Publications by authors named "Hiroaki Mon"

67 Publications

Production of scFv, Fab, and IgG of CR3022 Antibodies Against SARS-CoV-2 Using Silkworm-Baculovirus Expression System.

Mol Biotechnol 2021 Jul 25. Epub 2021 Jul 25.

Laboratory of Insect Genome Science, Faculty of Agriculture, Kyushu University, Motooka 744, Nishi-ku, Fukuoka, 819-0395, Japan.

COVID-19, caused by SARS-CoV-2, is currently spreading around the world and causing many casualties. Antibodies against such emerging infectious diseases are one of the important tools for basic viral research and the development of diagnostic and therapeutic agents. CR3022 is a monoclonal antibody against the receptor binding domain (RBD) of the spike protein (S protein) of SARS-CoV found in SARS patients, but it was also shown to have strong affinity for that of SARS-CoV-2. In this study, we produced large amounts of three formats of CR3022 antibodies (scFv, Fab and IgG) with high purity using a silkworm-baculovirus expression vector system. Furthermore, SPR measurements showed that the affinity of those silkworm-produced IgG antibodies to S protein was almost the same as that produced in mammalian expression system. These results indicate that the silkworm-baculovirus expression system is an excellent expression system for emerging infectious diseases that require urgent demand for diagnostic agents and therapeutic agents.
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http://dx.doi.org/10.1007/s12033-021-00373-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8310559PMC
July 2021

Stable trimer formation of spike protein from porcine epidemic diarrhea virus improves the efficiency of secretory production in silkworms and induces neutralizing antibodies in mice.

Vet Res 2021 Jul 7;52(1):102. Epub 2021 Jul 7.

Laboratory of Insect Genome Science, Kyushu University Graduate School of Bioresource and Bioenvironmental Sciences, Motooka 744, Nishi-ku, Fukuoka, 819-0395, Japan.

Porcine epidemic diarrhea virus (PEDV) is a highly infectious pathogen of watery diarrhea that causes serious economic loss to the swine industry worldwide. Especially because of the high mortality rate in neonatal piglets, a vaccine with less production cost and high protective effect against PEDV is desired. The intrinsically assembled homotrimer of spike (S) protein on the PEDV viral membrane contributing to the host cell entry is a target of vaccine development. In this study, we designed trimerized PEDV S protein for efficient production in the silkworm-baculovirus expression vector system (silkworm-BEVS) and evaluated its immunogenicity in the mouse. The genetic fusion of the trimeric motif improved the expression of S protein in silkworm-BEVS. A small-scale screening of silkworm strains to further improve the S protein productivity finally achieved the yield of about 2 mg from the 10 mL larval serum. Mouse immunization study demonstrated that the trimerized S protein could elicit strong humoral immunity, including the S protein-specific IgG in the serum. These sera contained neutralizing antibodies that can protect Vero cells from PEDV infection. These results demonstrated that silkworm-BEVS provides a platform for the production of trimeric S proteins, which are promising subunit vaccines against coronaviruses such as PEDV.
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http://dx.doi.org/10.1186/s13567-021-00971-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8261802PMC
July 2021

Active Human and Murine Tumor Necrosis Factor α Cytokines Produced from Silkworm Baculovirus Expression System.

Insects 2021 Jun 2;12(6). Epub 2021 Jun 2.

Laboratory of Creative Science for Insect Industries, Kyushu University Graduate School of Bioresource and Bioenvironmental Sciences, Motooka 744, Nishi-ku, Fukuoka 819-0395, Japan.

The tumor necrosis factor α (TNFα) has been employed as a promising reagent in treating autoimmunity and cancer diseases. To meet the substantial requirement of TNFα proteins, we report in this study that mature types of recombinant human and murine TNFα proteins are successfully expressed in the baculovirus expression system using silkworm larvae as hosts. The biological activities of purified products were verified in culture murine L929 cells, showing better performance over a commercial -derived murine TNFα. By comparing the activity of purified TNFα with or without the tag removal, it is also concluded that the overall activity of purified TNFα cytokines could be further improved by the complete removal of C-terminal fusion tags. Collectively, our current attempt demonstrates an alternative platform for supplying high-quality TNFα products with excellent activities for further pharmaceutical and clinical trials.
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http://dx.doi.org/10.3390/insects12060517DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8230043PMC
June 2021

Quick and Easy Assembly of a One-Step qRT-PCR Kit for COVID-19 Diagnostics Using In-House Enzymes.

ACS Omega 2021 Mar 15;6(11):7374-7386. Epub 2021 Mar 15.

Laboratory of DNA Replication and Recombination, Biological and Environmental Sciences and Engineering Division, King Abdullah University of Science and Technology (KAUST), Thuwal 23955-6900, Saudi Arabia.

One-step reverse-transcription quantitative polymerase chain reaction (qRT-PCR) is the most widely applied method for COVID-19 diagnostics. Notwithstanding the facts that one-step qRT-PCR is well suited for the diagnosis of COVID-19 and that there are many commercially available one-step qRT-PCR kits in the market, their high cost and unavailability due to airport closures and shipment restriction became a major bottleneck that had driven the desire to produce the key components of such kits locally. Here, we provide a simple, economical, and powerful one-step qRT-PCR kit based on patent-free, specifically tailored versions of Moloney murine leukemia virus reverse transcriptase and DNA polymerase and termed R3T (Rapid Research Response Team) one-step qRT-PCR. We also demonstrate the robustness of our enzyme production strategies and provide the optimal reaction conditions for their efficient augmentation in a one-step approach. Our kit was routinely able to reliably detect as low as 10 copies of the synthetic RNAs of SARS-CoV-2. More importantly, our kit successfully detected COVID-19 in clinical samples of broad viral titers with similar reliability and selectivity to that of the Invitrogen SuperScript III Platinum One-step qRT-PCR and TaqPath one-step RT-qPCR kits. Overall, our kit has shown robust performance in both laboratory settings and the Saudi Ministry of Health-approved testing facility.
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http://dx.doi.org/10.1021/acsomega.0c05635DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7986002PMC
March 2021

Efficient production of recombinant SARS-CoV-2 spike protein using the baculovirus-silkworm system.

Biochem Biophys Res Commun 2020 08 9;529(2):257-262. Epub 2020 Jun 9.

Laboratory of Insect Genome Science, Faculty of Agriculture, Kyushu University, Motooka 744, Nishi-ku, Fukuoka, 819-0395, Japan.

In the case of a new viral disease outbreak, an immediate development of virus detection kits and vaccines is required. For COVID-19, we established a rapid production procedure for SARS-CoV-2 spike protein (S protein) by using the baculovirus-silkworm expression system. The baculovirus vector-derived S proteins were successfully secreted to silkworm serum, whereas those formed insoluble structure in the larval fat body and the pupal cells. The ectodomain of S protein with the native sequence was cleaved by the host furin-protease, resulting in less recombinant protein production. The S protein modified in furin protease-target site was efficiently secreted to silkworm serum and was purified as oligomers, which showed immunoreactivity for anti-SARS-CoV-2 S2 antibody. By using the direct transfection of recombinant bacmid to silkworms, we achieved the efficient production of SARS-CoV-2 S protein as fetal bovine serum (FBS)-free system. The resultant purified S protein would be useful tools for the development of immunodetection kits, antigen for immunization for immunoglobulin production, and vaccines.
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http://dx.doi.org/10.1016/j.bbrc.2020.06.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7280120PMC
August 2020

Expression and Purification of Vaccinia Virus DNA Topoisomerase IB Produced in the Silkworm-Baculovirus Expression System.

Mol Biotechnol 2019 Aug;61(8):622-630

Laboratory of DNA Replication and Recombination, Division of Biological and Environmental Sciences and Engineering, King Abdullah University of Science and Technology, 4700 KAUST Thuwal, Jeddah, 23955, Saudi Arabia.

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 Mg 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, Mg, Mn, Ca, 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.
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http://dx.doi.org/10.1007/s12033-019-00184-4DOI Listing
August 2019

Expression and purification of biologically active human granulocyte-macrophage colony stimulating factor (hGM-CSF) using silkworm-baculovirus expression vector system.

Protein Expr Purif 2019 07 24;159:69-74. Epub 2019 Mar 24.

Laboratory of Creative Science for Insect Industries, Kyushu University Graduate School of Bioresource and Bioenvironmental Sciences, Motooka 744, Nishi-ku, Fukuoka, 819-0395, Japan. Electronic address:

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.
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http://dx.doi.org/10.1016/j.pep.2019.03.010DOI Listing
July 2019

Identification of an insecticidal toxin produced by Enterobacter sp. strain 532 isolated from diseased Bombyx mori silkworms.

FEMS Microbiol Lett 2019 01;366(2)

Laboratory of Plant Pathology, Faculty of Agriculture, Graduate School, Kyushu University, Motooka 744, Nishi-ku, Fukuoka 819-0395, Japan.

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.
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http://dx.doi.org/10.1093/femsle/fny295DOI Listing
January 2019

Expression, Purification, and Characterization of Recombinant Human α-Antitrypsin Produced Using Silkworm-Baculovirus Expression System.

Mol Biotechnol 2018 Dec;60(12):924-934

Laboratory of Insect Genome Science, Kyushu University Graduate School of Bioresource and Bioenvironmental Science, Hakozaki 6-10-1, Higashi-ku, Fukuoka, 812-8581, Japan.

Human α-antitrypsin (AAT) is the most abundant serine proteinase inhibitor (serpin) in the human plasma. Commercially available AAT for the medications of deficiency of α-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.
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http://dx.doi.org/10.1007/s12033-018-0127-yDOI Listing
December 2018

A functional polypeptide N-acetylgalactosaminyltransferase (PGANT) initiates O-glycosylation in cultured silkworm BmN4 cells.

Appl Microbiol Biotechnol 2018 Oct 22;102(20):8783-8797. Epub 2018 Aug 22.

Laboratory of Insect Genome Science, Kyushu University Graduate School of Bioresource and Bioenvironmental Sciences, 6-10-1 Hakozaki Higashi-ku, Fukuoka, 812-8581, Japan.

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.
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http://dx.doi.org/10.1007/s00253-018-9309-6DOI Listing
October 2018

Purification and characterization of immunogenic recombinant virus-like particles of porcine circovirus type 2 expressed in silkworm pupae.

J Gen Virol 2018 07;99(7):917-926

Laboratory of Insect Genome Science, Kyushu University Graduate School of Bioresource and Bioenvironmental Sciences, Hakozaki 6-10-1, Higashi-ku, Fukuoka 812-8581, Japan.

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.
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http://dx.doi.org/10.1099/jgv.0.001087DOI Listing
July 2018

Molecular characterization of mitochondrial Zucchini and its relation to nuage-piRNA pathway components in Bombyx mori ovary-derived BmN4 cells.

Biochem Biophys Res Commun 2017 11 20;493(2):971-978. Epub 2017 Sep 20.

Laboratory of Insect Genome Science, Department of Bioresource Science, Graduate School of Bioresource and Bioenvironmental Sciences, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan. Electronic address:

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.
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http://dx.doi.org/10.1016/j.bbrc.2017.09.107DOI Listing
November 2017

Lipidation of BmAtg8 is required for autophagic degradation of p62 bodies containing ubiquitinated proteins in the silkworm, Bombyx mori.

Insect Biochem Mol Biol 2017 10 1;89:86-96. Epub 2017 Sep 1.

Laboratory of Insect Genome Science, Kyushu University Graduate School of Bioresource and Bioenvironmental Sciences, 6-10-1 Hakozaki Higashi-ku, Fukuoka 812-8581, Japan. Electronic address:

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.
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http://dx.doi.org/10.1016/j.ibmb.2017.08.006DOI Listing
October 2017

Characterization of Armitage and Yb containing granules and their relationship to nuage in ovary-derived cultured silkworm cell.

Biochem Biophys Res Commun 2017 08 5;490(2):134-140. Epub 2017 Jun 5.

Laboratory of Insect Genome Science, Kyushu University, Graduate School of Bioresource and Bioenvironmental Sciences, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan. Electronic address:

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.
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http://dx.doi.org/10.1016/j.bbrc.2017.06.008DOI Listing
August 2017

Characterization of Recombinant Thermococcus kodakaraensis (KOD) DNA Polymerases Produced Using Silkworm-Baculovirus Expression Vector System.

Mol Biotechnol 2017 Jun;59(6):221-233

Laboratory of Insect Genome Science, Kyushu University Graduate School of Bioresource and Bioenvironmental Sciences, 6-10-1 Hakozaki Higashi-ku, Fukuoka, 812-8581, Japan.

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.
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http://dx.doi.org/10.1007/s12033-017-0008-9DOI Listing
June 2017

Identification and functional analysis of outer kinetochore genes in the holocentric insect Bombyx mori.

Insect Biochem Mol Biol 2017 07 1;86:1-8. Epub 2017 May 1.

Laboratory of Insect Genome Science, Kyushu University Graduate School of Bioresource and Bioenvironmental Sciences, Hakozaki 6-10-1, Higashi-ku, Fukuoka, Japan. Electronic address:

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.
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http://dx.doi.org/10.1016/j.ibmb.2017.04.005DOI Listing
July 2017

Expression and Characterization of Human β-1, 4-Galactosyltransferase 1 (β4GalT1) Using Silkworm-Baculovirus Expression System.

Mol Biotechnol 2017 May;59(4-5):151-158

Laboratory of Insect Genome Science, Kyushu University Graduate School of Bioresource and Bioenvironmental Sciences, Hakozaki 6-10-1, Higashi-ku, Fukuoka, 812-8581, Japan.

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.
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http://dx.doi.org/10.1007/s12033-017-0003-1DOI Listing
May 2017

Alkaline protease contributes to pyocyanin production in Pseudomonas aeruginosa.

FEMS Microbiol Lett 2017 04;364(7)

Laboratory of Insect Pathology and Microbial Control, Institute of Biological Control, Faculty of Agriculture, Graduate School, Kyushu University, Fukuoka 812-8581, Japan.

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.
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http://dx.doi.org/10.1093/femsle/fnx051DOI Listing
April 2017

Proteasome inhibitor MG132 impairs autophagic flux through compromising formation of autophagosomes in Bombyx cells.

Biochem Biophys Res Commun 2016 Oct 28;479(4):690-696. Epub 2016 Sep 28.

Laboratory of Insect Genome Science, Kyushu University Graduate School of Bioresource and Bioenvironmental Sciences, 6-10-1 Hakozaki Higashi-ku, Fukuoka 812-8581, Japan. Electronic address:

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.
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http://dx.doi.org/10.1016/j.bbrc.2016.09.151DOI Listing
October 2016

Expression and Characterization of Recombinant Serratia liquefaciens Nucleases Produced with Baculovirus-mediated Silkworm Expression System.

Mol Biotechnol 2016 Jun;58(6):393-403

Laboratory of Insect Genome Science, Faculty of Agriculture, Graduate School, Kyushu University, Fukuoka, Japan.

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.
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http://dx.doi.org/10.1007/s12033-016-9937-yDOI Listing
June 2016

Effect of antibiotics on extracellular protein level in Pseudomonas aeruginosa.

Plasmid 2016 Mar-May;84-85:44-50. Epub 2016 Mar 17.

Laboratory of Insect Pathology and Microbial Control, Institute of Biological Control, Faculty of Agriculture, Graduate School, Kyushu University, Fukuoka, Japan. Electronic address:

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.
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http://dx.doi.org/10.1016/j.plasmid.2016.03.001DOI Listing
March 2017

CRISPR/Cas9-mediated knockout of factors in non-homologous end joining pathway enhances gene targeting in silkworm cells.

Sci Rep 2015 Dec 10;5:18103. Epub 2015 Dec 10.

Laboratory of Insect Genome Science, Kyushu University Graduate School of Bioresource and Bioenvironmental Sciences, Hakozaki 6-10-1, Fukuoka 812-8581, Japan.

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.
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http://dx.doi.org/10.1038/srep18103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4674802PMC
December 2015

Comparative proteomic analysis of hemolymph proteins from Autographa californica multiple nucleopolyhedrovirus (AcMNPV)-sensitive or -resistant silkworm strains during infections.

Comp Biochem Physiol Part D Genomics Proteomics 2015 Dec 4;16:36-47. Epub 2015 Aug 4.

Laboratory of Insect Genome Science, Kyushu University Graduate School of Bioresource and Bioenvironmental Sciences, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan. Electronic address:

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.
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http://dx.doi.org/10.1016/j.cbd.2015.07.003DOI Listing
December 2015

Roles of silkworm endoplasmic reticulum chaperones in the secretion of recombinant proteins expressed by baculovirus system.

Mol Cell Biochem 2015 Nov 12;409(1-2):255-62. Epub 2015 Aug 12.

Laboratory of Insect Genome Science, Kyushu University Graduate School of Bioresource and Bioenvironmental Sciences, 6-10-1 Hakozaki Higashi-ku, Fukuoka, 812-8581, Japan.

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.
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http://dx.doi.org/10.1007/s11010-015-2529-5DOI Listing
November 2015

Loqs depends on R2D2 to localize in D2 body-like granules and functions in RNAi pathways in silkworm cells.

Insect Biochem Mol Biol 2015 Sep 13;64:78-90. Epub 2015 Jul 13.

Laboratory of Silkworm Science, Kyushu University Graduate School of Bioresource and Bioenvironmental Sciences, Hakozaki 6-10-1, Fukuoka 812-8581, Japan. Electronic address:

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.
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http://dx.doi.org/10.1016/j.ibmb.2015.07.011DOI Listing
September 2015

Characterization of KfrA proteins encoded by a plasmid of Paenibacillus popilliae ATCC 14706(T).

Meta Gene 2015 Jun 20;4:29-44. Epub 2015 Mar 20.

Laboratory of Insect Pathology and Microbial Control, Institute of Biological Control, Faculty of Agriculture, Graduate School, Kyushu University, Japan.

A scaffold obtained from whole-genome shotgun sequencing of Paenibacillus popilliae ATCC 14706(T) shares partial homology with plasmids found in other strains of P. popilliae. PCR and sequencing for gap enclosure indicated that the scaffold originated from a 15,929-bp circular DNA. The restriction patterns of a plasmid isolated from P. popilliae ATCC 14706(T) were identical to those expected from the sequence; thus, this circular DNA was identified as a plasmid of ATCC 14706(T) 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.
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http://dx.doi.org/10.1016/j.mgene.2015.03.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4372654PMC
June 2015

Mass Production of an Active Peptide-N-Glycosidase F Using Silkworm-Baculovirus Expression System.

Mol Biotechnol 2015 Aug;57(8):735-45

Laboratory of Silkworm Science, Kyushu University Graduate School of Bioresource and Bioenvironmental Sciences, Hakozaki 6-10-1, Higashi-ku, Fukuoka, 812-8581, Japan.

The peptide-N (4)-(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.
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http://dx.doi.org/10.1007/s12033-015-9866-1DOI Listing
August 2015

Biochemical characterization of maintenance DNA methyltransferase DNMT-1 from silkworm, Bombyx mori.

Insect Biochem Mol Biol 2015 Mar 23;58:55-65. Epub 2015 Jan 23.

Laboratory of Insect Genome Science, Kyushu University Graduate School of Bioresource and Bioenvironmental Sciences, Fukuoka, Japan. Electronic address:

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 Zn(2+) and Mn(2+). 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.
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http://dx.doi.org/10.1016/j.ibmb.2015.01.008DOI Listing
March 2015

Dynamics of polycomb proteins-mediated histone modifications during UV irradiation-induced DNA damage.

Insect Biochem Mol Biol 2014 Dec 13;55:9-18. Epub 2014 Oct 13.

Laboratory of Silkworm Science, Kyushu University Graduate School of Bioresource and Bioenvironmental Sciences, Hakozaki 6-10-1, Fukuoka 812-8581, Japan. Electronic address:

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.
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http://dx.doi.org/10.1016/j.ibmb.2014.10.001DOI Listing
December 2014

Differential contribution of the Fanconi anemia-related proteins to repair of several types of DNA damage in cultured silkworm cells.

FEBS Lett 2014 Nov 19;588(21):3959-63. Epub 2014 Sep 19.

Laboratory of Silkworm Science, Kyushu University Graduate School of Bioresource and Bioenvironmental Sciences, 6-10-1 Hakozaki, Fukuoka 812-8581, Japan. Electronic address:

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.
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http://dx.doi.org/10.1016/j.febslet.2014.09.009DOI Listing
November 2014
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