Shigeo Imanishi

Transcriptional regulation of juvenile hormone-mediated induction of Krüppel homolog 1, a repressor of insect metamorphosis

The Krüppel homolog 1 gene (Kr-h1) has been proposed to play a key role in the repression of insect metamorphosis. Kr-h1 is assumed to be induced by juvenile hormone (JH) via a JH receptor, methoprene-tolerant (Met), but the mechanism of induction is unclear. To elucidate the molecular mechanism of Kr-h1 induction, we first cloned cDNAs encoding Kr-h1 (BmKr-h1) and Met (BmMet1 and BmMet2) homologs from Bombyx mori. In a B. mori cell line, BmKr-h1 was rapidly induced by subnanomolar levels of natural JHs. Reporter assays identified a JH response element (kJHRE), comprising 141 nucleotides, located ∼2 kb upstream from the BmKr-h1 transcription start site. The core region of kJHRE (GGCCTCCACGTG) contains a canonical E-box sequence to which Met, a basic helix–loop–helix Per-ARNT-Sim (bHLH–PAS) transcription factor, is likely to bind. In mammalian HEK293 cells, which lack an intrinsic JH receptor, ectopic expression of BmMet2 fused with Gal4DBD induced JH-dependent activity of an upstream activation sequence reporter. Meanwhile, the kJHRE reporter was activated JH-dependently in HEK293 cells only when cotransfected with BmMet2 and BmSRC, another bHLH–PAS family member, suggesting that BmMet2 and BmSRC jointly interact with kJHRE. We also found that the interaction between BmMet2 and BmSRC is dependent on JH. Therefore, we propose the following hypothesis for the mechanism of JH-mediated induction of BmKr-h1: BmMet2 accepts JH as a ligand, JH-liganded BmMet2 interacts with BmSRC, and the JH/BmMet2/BmSRC complex activates BmKr-h1 by interacting with kJHRE.

Genome-wide analysis of host gene expression in the silkworm cells infected with Bombyx mori nucleopolyhedrovirus.

The global transcriptional profile of host genes in the silkworm cell line during the early phase of Bombyx mori nucleopolyhedrovirus (BmNPV) infection was analyzed by oligonucleotide microarray. Our analysis showed 35 genes were significantly up-regulated and 17 genes were significantly down-regulated. This is the first report of changes in the expression of these genes in response to NPV infection. We further quantified the levels of mRNA expression by quantitative reverse transcriptase-polymerase chain reaction and confirmed that the expression of 13 (such as BmEts and BmToll10-3) genes significantly increased and 7 genes (such as Hsp20-1) significantly decreased after BmNPV infection. However, the expression levels of most genes were not dramatically changed except BmEts expression increased approximately 8.0-fold 12h after BmNPV infection.

Establishment of a Novel In Vivo Sex-Specific Splicing Assay System To Identify a trans-Acting Factor That Negatively Regulates Splicing of Bombyx mori dsx Female Exons

ABSTRACT The Bombyx mori homolog of doublesex, Bmdsx, plays an essential role in silkworm sexual development. Exons 3 and 4 of Bmdsx pre-mRNA are specifically excluded in males. To explore how this occurs, we developed a novel in vivo sex-specific splicing assay system using sexually differentiated cultured cells. A series of mutation analyses using a Bmdsx minigene with this in vivo splicing assay system identified three distinct sequences (CE1, CE2, and CE3) positioned in exon 4 as exonic splicing silencers responsible for male-specific splicing. Gel shift analysis showed that CE1 binds to a nuclear protein from male cells but not that from female cells. Mutation of UAA repeats within CE1 inhibited the binding of the nuclear protein to the RNA and caused female-specific splicing in male cells. We have identified BmPSI, a Bombyx homolog of P-element somatic inhibitor (PSI), as the nuclear factor that specifically binds CE1. Down-regulation of endogenous BmPSI by RNA interference significantly increased female-specific splicing in male cells. This is the first report of a PSI homolog implicated in the regulated sex-specific splicing of dsx pre-mRNA.

Novel Macula-Like Virus Identified in Bombyx mori Cultured Cells

ABSTRACT We identified a novel, 6,513-bp-long RNA, termed Bombyx mori macula-like latent virus (BmMLV) RNA, which abundantly expressed in B. mori cultured BmN cells. BmMLV RNA potentially encodes two proteins, putative RNA replicase and coat protein, which share structural features and sequence similarities with those of a plant RNA virus, the genus Maculavirus. Northern blot analysis showed that two transcripts were expressed in BmN cells: a 6.5-kb-long RNA, which contains both putative RNA replicase and coat protein genes, and a 1.2-kb-long RNA, which contains only a coat protein gene. Southern blot analysis showed that BmMLV RNA is not carried by the B. mori genome. RT-PCR analysis also revealed the presence of BmMLV RNA in several B. mori cell lines other than BmN cells, suggesting that BmMLV RNA latently exists in B. mori cultured cells. Infection studies showed that BmMLV virions were able to infect BmMLV-negative Spodoptera frugiperda Sf-9 cells and B. mori larvae. Electron microscopy and Northern blot analysis of a purified BmMLV revealed that isometric virions appear to be 28 to 30 nm in diameter and contain a 6.5-kb genomic RNA. These results showed that BmMLV is a novel macula-like virus infectious to and replicable in B. mori-derived cells.

Identification of a Male-Specific RNA Binding Protein That Regulates Sex-Specific Splicing of Bmdsx by Increasing RNA Binding Activity of BmPSI§

ABSTRACT Bmdsx is a sex-determining gene in the silkworm and is alternatively spliced in males and females. CE1 is a splicing silencer element responsible for the sex-specific splicing of Bmdsx. To identify sex-specific factors implicated in the sex-specific splicing of Bmdsx, we performed RNA affinity chromatography using CE1 RNA as a ligand. We have identified BmIMP, a Bombyx homolog of IGF-II mRNA binding protein (IMP), as a male-specific factor that specifically binds to CE1. The gene encoding BmIMP is localized on the Z chromosome and is male-specifically expressed in various tissues. Antisense inhibition of BmIMP expression increased female-specific splicing of Bmdsx pre-mRNA. Coimmunoprecipitation and glutathione S-transferase (GST) pulldown analyses demonstrated that BmIMP physically interacts with BmPSI, which has been identified as a factor implicated in the sex-specific splicing of Bmdsx, through the KH domains of BmIMP. The functional consequence of this interaction was examined using RNA mobility shift analysis. BmIMP increased BmPSI-CE1 RNA binding activity by decreasing the rate of BmPSI dissociation from CE1 RNA. Truncation analysis of BmIMP suggested that the KH domains are responsible for enhancing BmPSI-CE1 RNA binding activity. These results suggest that BmIMP may enhance the male-specific splicing of Bmdsx pre-mRNA by increasing RNA binding activity of BmPSI.

Lipopolysaccharide elicits expression of immune-related genes in the silkworm, Bombyx mori.

Lipopolysaccharide (LPS), a major cell wall component of Gram‐negative bacteria, was found to be unable to activate immune‐related genes in Drosophila melanogaster. In contrast, highly purified LPS elicited immune‐related gene expression in the fat body of Bombyx mori. However, the level of activation by highly purified LPS was lower than crude LPS and peptidoglycan. Furthermore, synthetic lipid A also activated these genes, suggesting that B. mori possesses unknown signal pathways to activate immune‐related genes by LPS. Up‐regulation of antimicrobial peptide genes by highly purified LPS was not confirmed in the immune‐responsive cell line, NIAS‐Bm‐aff3, suggesting that some factors necessary for signal transduction activated by LPS are deficient in this cell line.

A novel cell-free translation/glycosylation system prepared from insect cells

A cell-free translation/glycosylation system derived from lepidopteran (Sf21) cells, which are widely used to express high yields of foreign active proteins that have post-translational modifications, was constructed. The insect cell extract was prepared using a Mini-Bomb cell disruption chamber by nitrogen pressure treatment, which stably retains translational and post-translational components. The gp120 mRNA was transcribed from the human immunodeficiency virus type-1 envelope glycoprotein gp120 gene with T7 RNA polymerase. When the gp120 mRNA was translated in the insect cell-free system, gp120 having a molecular mass of 100 kDa was detected by Western blot analysis. Synthesized gp120 and gp120 expressed in the intracellular fraction of recombinant-baculovirus-infected Sf21 cells had the same molecular mass, and they both had reduced mobility compared with gp120 secreted by recombinant baculovirus-infected Sf21 cells. In contrast, the 56-kDa gp120 protein, which corresponds to the polypeptide backbone of gp120, was synthesized in wheat germ and rabbit reticulocyte systems. The molecular mass of synthesized gp120 decreased from 100 kDa to 61 kDa after endoglycosidase H treatment, indicating that synthesized gp120 had been glycosylated with N-linked oligosaccharides. Furthermore, glycosylated gp120 was bound to human CD4 molecules expressed on the surface of quail cells. These results revealed that the insect cell-free system can synthesize gp120 that is folded in the proper conformation to provide a CD4-binding domain.

Ecdysone-inducible foreign gene expression in stably-transformed lepidopteran insect cells.

SummaryCultured cell lines that can be stably transformed with inducible gene constructs could prove extremely valuable for the continuous and economical production of recombinant proteins. Toward this goal, we have established 11 clones (designated NISES-BoMo-DK1 to 11) from a previously reported silkworm cell line, NISES-BoMo-DZ. Nine of these clonal lines showed a distinct morphological change, i.e., cell aggregation, in response to treatment with 1 μM 20-hydroxyecdysone (20E). DK10 cells transfected with various reporter assay plasmids under optimal conditions (i.e., 20–30% transfection efficiency) showed inducibility of gene expression by 20E. The 20E treatment of the prototypical DK10 cells resulted in a simultaneous, transient increase of the nuclear ecdysone (E) receptor levels. Further, this inducibility was also observed in a DK10 cell line stably transformed with the reporter plasmid that carries the hygromycin-resistance gene. This offers an opportunity to achieve efficient, continuous production of recombinant proteins. It could also allow high throughput screening for potential E agonists.

Differentially expressed genes in silkworm cell cultures in response to infection by Wolbachia and Cardinium endosymbionts.

Wolbachia and Cardinium are bacterial endosymbionts that are widely distributed amongst arthropods. Both cause reproductive alterations, such as cytoplasmic incompatibility, parthenogenesis and feminization. Here we studied differentially expressed genes in Wolbachia‐ and Cardinium‐infected Bm‐aff3 silkworm cells using a silkworm microarray. Wolbachia infection did not alter gene expression or induce or suppress immune responses. In contrast, Cardinium infection induced many immune‐related genes, including antimicrobial peptides, pattern recognition receptors and a serine protease. Host immune responses differed, possibly because of the different cell wall structures of Wolbachia and Cardinium because the former lacks genes encoding lipopolysaccharide components and two racemases for peptidoglycan formation. A few possibly non‐immune‐related genes were differentially expressed, but their involvement in host reproductive alteration was unclear.

Isolation of p10 gene fromBombyx mori nuclear polyhedrosis virus and study of its promoter activity in recombinant baculovirus vector system

A homologue ofAutographa californica NPV (AcNPV) p10 gene was identified and cloned fromBombyx mori NPV (BmNPV). BmNPV p10 gene encodes truncated protein of 70 amino acid residues that lacks carboxyl terminus comparing with the p10 protein encoded by AcNPV. The putative TATA box sequence and the ATAAG motif which is the consensus sequence of baculovirus very late promoter were conserved. A transfer vector, pBNT1, which includes the p10 promoter region of BmNPV for foreign gene expression was constructed. By using pBNT1, a recombinant BmNPV, Bmp10-Luc, in which the p10 gene was replaced by the firefly luciferase gene, was obtained. We also obtained another recombinant virus, BmPH-Luc, in which the polyhedrin gene was replaced by the luciferase gene. The luciferase activity detected in BoMo-15AIIc insect cells infected with Bmp10-Luc was approximately 50% of that infected with BmPH-Luc, suggesting that although both the p10 and polyhedrin promoters of BrnNPV are effective in high-level expression of foreign gene, the p10 promoter is not so strong as the polyhedrin promoter.