Masashi Iwanaga

βFTZ-F1 and Broad-Complex positively regulate the transcription of the wing cuticle protein gene, BMWCP5, in wing discs of Bombyx mori.

The present study was undertaken to clarify the mechanism regulating cuticle protein gene expression. Expression of BMWCP5 was strong at around pupation and weak at the mid-pupal stage in wing tissues of Bombyx mori. We analyzed the upstream region of the BMWCP5 gene using a transient reporter assay with a gene gun system to identify the regulatory elements responsible for its unique expression pattern. We identified two betaFTZ-F1 binding sites to be important cis-acting elements for the transcription activation of the luciferase reporter gene by an ecdysone pulse. Site-directed mutagenesis of these sites, followed by introduction into wing discs, significantly decreased the reporter activity. We also found that the regions carrying the binding sites for the ecdysone-responsive factor BR-C Z4 (BR-Z4) were responsible for the hormonal enhancement of the reporter gene activity in wing discs. Mutation of the BR-Z4 binding sites decreased the reporter activity. The nuclear proteins that bound to these betaFTZ-F1 and BR-Z4 sites were identified by an electrophoretic mobility shift assay (EMSA). The results demonstrate for the first time that the BR-Z4 isoform can bind to the upstream region of the cuticle protein gene, BMWCP5, and activate its expression. The results also suggest that the BMWCP5 transcription is primarily regulated by the ecdysone pulse through betaFTZ-F1, and the stage-specific enhancement is brought about through BR-Z4.

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.

Analysis of ecdysone-pulse responsive region of BMWCP2 in wing disc of Bombyx mori

The present study was undertaken to clarify the regulation of cuticle-protein gene expression. Bombyx BAC library was screened to obtain the sequences of regulatory regions of cuticle protein genes isolated from wing discs of Bombyx mori. Two kb upstream of BMWCP2 was cloned into a reporter plasmid, and a reporter assay was operated. Plasmids were introduced into wing discs and wing tissues using a gene gun. DNA introduction into wing discs was confirmed with plasmid pA3GFP. The upstream region of BMWCP2 showed stage-specific activity: strongest at P0. EMSA analysis indicated the binding of BmbetaFTZ-F1. Ecdysone pulse-responsive sequences were examined in vitro. A luciferase assay was performed using reporter plasmids that contained different length upstream-regions of BMWCP2. With this method, we identified the ecdysone-responsive region. With deletion of the BMWCP2 upstream region, mutagenesis of the BmbetaFTZ-F1 binding site and EMSA analysis, it was confirmed that the BMWCP2 expression was regulated by BmbetaFTZ-F1 through the ecdysone pulse. This is first to apply the introduction of reporter plasmids into small organs to examine the developmental and hormonal regulation of the cuticle protein gene expression. We demonstrated that the binding of BmbetaFTZ-F1 facilitated the promoter activity of the BMWCP2 cuticle protein gene in vitro.

Ecdysone directly and indirectly regulates a cuticle protein gene, BMWCP10, in the wing disc of Bombyx mori

The hormonal regulation of cuticle protein genes is a good model to study the molecular mechanism of signaling by ecdysteroids, which initiates each of the major developmental transitions in insects. This study was conducted to clarify the regulation of the expression of an ecdysone-inducible cuticle protein gene, BMWCP10. Induction of the BMWCP10 transcript by ecdysone was partly inhibited in the presence of cycloheximide, which implies that the BMWCP10 promoter is directly and indirectly activated by ecdysone. Using electrophoretic mobility shift analysis and a competition experiment, we identified a putative ecdysone response element (EcRE1) located at positions -93 to -81 relative to the transcription start site. Site-directed mutagenesis of this site, followed by introduction into wing discs, dramatically abolished the reporter activity. This EcRE1 is necessary for the activation of the promoter by 20-hydroxyecdysone (20E) in the wing disc, since the mutation of EcRE1 caused loss of responsiveness to 20E. Collectively, the data obtained in our current and previous work indicate that ecdysone receptor and Broad-Complex Z2 (BR-Z2) are required for maximal BMWCP10 expression in wing disc.

Ecdysone-responsive transcription factors determine the expression region of target cuticular protein genes in the epidermis of Bombyx mori

In the present study, we found that different ecdysone-responsive transcription factors were expressed differentially in different regions of the epidermis at around pupation. βFTZ-F1 transcripts were strongly but E74A transcripts were barely observed in the thoracic region of the epidermis, and vice versa in the abdominal region. Transcripts of all the examined transcription factors were observed in wing disc. Transcript of a cuticular protein gene, BMWCP4, which does not have a βFTZ-F1 binding site in the 2-kb upstream region, was not observed in the thoracic region of the epidermis. Transcript of BMWCP9, which does not have an E74 binding site in the 2-kb upstream region, was not observed in the abdominal region of the epidermis. BMWCP2 has all the transcription factor binding sites examined and was expressed in the thoracic and abdominal region of the epidermis. Thus, it is suggested that ecdysone-responsive transcription factors determined the space where the cuticular protein genes were expressed, which, in turn, determined the character of the cuticle that was characterized by the combination of cuticular proteins.

Involvement of HSC70-4 and other inducible HSPs in Bombyx mori nucleopolyhedrovirus infection

Heat shock proteins (HSPs) and heat shock cognate proteins (HSCs) function as molecular chaperones under normal cellular conditions. In this report, we describe the role of Bombyx mori heat shock cognate protein 70-4 (BmHSC70-4), which is a constitutively expressed member of the heat shock protein 70 (HSP70) family, in B. mori nucleopolyhedrovirus (BmNPV) infection. We first generated the BmHSC70-4 antibody, which can react specifically with an endogenous BmHSC70 from BmN cells. Immunohistochemistry has demonstrated that BmHSC70-4 was expressed at steady-state levels throughout the BmNPV infection and was accumulated in the nucleus of BmNPV-infected cells at a very late phase of infection. Western blot experiments have also shown that BmHSC70-4 is a novel component protein of budded virus (BV) and occlusion-derived virus (ODV). Next, we investigated the effect of KNK437, a known inhibitor of inducible HSPs, in BmNPV-infected BmN cells and found that both reduced BV production and delayed viral DNA replication were observed in virus-infected cells treated with KNK437. Furthermore, the formation of occlusion bodies (OBs) was not observed in KNK437-treated cells because this compound reduced the promoter activity of the polyhedrin gene severely. Collectively, the present results suggest that BmHSC70-4 is a novel structural protein of BmNPV and may have important roles in BmNPV propagation.

Infection study of Bombyx mori macula-like virus (BmMLV) using a BmMLV-negative cell line and an infectious cDNA clone

Previously, a novel macula-like virus was identified from Bombyx mori cultured cell line BmN and termed B. mori macula-like virus (BmMLV). BmMLV encodes a 6.5-kb-long positive, single-strand RNA genome, which contains putative RNA-dependent RNA polymerase (RdRp), coat protein (cp) and p15 genes. In this study, CP expression in several B. mori-derived cell lines was examined by using the CP antibody. Surprisingly, Western blot analysis revealed that all of the cell lines tested have already been infected with BmMLV. To perform reverse genetic studies in BmMLV, a new BmMLV-negative cell line, designated as BmVF from the embryos of B. mori was established. Infection studies showed that BmVF cells were permissive to BmMLV persistent infection. In addition, a full-length infectious cDNA clone of BmMLV, termed pHMLV was developed. Upon transfection of pHMLV into BmMLV-negative BmVF cells, viral CP was detected in both cells and conditioned medium. When the cDNA-derived virus in conditioned medium was inoculated onto BmVF cells, efficient propagation of BmMLV was observed. Collectively, these results indicate that the new BmMLV-negative cell line and the infectious cDNA clone of BmMLV will be useful for elucidation of the mechanism of BmMLV replication and the functional roles of BmMLV genes.

Isolation and characterization of calmodulin in the pheromone gland of the silkworm, Bombyx mori

Production of the sex pheromone bombykol in the silkworm, Bombyx mori, is regulated by a neurohormone termed pheromone biosynthesis activating neuropeptide (PBAN). It has been suggested that the external signal of PBAN in this species is transmitted to the intracellular cascade reactions consisting of Ca2+/calmodulin (CaM) complex and phosphoprotein phosphatase. To demonstrate the molecular mechanisms regulated by PBAN, we attempted to characterize CaM in the pheromone gland of B. mori. By using ion-exchange and RP-HPLC, B. mori CaM was purified from the cytosolic fraction of the pheromone gland. The primary structure was deduced by composition/sequence analysis and mass spectrometric analysis of the fragment peptides obtained from enzymatic and chemical fragmentations. The amino acid sequence of B. mori CaM was identical with Drosophila CaM deduced from the CaM gene of D. melanogaster, suggesting that insects have well conserved the molecule of CaM.

Cuticular protein and transcription factor genes expressed during prepupal–pupal transition and by ecdysone pulse treatment in wing discs of Bombyx mori

We aimed to understand the underlying mechanism that regulates successively expressed cuticular protein (CP) genes around pupation in Bombyx mori. Quantitative PCR was conducted to clarify the expression profile of CP genes and ecdysone‐responsive transcription factor (ERTF) genes around pupation. Ecdysone pulse treatment was also conducted to compare the developmental profiles and the ecdysone induction of the CP and ERTF genes. Fifty‐two CP genes (RR‐1 13, RR‐2 18, CPG 8, CPT 3, CPFL 2, CPH 8) in wing discs of B. mori were examined. Different expression profiles were found, which suggests the existence of a mechanism that regulates CP genes. We divided the genes into five groups according to their peak stages of expression. RR‐2 genes were expressed until the day of pupation and RR‐1 genes were expressed before and after pupation and for longer than RR‐2 genes; this suggests different construction of exo‐ and endocuticular layers. CPG, CPT, CPFL and CPH genes were expressed before and after pupation, which implies their involvement in both cuticular layers. Expression profiles of ERTFs corresponded with previous reports. Ecdysone pulse treatment showed that the induction of CP and ERTF genes in vitro reflected developmental expression, from which we speculated that ERTFs regulate CP gene expression around pupation.

Ecdysone-responsive transcriptional regulation determines the temporal expression of cuticular protein genes in wing discs of Bombyx mori.

The present study was conducted to clarify the regulatory mechanism of cuticular protein genes of Bombyx mori expressed in wing discs in the prepupal stage. BHR3, BHR4, E74A, and ßFTZ-F1 were successively expressed in wing discs at the pre-pupal stage. BHR3 showed different ecdysone responsiveness from other ecdysone-responsive transcription factors (ERTFs) and was induced by ecdysone addition but showed decrease by ecdysone removal after treatment (ecdysone pulse). BHR4 and E74A were induced by the ecdysone addition and by the ecdysone pulse. ßFTZ-F1 was not induced by the ecdysone addition but was induced by the ecdysone pulse. Thus, ERTFs showed different hormone responsiveness, resulting in the different expression timing. We selected cuticular protein genes that showed the same expression stage with each transcription factor and examined their ecdysone responsiveness. The developmental expression and ecdysone responsiveness of BmorCPH5, BmorCPR34, BmorCPR23, and BmorCPR99 resembled those of BHR3, BHR4, E74A, and ßFTZ-F1, respectively. The results of the transient reporter assay strongly suggested the regulation of each cuticular protein promoter by ERTF. These ERTFs regulated different cuticular protein genes and determined their expression timing and probably the nature of the cuticle layer of insects.