Kyushu University Academic Staff Educational and Research Activities Database
List of Papers
Hiroaki Mon Last modified date:2020.02.06

Associate Professor / Agricultural Bioresource Sciences / Department of Bioresource Sciences / Faculty of Agriculture


Papers
1. Masahiko Kobayashi, Jian Xu, Kohei Kakino, Akitsu Masuda, Masato Hino,Naoki Fujimoto, Kosuke Minamihata, Noriho Kamiya, Hiroaki Mon, HiroshiIida, Masateru Takahashi, Takahiro Kusakabe, Jae Man Lee, Optimal silkworm larva host for high-level production of Mus musculus IL-4 using a baculovirus expression vector system., Journal of Asia-Pacific Entomology, https://doi.org/10.1016/j.aspen.2019.12.014, 2019.12.
2. Jian Xu, Jae Man Lee, Tuneyuki Tatsuke, Takeru Ebihara, Akitsu Masuda, Masato Hino, Daisuke Morokuma, Ryosuke Fujita, Hiroaki Mon, Takahiro Kusakabe, Masateru Takahashi, Expression and Purification of Vaccinia Virus DNA Topoisomerase IB Produced in the Silkworm–Baculovirus Expression System, Molecular Biotechnology, 10.1007/s12033-019-00184-4, 61, 8, 622-630, 2019.08, Type IB DNA topoisomerases are enzymes to change the topological state of DNA molecules and are essential in studying replication, transcription, and recombination of nucleic acids in vitro. DNA topoisomerase IB from Vaccinia virus (vTopIB) is a 32 kDa, type I eukaryotic topoisomerase, which relaxed positively and negatively supercoiled DNAs without Mg2+ and ATP. Although vTopIB has been effectively produced in E. coli expression system, no studies remain available to explore an alternative platform to express recombinant vTopIB (rvTopIB) in a higher eukaryote, where the one can expect post-translational modifications that affect the activity of rvTopIB. Here in this study, rvTopIB with N-terminal tags was constructed and expressed in a silkworm–baculovirus expression vector system (silkworm–BEVS). We developed a simple two consecutive chromatography purification to obtain highly pure rvTopIB. The final yield of rvTopIB obtained from a baculovirus-infected silkworm larva was 83.25 μg. We also evaluated the activity and function of rvTopIB by the DNA relaxation activity assays using a negatively supercoiled pUC19 plasmid DNA as a substrate. With carefully assessing optimized conditions for the reaction buffer, we found that divalent ions, Mg2+, Mn2+, Ca2+, as well as ATP stimulate the DNA relaxation activity by rvTopIB. The functional and active form of rvTopIB, together with the yields of the protein we obtained, suggests that silkworm–BEVS would be a potential alternative platform to produce eukaryotic topoisomerases on an industrial scale..
3. Yurie Kinoshita, Jian Xu, Akitsu Masuda, Kosuke Minamihata, Noriho Kamiya, Hiroaki Mon, Ryosuke Fujita, Takahiro Kusakabe, Jae Man Lee, Expression and purification of biologically active human granulocyte-macrophage colony stimulating factor (hGM-CSF) using silkworm-baculovirus expression vector system, Protein Expression and Purification, 10.1016/j.pep.2019.03.010, 159, 69-74, 2019.07, Human granulocyte-macrophage colony stimulating factor (hGM-CSF) is a hematopoietic growth factor. It is widely employed as a therapeutic agent targeting neutropenia in cancer patients undergoing chemotherapy and in patients with AIDS or after bone marrow transplantation. In this study, we constructed the recombinant baculoviruses for the expression of recombinant hGM-CSF (rhGM-CSF) with two small affinity tags (His-tag and Strep-tag) at the N or C-terminus. Compared to N-tagged rhGM-CSF, C-tagged rhGM-CSF was highly recovered from silkworm hemolymph. The purified rhGM-CSF proteins migrated as a diffuse band and were confirmed to hold N-glycosylations. A comparable activity was achieved when commercial hGM-CSF was tested as a control. Considering the high price of hGM-CSF in the market, our results and strategies using silkworm-baculovirus system can become a great reference for mass production of the active rhGM-CSF at a lower cost..
4. Takumi Yano, Jae Man Lee, Jian Xu, Yoshiki Morifuji, Akitsu Masuda, Masato Hino, Daisuke Morokuma, Ryosuke Fujita, Masateru Takahashi, Takahiro Kusakabe, Hiroaki Mon, Expression of the thermostable Moloney murine leukemia virus reverse transcriptase by silkworm-baculovirus expression system, Journal of Asia-Pacific Entomology, 10.1016/j.aspen.2019.02.008, 22, 2, 453-457, 2019.06, Reverse transcriptase from Moloney murine leukemia virus (MMLVRT) is an RNA dependent DNA polymerase, which has been used as a fundamental tool for molecular biology and biotechnology. The secondary structures formed in the RNA templates decrease the accessibility of the reverse transcriptase to the RNA templates; it is important to unfold the RNA secondary structure by increasing the reaction temperature to perform the successful transcription. In this study, we applied silkworm baculovirus expression vector system (silkworm-BEVS) to mass-produce and purify the recombinant MMLVRTs with the N- or C- terminal tandem tags. We confirmed that both of the recombinant MMLVRT enzymes have intact DNA polymerase activity. It is notable that C- terminal tagged MMLVRT outperformed MMLVRT obtained from the E. coli expression system in terms of thermostability and sensitivity to low quantities of RNA template. Taken together, these results demonstrate that silkworm-BEVS is a promising alternative strategy to produce the functional and thermostable reverse transcriptase..
5. Akihiro Morio, Jian Xu, Akitsu Masuda, Yurie Kinoshita, Masato Hino, Daisuke Morokuma, Hatsumi M. Goda, Nozomu Okino, Makoto Ito, Hiroaki Mon, Ryosuke Fujita, Takahiro Kusakabe, Jae Man Lee, Expression, purification, and characterization of highly active endo-α-N-acetylgalactosaminidases expressed by silkworm-baculovirus expression system, Journal of Asia-Pacific Entomology, 10.1016/j.aspen.2019.01.009, 22, 2, 404-408, 2019.06, The O-glycosidase, endo-α-N-acetylgalactosaminidase from Enterococcus faecalis (endoEF) catalyzes the cleavage of core 1 and core 3 type O-linked disaccharides between GalNAc and serine or threonine residues from glycoproteins. The endoEF has broad substrate specificity and thus is extensively utilized for the structural and functional analysis of the O-linked glycans. In this study, we expressed and purified the recombinant endoEF (rEndoEF) by using the silkworm-baculovirus expression vector system (Silkworm-BEVS) and confirmed the deglycosylation activity of rEndoEF targeting reporter glycoproteins, which was equivalent to the commercial O-glycosidase. Thus, our study provides important clues to produce highly active rEndoEF O-glycosidases employing silkworm-BEVS as an alternative..
6. Mai Morishita, Akitsu Masuda, Hiroaki Mon, Jae Man Lee, Takahiro Kusakabe, Kosuke Tashiro, Chisa Yasunaga-Aoki, Kazuhiro Iiyama, Identification of an insecticidal toxin produced by Enterobacter sp. strain 532 isolated from diseased Bombyx mori silkworms, FEMS microbiology letters, 10.1093/femsle/fny295, 366, 2, 2019.01, Although Enterobacter sp. 532 shows pathogenicity in Bombyx mori, the insecticidal mechanisms are unclear. Here, we identified and characterised an insecticidal protein from Enterobacter. The insecticidal protein was purified from the strain and inoculated into B. mori larvae. Intracellular proteins were prepared, purified and separated by preparative native polyacrylamide gel electrophoresis (PAGE); one protein band had insecticidal activity. Sodium dodecyl sulfate-PAGE showed the presence of several bands, indicating that the insecticidal protein formed a complex. Peptide mass fingerprinting of a prominent 255.3-kDa band revealed 64 peptides that matched one protein with 33.0% sequence coverage. This protein was a homologue of the A component of the toxin complex (Tc), and the VRP1 domain was conserved; thus, the gene was named itcA (insecticidal toxin complex A). In the itcA downstream region, B and C component gene homologues were found, and these genes were located on an 86.2-kb contig sequence. Two repA genes and 27 genes related to conjugation transfer of plasmids were located on the contig, suggesting that the contig originated from a mobilisable plasmid. Therefore, these findings suggested that the strain may have acquired the Tc genes by horizontal transfer. This is the first description of Tc produced by the genus Enterobacter..
7. Mai Morishita, Hiroaki Mon, Jae Man Lee, Takahiro Kusakabe, Maximiano Corrêa Cassal, Chisa Yasunaga-Aoki, Kazuhiro Iiyama, Toxin complex is a major virulence determinant of Enterobacter sp. 532 against the silkworm, Bombyx mori, Journal of Insect Biotechnology and Sericology, 10.11416/jibs.88.1_021, 88, 1, 1_021-1_025, 2019.01.
8. Jian Xu, Akihiro Morio, Daisuke Morokuma, Yudai Nagata, Masato Hino, Akitsu Masuda, Zhiqing Li, Hiroaki Mon, Takahiro Kusakabe, Jae Man Lee, A functional polypeptide N-acetylgalactosaminyltransferase (PGANT) initiates O-glycosylation in cultured silkworm BmN4 cells, Applied Microbiology and Biotechnology, 10.1007/s00253-018-9309-6, 102, 20, 8783-8797, 2018.10, Mucin-type O-glycosylation is initiated by UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-Ts or PGANTs), attaching GalNAc to serine or threonine residue of a protein substrate. In the insect model from Lepidoptera, silkworm (Bombyx mori), however, O-glycosylation pathway is totally unexplored and remains largely unknown. In this study, as the first report regarding protein O-glycosylation analysis in silkworms, we verified the O-glycan profile that a common core 1 Gal (β1-3) GalNAc disaccharide branch without terminally sialylated structure is mainly formed for a baculovirus-produced human proteoglycan 4 (PRG4) protein. Intriguingly, functional screenings in cultured silkworm BmN4 cells for nine Bmpgants reveal that Bmpgant2 is the solo functional BmPGANT for PRG4, implying that Bmpgants may have unique cell/tissue or protein substrate preferences. Furthermore, a recombinant BmPGANT2 protein was successfully purified from silkworm-BEVS and exhibited a high ability to transfer GalNAc for both peptide and protein substrates. Taken together, the present results clarified the functional BmPGANT2 in cultured silkworm cells, providing crucial fundamental insights for future studies dissecting the detailed silkworm O-glycosylation pathways and productions of glycoproteins with O-glycans..
9. Akitsu Masuda, Jian Xu, Kosuke Minamihata, Genki Kagawa, Yusei Hamada, Yoshiki Morifuji, Takumi Yano, Masato Hino, Daisuke Morokuma, Noriko Karasaki, Hiroaki Mon, Noriho Kamiya, Takahiro Kusakabe, Jae Man Lee, Production of a biologically active human basic fibroblast growth factor using silkworm-baculovirus expression vector system, Journal of Asia-Pacific Entomology, 10.1016/j.aspen.2018.05.002, 21, 2, 716-720, 2018.06, As a therapeutic treatment, recombinant human basic fibroblast growth factor (rhbFGF) is usually employed in tissue regeneration, and as an essential component in culture medium for maintaining the induced pluripotent stem (iPS) cell and embryonic stem (ES) cell in an undifferentiated state. Therefore, a large amount of biologically active rhbFGF is required. In this study, silkworm-baculovirus expression vector system (silkworm-BEVS) is employed to achieve a high productivity of recombinant rhbFGF with two small affinity tags (His-tag and STREP-tag) at the N or C-terminus. It is observed that rhbFGF with 30 K signal peptide of silkworm were successfully expressed but are not sufficiently secreted into the culture medium of cultured insect cells. Then we purified the N- or C-tagged intracellular rhbFGF protein and obtained a yield of about 0.7 mg/larva and 1.2 mg/larva, respectively. Although the final yield of the C-tagged rhbFGF is higher than that of the N-tagged, rhbFGF with N-tag demonstrated promising and comparable biological activity, which is evaluated through a mammalian cell proliferation assay. Taken together, these results indicate that silkworm-BEVS could contribute to the mass-production of the biologically active rhbFGF for medical uses..
10. Yoshiki Morifuji, Jian Xu, Noriko Karasaki, Kazuhiro Iiyama, Daisuke Morokuma, Masato Hino, Akitsu Masuda, Takumi Yano, Hiroaki Mon, Takahiro Kusakabe, Jae Man Lee, Expression, Purification, and Characterization of Recombinant Human α1-Antitrypsin Produced Using Silkworm–Baculovirus Expression System, Molecular Biotechnology, 10.1007/s12033-018-0127-y, 2018.01, Human α1-antitrypsin (AAT) is the most abundant serine proteinase inhibitor (serpin) in the human plasma. Commercially available AAT for the medications of deficiency of α1-antitrypsin is mainly purified from human plasma. There is a high demand for a stable and low-cost supply of recombinant AAT (rAAT). In this study, the baculovirus expression vector system using silkworm larvae as host was employed and a large amount of highly active AAT was recovered from the silkworm serum (~ 15 mg/10 ml) with high purity. Both the enzymatic activity and stability of purified rAAT were comparable with those of commercial product. Our results provide an alternative method for mass production of the active rAAT in pharmaceutical use..
11. Akitsu Masuda, Kosuke Minamihata, Masato Hino, Daisuke Morokuma, Noriko Karasaki, Hiroaki Mon, Noriho Kamiya, Takahiro Kusakabe, Jae Man Lee, Modular surface display for porcine circovirus type 2 virus-like particles mediated by microbial transglutaminase, Journal of Insect Biotechnology and Sericology, 10.11416/jibs.87.2_053, 87, 2, 53-60, 2018.01, Virus-like particles (VLPs) are multi-protein complex, which mimic viral particles without viral genomes. Recently various applications of VLPs for medical and pharmaceutical fields are developed. Furthermore, surface modification of VLPs is expected to extend their functional versatility. As an enzymatic strategy for the modification, microbial transglutaminase (MTG) that catalyzes the covalent bond between a glutamine residue and a ly-sine residue or a primary amine is suitable for efficient protein ligation. In the previous study, we demonstrated the efficient production of immunogenic VLPs of porcine circovirus type 2 (PCV2) using silkworm-baculovirus expression vector system (silkworm-BEVS). Herein, we established the display system of exotic molecules to VLPs by enzymatic covalent cross-linking using MTG. For the target modification, Q-tag (YPLQMRG) that is MTG reactive sequence were introduced into the N-terminus, C-terminus, or loop BC of capsid protein of PCV2 and successfully expressed as VLPs in silkworm pupae. Of these, PCV2 VLPs with Q-tag at the loop BC and C-terminus were efficiently conjugated with FITC-cadaverine and K-tagged (MRHKGS) EGFP by MTG. These results showed that flexible surface modification of VLPs mediated by MTG could be used for development of several therapeutic tools such as multivalent vaccines..
12. Akitsu Masuda, Jae Man Lee, Takeshi Miyata, Tetsuo Sato, Shizuka Hayashi, Masato Hino, Daisuke Morokuma, Noriko Karasaki, Hiroaki Mon, Takahiro Kusakabe, Purification and characterization of immunogenic recombinant virus-like particles of porcine circovirus type 2 expressed in silkworm pupae, Journal of General Virology, 10.1099/jgv.0.001087, 99, 7, 917-926, 2018.01, Porcine circovirus type 2 (PCV2) is a primary causative agent of postweaningmultisystemic wasting syndrome (PMWS), which has a significant economic impact on the swine industry. The capsid protein (Cap) encoded by ORF2 of the viral genome has been used effectively as a vaccine against PCV2 infection. The Cap protein can spontaneously assemble into virus-like particles (VLPs) that are safe and highly immunogenic for vaccine applications. Several expression systems, including bacteria, yeast and insect cells, have been utilized to produce PCV2 VLPs. However, in some cases, the recombinant Cap (rCap) proteins produced in bacteria and yeast do not assemble spontaneously. In this study, we expressed rCap protein using a silkworm–baculovirus expression vector system (silkworm–BEVS) for mass production of PCV2 VLPs and established a simple three-step protocol for its purification from pupae: extraction by detergent, ammonium sulfate precipitation and anion exchange column chromatography. Size-exclusion chromatography (SEC) analysis and transmission electron microscope (TEM) observation showed that purified rCap proteins formed VLPs with a similar morphology to that of the original virus. Furthermore, the VLPs produced in silkworms were capable of inducing neutralizing antibodies against PCV2 in mice. Our results demonstrated that the silkworm system is a powerful tool for the production of PCV2 VLPs and will be useful for the development of a reliable and cost-effective PCV2 vaccine..
13. Anandrao Ashok Patil, Tsuneyuki Tatsuke, Hiroaki Mon, Jae Man Lee, Daisuke Morokuma, Masato Hino, Takahiro Kusakabe, Molecular characterization of mitochondrial Zucchini and its relation to nuage-piRNA pathway components in Bombyx mori ovary-derived BmN4 cells, Biochemical and Biophysical Research Communications, 10.1016/j.bbrc.2017.09.107, 493, 2, 971-978, 2017.11, Piwi-interacting RNAs (piRNAs) are a class of small non-coding RNAs that associate with PIWI subfamily proteins, which play an important role in transposon silencing in animal germ cell. The piRNAs biogenesis is divided into two major pathways: primary and secondary, and both pathways are independent of double-stranded RNA-processing enzyme Dicer, which processes the single-stranded RNA transcripts in microRNA (miRNA) and siRNA (small interfering RNA) pathway. Primary piRNAs are processed from long non-coding RNA precursors transcribed from piRNA clusters. Zucchini (Zuc), a mitochondrial phospholipase D (PLD) superfamily protein is conserved among the animals and involved in piRNA biogenesis. Recent studies showed that the Zucchini is an endoribonuclease essential for primary piRNA maturation and production of phased piRNA in secondary piRNA biogenesis of drosophila germ cell. Based on these reports, here we identified and studied the silkworm Zucchini (BmZuc) at subcellular level in ovary-derived BmN4 cell. The silkworm Zuc specifically expressed in germ-related tissues and localized on mitochondria and partially co-localized with perinuclear nuage-piRNA pathway components and nuage marker protein BmVasa. Molecular dissection analyses revealed that the conserved mitochondrial localization sequence, RGV motif, PLDc 2 domain and HKD motif are important for the BmZuc mitochondrial localization. Moreover, the knockdown analyses showed that the piRNA pathway components are independent on BmZuc for their nuage localization, whereas BmZuc depend on piRNA pathway components for the proper localization. Our data provides vital information on mitochondrial BmZuc and its relationship to “nuage” in ovary-derived BmN4 cell..
14. Ming Ming Ji, Jae Man Lee, Hiroaki Mon, Kazuhiro Iiyama, Tsuneyuki Tatsuke, Daisuke Morokuma, Masato Hino, Mami Yamashita, Kazuma Hirata, Takahiro Kusakabe, Lipidation of BmAtg8 is required for autophagic degradation of p62 bodies containing ubiquitinated proteins in the silkworm, Bombyx mori, Insect Biochemistry and Molecular Biology, 10.1016/j.ibmb.2017.08.006, 89, 86-96, 2017.10, p62/Sequestosome-1 (p62/SQSTM1, hereafter referred to as p62) is a major adaptor that allows ubiquitinated proteins to be degraded by autophagy, and Atg8 homologs are required for p62-mediated autophagic degradation, but their relationship is still not understood in Lepidopteran insects. Here it is clearly demonstrated that the silkworm homolog of mammalian p62, Bombyx mori p62 (Bmp62), forms p62 bodies depending on its Phox and Bem1p (PB1) and ubiquitin-associated (UBA) domains. These two domains are associated with Bmp62 binding to ubiquitinated proteins to form the p62 bodies, and the UBA domain is essential for the binding, but Bmp62 still self-associates without the PB1 or UBA domain. The p62 bodies in Bombyx cells are enclosed by BmAtg9-containing membranes and degraded via autophagy. It is revealed that the interaction between the Bmp62 AIM motif and BmAtg8 is critical for the autophagic degradation of the p62 bodies. Intriguingly, we further demonstrate that lipidation of BmAtg8 is required for the Bmp62-mediated complete degradation of p62 bodies by autophagy. Our results should be useful in future studies of the autophagic mechanism in Lepidopteran insects..
15. Anandrao Ashok Patil, Tsuneyuki Tatsuke, Hiroaki Mon, Jae Man Lee, Daisuke Morokuma, Masato Hino, Takahiro Kusakabe, Characterization of Armitage and Yb containing granules and their relationship to nuage in ovary-derived cultured silkworm cell, Biochemical and Biophysical Research Communications, 10.1016/j.bbrc.2017.06.008, 490, 2, 134-140, 2017.08, PIWI-interacting RNAs (piRNAs) are a class of endogenous small non-coding RNAs, which are mostly 24–32 nucleotides in length and interact specifically with PIWI subfamily of argonaute proteins. Despite the significant research progress in germ line piRNA pathway, its role in somatic cell is not well known. In Drosophila ovarian somatic cell, maturation of primary piRNA and its loading onto Piwi occurs at perinuclear Yb body. The Armitage (Armi) and Yb proteins are the major components of Yb body and specially expressed in ovarian somatic cell. Based on the reports, here we studied the BmArmi and BmYb in Bombyx mori ovary-derived BmN4 cells expressing BmVasa. In this study, we show that BmArmi and BmYb co-localized with BmVasa at nuage. The helicase domains of BmArmi and BmYb are important for nuage localization. Moreover, RNAi of piRNA components reveal that BmArmi depend on BmAgo3 for nuage localization, and BmArmi and BmYb form cytoplasmic granules independently in the absence of BmVasa. Our results provide evidence that the BmArmi and BmYb coexist with BmVasa and play an important role in perinuclear nuage granules formation in ovary-derived BmN4 cell..
16. Hiroaki Mon, Jae Man Lee, Masanao Sato, Takahiro Kusakabe, Identification and functional analysis of outer kinetochore genes in the holocentric insect Bombyx mori, Insect Biochemistry and Molecular Biology, 10.1016/j.ibmb.2017.04.005, 86, 1-8, 2017.07, The kinetochore creates chromosomal attachment sites for microtubules. The kinetochore-microtubule interface plays an important role in ensuring accurate transmission of genetic information to daughter cells. Bombyx mori is known to possess holocentric chromosomes, where spindle microtubules attach along the entire length of the chromosome. Recent evidence suggests that CENP-A and CENP-C, which are essential for centromere structure and function in other species, have lost in holocentric insects, implying that B. mori is able to build its kinetochore regardless of the lack of CENP-A and CENP-C. Here we report the identification of three outer kinetochore genes in the silkworm B. mori by using bioinformatics and RNA interference-based screening. While the homologs of Ndc80 and Mis12 have strong similarity with those of other organisms, the five encoded proteins (BmNuf2, BmSpc24, BmSpc25, BmDsn1 and BmNnf1) are highly diverged from their counterparts in other species. Microscopic studies show that the outer kinetochore protein is distributed along the entire length of the chromosomes, which is a key feature of holocentric chromosomes. We also demonstrate that BmDsn1 forms a heterotrimeric complex with BmMis12 and BmNnf1, which acts as a receptor of the Ndc80 complex. In addition, our study suggests that a small-scale RNAi-based candidate screening is a useful approach to identify genes which may be highly divergent among different species..
17. Mami Yamashita, Jian Xu, Daisuke Morokuma, Kazuma Hirata, Masato Hino, Hiroaki Mon, Masateru Takahashi, Samir M. Hamdan, Kosuke Sakashita, Kazuhiro Iiyama, Yutaka Banno, Takahiro Kusakabe, Jae Man Lee, Characterization of Recombinant Thermococcus kodakaraensis (KOD) DNA Polymerases Produced Using Silkworm-Baculovirus Expression Vector System, Molecular Biotechnology, 10.1007/s12033-017-0008-9, 59, 6, 221-233, 2017.06, The KOD DNA polymerase from Thermococcus kodakarensis (Tkod-Pol) has been preferred for PCR due to its rapid elongation rate, extreme thermostability and outstanding fidelity. Here in this study, we utilized silkworm-baculovirus expression vector system (silkworm-BEVS) to express the recombinant Tkod-Pol (rKOD) with N-terminal (rKOD-N) or C-terminal (rKOD-C) tandem fusion tags. By using BEVS, we produced functional rKODs with satisfactory yields, about 1.1 mg/larva for rKOD-N and 0.25 mg/larva for rKOD-C, respectively. Interestingly, we found that rKOD-C shows higher thermostability at 95 °C than that of rKOD-N, while that rKOD-N is significantly unstable after exposing to long period of heat-shock. We also assessed the polymerase activity as well as the fidelity of purified rKODs under various conditions. Compared with commercially available rKOD, which is expressed in E. coli expression system, rKOD-C exhibited almost the same PCR performance as the commercial rKOD did, while rKOD-N did lower performance. Taken together, our results suggested that silkworm-BEVS can be used to express and purify efficient rKOD in a commercial way..
18. Daisuke Morokuma, Jian Xu, Masato Hino, Hiroaki Mon, Jasmeen S. Merzaban, Masateru Takahashi, Takahiro Kusakabe, Jae Man Lee, Expression and Characterization of Human β-1, 4-Galactosyltransferase 1 (β4GalT1) Using Silkworm–Baculovirus Expression System, Molecular Biotechnology, 10.1007/s12033-017-0003-1, 59, 4-5, 151-158, 2017.05, Baculovirus expression vector system (BEVS) is widely known as a mass-production tool to produce functional recombinant glycoproteins except that it may not be always suitable for medical practice due to the differences in the structure of N-linked glycans between insects and mammalian. Currently, various approaches have been reported to alter N-linked glycan structures of glycoproteins derived from insects into terminally sialylated complex-type N-glycans. In the light of those studies, we also proposed in vitro maturation of N-glycan with mass-produced and purified glycosyltransferases by silkworm–BEVS. β-1,4-Galactosyltransferase 1 (β4GalT1) is known as one of type II transmembrane enzymes that transfer galactose in a β-1, 4 linkage to accepter sugars, and a key enzyme for further sialylation of N-glycans. In this study, we developed a large-scale production of recombinant human β4GalT1 (rhβ4GalT1) with N- or C-terminal tags in silkworm–BEVS. We demonstrated that rhβ4GalT1 is N-glycosylated and without mucin-type glycosylation. Interestingly, we found that purified rhβ4GalT1 from silkworm serum presented higher galactosyltransferase activity than that expressed from cultured mammalian cells. We also validated the UDP-galactose transferase activity of produced rhβ4GalT1 proteins by using protein subtracts from silkworm silk gland. Taken together, rhβ4GalT1 from silkworms can become a valuable tool for producing high-quality recombinant glycoproteins with mammalian-like N-glycans..
19. Kazuhiro Iiyama, Eigo Takahashi, Jae Man Lee, Hiroaki Mon, Mai Morishita, Takahiro Kusakabe, Chisa Yasunaga-Aoki, Alkaline protease contributes to pyocyanin production in Pseudomonas aeruginosa, FEMS microbiology letters, 10.1093/femsle/fnx051, 364, 7, 2017.04, The role of the alkaline protease (AprA) in pyocyanin production in Pseudomonas aeruginosa was investigated. AprA was overproduced when a plasmid carrying the aprA gene was introduced to an aprA-deletion mutant strain, EG03; thus, aprA-complemented EG03 was used as an overproducing strain. The complemented strain produced higher pyocyanin than the mutant strain in all commercially available media evaluated. Particularly, pyocyanin production was higher in the complemented than in the parental strain in brain-heart infusion and tryptic soy broths. These results suggested that protein degradation products by AprA were utilized for pyocyanin production. Protein-rich media were used in subsequent validation studies. Similar results were obtained when the basal medium was supplemented with casein or skim milk as the sole organic nitrogen source. However, gelatin failed to induce abundant pyocyanin production in the complemented strain, despite the presence of protein degradation products by AprA as assessed by SDS-PAGE. Thus, gelatin degradation products may not be suitable for pyocyanin synthesis. In conclusion, AprA could contribute to pyocyanin production in the presence of several proteins or peptides..
20. Kazuhiro Iiyama, Mai Morishita, Jae Man Lee, Hiroaki Mon, Takahiro Kusakabe, Kosuke Tashiro, Taiki Akasaka, Chisa Yasunaga-Aoki, Kazuhisa Miyamoto, A reconsideration of the taxonomic position of two bacterial strains isolated from Flacherie-Diseased silkworms in 1965, Journal of Insect Biotechnology and Sericology, 10.11416/jibs.86.2_035, 86, 2, 35-41, 2017.01, Recent advances in bacterial characterization methodologies have made taxonomic categorization significantly more accurate. Here, we re-evaluated the position of bacterial strains (532 and 652) belonging to the genus Hafnia, isolated from flacherie-diseased silkworms in 1965. Phylogenetic analysis based on the 16S rRNA gene sequences of these strains suggests that they belong to genus Enterobacter. Using multilocus sequence analysis (MLSA), these strains were further classified to MLSA group A, which is a “core” group of Enterobacter containing E. cloacae (the type species of the genus). Although these strains were closely related to E. mori, E. tabaci, and E. asburiae, they also had other MLSA characteristics that distinguished them from these neighboring bacterial species. These data were supported by further biochemical analysis. Thus, it appears that the 532 and 652 strains isolated almost half a century ago belong to genus Enterobacter, and their unique characteristics strongly suggest that they are a novel bacterial species..
21. Ming Ming Ji, Jae Man Lee, Hiroaki Mon, Jian Xu, Tsuneyuki Tatsuke, Takahiro Kusakabe, Proteasome inhibitor MG132 impairs autophagic flux through compromising formation of autophagosomes in Bombyx cells, Biochemical and Biophysical Research Communications, 10.1016/j.bbrc.2016.09.151, 479, 4, 690-696, 2016.10, MG132 has been used as a proteasome inhibitor on Bombyx cells, but its physiological effects on autophagy still have not been elucidated. In this study, we find that the lipidated BmAtg8, BmAtg8-PE as an autophagosomal marker protein, is only localized to membranes. Then we established systems to monitor autophagic flux in Bombyx cells: Induction of autophagy reduces exogenous BmAtg8 and exogenous BmAtg8-PE, facilitates formation of autophagosomes indicated by green EGFP-BmAtg8 puncta after cotreatment by Rapamycin and Bafilomycin A1, and causes accumulation of free EGFP from EGFP-BmAtg8 cleavage in autolysosomes. Using these established systems, we find that exposure of MG132 inhibits both basal and Rapamycin-induced autophagy when polyubiquitinated proteins are accumulated markedly in Bombyx cells. Interestingly, we reveal that attenuation of autophagy in these cells is ascribed as distinct suppression of formation of autophagosomes after MG132 treatment..
22. Jianping Chen, Jian Xu, Masato Hino, Mami Yamashita, Kazuma Hirata, Anandrao Ashok Patil, Tsuneyuki Tatsuke, Hiroaki Mon, Yutaka Banno, Takahiro Kusakabe, Jae Man Lee, Co-expression of silkworm allatostatin-C receptor BNGR-A1 with its cognate G protein subunits enhances the GPCR display on the budding baculovirus, Journal of Asia-Pacific Entomology, 10.1016/j.aspen.2016.07.007, 19, 3, 753-760, 2016.09, G protein-coupled receptors (GPCRs) are seven transmembrane proteins, which play an essential role in transmitting various extracellular signals into cells. For functional and structural analysis of GPCRs, it is necessary to produce active GPCRs in high quantities with outstanding purity. Fortunately, earlier baculovirus expression vector system has been reported as a proven functional GPCR mass-production tool. Therefore, in this study, we selected Bombyx mori allatostatin-C neuropeptide receptor BNGR-A1 as a GPCR reporter protein, which has been already proved successful binding of ligand. We confirmed its expression profile in various silkworm tissues and cell lines and then verified its plasma membrane subcellular localization in cultured silkworm BmN4 cells. In addition, we constructed recombinant baculoviruses for BNGR-A1, its ligand allatostatin-C (BmAST-C) and related eight G proteins (Gs, G12, Gα4, Gq, Gβ2, Gβ3, Gβ5 and Gγ) and subsequently monitored the extracellular or intracellular expression of BNGR-A1 by co-infection with its ligand and cognate G proteins-expressing viruses. It is interesting to observe that different combinations of G proteins could result in changes or even undetectable of final yields of BNGR-A1, suggesting the essential roles of G proteins involved in the GPCR expression or stabilization. The present study demonstrated that co-infection of recombinant viruses expressing Gα4β3γ trimer enhanced the production of BNGR-A1 on BV fractions. To our knowledge, this is a fine strategy for identifying the specific G protein partner responsible for certain GPCR of interest. These studies would provide a novel idea for improving GPCR expression in the silkworm by baculovirus expression vector system..
23. Kazuhiro Iiyama, Jae Man Lee, Tuneyuki Tatsuke, Hiroaki Mon, Takahiro Kusakabe, Expression and Characterization of Recombinant Serratia liquefaciens Nucleases Produced with Baculovirus-mediated Silkworm Expression System, Molecular Biotechnology, 10.1007/s12033-016-9937-y, 58, 6, 393-403, 2016.06, Baculovirus-Bombyx mori protein expression system has mainly been used for translation of eukaryotic proteins. In contrast, information pertaining to bacterial protein expression using this system is not sufficient. Therefore, recombinant nucleases from Serratia liquefaciens (rSlNucAs) were expressed in a Baculovirus-B. mori protein expression system. rSlNucAs containing the native signal peptide (rSlNucA-NSP) or silkworm 30-K signal peptide (rSlNucA-30K) at the NH2-terminus were constructed to enable secretion into the extracellular fraction. Both rSlNucA-30K and rSlNucA-NSP were successfully secreted into hemolymph of B. mori larvae. Affinity-purified rSlNucAs showed high nuclease activity. Optimum pH was 7.5 and half of maximum activity was maintained between pH 7.0 and 9.5. Optimum temperature was 35 °C. rSlNucAs showed sufficient activity in twofold-diluted radioimmunoprecipitation assay buffer and undiluted, mild lysis buffer. Genomic DNA of Escherichia coli was efficiently digested by rSlNucAs in the bacterial lysate. The results in this study suggest that rSlNucAs expressed by the Baculovirus-B. mori protein expression system will be a useful tool in molecular biology. Functional recombinant protein of bacteria was produced by Baculovirus-B. mori protein expression system. This system may be highly suitable for bacterial extracellular protein secreted via Sec pathway..
24. Masato Hino, Takuji Kawanami, Jian Xu, Daisuke Morokuma, Kazuma Hirata, Mami Yamashita, Noriko Karasaki, Tuneyuki Tatsuke, Hiroaki Mon, Kazuhiro Iiyama, Noriho Kamiya, Yutaka Banno, Takahiro Kusakabe, Jae Man Lee, High-level expression and purification of biologically active human IL-2 using silkworm-baculovirus expression vector system, Journal of Asia-Pacific Entomology, 10.1016/j.aspen.2016.03.014, 19, 2, 313-317, 2016.06, Interleukin 2 (IL-2) is a pharmacologically vital cytokine secreted mainly by activated CD4+ T lymphocyte. Recombinant human IL-2 (rhIL-2) protein has already been globally applied as an immune-therapeutic reagent for various diseases. Therefore, there is great interest in developing an active form of rhIL-2 in very large amounts and with excellent purity for clinical use. In this study, we successfully mass-expressed and purified N- or C-terminal tandem tag-fused rhIL-2 in a baculovirus expression vector system (BEVS) using silkworm larvae as factories. We confirmed that the intrinsic instability of hIL-2 causes the loss and low recovery of N-tagged rhIL-2 and that C-tagged rhIL-2 is more suitable for mass production. Furthermore, the activity of purified rhIL-2s was further validated by a cell proliferation assay of a human natural killer cell line, and both rhIL-2 proteins produced in the silkworm-BEVS exhibited comparable activity to that of the commercial E.coli-derived rhIL-2. Taken together, our results and strategies could contribute greatly to the mass production of active rhIL-2..
25. Erika Taira, Hiroaki Mon, Jae Man Lee, Takahiro Kusakabe, Chisa Yasunaga-Aoki, Kazuhiro Iiyama, Virulence of lipopolysaccharide-deficient mutants of serratia liquefaciens toward the silkworm, Bombyx mori, Journal of Insect Biotechnology and Sericology, 10.11416/jibs.85.1_007, 85, 1, 7-14, 2016.04, We aimed to identify virulence-associated genes of Serratia liquefaciens FK01 against Bombyx mori. Among 1,200 transconjugants from a transposon library, 4 (ET0234, ET0373, ET0418, and ET0964) showed decreased virulence towards B. mori. Southern hybridization revealed that the transposon was inserted at a single site of the S. liquefaciens genome. The flanking sequences of the transposon indicated that it disrupted the lipopolysaccharide (LPS) synthesis gene in all mutants. The complemented strain restored virulence completely or partially. Thus, LPS contributes to the virulence of S. liquefaciens against B. mori. Serum killing assays indicated that the bacterium was probably killed by the complement system. Since the innate immunity of a host is triggered by bacterial recognition, LPS might inhibit recognition by modifying the bacterial surface in B. mori..
26. Eigo Takahashi, Jae Man Lee, Hiroaki Mon, Yuuka Chieda, Chisa Yasunaga-Aoki, Takahiro Kusakabe, Kazuhiro Iiyama, Effect of antibiotics on extracellular protein level in Pseudomonas aeruginosa, Plasmid, 10.1016/j.plasmid.2016.03.001, 84-85, 44-50, 2016.03, Pseudomonas aeruginosa PAO1 organisms harbouring different plasmids were cultured in broths containing appropriate antibiotic(s). Extracellular proteins were more abundant in the presence of tetracycline or kanamycin than in the presence of other antibiotics. Zymography revealed that alkaline protease (AprA) production was interfered by these antibiotics. Extracellular proteins were not observed at the same level when AprA-deficient EG03 strains were cultured in the presence of different antibiotics. The extracellular protein levels were dependent on the antibiotics and plasmid derivative groups. Levels of extracellular protein were not significantly different between PAO1 (pBBR1MCS-5) and EG03 (pAprcomp-MCS5), and profiles of the extracellular proteome were comparable. In contrast, the level of EG03 (pBBR1MCS-MCS5) extracellular protein was higher than those observed in the other two strains. These results suggested that although AprA partially contributes to the alteration of extracellular protein level, the effect is limited..
27. Masato Hino, Daisuke Morokuma, Hiroaki Mon, Jae Man Lee, Takahiro Kusakabe, Characterization of the roles of DNA polymerases, clamp, and clamp loaders during s-phase progression and cell cycle regulation in the silkworm, bombyx mori, Journal of Insect Biotechnology and Sericology, 10.11416/jibs.85.2_021, 85, 2, 21-29, 2016.01, DNA replication is one of key event in cell-cycle progression, yet due to their importance and lethality, the chronological phenotypes of DNA synthesis machineries after the depletion of corresponding genes have proved difficult to study. In the present study, mRNAs for three DNA polymerases, a clamp, and three clamp loaders were gradually depleted from cultured silkworm cells by soaking RNAi. Interestingly, the depletion of these DNA synthesis factors had different effects on the cell growth rate and arrest of cell-cycle progression during time-lapse observation. The depletion of DNA polymerases immediately arrested the cell-cycle progression at the S phase, while that of PCNA, a DNA clamp, required more time to slow cell growth and finally induced apoptosis. Surprisingly, silkworm cells continued to undergo several rounds of cell division when the components of clamp loaders were knocked down..
28. Rina Hamajima, Chisa Yasunaga-Aoki, Masashi Iwanaga, Shigeo Imanishi, Jun Kobayashi, Kuni Sasaki, Takahiro Kusakabe, Jae Man Lee, Hiroaki Mon, Michihiro Kobayashi, Motoko Ikeda, rRNA degradation in Bombyx mori and Bombyx mandarina cells infected witv veterologous nucleopolyvedroviruses, Journal of Insect Biotechnology and Sericology, 85, 3, 73-77, 2016.01, We previously found that rRNA of BM-N cells derived from the silkworm Bombyx mori undergoes rapid and extensive degradation through site-specific cleavage during abortive infection with nucleopolyhedroviruses (NPVs) of Autographa californica (AcMNPV), Hyphantria cunea (HycuMNPV), Spodoptera exigua and S. litura. Here, we demonstrated that rRNA degradation also occurs in Bme21 and Bm-aff3 cells, which are derived from B. mori embryo and fat body, respectively, during infection with AcMNPV. rRNA degradation in Bme21 cells was also observed following HycuMNPV infection, but was not detected in Bm-aff3 cells. We further showed that rRNA in a cell line derived from B. mandarina, an ancestor of B. mori, underwent degradation in response to cellular infection with AcMNPV and HycuMNPV. In contrast, no rRNA degradation was observed in a cell line derived from Antheraea pernyi. Taken together, these results indicate that NPV-triggered rRNA degradation represents a mechanism of innate antiviral immunity that is unique to B. mori and B. mandarina cells..
29. Li Zhu, Hiroaki Mon, Jian Xu, Jae Man Lee, Takahiro Kusakabe, CRISPR/Cas9-mediated knockout of factors in non-homologous end joining pathway enhances gene targeting in silkworm cells, Scientific reports, 10.1038/srep18103, 5, 2015.12, Gene targeting can be achieved by precise genetic modifications through homology-directed repair (HDR) after DNA breaks introduced by genome editing tools such as CRISPR/Cas9 system. The most common form of HDR is homologous recombination (HR). Binding to the DNA breaks by HR factors is thought to compete with non-homologous end joining (NHEJ), an alternative DNA repair pathway. Here, we knocked out the factors in NHEJ by CRISPR/Cas9 system in silkworm cells, so that increased the activities of HR up to 7-fold. Also efficient HR-mediated genome editing events occurred between the chromosomal BmTUDOR-SN gene and donor DNA sequences with an EGFP gene in the middle of two homologous arms for the target gene. Utilizing the NHEJ-deficient silkworm cells, we found that homologous arms as short as 100 bp in donor DNA could be designed to perform precise genome editing. These studies should greatly accelerate investigations into genome editing of silkworm..
30. Jian Xu, Pingbo Zhang, Takahiro Kusakabe, Hiroaki Mon, Zhiqing Li, Li Zhu, Kazuhiro Iiyama, Yutaka Banno, Daisuke Morokuma, Jae Man Lee, Comparative proteomic analysis of hemolymph proteins from Autographa californica multiple nucleopolyhedrovirus (AcMNPV)-sensitive or -resistant silkworm strains during infections, Comparative Biochemistry and Physiology - Part D: Genomics and Proteomics, 10.1016/j.cbd.2015.07.003, 16, 36-47, 2015.12, Abstract We reported previously that baculovirus AcMNPV host-ranges in silkworm strains are controlled by a novel third chromosomal locus. To further isolate the potential host factor and uncover the functional pathway involved, in this study we analyzed hemolymph proteins from AcMNPV-resistant or -sensitive silkworm strains infected with baculoviruses. All the protein spots from 2D electrophoresis were characterized by MALDI-TOF MS and further systematically assessed for differentially regulated proteins at different stages of infection. Subsequently, six candidates were selected for functional analysis using Bm5 cells, where the candidates were knocked-down or overexpressed. We observed that mRNA expression levels of beta-N-acetylglucosaminidase and prophenoloxidase subunit 2 are significantly upregulated during AcMNPV infections in Bm5 cells. Ultimately, we found that RNA interference of ribosomal protein RpL34 causes serious damages to cell viability as well as abortive infection, indicating that ribosomal components are essential for productive baculovirus infection..
31. Daisuke Morokuma, Hiroaki Mon, Yutaka Banno, Takahiro Kusakabe, Jae Man Lee, Differential N-Glycan modifications of human alpha 1-acid glycoprotein (α1AGP) produced in different silkworm strains using the baculovirus expression system, Journal of Insect Biotechnology and Sericology, 10.11416/jibs.84.2_049, 84, 2, 49-53, 2015.11, N-glycosylation plays an important role in various biological activities and in the structural stability of serum glycoproteins. The baculovirus expression system (BES) is widely used to produce recombinant proteins but in some case it is not suitable for medical use because of the differences in N-linked glycans between insects and mammals. We reported that human serum protein alpha 1-acid protein (α1AGP) is effectively used as a model protein for evaluating the validity of engineering the insect-type N-glycosylation pathway. Using this protein, the productivity and N-linked glycan structures were compared among the 37 different silkworm strains. Interestingly, there was no difference in N-linked glycan structure among the silkworm strains, but there was difference in the degree of N-glycosylation..
32. Saki Imai, Takahiro Kusakabe, Jian Xu, Zhiqing Li, Shintaro Shirai, Hiroaki Mon, Daisuke Morokuma, Jae Man Lee, Roles of silkworm endoplasmic reticulum chaperones in the secretion of recombinant proteins expressed by baculovirus system, Molecular and cellular biochemistry, 10.1007/s11010-015-2529-5, 409, 1-2, 255-262, 2015.11, Baculovirus expression vector system (BEVS) is widely used for production of recombinant eukaryotic proteins in insect larvae or cultured cells. BEVS has advantages over bacterial expression system in producing post-translationally modified secreted proteins. However, for some unknown reason, it is very difficult for insects to secrete sufficiently for certain proteins of interest. To understand the reasons why insect cells fail to secrete some kinds of recombinant proteins, we here employed three mammalian proteins as targets, EPO, HGF, and Wnt3A, with different secretion levels in BEVS and investigated their mRNA transcriptions from the viral genome, subcellular localizations, and interactions with silkworm ER chaperones. Moreover, we observed that no significantly influence on the secretion amounts of all three proteins when depleting or overexpressing most endogenous ER chaperone genes in cultured silkworm cells. However, among all detected ER chaperones, the depletion of BiP severely decreased the recombinant protein secretion in BEVS, indicating the possible central role of Bip in silkworm secretion pathway..
33. Li Zhu, Tsuneyuki Tatsuke, Jian Xu, Zhiqing Li, Hiroaki Mon, Jae Man Lee, Takahiro Kusakabe, Loqs depends on R2D2 to localize in D2 body-like granules and functions in RNAi pathways in silkworm cells, Insect Biochemistry and Molecular Biology, 10.1016/j.ibmb.2015.07.011, 64, 78-90, 2015.09, The phenomenon of RNA interference (RNAi) has been found in various organisms. However, the proteins implicated in RNAi pathway in different species show distinct roles. Knowledge on the underlying mechanism of lepidopteron RNAi is quite lacking such as the roles of Loquacious (Loqs) and R2D2, the dsRNA-binding proteins in silkworm RNAi pathway. Here, we report that Loqs and R2D2 protein depletion affected efficiency of dsRNA-mediated RNAi pathway. Besides, Loqs was found to co-localize with Dicer2 to some specific cytoplasmic foci, which were looked like D2-bodies marked by R2D2 and Dicer2 in Fly cells, thereby calling the foci as D2 body-like granules. Using RNAi methods, Loqs was found to be the key protein in these granules, although R2D2 determined the localization of Loqs in D2 body-like granules. Interestingly, in the R2D2-depeted silkworm cells, the formation of processing bodies, another cytoplasmic foci, was affected. These data indicated R2D2 regulated these two kinds of cytoplasmic foci. Domain deletion analysis demonstrated that dsRBD 1 and 2 were required for Loqs in D2 body-like granules and dsRBD 2 and 3 were required for Loqs to interact with R2D2 and Ago1, respectively. Altogether, our observations provide important information for further study on D2 body-like granules, the newly found cytoplasmic foci in silkworm cells..
34. Atsushi Masuda, Jian Xu, Takumi Mitsudome, Yudai Nagata, Daisuke Morokuma, Hiroaki Mon, Yutaka Banno, Takahiro Kusakabe, Jae Man Lee, Mass Production of an Active Peptide-N-Glycosidase F Using Silkworm-Baculovirus Expression System, Molecular Biotechnology, 10.1007/s12033-015-9866-1, 57, 8, 735-745, 2015.08, The peptide-N4-(N-acetyl-β-d-glucosaminyl) asparagine amidase F (PNGase F) catalyzes the cleavage of N-linked oligosaccharides between the innermost GlcNAc and asparagine residues of high mannose, hybrid and complex oligosaccharides from glycoproteins. The PNGase F has broad substrate specificity and thus is extensively used for the structural and functional studies of the glycoproteins. In this study, we tried to produce active recombinant PNGase F as secreted and intracellular-expressed forms using baculovirus expression vector system (BEVS) through silkworm larvae or cultured cells. PNGase F itself contains potential N-linked glycosylation sites and we found that it was N-glycosylated when PNGase F secreted from silkworm cells. Intriguingly, the secreted recombinant PNGase F has the lower catalytic activity and self-digests its N-linked glycans and therefore this secreted form of this enzyme produced from BEVS is not appropriate for carbohydrate chain analysis. Instead, we successfully mass-produced (2.1 mg/20 silkworm larvae) and purified active recombinant PNGase F as an intracellular protein without N-glycosylations. Besides, we confirmed by directed mutagenesis that several amino acid residues are crucial for the function of PNGase F. Our results provide an alternative method for the mass production of active enzymes involved in the study of glycoproteins..
35. Kazuhiro Iiyama, Hiroaki Mon, Kazuki Mori, Takumi Mitsudome, Jae Man Lee, Takahiro Kusakabe, Kousuke Tashiro, Shin ichiro Asano, Chisa Yasunaga-Aoki, Characterization of KfrA proteins encoded by a plasmid of Paenibacillus popilliae ATCC 14706T, Meta Gene, 10.1016/j.mgene.2015.03.001, 4, 29-44, 2015.06, A scaffold obtained from whole-genome shotgun sequencing of Paenibacillus popilliae ATCC 14706T shares partial homology with plasmids found in other strains of P. popilliae. PCR and sequencing for gap enclosure indicated that the scaffold originated from a 15,929-bp circular DNA. The restriction patterns of a plasmid isolated from P. popilliae ATCC 14706T were identical to those expected from the sequence; thus, this circular DNA was identified as a plasmid of ATCC 14706T and designated pPOP15.9. The plasmid encodes 17 putative open reading frames. Orfs 1, 5, 7, 8, and 9 are homologous to Orfs 11, 12, 15, 16, and 17, respectively. Orf1 and Orf11 are annotated as replication initiation proteins. Orf8 and Orf16 are homologs of KfrA, a plasmid-stabilizing protein in Gram-negative bacteria. Recombinant Orf8 and Orf16 proteins were assessed for the properties of KfrA. Indeed, they formed multimers and bound to inverted repeat sequences in upstream regions of both orf8 and orf16. A phylogenetic tree based on amino acid sequences of Orf8, Orf16 and Kfr proteins did not correlate with species lineage..
36. Daisuke Morokuma, Jian Xu, Hiroaki Mon, Kazuma Hirata, Masato Hino, Shoko Kuboe, Mami Yamashita, Takahiro Kusakabe, Jae Man Lee, Human alpha 1-acid glycoprotein as a model protein for glycoanalysis in baculovirus expression vector system, Journal of Asia-Pacific Entomology, 10.1016/j.aspen.2015.03.006, 18, 2, 303-309, 2015.06, Glycosylation is an important post-translational modification that confers various biological activities, structural stability, and inter-molecular interactions to proteins. Baculovirus expression vector system (BEVS) is widely used to produce recombinant glycoproteins, which may not be suitable for clinical use due to differences in the N-linked glycan structure between insects and mammals. It is necessary to develop an appropriate model protein-base platform for glycoanalysis to engineer the insect-type N-glycosylation pathway into human type efficiently. In this study, we employed human plasma protein alpha 1-acid glycoprotein (α1AGP). It was highly secreted from cultured silkworm cells and larvae when using the BEVS and glycosylated with insect type N-linked glycans. Interestingly, when separated on SDS-PAGE, the purified recombinant α1AGP secreted into silkworm haemolymph generated six distinct products from three alternative translates, suggesting that α1AGP has variations for the recognition or choice of glycosylation sites..
37. Takumi Mitsudome, Hiroaki Mon, Jian Xu, Zhiqing Li, Jae Man Lee, Anandrao Ashok Patil, Atsushi Masuda, Kazuhiro Iiyama, Daisuke Morokuma, Takahiro Kusakabe, Biochemical characterization of maintenance DNA methyltransferase DNMT-1 from silkworm, Bombyx mori, Insect Biochemistry and Molecular Biology, 10.1016/j.ibmb.2015.01.008, 58, 55-65, 2015.03, DNA methylation is an important epigenetic mechanism involved in gene expression of vertebrates and invertebrates. In general, DNA methylation profile is established by de novo DNA methyltransferases (DNMT-3A, -3B) and maintainance DNA methyltransferase (DNMT-1). DNMT-1 has a strong substrate preference for hemimethylated DNA over the unmethylated one. Because the silkworm genome lacks an apparent homologue of de novo DNMT, it is still unclear that how silkworm chromosome establishes and maintains its DNA methylation profile. As the first step to unravel this enigma, we purified recombinant BmDNMT-1 using baculovirus expression system and characterized its DNA-binding and DNA methylation activity. We found that the BmDNMT-1 preferentially methylates hemimethylated DNA despite binding to both unmethylated and hemimethylated DNA. Interestingly, BmDNMT-1 formed a complex with DNA in the presence or absence of methyl group donor, S-Adenosylmethionine (AdoMet) and the AdoMet-dependent complex formation was facilitated by Zn2+ and Mn2+. Our results provide clear evidence that BmDNMT-1 retained the function as maintenance DNMT but its sensitivity to metal ions is different from mammalian DNMT-1..
38. Atsushi Masuda, Jian Xu, Takumi Mitsudome, Daisuke Morokuma, Hiroaki Mon, Yutaka Banno, Takahiro Kusakabe, Jae Man Lee, Improvement of Endo-β-N-acetylglucosaminidase H production using silkworm-baculovirus protein expression system, Journal of Asia-Pacific Entomology, 10.1016/j.aspen.2015.01.006, 18, 2, 175-180, 2015.01, Endo-β-. N-acetylglucosaminidase H (Endo H) catalyzes cleavage between the GlcNAc residues of the chitobiose core of N-linked glycans, leaving one GlcNAc residues attached to asparagine. Endo H cleaves high mannose and hybrid, but not complex, N-linked oligosaccharides on glycoproteins. Because of its unique specificity, Endo H is widely used for the structural and functional analyses of glycoproteins. In our previous study, the recombinant Endo H was produced as a secreted protein using silkworm-baculovirus expression system, but the yield was low (30. μg Endo H/10. ml larval hemolymph) compared to that of Escherichia coli. In this study, we purified active recombinant Endo H as an intracellular protein from fat body of silkworm infected with the recombinant baculovirus expressing Endo H without the exogenous signal peptide. Remarkably, the yield (9.3. mg from 20 silkworm larvae) was about 310-fold higher than that secreted into larval hemolymph as reported previously. In addition, we screened the silkworm strains maintained in Kyushu University and identified n17 as a high-level expression strain for Endo H..
39. Zhiqing Li, Hiroaki Mon, Hitoshi Mitsunobu, Li Zhu, Jian Xu, Jae Man Lee, Takahiro Kusakabe, Dynamics of polycomb proteins-mediated histone modifications during UV irradiation-induced DNA damage, Insect Biochemistry and Molecular Biology, 10.1016/j.ibmb.2014.10.001, 55, 9-18, 2014.12, Polycomb group (PcG) complexes are known to be chromatin modifiers and transcriptional repressors. In this work, we reported that the histone-modifying PcG complexes are able to participate in the repair process of ultraviolet (UV)-induced DNA lesions in the silkworm, Bombyx mori. The silkworm cells with depletion of PcG genes showed hypersensitive to UV-C irradiation and increased inhibition of cell proliferation. Interestingly, an SQ site in the silkworm-human chimeric H2A protein synthesized here was phosphorylated rapidly upon UV-C exposure, which could be used as a marker for monitoring the response to DNA damage in silkworm cells. Under these UV-C irradiated conditions, we found that PRC1-mediated ubiquitylation of H2AX, but not of H2AZ, were decreased and this deubiquitylation was independent of its phosphorylation event. In contrast, UV-C irradiation induced the increase of trimethylation of lysine 27 on histone H3 (H3K27me3), a mark of transcriptionally silent chromatin catalyzed by another PcG subcomplex, PRC2. Collectively, we provided the first evidence on chromatin remodeling in response to UV-C lesion in silkworm and revealed another layer role for PcG complexes-mediated histone modifications in contributing to creating an open chromatin structure for the efficient repair of DNA damages..
40. Ryohei Sugahara, Hiroaki Mon, Jae Man Lee, Takahiro Shiotsuki, Takahiro Kusakabe, Differential contribution of the Fanconi anemia-related proteins to repair of several types of DNA damage in cultured silkworm cells, FEBS Letters, 10.1016/j.febslet.2014.09.009, 588, 21, 3959-3963, 2014.11, The silkworm Fanconi anemia (FA) pathway is required for normal cellular resistance to mitomycin C (MMC) in silkworms, but little is known about the requirement for repair of other types of DNA damage. Here we report that silkworm cells deficient for FA proteins FancD2 and FancM exhibit normal sensitivities to hydroxyurea (HU) and camptothecin (CPT), although FancM-dependent FancD2 monoubiquitination is induced upon these treatments. Similar results were observed in cells depleted for Rmi1 and Mhf1, which interact with the FancM protein. We also found that Rad51-knockdown cells exhibited normal sensitivity to HU despite induction of double-strand breaks by HU treatment..
41. Zhiqing Li, Hiroaki Mon, Jian Xu, Li Zhu, Jae Man Lee, Takahiro Kusakabe, A conserved SUMOylation signaling for cell cycle control in a holocentric species Bombyx mori, Insect Biochemistry and Molecular Biology, 10.1016/j.ibmb.2014.05.008, 51, 1, 71-79, 2014.08, SUMOylation is an essential post-translational modification that regulates a variety of cellular processes including cell cycle progression. Although the SUMOylation pathway has been identified and investigated in many eukaryotes, the mechanisms of SUMOylation in regulating the functions of various substrates are still poorly understood. Here, we utilized a model species, the silkworm Bombyx mori that possesses holocentric chromosomes, to exploit the role of the SUMOylation system in cell cycle regulation. We identified all the components that are involved in the SUMOylation pathway in the silkworm genome. Our data revealed a cell cycle-dependent transcription of the SUMOylation genes, localization of the SUMOylation proteins, and abundance of the SUMOylation substrates in cultured silkworm cells. Importantly, the proliferation of the silkworm cells was strikingly inhibited by interference with SUMOylation genes expression, possibly due to an arrest of the SUMOylation-deficient cells at the G2/M phase. Furthermore, disruption of the SUMOylation genes induced the defects of holocentric chromosome congression and segregation during mitosis, which was consistent with high expressions of the SUMOylation genes and high enrichments of global SUMOylation at this stage, suggesting that the SUMOylation system in silkworm is essential for cell cycle regulation, with one particular role in mitosis..
42. Sun Mee Hong, Hiroaki Mon, Jae Man Lee, Takahiro Kusakabe, Characterization and recombinant protein expression of ferritin light chain homologue in the silkworm, Bombyx mori, Insect Science, 10.1111/1744-7917.12031, 21, 2, 135-146, 2014.04, The silkworm genome encodes three iron storage proteins or ferritins, Fer1HCH, Fer2LCH, and Fer3HCH. Probing our EST library constructed from 1-day-old silkworm eggs revealed only Fer2LCH mRNA, which encoded for a protein with a predicted putative N-glycosylation site. Developmental and tissue expression analyses during embryogenesis revealed that Fer2LCH mRNA was abundant from 6 h to 6 days after oviposition. Transcriptional expression of Fer2LCH during the postembryonic stage is also high in the larval fat body and mid-gut, and then is upregulated in all pupal tissues tested. We found that Fer2LCH mRNA contains an iron-responsive element, suggesting this ferritin subunit is subject to translational control. Although ferritin expression has been shown to increase following immune challenge in other insects, the levels of Fer2LCH mRNA were not significantly induced following viral or bacterial infection of Bombyx mori. Using a baculovirus expression system we expressed recombinant BmFer2LCH protein, which was detectable in the cytoplasmic fraction, likely in a compartment of the secretory pathway, and was shown to undergo posttranslational modifications including N-glycosylation. In particular, rBmFer2LCH carbohydrate chains were composed of mannose and GlcNAc. We suggest that Fer2LCH is important for iron homeostasis and maintaining normal organ function in silkworms..
43. R. Sugahara, H. Mon, J. M. Lee, T. Kusakabe, Middle region of FancM interacts with Mhf and Rmi1 in silkworms, a species lacking the Fanconi anaemia (FA) core complex, Insect Molecular Biology, 10.1111/imb.12072, 23, 2, 185-198, 2014.04, The Fanconi anaemia (FA) pathway is responsible for interstrand crosslink (ICL) repair. Among the FA core complex components, FANCM is believed to act as a damage sensor for the ICL-blocked replication fork and also as a molecular platform for FA core complex assembly and interaction with Bloom's syndrome (BS) complex that is thought to play an important role in the processing of DNA structures such as stalled replication forks. In the present study, we found that in silkworms, Bombyx mori, a species lacking the major FA core complex components (FANCA, B, C, E, F, and G), FancM is required for FancD2 monoubiquitination and cell proliferation in the presence of mitomycin C (MMC). Silkworm FancM (BmFancM) was phosphorylated in the middle regions, and the modification was associated with its subcellular localization. In addition, BmFancM interacted with Mhf1, a histone-fold protein, and Rmi1, a subunit of the BS complex, in the different regions. The interaction region containing at least these two protein-binding domains played an essential role in FancM-dependent resistance to MMC. Our results suggest that BmFancM also acts as a platform for recruitment of both the FA protein and the BS protein, although the silkworm genome seems to lose FAAP24, a FancM-binding partner protein in mammals..
44. Jae Man Lee, Jian Xu, Hiroaki Mon, Takumi Mitsudome, Atsushi Masuda, Kaito Yoshimura, Kazuhiro Iiyama, Yuuka Chieda, Takahiro Kusakabe, Baculovirus-mediated gene transfer systems in silkworm larvae using constitutive host promoters, Journal of Asia-Pacific Entomology, 10.1016/j.aspen.2013.10.009, 17, 1, 73-78, 2014.03, Baculoviruses serve as efficient viral vectors for gene delivery into vertebrate and invertebrate cells. The identification and characterization of the functional promoters in different baculovirus-infected hosts are essential for the efficient gene expression. To establish a baculovirus-mediated gene transfer system in the silkworm, Bombyx mori, we investigated the activities of silkworm-derived TCTP, ACTIN3, and HSC70-4 promoters delivered by AcNPV or BmNPV in various tissues of silkworm. In many of the tested silkworm tissues, the BmHSC70-4 promoter exhibited a higher transcription activity than those of BmTCTP or BmACTIN3 promoters when delivered by AcNPV, which is reported to be incapable of replicating in silkworms. In contrast, the BmACTIN3 promoter was found to be the strongest promoters when delivered by BmNPV. The present results indicate that the BmHSC70-4 promoter is potentially useful for the stable gene expression by the non-replicating AcNPV vector for gene function analysis in the silkworm..
45. Jian Xu, Kaito Yoshimura, Hiroaki Mon, Zhiqing Li, Li Zhu, Kazuhiro Iiyama, Takahiro Kusakabe, Jae Man Lee, Establishment of Caenorhabditis elegans SID-1-dependent DNA delivery system in cultured silkworm cells, Molecular Biotechnology, 10.1007/s12033-013-9694-0, 56, 3, 193-198, 2014.03, The systemic RNA interference defective-1 (SID-1) can transport double-stranded RNA (dsRNA) into cytosol across the cytoplasmic membrane. We report here that ectopic expression of Caenorhabditis elegans SID-1 allows BmN4 cells to import extracellular plasmid dsDNA into cells via the direct soaking method. Interestingly, BmN4-SID1 cells incorporate dsRNA and plasmid DNA simultaneously. Furthermore, the ectopic SID-1 allows us to establish a stably transformed cell line by the simple soaking method. Our results provide an alternative method for silkworm gene function analysis with low cost and low cell toxicity..
46. Tsuneyuki Tatsuke, Li Zhu, Zhiqing Li, Hitoshi Mitsunobu, Kaito Yoshimura, Hiroaki Mon, Jae Man Lee, Takahiro Kusakabe, Roles of piwi proteins in transcriptional regulation mediated by HP1s in cultured silkworm cells, PloS one, 10.1371/journal.pone.0092313, 9, 3, 2014.03, Piwi proteins are part of a superfamily of Argonaute proteins, which are one of the core components of the RNA silencing pathway in many eukaryotes. Piwi proteins are thought to repress the transposon expression both transcriptionally and post-transcriptionally. Recently, Drosophila melanogaster Piwi was recently reported to associate with chromatin and to interact directly with the Heterochromatin Protein 1 (HP1a). However, similar interactions have not been reported in other higher eukaryotes. Here we show that silkworm Piwi proteins interact with HP1s in the nucleus. The silkworm, Bombyx mori, has two Piwi proteins, Ago3 and Siwi, and two typical HP1 proteins, HP1a and HP1b. We found that HP1a plays an important role in the interaction between Ago3/Siwi and HP1b in the ovary-derived BmN4 cell line. We also found that Ago3/Siwi regulates the transcription in an HP1-dependent manner. These results suggest that silkworm Piwi proteins function as a chromatin regulator in collaboration with HP1a and HP1b. Copyright:.
47. Hiroaki Mon, Jae Man Lee, Kazuei Mita, Marian R. Goldsmith, Takahiro Kusakabe, Chromatin-induced spindle assembly plays an important role inmetaphase congression of silkworm holocentric chromosomes, Insect Biochemistry and Molecular Biology, 10.1016/j.ibmb.2013.11.007, 45, 1, 40-50, 2014.02, The kinetochore plays important roles in cell cycle progression. Interactions between chromosomes and spindle microtubules allow chromosomes to congress to the middle of the cell and to segregate the sister chromatids into daughter cells in mitosis. The chromosome passenger complex (CPC), composed of the Aurora B kinase and its regulatory subunits INCENP, Survivin, and Borealin, plays multiple roles in these chromosomal events. In the genome of the silkworm, Bombyx mori, which has holocentric chromosomes, the CPC components and their molecular interactions were highly conserved. In contrast to monocentric species, however, the silkworm CPC co-localized with the chromatin-driven spindles on the upper side of prometaphase chromosomes without forming bipolar mitotic spindles. Depletion of the CPC by RNAi arrested the cell cycle progression at prometaphase and disrupted the microtubule network of the chromatin-driven spindles. Interestingly, depletion of mitotic centromere-associated kinesin (MCAK) recovered formation of the microtubule network but did not overcome the cell cycle arrest at prometaphase. These results suggest that the CPC modulates the chromatin-induced spindle assembly and metaphase congression of silkworm holocentric chromosomes..
48. Jian Xu, Takahiro Kusakabe, Kimiko Yamamoto, Yoshitaka Suetsugu, Hiroaki Mon, Zhiqing Li, Li Zhu, Kazuhiro Iiyama, Yutaka Banno, Kaito Yoshimura, Jae Man Lee, A novel third chromosomal locus controls susceptibility to Autographa californica multiple nucleopolyhedrovirus in the silkworm, Bombyx mori, Applied Microbiology and Biotechnology, 10.1007/s00253-013-5437-1, 98, 7, 3049-3058, 2014.01, Baculovirus demonstrates specific infection spectrums and thus one certain host exhibits particular response to single baculovirus isolate. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is considered to be not an innate pathogen to Bombyx mori, but some silkworm strains have been identified to be permissive to AcMNPV, indicating the positive or negative involvement of certain host factors in baculovirus replications in vivo. To provide a fundamental knowledge of this process, we performed large-scale screening to investigate the responses of 448 silkworm strains against recombinant AcMNPV inoculation. By genetic analysis between permissive and resistant strains identified, we further confirmed that a potential corresponding locus on chromosome 3 regulates host responses to AcMNPV in silkworm. Additionally, we found that it is available for AcMNPV-silkworm baculovirus expression vector system to produce proteins of interest..
49. Erika Taira, Kazuhiro Iiyama, Hiroaki Mon, Kazuki Mori, Taiki Akasaka, Kousuke Tashiro, Chisa Yasunaga-Aoki, Jae Man Lee, Takahiro Kusakabe, Draft genome sequence of entomopathogenic Serratia liquefaciens strain FK01, Genome Announcements, 10.1128/genomeA.00609-14, 2, 3, 2014.01, In the present study, we determined the draft genome sequence of the entomopathogenic bacterium Serratia liquefaciens FK01, which is highly virulent to the silkworm. The draft genome is ~5.28 Mb in size, and the G+C content is 55.8%..
50. Takumi Mitsudome, Jian Xu, Yudai Nagata, Atsushi Masuda, Kazuhiro Iiyama, Daisuke Morokuma, Zhiqing Li, Hiroaki Mon, Jae Man Lee, Takahiro Kusakabe, Expression, purification, and characterization of endo-β-N- acetylglucosaminidase H using baculovirus-mediated silkworm protein expression system, Applied Biochemistry and Biotechnology, 10.1007/s12010-014-0814-5, 172, 8, 3978-3988, 2014.01, Endo-β-N-acetylglucosaminidase (Endo H) from Streptomyces plicatus hydrolyzes the core di-GlcNAc units of asparagine-linked oligosaccharides and is regarded as an important tool for glycobiology research. In the present study, we established a large-scale system to produce secreted Endo H using a silkworm-baculovirus expression system (silkworm-BES). The recombinant Endo H purified from silkworm hemolymph had activity comparable to that from recombinant Escherichia coli. As well as its well-characterized substrate RNase B, the Endo H from silkworm-BES was able to deglycosylate the high-mannose glycoproteins from silkworm hemolymph. Interestingly, the secretion amount of recombinant Endo H was significantly varied among the different silkworm strains, which could provide valuable information for larger-scale protein productions from silkworm-BES..
51. Kazuhiro Iiyama, Masahiro Otao, Kazuki Mori, Hiroaki Mon, Jae Man Lee, Takahiro Kusakabe, Kousuke Tashiro, Shin Ichiro Asano, Chisa Yasunaga-Aoki, Phylogenetic relationship of Paenibacillus species based on putative replication origin regions and analysis of an yheCD-like sequence found in this region, Bioscience, Biotechnology and Biochemistry, 10.1080/09168451.2014.905188, 78, 5, 891-897, 2014.01, To determine the phylogenetic relationship among Paenibacillus species, putative replication origin regions were compared. In the rsmG-gyrA region, gene arrangements in Paenibacillus species were identical to those of Bacillus species, with the exception of an open reading frame (orf14) positioned between gyrB and gyrA, which was observed only in Paenibacillus species. The orf14 product was homologous to the endospore-associated proteins YheC and YheD of Bacillus subtilis. Phylogenetic analysis based on the YheCD proteins suggested that Orf14 could be categorized into the YheC group. In the Paenibacillus genome, DnaA box clusters were found in rpmH-dnaA and dnaA-dnaN intergenic regions, known as box regions C and R, respectively; this localization was similar to that observed in B. halodurans. A phylogenetic tree based on the nucleotide sequences of the whole replication origin regions suggested that P. popilliae, P. thiaminolyticus, and P. dendritiformis are closely related species..
52. Saki Imai, Zhiqing Li, Kazuhiro Iiyama, Yoshitaka Miyagawa, Masashi Toyoda, Akihiro Umezawa, Hiroaki Mon, Takahiro Kusakabe, Kaito Yoshimura, Jae Man Lee, Biologically active human bone morphogenetic protein 4 fused to collagen-binding domain produced in silkworm-baculovirus expression system, Journal of Insect Biotechnology and Sericology, 10.11416/jibs.82.2_039, 82, 2, 39-44, 2013.12, Human bone morphogenetic protein 4 (BMP4) fused to the collagen-binding domain (CBD) of fbronectin was constructed and expressed in a silkworm-baculovirus expression system. When recombinant BmNPV was injected into the silkworm larvae and harvested after approximately 4 days, 0.22 mg/ml (0.1 mg/larva) of recombinant CBD-BMP4 was secreted into the silkworm haemolymph. Interestingly, cultured silkworm cells infected with the same recombinant virus could not secrete the recombinant CBD-BMP4 into culture media. The purifed rCBD-BMP4 showed Smad- and MAPK-stimulating activities in human UET-13 cells..
53. Kazuhiro Iiyama, Kazuki Mori, Hiroaki Mon, Yuuka Chieda, Jae Man Lee, Takahiro Kusakabe, Kousuke Tashiro, Shin Ichiro Asano, Chisa Yasunaga-Aoki, Susumu Shimizu, Draft genome sequence of Paenibacillus popilliae ATCC 14706T, Journal of Insect Biotechnology and Sericology, 10.11416/jibs.82.2_045, 82, 2, 45-48, 2013.12.
54. Jian Xu, Hiroaki Mon, Takahiro Kusakabe, Zhiqing Li, Li Zhu, Kazuhiro Iiyama, Atsushi Masuda, Takumi Mitsudome, Jae Man Lee, Establishment of a soaking RNA interference and Bombyx mori nucleopolyhedrovirus (BmNPV)-hypersensitive cell line using Bme21 cell, Applied Microbiology and Biotechnology, 10.1007/s00253-013-5279-x, 97, 24, 10435-10444, 2013.12, The double-stranded RNA (dsRNA) mediated RNA interference (RNAi) is widely employed in silkworm and its tissue-derived cell lines for gene function analysis. Baculovirus expression vector system (BEVS) has an advantage for large-scale protein expression. Previously, combining these useful tools, we improved traditional AcMNPV-Sf9 BEVS to produce modified target glycoproteins, where the ectopic expression of Caenorhabditis elegans systemic RNAi defective-1 (SID-1) was found to be valuable for soaking RNAi. In current study, we applied CeSID-1 protein to a Bombyx mori NPV (BmNPV)-hypersensitive Bme21 cell line and investigated its properties both in soaking RNAi ability and recombinant protein expression. The soaking RNAimediated suppression in the Bme21 cell enables us to produce modified glycoproteins of interest in BmNPV-Bme21 BEVS..
55. Hiroaki Mon, Zhiqing Li, Isao Kobayashi, Shuichiro Tomita, Jae Man Lee, Hideki Sezutsu, Toshiki Tamura, Takahiro Kusakabe, Soaking RNAi in Bombyx mori BmN4-SID1 cells arrests cell cycle progression, Journal of Insect Science, 10.1673/031.013.15501, 13, 2013.12, RNA interference (RNAi) is an evolutionarily conserved mechanism for sequence-specific gene silencing. Previously, the BmN4-SID1 cell expressing Caenorhabditis ele gans SID-1 was established, in which soaking RNAi could induce effective gene silencing. To establish its utility, 6 cell cycle progression related cDNAs, CDK1, MYC, MYB, RNRS, CDT1, and GEMININ, were isolated from the silkworm, Bombyx mori L. (Lepidoptera: Bombycidae), and their expressions were further silenced by soaking RNAi in the BmN4-SID1 cells. The cell cycle progression analysis using flow cytometer demonstrated that the small amount of double stranded RNA was enough to arrest cell cycle progression at the specific cell phases. These data suggest that RNAi in the BmN4-SID1 cells can be used as a powerful tool for loss-of-function analysis of B. mori genes..
56. Li Zhu, Tsuneyuki Tatsuke, Hiroaki Mon, Zhiqing Li, Jian Xu, Jae Man Lee, Takahiro Kusakabe, Characterization of tudor-sn-containing granules in the silkworm, bombyx mori, Insect Biochemistry and Molecular Biology, 10.1016/j.ibmb.2013.04.004, 43, 8, 664-674, 2013.08, The Tudor-sn protein, which contains four staphylococcal nuclease domains and a Tudor domain, is a ubiquitous protein found in almost all organisms. It has been reported that Tudor-sn in mammals participates in various cellular pathways involved in gene regulation, cell growth, and development. In insects, we have previously identified a Tudor-sn ortholog in the silkworm, Bombyx mori, and detected its interactions between with Argonaute proteins. The role of Tudor-sn in silkworm, however, still remains largely unknown. In this study, we demonstrated that silkworm Tudor-sn is a stress granule (SG) protein, and determined its interactions with other SG proteins using Bimolecular Fluorescence Complementation assay and Insect Two-Hybrid method. Depletions of Argonaute proteins and SG-marker protein Tia1 by RNAi impaired the involvement of Tudor-sn in the SG formation. Protein domain deletion analysis of Tudor-sn demonstrated that SN2 is the key domain required for the aggregation of Tudor-sn in SGs..
57. Li Zhu, Zhiqing Li, Tsuneyuki Tatsuke, Daojun Cheng, Jian Xu, Kaito Yoshimura, Hiroaki Mon, Kazuhiro Iiyama, Jae Man Lee, Qingyou Xia, Takahiro Kusakabe, Genome-wide identification of Argonaute 1- and Argonaute 2-regulating genes revealed an inhibition of macula-like virus by RNAi pathway in the silkworm, Bombyx mori, Journal of Insect Biotechnology and Sericology, 10.11416/jibs.82.1_019, 82, 1, 19-23, 2013.08, Using a cDNA microarray, we monitored global gene expression profiles in silkworm BmN4-SID1 cells after the knockdown of BmAGO1 and BmAGO2 genes. Interestingly, there was a significant overlap between the target genes up-regulated by BmAgo1 (292 out of 481) and BmAgo2 (292 out of 361). Of these, the Replicase gene of macula-like virus (BmMLV) was highly induced in both of BmAgo1- and BmAgo2-depleted cells. RT-PCR analysis confirmed that the knockdown of silkworm RNAi pathway-related genes increased the expression of the Replicase gene. These data strongly suggested that the RNAi pathway negatively regulated BmMLV proliferation in silkworm BmN4 cells..
58. Kazuhiro Iiyama, Oumi Nishi, Hiroaki Mon, Jae Man Lee, Takahiro Kusakabe, Shin Ichiro Asano, Chisa Yasunaga-Aoki, Susumu Shimizu, Phylogenetic analysis of Paenibacillus popilliae and its related taxa based on housekeeping genes, Journal of Insect Biotechnology and Sericology, 10.11416/jibs.82.1_001, 82, 1, 1-11, 2013.08, Four housekeeping genes including gapA, groEL, gyrA and pgi were used in order to reveal a phylogenetic relationship among Paenibacillus species. Phylogenetic analysis and end-to-end pairwise analysis were carried out using nucleotide and amino acid sequences of the housekeeping genes. A monophylogenetic clade including P. popilliae, P. thiaminolyticus and P. dendritiformis formed in all phylogenetic trees. The results in both analyses indicated that the relationship between P. larvae subsp. larvae and P. popilliae was comparatively far. The result of the end-to-end pairwise analysis also showed that, among the Paenibacillus species used in this study, the closest species to P. popilliae was P. dendritiformis..
59. Hiroaki Mon, Jaeman Lee, Mai Fukushima, Yudai Nagata, Mie Fujii, Jian Xu, Oumi Nishi, Kazuhiro Iiyama, Takahiro Kusakabe, Production and characterization of the celery mismatch endonuclease CEL II using baculovirus/silkworm expression system, Applied Microbiology and Biotechnology, 10.1007/s00253-012-4583-1, 97, 15, 6813-6822, 2013.08, Mutation and polymorphism detection by nucleases has become a more important tool in clinical and biological researches. There are several kinds of single-stranded nucleases for detecting mismatched DNAs. One of them, CEL II, was isolated from Apium graveolens and cleaves DNA with high specificity at sites of mismatch. High-throughput mutation scanning requires large quantity of CEL II endonuclease. Here, we demonstrate high-level expression of CEL II using silkworm-baculovirus system. The recombinant CEL II secreted in silkworm hemolymph was glycosylated and susceptible to N-glycosidase F. Additionally, larger metal ions such as Ca2+ and Sr2+ were able to replace Mg2+ and enhanced mismatch cleavage activity of CEL II. These results indicate that the silkworm-baculovirus platform is a good alternative system to obtain the functional CEL II..
60. Mai Fukushima, Kazuhiro Iiyama, Jun Yamashita, Masutaka Furue, Gaku Tsuji, Shigeo Imanishi, Hiroaki Mon, Jae Man Lee, Takahiro Kusakabe, Production of small antibacterial peptides using silkworm-baculovirus protein expression system, Preparative Biochemistry and Biotechnology, 10.1080/10826068.2012.762717, 43, 6, 565-576, 2013.08, The recombinant proteins with strong antimicrobial activity are known to be very difficult to express using bacterial expression system. Here, human β-defensin (DEFB) 1, DEFB2, and DEFB3 were successfully produced using a silkworm-baculovirus protein expression system. We have generated four baculoviruses for each DEFB protein to compare the effect of different peptide tags in secretion into silkworm larval hemolymph. Interestingly, the best performing peptide tags for the secretion were different among DEFBs: C-terminal GST-H8 tag for DEFB1, N-terminal H8 tag for DEFB2, and C-terminal H8 tag for DEFB3, respectively. In addition, the colony count assay demonstrated that the recombinant DEFB2 s showed antimicrobial activities against Escherichia coli, Pseudomonas aeruginosa, and Paenibacillus thiaminolyticus..
61. Yudai Nagata, Jae Man Lee, Hiroaki Mon, Shigeo Imanishi, Sun Mee Hong, Shoji Komatsu, Yuji Oshima, Takahiro Kusakabe, RNAi suppression of β-N-acetylglucosaminidase (BmFDL) for complex-type N-linked glycan synthesis in cultured silkworm cells, Biotechnology letters, 10.1007/s10529-013-1183-9, 35, 7, 1009-1016, 2013.07, Glycoproteins have various biological functions including enzymatic activity, protein stability and others. Due to the presence of paucimannosidic N-linked glycans, recombinant proteins from an insect cell expression system may not be suitable for therapeutic use. Because baculovirus expression systems (BESs) are used to produce recombinant proteins, it is of interest to modify the endogenous N-glycosylation pathway in insects to mimic that of mammals. Using a soaking RNAi sensitive cell line, BmN4-SID1, has enabled us to suppress Bombyx mori FDL (BmFDL), an N-linked glycan-specific β-N-acetylglucosaminidase. Western blotting and MALDI-TOF MS demonstrated that the BmFDL depletion almost completely converted the paucimannosidic structures of the recombinant proteins produced by BES into a complex-type structure. This highly efficient, simple and low-cost method can be used for mass production of secretion proteins with complex-type N-linked glycans..
62. Jian Xu, Yudai Nagata, Hiroaki Mon, Zhiqing Li, Li Zhu, Kazuhiro Iiyama, Takahiro Kusakabe, Jae Man Lee, Soaking RNAi-mediated modification of Sf9 cells for baculovirus expression system by ectopic expression of Caenorhabditis elegans SID-1, Applied Microbiology and Biotechnology, 10.1007/s00253-013-4785-1, 97, 13, 5921-5931, 2013.07, RNA interference (RNAi) is a biological phenomenon that silences the expression of genes of interest. Passive double-stranded RNA (dsRNA) uptake has been uniquely observed in Caenorhabditis elegans due to the expression of systemic RNAi defective-1 (SID-1). We report that ectopic expression of CeSID-1 endows the Sf9 cells with a capacity for soaking RNAi. Soaking the Sf9-SID1 with dsRNA corresponding to either exogenous or endogenous target genes induced a significant decrease in the amount of mRNA or protein. These results enabled us to modify the target proteins of baculovirus expression vector system in both quantities and posttranslational modifications. The current low-cost and high-efficiency RNAi system is useful for high-throughput gene function analysis and mass production of recombinant protein..
63. L. Zhu, Y. Masaki, T. Tatsuke, Z. Li, H. Mon, J. Xu, J. M. Lee, T. Kusakabe, A MC motif in silkworm Argonaute 1 is indispensible for translation repression, Insect Molecular Biology, 10.1111/imb.12023, 22, 3, 320-330, 2013.06, Small RNA-mediated gene silencing is a fundamental gene regulatory mechanism, which is conserved in many organisms. Argonaute (Ago) family proteins in the RNA-induced silencing complex (RISC) play crucial roles in RNA interference (RNAi) pathways. In the silkworm Bombyx mori, four Ago proteins have been identified, named as Ago1, Ago2, Ago3 and Siwi. Ago2 participates in double-stranded RNA (dsRNA)-induced RNAi, whereas Ago3 and Siwi are involved in the Piwi-interacting RNA (piRNA) pathway. However, there is no experimental evidence concerning silkworm Ago1 (BmAgo1) in the RNAi mechanism. In the present study, we analysed the function of BmAgo1 in the microRNA (miRNA)-mediated RNAi pathway using tethering and miRNA sensor reporter assays. These results clearly demonstrate that BmAgo1 plays an indispensable role in translation repression in silkworm. Moreover, coimmunoprecipitation data indicated that BmAgo1 interacts with BmDcp2, an orthologue of mRNA-decapping enzyme 2 (Dcp2) protein in the Drosophila processing-bodies (P-bodies). Substitutions of two conserved phenylalanines (F522 and F557) by valines in the MC motif strongly impaired the function of BmAgo1 in translation repression and its localization in P-bodies, suggesting that these two amino acid residues in the MC motif of BmAgo1 are prerequisites for mRNA translation repression in B. mori..
64. Zhiqing Li, Li Zhu, Jian Xu, Hiroaki Mon, Jae Man Lee, Takahiro Kusakabe, Amino acid deprivation-induced expression of asparagine synthetase regulates the growth and survival of cultured silkworm cells, Archives of insect biochemistry and physiology, 10.1002/arch.21091, 83, 2, 57-68, 2013.06, Expression of Bombyx mori Asparagine synthetase (BmASNS), one gene that encodes an enzyme catalyzing asparagine biosynthesis, is transcriptionally induced following amino acid deprivation. Previous transcriptional analysis of the BmASNS gene showed the involvement of Polycomb proteins, epigenetic repressors, in suppressing BmASNS expression in a cell cycle-dependent manner. However, the role of BmAsns protein in these cellular processes remains unclear. The present study thus exploited the potential function of BmAsns protein in cultured silkworm cells. Our results showed that ectopic overexpression of BmASNS gene effectively inhibited cell growth in silkworm cells, whereas its overexpression could rescue cell growth upon amino acid deprivation treatment. We found that the cells expressing BmAsns protein were capable of influencing the formation of autophagic vacuoles stimulated by amino acid deprivation. We speculated that the recovery of cell growth by overexpressed BmAsns protein is due to the rapid turnover of autophagic vacuoles in the cells. To further assess the effects of BmAsns on cell development, we used RNA interference to silence BmASNS expression in silkworm cells in the presence or absence of amino acids. Our results revealed a significant change of cell proliferation as well as cell cycle distribution after knockdown of BmASNS. Importantly, silkworm cells lacking BmASNS under the condition of amino acid deprivation showed severely impaired proliferation. Altogether, we concluded that the up-regulated expression of BmASNS would be able to protect cells from impairment induced by amino acid deprivation, which in turn facilitates cell growth and survival..
65. Jae Man Lee, Yoshito Kojin, Tsuneyuki Tatsuke, Hiroaki Mon, Yoshitaka Miyagawa, Takahiro Kusakabe, Coexpression of Escherichia coli RNase III in silkworm cells improves the efficiency of RNA interference induced by long hairpin dsRNAs, Insect Science, 10.1111/j.1744-7917.2012.01569.x, 20, 1, 69-77, 2013.02, Long hairpin dsRNA transcribed from chromosomal DNA can induce RNA interference in Bombyx mori cells, although its gene silencing efficiency is lower than that of exogenously introduced double-stranded RNAs (dsRNAs). To solve this problem, we monitored the nuclear cytoplasmic translocation of the transcribed hairpin dsRNA and analyzed the processing efficiency into mature small interfering RNA (siRNA). Northern blot analysis revealed that the transcribed hairpin dsRNAs were spliced and transported into the cytoplasm, but were not effectively diced into siRNAs. Interestingly, RNAi with hairpin dsRNAs from genome-integrated IR transgene was stimulated by the coexpression of Escherichia coli RNase III, although this exogenous enzyme seemed to bring about nonspecific cleavage of cellular mRNA..
66. Zhiqing Li, Daojun Cheng, Hiroaki Mon, Li Zhu, Jian Xu, Tsuneyuki Tatsuke, Jae Man Lee, Qingyou Xia, Takahiro Kusakabe, Cell Cycle-Dependent Recruitment of Polycomb Proteins to the ASNS Promoter Counteracts C/ebp-Mediated Transcriptional Activation in Bombyx mori, PloS one, 10.1371/journal.pone.0052320, 8, 1, 2013.01, Epigenetic modifiers and transcription factors contribute to developmentally programmed gene expression. Here, we establish a functional link between epigenetic regulation by Polycomb group (PcG) proteins and transcriptional regulation by C/ebp that orchestrates the correct expression of Bombyx mori asparagine synthetase (BmASNS), a gene involved in the biosynthesis of asparagine. We show that the cis-regulatory elements of YY1-binding motifs and the CpG island present on the BmASNS promoter are required for the recruitment of PcG proteins and the subsequent deposition of the epigenetic repression mark H3K27me3. RNAi-mediated knockdown of PcG genes leads to derepression of the BmASNS gene via the recruitment of activators, including BmC/ebp, to the promoter. Intriguingly, we find that PcG proteins and BmC/ebp can dynamically modulate the transcriptional output of the BmASNS target in a cell cycle-dependent manner. It will be essential to suppress BmASNS expression by PcG proteins at the G2/M phase of the cell cycle in the presence of BmC/ebp activator. Thus, our results provide a novel insight into the molecular mechanism underlying the recruitment and regulation of the PcG system at a discrete gene locus in Bombyx mori..
67. Yasuhiko Soejima, Jae Man Lee, Yudai Nagata, Hiroaki Mon, Kazuhiro Iiyama, Hajime Kitano, Michiya Matsuyama, Takahiro Kusakabe, Comparison of signal peptides for efficient protein secretion in the baculovirus-silkworm system, Central European Journal of Biology, 10.2478/s11535-012-0112-6, 8, 1, 1-7, 2013.01, The baculovirus-silkworm expression system is widely used as a mass production system for recombinant secretory proteins. However, the final yields of some recombinant proteins are not sufficient for industrial use. In this study, we focused on the signal peptide as a key factor for improving the efficiency of protein production. Endoplasmic reticulum (ER) translocation of newly synthesized proteins is the first stage of the secretion pathway; therefore, the selection of an efficient signal peptide would lead to the efficient secretion of recombinant proteins. The Drosophila Bip and honeybee melittin signal peptides have often been used in this system, but to the best of our knowledge, there has been no study comparing secretion efficiency between exogenous and endogenous signal peptides. In this study we employed signal peptides from 30K Da and SP2 proteins as endogenous signals, and compared secretion efficiency with those of exogenous or synthetic origins. We have found that the endogenous secretory signal from the 30K Da protein is the most efficient for recombinant secretory protein production in the baculovirus-silkworm expression system..
68. Jae Man Lee, Yoshito Kojin, Tsuneyuki Tatsuke, Hiroaki Mon, Yoshitaka Miyagawa, Takahiro Kusakabe, RNA interference induction by long hairpin dsRNAs expressed from chromosomal DNA of Bombyx mori cells, Journal of the Faculty of Agriculture, Kyushu University, 57, 2, 441-445, 2012.09, RNA interference (RNAi) triggered by long double-stranded RNA (dsRNA) transcribed from chromosomal DNA has a lot of advantages in high-throughput analyses of gene function. In this report, we have constructed Gateway®-based RNAi induction vectors, by which we efficiently integrated expression cassettes for long hairpin dsRNA into chromosomal DNA of Bombyx mori cells. RNAi induced by the hairpin dsRNAs using our constructs decreased a reporter gene activity by approximately 70-80% in cultured B. mori cells..
69. Li Zhu, Tsuneyuki Tatsuke, Zhiqing Li, Hiroaki Mon, Jian Xu, Jae Man Lee, Takahiro Kusakabe, Molecular cloning of BmTUDOR-SN and analysis of its role in the RNAi pathway in the silkworm, Bombyx mori (Lepidoptera
Bombycidae), Applied Entomology and Zoology, 10.1007/s13355-012-0109-7, 47, 3, 207-215, 2012.08, Tudor-sn, a conserved nuclease, was first isolated from RNA-induced silencing complex (RISC) and was subsequently implicated in the RNA interference (RNAi) pathway in humans, flies and nematodes. However, in the silkworm, Bombyx mori L, the RNAi mechanism and the components of RISC were quite unclear. Here, we cloned the full-length cDNA of TUDOR-SN (BmTUDOR-SN) from the silkworm. Phylogenetic analysis revealed that BmTudor-sn had a high homology with Tudor-sn proteins in other insects. Fluorescent microscopic observation indicated that the subcellular localization of enhanced green fluorescent protein fused BmTudor-sn was mainly in the cytoplasm of silkworm BmN4 cells. Knockdown of BmTUDOR-SN did not, however, affect the RNAi efficiency in BmN4 cells..
70. Ryohei Sugahara, Hiroaki Mon, Jae Man Lee, Takahiro Kusakabe, Monoubiquitination-dependent chromatin loading of FancD2 in silkworms, a species lacking the FA core complex, Gene, 10.1016/j.gene.2012.03.071, 501, 2, 180-187, 2012.06, The Fanconi anemia (FA) pathway is required for activation and operation of the DNA interstrand cross-link (ICL) repair pathway, although the precise mechanism of the FA pathway remains largely unknown. A critical step in the FA pathway is the monoubiquitination of FANCD2 catalyzed by a FA core complex. This modification appears to allow FANCD2 to coordinate ICL repair with other DNA repair proteins on chromatin. Silkworm, . Bombyx mori, lacks apparent homologues of the FA core complex. However, BmFancD2 and BmFancI, the putative substrates of the complex, and BmFancL, the putative catalytic E3 ubiquitin ligase, are conserved. Here, we report that the silkworm FancD2 is monoubiquitinated depending on FancI and FancL, and stabilized on chromatin, following MMC treatment. A substitution of BmFancD2 at lysine 519 to arginine abolishes the monoubiquitination, but not the interaction between the FancD2 and FancI. In addition, we demonstrated that depletion of BmFancD2, BmFancI or BmFancL had effects on cell proliferation in the presence of MMC. These results suggest that the FA pathway in . B. mori works in the same manner as that in vertebrates..
71. Zhiqing Li, Tsuneyuki Tatsuke, Kosuke Sakashita, Li Zhu, Jian Xu, Hiroaki Mon, Jae Man Lee, Takahiro Kusakabe, Identification and characterization of polycomb group genes in the silkworm, bombyx mori, Molecular biology reports, 10.1007/s11033-011-1362-5, 39, 5, 5575-5588, 2012.05, Polycomb group (PcG) proteins are involved in chromatin modifications for maintaining gene repression that play important roles in the regulation of gene expression, tumorigenesis, chromosome X-inactivation, and genomic imprinting in Drosophila melanogaster, mammals, and even plants. To characterize the orthologs of PcG genes in the silkworm, Bombyx mori, 13 candidates were identified from the updated silkworm genome sequence by using the fruit fly PcG genes as queries. Comparison of the silkworm PcG proteins with those from other insect species revealed that the insect PcG proteins shared high sequence similarity. High-level expressions of all the silkworm PcG genes were maintained through day 2 to day 7 of embryogenesis, and tissue microarray data on day 3 of the fifth instar larvae showed that their expression levels were relatively low in somatic tissues, except for Enhancer of zeste (E(Z)). In addition, knockdown of each PRC2 component, such as E(Z), Extra sex combs (ESC), and Suppressor of zeste 12 (SU(Z)12), considerably decreased the global levels of H3K27me3 but not of H3K27me2. Taken together, these results suggest that insect PcG proteins are highly conserved during evolution and might play similar roles in embryogenesis..
72. Zhiqing Li, Daojun Cheng, Hiroaki Mon, Tsuneyuki Tatsuke, Li Zhu, Jian Xu, Jae Man Lee, Qingyou Xia, Takahiro Kusakabe, Genome-wide identification of polycomb target genes reveals a functional association of pho with scm in bombyx mori, PloS one, 10.1371/journal.pone.0034330, 7, 4, 2012.04, Polycomb group (PcG) proteins are evolutionarily conserved chromatin modifiers and act together in three multimeric complexes, Polycomb repressive complex 1 (PRC1), Polycomb repressive complex 2 (PRC2), and Pleiohomeotic repressive complex (PhoRC), to repress transcription of the target genes. Here, we identified Polycomb target genes in Bombyx mori with holocentric centromere using genome-wide expression screening based on the knockdown of BmSCE, BmESC, BmPHO, or BmSCM gene, which represent the distinct complexes. As a result, the expressions of 29 genes were up-regulated after knocking down 4 PcG genes. Particularly, there is a significant overlap between targets of BmPho (331 out of 524) and BmScm (331 out of 532), and among these, 190 genes function as regulator factors playing important roles in development. We also found that BmPho, as well as BmScm, can interact with other Polycomb components examined in this study. Further detailed analysis revealed that the C-terminus of BmPho containing zinc finger domain is involved in the interaction between BmPho and BmScm. Moreover, the zinc finger domain in BmPho contributes to its inhibitory function and ectopic overexpression of BmScm is able to promote transcriptional repression by Gal4-Pho fusions including BmScm-interacting domain. Loss of BmPho expression causes relocalization of BmScm into the cytoplasm. Collectively, we provide evidence of a functional link between BmPho and BmScm, and propose two Polycomb-related repression mechanisms requiring only BmPho associated with BmScm or a whole set of PcG complexes..
73. H. Mitsunobu, M. Izumi, H. Mon, T. Tatsuke, J. M. Lee, T. Kusakabe, Molecular characterization of heterochromatin proteins 1a and 1b from the silkworm, Bombyx mori, Insect Molecular Biology, 10.1111/j.1365-2583.2011.01115.x, 21, 1, 9-20, 2012.02, Heterochromatin protein 1s (HP1s) are nonhistone chromosomal proteins that play a direct role in the formation and maintenance of heterochromatin structure. Similarly to Caenorhabditis elegans, silkworms possess holocentric chromosomes, in which diffused kinetochores extend along the length of each chromosome. We have isolated two silkworm HP1 homologues, BmHP1a and BmHP1b. Cytological analysis showed a unique localization of BmHP1s during cell division, in which these proteins first appear to dissociate from the chromosomes, but then return to enclose the chromosomes during metaphase. BmHP1s formed homo- and hetero-dimers and interacted with BmSu(var)3-9, which is a methyltransferase for histone H3 lysine 9 (H3K9). We further showed, using a silkworm cell-based reporter system, that BmHP1b had higher transcriptional repression activity than BmHP1a, whereas BmHP1a interacted more strongly with BmSu(var)3-9 than did BmHP1b. These results suggest that silkworm HP1a and HP1b may play different roles in heterochromatin formation in holocentric silkworm chromosomes..
74. Mie Fujii, Masateru Takahashi, Hiroaki Mon, Tsuneyuki Tatsuke, Jae Man Lee, Takahiro Kusakabe, Molecular cloning of the silkworm p53r2-like gene, Journal of the Faculty of Agriculture, Kyushu University, 57, 1, 79-82, 2012.02, The p53R2 is a homologue of the small subunit of mammalian ribonucleotide reductase. In human, the transcription of p53R2 is induced clearly by active form of p53 and required for DNA synthesis during cell division and DNA repair. In this study, we have isolated and determined cDNA sequence for Bombyx mori p53R24ike (p53R2-L) protein. The Bmp53R2-like protein had high homology to the Hsp53R2, but had histidine residue at a position corresponding to Y241 of Hsp53R2, which is a key residue in discriminating human p53R2 and R2. Bmp53R2-L expressed strongly in the silk grand, gonad and blood cell, in which cell division occurs actively and DNA synthesis is required. The knockdown of the Bmp53R2-L in BmN4-SIDl led to a substantial arrest in Gl/S. This Gl/S arrest may be caused by the retardation of DNA synthesis due to the depletion of dNTPs in nucleus under the conditions of the knockdown of Bmp53R2-L. Thus, Bmp53R2-L is a functional homolog of human R2 rather than p53R2, and considered to be indispensable for DNA synthesis and cell cycle progression in a DNA damage independent manner..
75. Hiroaki Mon, Isao Kobayashi, Shinji Ohkubo, Shuichiro Tomita, Jae Man Lee, Hideki Sezutsu, Toshiki Tamura, Takahiro Kusakabe, Effective RNA interference in cultured silkworm cells mediated by overexpression of Caenorhabditis elegans SID-1, RNA biology, 10.4161/rna.9.1.18084, 9, 1, 40-46, 2012.01, RNA interference (RNAi) is a conserved mechanism that catalyzes sequence-specific gene silencing, and has been used for loss-of-function genetic screens in many organisms. Here, we demonstrated that the expression of Caenorhabditis elegans SID-1 (CeSID-1) could trigger effective gene silencing in the cultured silkworm cell line, BmN4 (BmN4-SID1). Soaking the BmN4-SID1 in dsRNA corresponding to endogenous target genes induced a significant decrease of the amount of mRNA or protein. A small amount of dsRNA was enough to silence the target gene in a few days. Overexpression of CeSID-1 did not affect the cell viability. Our results suggest that BmN4-SID1 can be used in many applications in silkworm cells and will become a valuable resource for gene analysis..
76. Jae Man Lee, Naoya Kawakami, Hiroaki Mon, Hitoshi Mitsunobu, Kazuhiro Iiyama, Satoshi Ninaki, Katsumi Maenaka, Enoch Y. Park, Takahiro Kusakabe, Establishment of a Bombyx mori nucleopolyhedrovirus (BmNPV) hyper-sensitive cell line from the silkworm e21 strain., Biotechnology letters, 10.1007/s10529-012-0971-y, 34, 10, 1773-1779, 2012.01, Baculoviral expression systems, including those of Autographa californica multiple nucleopolyhedrovirus Bombyx mori nucleopolyhedrovirus (BmNPV), are used for recombinant protein production. Four B. mori-derived (BmN4, Bm5, Bmc140, and Bme21) cell lines were infected with recombinant BmNPV viruses expressing firefly luciferase or EGFP as reporters under the control of a viral polyhedrin promoter. Bme21 exhibited significantly higher (100-fold) luciferase activity than BmN4 and Bm5. With the EGFP reporter protein, Bme21 cells showed a marked increase in the ratio of EGFP-positive cells, reaching 90 % on day 4 post-infection, while Bm5 and BmN4 cells had a slow increase in the ratio of their EGFP-positive population. The viral titer in a supernatant of Bme21 cell culture increased faster than those of Bm5 and BmN4 cells. This susceptibility indicates that the Bme21 cell line is useful for large-scale protein expression using BmNPV..
77. Hiroaki Mon, Makiko Izumi, Hitoshi Mitsunobu, Tsuneyuki Tatsuke, Kazuhiro Iiyama, Hiroyuki Jikuya, Jae Man Lee, Takahiro Kusakabe, Post-translational modifications of the N-terminal tail of histone H3 in holocentric chromosomes of Bombyx mori, Insect Biochemistry and Molecular Biology, 10.1016/j.ibmb.2011.08.004, 41, 11, 902-908, 2011.11, Epigenetic information is encoded in post-translational modifications (PTMs) of histones. Various combinations of these marks contribute to the regulation of chromatin-templated DNA metabolisms. The histone code is gradually translated into biological responses in model organisms. However, in the silkworm, the modifications of histones with unique holocentric chromosomes have not yet been analyzed. TAU-PAGE analysis of the silkworm histone variants H2A, H2B, and H3, separated by RP-HPLC, suggested silkworm specific modification. Detailed mass spectrometry analyses of the peptides derived from the N-terminus of the silkworm H3.2 generated by glutamyl endopeptidase, lysyl endopeptidase, and trypsin digestions revealed global modifications around H3K9..
78. Hiroaki Mon, Jaeman Lee, Yutaka Kawaguchi, Takahiro Kusakabe, Double-strand breaks repair by gene conversion in silkworm holocentric chromosomes, Molecular Genetics and Genomics, 10.1007/s00438-011-0640-1, 286, 3-4, 215-224, 2011.10, Maintenance of genome stability relies on the accurate repair of DNA double-strand breaks (DSBs) that arise during DNA replication or introduced by DNA-damaging agents. Failure to repair such breaks can lead to the introduction of mutations and chromosomal translocations. Several pathways, homologous recombination, single-strand annealing and nonhomologous end-joining, are known to repair DSBs. So far in the silkworm Bombyx mori, these repair pathways have been analyzed using extrachromosomal plasmids in vitro or in cultured cells. To elucidate the precise nature of the chromosomal DSB repair pathways in cultured silkworm cells, we developed a luciferase-based assay system for measuring the frequency of chromosomal homologous recombination and SSA. An I-SceI-induced DSB, within a nonfunctional luciferase gene, could be efficiently repaired by HR. Additionally, the continuous expression of the I-SceI endonuclease in the HR reporter cell allowed us to investigate the interrelationship between HR, SSA and NHEJ. In this study, we demonstrated that chromosome DSBs were mainly repaired by NHEJ and HR, whereas SSA was unlikely to be a dominant repair pathway in cultured silkworm cell. These results indicate that the assay system presented here will be useful to analyze the mechanisms of DSB repair in insect cells..
79. Sun Mee Hong, Jun Yamashita, Hitoshi Mitsunobu, Keiro Uchino, Isao Kobayashi, Hideki Sezutsu, Toshiki Tamura, Hideki Nakajima, Yoshitaka Miyagawa, Jae Man Lee, Hiroaki Mon, Yoshihiko Miyata, Yutaka Kawaguchi, Takahiro Kusakabe, Efficient soluble protein production on transgenic silkworms expressing cytoplasmic chaperones, Applied Microbiology and Biotechnology, 10.1007/s00253-010-2617-0, 87, 6, 2147-2156, 2010.08, Baculovirus expression systems (BES) are widely used for recombinant protein production in lepidopteran cells or larvae. However, even in BES, the insolubility of recombinant proteins sometimes makes their expression difficult. In this study, to improve the solubility and yield of foreign proteins, we constructed transgenic silkworms using silkworm heat-shock proteins, Hsp70 and Hsp40, or Hsc70 and Hsp90 co-chaperone Hop. In these transgenic silkworms, the expression levels of the transgenes were under the control of a UAS•hsp mini-promoter driven by a Gal4NFkBp65 activator. When the transgenic silkworm with HSP70 and 40 (TGS-HSP70/40) was infected with BmNPV carrying mC3d and Gal4NFkBp65 under the control of baculovirus polyhedrin or p10 promoters, respectively, the soluble fraction of the His- or His•GST-tagged mC3d increased significantly. Similarly, the transgenic silkworm with HSC70 and HOP (TGS-HOP7) was effective for the expression of a steroid hormone receptor, USP2. In conclusion, the His-tagged baculovirus expression system featuring the chaperone effect TGS-HSP70/40 and TGS-HOP7 silkworms is effective for increasing the yields of soluble and functional foreign gene products..
80. Hitoshi Mitsunobu, Makiko Izumi, Kazuhiro Iiyama, Hiroyuki Jikuya, Jae Man Lee, Hiroaki Mon, Yutaka Kawaguchi, Takahiro Kusakabe, Molecular characterization of core histones in the silkworm, Bombyx mori, Journal of Insect Biotechnology and Sericology, 79, 3, 75-83, 2010, In eukaryotes, genomic DNA is wrapped around an octamer of the core histone proteins H2A, H2B, H3 and H4. Silkworms have been known to bear holocentric chromosomes and their regulatory mechanisms of chroma-tin remodeling remain unclear. We have cloned the silkworm canonical core histones and their variants. The H2A variants, H2AX and H2AZ, were highly conserved in the silkworm, whereas the fy has only one H2A variant, H2Av, which is a chimera of H2AX and H2AZ with the function of both molecules. In the silkworm, all histones except for H2AX were ubiquitously expressed in all tested tissues, and H2AX was expressed only in the genital organs. A subcellular localization analysis of the cloned histones using an EGFP fusion construction demonstrated that the behaviors of the histones are the same as those of genomic DNA throughout the cell cycle. The fuo-rescence intensities of H2AX and H3.3 were weaker than those of the other histones. The incorporation of these histones into chromatin might be restricted due to their specifc function in DNA repair and transcription activity. There were two H3 variants, H3.2 and H3.3, in the silkworm; H3.1 was not present. Lysine 9 on H3 was methylated and acetylated in silkworms bearing holocentric chromosomes..
81. Tsuneyuki Tatsuke, Sun Mee Hong, Hiromi Tobata, Hiroaki Mon, Jae Man Lee, Yutaka Kawaguchi, Takahiro Kusakabe, Construction of piggyBac-based vectors using visible and drug-resistance marker for introducing foreign genes into silkworm cultured cells, Journal of the Faculty of Agriculture, Kyushu University, 54, 2, 397-400, 2009.10, Transgenic organisms have been indispensable for modern genetic analysis, such as over expression or knocked-down of the genes of interest. Transposon is one of the most efficient tools for introducing foreign DNA sequences into host genome. DNA transposon, such as piggyBac, Hermes, Minos, hobo, and mariner, have been identified in insects and have been used successfully as vectors for germline tarsnformation in various insect species. piggyBac-based transformation vectors have been broadly used in generating transgenic silkworm. However, there are few studies reporting vectors for transformation of cultured B. mori cells. In this study, we constructed new piggyBac-based vectors pPigGate, which have visible and drug selectable marker, PuroDsRed or GFPZeo in cultured cells. In order to access the utility of these vectors, we introduced BmHop2 and BmMnd1, which are meiosis specific recombination proteins, into cultured B. mori cells using the pPigGate..
82. Hiroaki Mon, Ryohei Sugahara, Sun Mee Hong, Jae Man Lee, Yusuke Kamachi, Yutaka Kawaguchi, Takahiro Kusakabe, Analysis of protein interactions with two-hybrid system in cultured insect cells, Analytical Biochemistry, 10.1016/j.ab.2009.05.033, 392, 2, 180-182, 2009.09, Many biological processes are usually coupled to the formation of protein complexes. The yeast two-hybrid system is a powerful tool for analyzing protein-protein interactions. Different patterns of protein modifications, such as glycosylation, phosphorylation, and acetylation, may affect the ability of proteins to interact. In this study, we developed the two-hybrid system that can be used in insect cells. To validate the insect two-hybrid (I2H) system, we analyzed and confirmed the known oligomer or dimer formation of silkworm Rad51 or RPA2-RPA3, respectively. The results established the feasibility of the I2H system for efficient analysis of protein interaction under conditions that closely reflect the normal physiological environment..
83. Sun Mee Hong, Si Kab Noh, Kyung A. Kim, Hitoshi Mitsunobu, Hiroaki Mon, Jae Man Lee, Yutaka Kawaguchi, Takahiro Kusakabe, Molecular characterization, localization, and distribution of innexins in the silkworm, bombyx mori, Molecular Biotechnology, 10.1007/s12033-009-9175-7, 43, 1, 52-58, 2009.09, Gap junctions that allow for a direct exchange of second messenger and ions are the most conserved cellular structures in multicellular organisms. We have isolated and characterized a Bombyx mori gene innexin3 that encodes a new member of the innexin family required for the early embryonic development. The BmINX3 mRNA was 1,814 nucleotide residues in length, and the deduced amino acid sequence of BmInx3 shared 74% similarity with Apis melifera innexin3. The expression profile of the BmINX3 mRNA is similar to that of previously described BmINX2, expressed in ovary and testis after 5th instar larvae and in fat body after gut purge. However, during embryogenesis, the expression of BmINX3 mRNA is restricted to the blastokinesis stage. Microscopic observation of the BmInx2 and BmInx3 fused to fluorescent proteins showed an overlapping cytoplasmic expression, whereas the BmInx4 is accumulated in the cytoplasmic surface at which two cells have physical contact. This finding of innexins distribution in silkworm would provide an essential basis for future studies of the functions and interactions of innexins..
84. Noriko Karasaki, Hiroaki Mon, Masateru Takahashi, Jae Man Lee, Katsumi Koga, Yutaka Kawaguchi, Takahiro Kusakabe, Establishment of tetracycline-inducible gene expression systems in the silkworm, Bombyx mori, Biotechnology letters, 10.1007/s10529-008-9898-8, 31, 4, 495-500, 2009.04, Tetracycline-inducible gene expression (Tet-on) system has become one of the first choices for the control of transgenes expression in mammal and drosophila. However, the Tet-on systems that have been established in mammalian system or tuned into drosophila do not function in the silkworm, Bombyx mori. To construct a functional Tet-on system in B. mori, we modified rtTA by introducing a transcription activation domain of immediate-early gene 1 of Autographa californica nuclear polyhedrosis virus and nuclear localization signal of SV40 large T-antigen. The modified rtTA can activate the transcription from 9 × tetO promoter in the silkworm cells up to 250-fold in the presence of doxycycline..
85. Naoya Kawakami, Jae Man Lee, Hiroaki Mon, Yuji Kubo, Yutaka Banno, Yutaka Kawaguchi, Katsumi Maenaka, Enoch Y. Park, Katsumi Koga, Takahiro Kusakabe, Efficient protein expression in Bombyx mori larvae of the strain d17 highly sensitive to B. mori nucleopolyhedrovirus, Molecular Biotechnology, 10.1007/s12033-008-9074-3, 40, 2, 180-185, 2008.10, The recombinant protein expression by Bombyx mori nucleopolyhedrovirus (BmNPV) infecting silkworm larvae or pupae may endow us with a potent system for the production of large eukaryotic proteins. However, the screening of silkworm strains ideally suited to this method has scarcely been conducted. In the present study, we injected recombinant BmNPV containing a reporter gene, luciferase or DsRed, into hemocoel of fifth instar larvae of selected 12 silkworm strains. Among them, the strain d17 is found to be the highest in reporter expression from the intrinsic polyhedrin promoter of Autographa californica NPV or the silkworm actin A3 promoter. These results suggest that the d17 strain is highly permissive for BmNPV replication and is the most likely candidate of a "factory" for large-scale expression using the BmNPV bacmid system..
86. Jae Man Lee, Masateru Takahashi, Hiroaki Mon, Hitoshi Mitsunobu, Katsumi Koga, Yutaka Kawaguchi, Yumiko Nakajima, Takahiro Kusakabe, Construction of gene expression systems in insect cell lines using promoters from the silkworm, Bombyx mori, Journal of Biotechnology, 10.1016/j.jbiotec.2007.08.033, 133, 1, 9-17, 2008.01, The promoter regions of the Bombyx mori HSC70-4 and B. mori TCTP genes characterized previously were used for the construction of a series of constitutive gene expression systems active in cultured cells. The relative abilities of these promoters were evaluated by comparing those of a silkworm actin A3 (BmActin3) promoter, which is used widely as the first choice. A series of constitutive expression systems constructed were assayed for the transcription efficiency by connecting four reporter cDNAs, firefly luciferase, 3GFP, Ds-Red, and β-galactosidase gene using the Gateway LR reaction. The insertion of an intron enhancer into the site between the TCTP promoter and gene increased the transcription of the BmTCTP promoter by 10-fold. The insertion of the IE-1 gene and HR3 enhancer to the all three promoters were found to increase the transcription up to 560 times..
87. Jae Man Lee, Hiroaki Mon, Chisa Yasunaga-Aoki, Masateru Takahashi, Naoya Kawakami, Hitoshi Mitsunobu, Yutaka Banno, Katsumi Koga, Keiro Uchino, Yutaka Kawaguchi, Takahiro Kusakabe, Erratum
"Screening of high-permissive silkworm strains for efficient recombinant protein production in Autographa californica nuclear polyhedrosis virus (AcNPV)" (Journal of Insect Biotechnology and Sericology vol. 76 (2) (101-105)), Journal of Insect Biotechnology and Sericology, 76, 3, 2007.10.
88. Ryohei Sugahara, Hiroaki Mon, Jun Yamashita, Hitoshi Mitsunobu, Jae Man Lee, Yutaka Kawaguchi, Katsumi Koga, Takahiro Kusakabe, Heterotrimeric complex of replication protein A, a single-stranded DNA binding protein, from the silkworm, Bombyx mori, Journal of Insect Biotechnology and Sericology, 76, 3, 129-135, 2007.10, Replication protein A (Rpa) is a heterotrimeric protein complex, composed of three tightly associated subunits of RPA70 (Rpa1), RPA32 (Rpa2) and RPA14 (Rpa3) in the case of human cells. The Rpa is known to be required for almost all aspects of cellular DNA metabolism. In the present study, we isolated and characterized the cDNAs orthologous to the Homo sapience RPA2 and RPA3, BmRPA2 and BmRPA3, respectively, from the silkworm, Bombyx mori. Each gene has a single conserved OB-fold domain. Although there was a higher level of homology among Rpa2s from different species, those of Rpa3s were very low. Furthermore, the analysis using yeast two-hybrid system revealed that the BmRpa2 and BmRpa3 form a tight heterotrimeric complex with BmRpa1. In addition, we confirmed the silkworm Rpa complex binds single-stranded DNA, but not double-stranded DNA..
89. Jun Yamashita, Yoshitaka Miyagawa, Ryohei Sugahara, Hiroaki Mon, Hitoshi Mitsunobu, Jae Man Lee, Yutaka Kawaguchi, Takahiro Kusakabe, Molecular cloning of silkworm Cdc37 and its interaction with Hsp90 chaperone, Journal of Insect Biotechnology and Sericology, 76, 3, 137-143, 2007.10, Hsp90-Cdc37 chaperone complex facilitates the folding and activation of numerous protein kinases. In this report, we have isolated a cDNA clone coding for the Bombyx mori Cdc37 homologue, BmCdc37, and determined its nucleotide sequence. Its mRNA encodes a polypeptide of 373 amino acid residues, which shares 52% amino acid identity with Drosophila melanogaster Cdc37. RT-PCR analysis revealed that the expression of BmCDC37 mRNA occurred mainly in the testis. Direct interaction of the HA-tagged BmCdc37 with endogenous BmHsp90 was demonstrated by co-immunoprecipitation assay from the cell lysates. Subcellular localization site of HA-BmHsp90 and HA-BmCdc37 was exclusively cytoplasmic. However, anomalous nuclear localization of DsRed-BmHsp90, probably due to interaction with EGFP-fused BmCdc37, was re-adjusted to cytoplasmic localization by heat stress. These results suggested that BmCdc37 interacts with BmHsp90 in vivo and tends to be transported to cytoplasm by stress-induced cellular mechanisms..
90. Jae Man Lee, Hiroaki Mon, Masateru Takahashi, Naoya Kawakami, Hitoshi Mitsunobu, Yutaka Banno, Katsumi Koga, Keiro Uchino, Yutaka Kawaguchi, Takahiro Kusakabe, Screening of high-permissive silkworm strains for efficient recombinant protein production in Autographa californica nuclear polyhedrosis virus (AcNPV), Journal of Insect Biotechnology and Sericology, 76, 2, 101-105, 2007.06, The baculovirus expression system (BES) using Autographa california nuclear polyhedrosis virus (AcNPV) has been extensively utilized for the high-level expression of sufficient quantities of recombinant proteins in a broad taxonomic range of insect cell lines and insect larvae. In the case of the silkworm, Bombyx mori, however, the BES using AcNPV tends to be much less exploited because AcNPV is believed to be inefficient in the replication of B. mori and cell lines derived from it. In this study, we have searched for high-permissive silkworm strains with higher production levels of a recombinant protein, by screening the susceptibility of 163 silkworm strains to the recombinant AcNPV, which expresses firefly luciferase. Based on their relative luciferase expression levels in larval hemocytes, the silkworm strains tested were divided into 3 groups: 5 high-permissive strains, 74 middle-permissive strains, and 84 low-permissive strains. Among the 5 high-permissive strains (c11, d17, f10, f38, and 1312), d17 was the most productive, showing the luciferase activity of 3.5 × 104 ± 1,764 relative light units (RLU) per microgram of cell proteins. This remarkable susceptibility indicated that the silkworm d17 strain is very useful for large-scale protein production by the BES using AcNPV..
91. Hitoshi Mitsunobu, Kosuke Sakashita, Hiroaki Mon, Hiroko Yoshida, Jae Man Lee, Yutaka Kawaguchi, Katsumi Koga, Takahiro Kusakabe, Construction of gateway-based destination vectors for detecting subcellular localization of proteins in the silkworm, Bombyx mori, Journal of Insect Biotechnology and Sericology, 75, 3, 141-145, 2006.10, We have constructed 3 kinds of Gateway-based vectors applicable for the subcellular localization analysis of proteins in cultured Bombyx mori cells. Two were designed to visualize the location of an N- or C-terminal fusion protein with EGFP, expressed under the control of the baculoviral IE2 promoter. Another could express an N-terminal fusion protein with DsRed, similarly controlled by IE2. In order to test the applicability, the vectors were individually modified to have a cDNA encoding B. mori Actin1, histone H3 or TMS-1 (ubiquitous family of membrane protein), as well as firefly luciferase. As expected, these proteins showed cytosolic, nuclear, plasma membranous and putative peroxisomal distribution, respectively..
92. Masateru Takahashi, Jae Man Lee, Hiroaki Mon, Hiroko Yoshida, Yutaka Kawaguchi, Hideaki Maekawa, Katsumi Koga, Takahiro Kusakabe, Radiation resistance and its inheritance in the silkworm, Bombyx mori, Journal of the Faculty of Agriculture, Kyushu University, 51, 2, 261-264, 2006.10, Exposure to radiation causes various biological effects, which can be detected both in the exposed animals and their offspring. In the silkworm, Bombyx mori, many studies in somatic and genetic effects of radiation have been conducted using various doses and qualities of radiation sources, because of their usability for mutation induction. However, differences in sensitivity to radiation among silkworm strains remain unknown. In the present study, we have identified two resistant strains (p50 and m042) and two sensitive strains (Crossed D sign2 and m91) for γ-irradiation by screening genetic stocks of B. mori maintained in Kyushu University Graduate School. In reciprocal mating experiments, there is liability that F1 progeny became more sensitive to γ-irradiation when the susceptible strains were used as the female parent. Even though details remain to be determined, it appears from the current studies that radiation sensitivity in the silkworm is controlled, at least in part, by maternal cytoplasmic inheritance..
93. Haruna Tsukioka, Masateru Takahashi, Hiroaki Mon, Kazuhiro Okano, Kazuei Mita, Toru Shimada, Jae Man Lee, Yutaka Kawaguchi, Katsumi Koga, Takahiro Kusakabe, Role of the silkworm argonaute2 homolog gene in double-strand break repair of extrachromosomal DNA, Nucleic acids research, 10.1093/nar/gkj507, 34, 4, 1092-1101, 2006.03, The argonaute protein family provides central components for RNA interference (RNAi) and related phenomena in a wide variety of organisms. Here, we isolated, from a Bombyx mori cell, a cDNA clone named BmAGO2 which is homologous to Drosophila ARGONAUTE2 the gene encoding a repressive factor for the recombination repair of extrachromosomal double-strand breaks (DSBs). RNAi-mediated silencing of the BmAGO2 sequence markedly increased homologous recombination (HR) repair of DSBs in episomal DNA, but had no effect on that in chromosomes. Moreover, we found that RNAi for BmAGO2 enhanced the integration of linearized DNA into a silkworm chromosome via HR. These results suggested that BmAgo2 protein plays an indispensable role in the repression of extrachromosomal DSB repair..
94. Masateru Takahashi, Jae Man Lee, Hiroaki Mon, Yutaka Kawaguchi, Katsumi Koga, Takahiro Kusakabe, Cell cycle arrest induced by radiation in cultured silkworm cells, Journal of Insect Biotechnology and Sericology, 75, 1, 23-30, 2006.02, The silkworm, Bombyx mori, is known to be more resistant to ionizing radiation (IR) compared with mammals, but detailed processes underlying this resistance have remained largely unexplored. In this study, we have confirmed that silkworm larvae survived and showed no effect in the apparent reproduction ability after the irradiation of high doses of γ -ray. We have then observed the effects of γ -irradiation to cells of the silkworm cell line BmN4, which showed marked cell-cycle arrest at the G2/M phase, but not at the G1, or S phases. These cells did not undergo apoptosis after γ -ray irradiation in contrast to the mammalian cells wherein G1, arrests after IR causes apoptosis. After UV-C radiation to cells of BmN4 and Sf21 (a Spodoptera frugiperuda cell line), S-phase checkpoint activation was provoked. But there was also no observation of cell death in BmN4 cells, in contrast to Sf21 cells. Based on these results, we proposed that the extraordinary tolerance of induced DNA injury or the elimination of cell death programs after irradiation is a possible cause of the irradiation resistance in the silkworm cells..
95. Jae Man Lee, Masateru Takahashi, Hiroaki Mon, Katsumi Koga, Yutaka Kawaguchi, Takahiro Kusakabe, Efficient gene transfer into silkworm larval tissues by a combination of sonoporation and lipofection, Cell Biology International, 10.1016/j.cellbi.2005.07.007, 29, 11, 976-979, 2005.11, Sonoporation (ultrasound treatment) provides a new and attractive nonviral way of in vivo gene transfer. To access the applicability of this method to the silkworm, Bombyx mori, we have compared the efficiencies of gene transfer by means of lipofection (using an appropriate agent, PDD111), sonoporation (ditto, FluoroGene™), and lipofection followed by sonoporation. By these methods, a luciferase expression plasmid was found to be markedly transferred into the haemocoel of newly ecdysed fifth instar silkworm larvae, and also into other tissues although with lower rates compared with the haemocoel. In terms of luciferase activity, the efficiencies of transgene by lipofection plus sonoporation were approximately 6 (hemocytes), 20 (silk glands), 8 (mid-gut), 38 (fat body), 10 (Malpighian tubules), 33 (ovaries), and 16 (testes) times as high as those by lipofection or sonoporation alone. These results demonstrated that the present method is useful to introduce the exogenous DNA into insect organs in vivo..
96. Hiroaki Mon, Takahiro Kusakabe, Jae Man Lee, Yutaka Kawaguchi, Katsumi Koga, In vivo DNA double-strand breaks enhance gene targeting in cultured silkworm cells, Comparative Biochemistry and Physiology - B Biochemistry and Molecular Biology, 10.1016/j.cbpc.2004.06.013, 139, 1, 99-106, 2004.09, Alteration of genomic information through homologous recombination (HR) is a powerful tool for reverse genetics in bacteria, yeast, and mice. The low frequency of HR is, however, a major obstacle to achieve efficient gene targeting. In this study, we have developed an assay system for investigating the frequency of gene targeting in cultured silkworm cells using a firefly luciferase gene as a reporter. The introduction of a DNA double-strand break (DSB) either in the chromosomal target locus or in the targeting construct drastically increased the frequency of gene targeting. Interestingly, the inhibition of poly(ADP-ribose) polymerase (PARP), a protein known to play an important role in overall suppression of the HR pathway, stimulated the targeting efficiency, whereas the overexpression of two silkworm RecA homologs, BmRad51 and BmDmc1, had no effect. The presently devised assay system may serve as a useful tool to improve the gene targeting efficiency in the silkworm (Bombyx mori)..
97. Jae Man Lee, Takahiro Kusakabe, Yutaka Kawaguchi, Yoshitaka Miyagawa, Masateru Takahashi, Hiroaki Mon, Si Kab Nho, Katsumi Koga, Molecular cloning and characterization of the translationally controlled tumor protein gene in Bombyx mori, Comparative Biochemistry and Physiology - B Biochemistry and Molecular Biology, 10.1016/j.cbpc.2004.06.004, 139, 1, 35-43, 2004.09, Translationally controlled tumor protein (Tctp/p23) is known to be synthesized preferentially in cells during the early growth phase of tumors, but is also expressed in normal cells. To elucidate its molecular basis of the expression and physiological significance, a cDNA encoding for the Bombyx mori Tctp (BmTctp) was deduced by editing the partial cDNA sequences registered in a Bombyx EST database. RT-PCR analyses indicated that the BmTCTP mRNA was transcribed in all larval organs examined and was present constantly during the cell cycle of BmN4 cells. A genomic clone of 4255 nucloetide residues produced by inverse PCR contained the 5′-flanking region, two introns and three exons of the BmTCTP gene. Sequence analysis of the 5′-flanking region indicated that a putative promoter region contains several canonical transcription elements such as GATA box, CCAAT motif, MEF2, E4BP4.01 and AP-1, but lacks a TATA box element. Luciferase reporter assay of the deletion constructs of the 5′-flanking region revealed that the -676 to +66 region enhanced the promoter activity the most markedly. In addition to this, there were at least two enhancer-like elements and several repressor elements..
98. T. Kusakabe, K. Kido, K. Kita, Y. Banno, H. Mon, Y. Kawaguchi, K. Koga, Analysis of artificial and spontaneous parthenogenetic development in mosaic mutations and the parthenogenetic strain of bombyx mori, Invertebrate Reproduction and Development, 10.1080/07924259.2004.9652579, 45, 2, 101-108, 2004.01, Interesting phenotypes found in male-female mosaic mutations of the silkworm are thought to result from the abnormal activation of a polar body due to the defect on the inactivation system. In addition to these mosaic mutations, a parthenogenetic strain (m90) of silkworm has been identified. The precise mechanism of parthenogenetic development in this strain is unknown, but it is possible that a defect of the polar body inactivation system plays a crucial role here, too. To understand the molecular mechanisms of polar body inactivation, we investigated the process of artificial and spontaneous parthenogenetic development using the mosaic mutations and the m90 strain. Both the pigmentation and the increase in DNA content were used to monitor development. In order to determine whether parthenogenesis in these strains is caused by incomplete meiotic division or fertilization of a polar body with the egg nucleus, the chromosome compositions were analyzed using an insertion sequence in the testis-specific tektin gene as a molecular marker. It was confirmed that parthenogenesis in the mosaic mutations was partly caused by the fertilization of a polar body nucleus with the egg nucleus. In contrast, parthenogenetic development in the m90 strain is apparently caused by incomplete meiotic division..
99. Hiroaki Mon, Takahiro Kusakabe, Hisanori Bando, Katsura Kojima, Yutaka Kawaguchi, Katsumi Koga, Analysis of extrachromosomal homologous recombination in cultured silkworm cells, Biochemical and Biophysical Research Communications, 10.1016/j.bbrc.2003.10.169, 312, 3, 684-690, 2003.12, Double-strand breaks (DSBs) are potentially lethal lesions causing the loss of chromosomal information. Eukaryotic cells have evolved the error-free repair systems of DSBs by homologous recombination (HR) through gene conversion with or without crossing over. In this study, we have developed a rapid assay system for extrachromosomal HR events in the cultured silkworm BmN4 cells. When HR occurs within the disrupted luciferase gene, an enzymatically active luciferase is restored and expressed. Our results strongly suggest that error-prone single strand annealing (SSA) accounts for the majority of extrachromosomal recombination processes in the cells. However, upon the substrates which cannot be repaired through SSA, DSBs were efficiently repaired though gene conversion. The rapid and sensitive HR assay system developed in the present study is expected to be a powerful tool for the identification and analysis of HR-related genes in the silkworm..