| 1. |
Analysis of artificial and spontaneous parthenogenetic development in mosaic mutations and the parthenogenetic strain of bombyx mori
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. © 2004 Taylor & Francis Group, LLC.. |
| 2. |
Construction of gateway-based destination vectors for detecting subcellular localization of proteins in the silkworm, Bombyx mori
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.. |
| 3. |
Cell cycle arrest induced by radiation in cultured silkworm cells
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.. |
| 4. |
Molecular cloning of silkworm Cdc37 and its interaction with Hsp90 chaperone
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.. |
| 5. |
Heterotrimeric complex of replication protein A, a single-stranded DNA binding protein, from the silkworm, Bombyx mori
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.. |
| 6. |
Molecular characterization of core histones in the silkworm, Bombyx mori
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.. |
| 7. |
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
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.. |
| 8. |
Biologically active human bone morphogenetic protein 4 fused to collagen-binding domain produced in silkworm-baculovirus expression system
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.. |
| 9. |
A MC motif in silkworm Argonaute 1 is indispensible for translation repression
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. © 2013 Royal Entomological Society.. |
| 10. |
Middle region of FancM interacts with Mhf and Rmi1 in silkworms, a species lacking the Fanconi anaemia (FA) core complex
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. © 2013 The Royal Entomological Society.. |
| 11. |
Differential N-Glycan modifications of human alpha 1-acid glycoprotein (α1AGP) produced in different silkworm strains using the baculovirus expression system
© 2015, Japanese Society of Sericultural Sciences. All rights reserved. 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.. |
| 12. |
Virulence of lipopolysaccharide-deficient mutants of serratia liquefaciens toward the silkworm, Bombyx mori
© 2016, Japanese Society of Sericultural Sciences. All rights reserved. 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.. |
| 13. |
Modular surface display for porcine circovirus type 2 virus-like particles mediated by microbial transglutaminase
© 2018, Japanese Society of Sericultural Sciences. All rights reserved. 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.. |
| 14. |
A reconsideration of the taxonomic position of two bacterial strains isolated from Flacherie-Diseased silkworms in 1965
© 2017, Japanese Society of Sericultural Sciences. All rights reserved. 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.. |
| 15. |
Characterization of the roles of DNA polymerases, clamp, and clamp loaders during s-phase progression and cell cycle regulation in the silkworm, bombyx mori
© 2016, Journal of Insect Biotechnology and Sericology. All rights reserved. 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.. |
