are almost identical in type. The Zebra-W strain (T(W;3)Ze chromosome) lacked the W-Samurai and W-Mikan RAPD markers and the Black-egg-W strain (T(W;10)+(w-2) chromosome) lacked the W-Mikan RAPD marker. These results strongly indicate that the regions containing the W-Samurai and W-Mikan RAPD markers or the W-Mikan RAPD marker were deleted in the T(W;3)Ze and T(W;10)+(w-2) chromosomes, respectively, due to reciprocal translocation between the W chromosome and the autosome. This deletion apparently does not affect the expression of Fem; therefore, this deleted region of the W chromosome does not contain the putative Fem gene.. |
| 53. |
Tsuguru Fujii, Takeshi Yokoyama, Hiroaki Abe, Osamu Ninagi, Toshikazu Oshiki, The sperm from two male parents can enter an egg and fuse with both the egg nucleus and the polar body by double copulation in the silkworm, Bombyx mori, Journal of Insect Biotechnology and Sericology, 74, 1, 27-28, 2005.02. |
