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Toru Shimada

Publications and source records attributed to Toru Shimada.

33 records · Page 2Linked to original sources
PubMed2005

The genetics and genomics of the silkworm, Bombyx mori.

We review progress in applying molecular genetic and genomic technologies to studies in the domesticated silkworm, Bombyx mori, highlighting its use as a model for Lepidoptera, and in sericulture and biotechnology. Dense molecular linkage maps are being integrated with classical linkage maps for positional cloning and marker-assisted selection. Classical mutations have been identified by a candidate gene approach. Cytogenetic and sequence analyses show that the W chromosome is composed largely of nested full-length long terminal repeat retrotransposons. Z-chromosome-linked sequences show a lack of dosage compensation. The downstream sex differentiation mechanism has been studied via the silkworm homolog of doublesex. Expressed sequence tagged databases have been used to discover Lepidoptera-specific genes, provide evidence for horizontal gene transfer, and construct microarrays. Physical maps using large-fragment bacterial artificial chromosome libraries have been constructed, and whole-genome shotgun sequencing is underway. Germline transformation and transient expression systems are well established and available for functional studies, high-level protein expression, and gene silencing via RNA interference.

Animals↗
PubMed2004

Change in the expressed gene patterns of the wing disc during the metamorphosis of Bombyx mori.

The present study was conducted to clarify what occurs during the metamorphosis of the imaginal disc in insects. To understand the metamorphosis on a molecular level, the changes in expression profiles in the imaginal disc during metamorphosis were investigated. For this purpose, we constructed cDNA libraries from four different stages of wing discs of Bombyx mori, sequenced about 1000 cDNAs randomly collected from each library, and constructed a database of expressed sequence tags (EST). The morphological changes and expression profiles from EST were compared during those four stages. Microarray analysis was applied to quantify the expression of each gene in each stage in order to confirm whether the expression of the genes identified from EST was induced by 20-hydroxyecdysone (20E) in a stage-specific manner. Wing discs showed dynamic morphogenesis in 4-5 days during the preparatory stage of metamorphosis, which was under the control of an ecdysteroid. Different expressed profiles were observed in each of the four different stages by comparison of each EST clone. These profiles reflected the morphological changes of the Bombyx wing disc during metamorphosis. The results of expression profiles from the four stages suggested that the V4 stage was cell proliferating; W0, proliferating and the beginning of differentiation; W2, morphologically changing; W3, cuticle secreting. Microarray analysis showed the effectiveness of its application on 20E induction of genes in wing discs. The wing disc of B. mori is an exceptionally suitable system for understanding the relationship between morphological changes and the distribution of mRNA.

Animals↗
PubMed2004

Expression profiling of baculovirus genes in permissive and nonpermissive cell lines.

The baculoviruses Bombyx mori nucleopolyhedrovirus (BmNPV) and Autographa californica multicapsid NPV (AcMNPV) are highly homologous at the genomic level, but they have essentially nonoverlapping host ranges. In order to characterize baculovirus replication in permissive and nonpermissive cell lines, the expression profiles of baculovirus-specific genes (at 2, 6, 12, 24, 36, 48 or 72 h post-infection) were examined in BmN (BmNPV-permissive) or Sf-9 (AcMNPV-permissive) cells that were inoculated with BmNPV or AcMNPV. Surprisingly, nearly all of the 154 genes of AcMNPV appeared to be expressed in both Sf-9 and BmN cells although the peak expression levels of these genes were delayed by roughly 12 h in the nonpermissive BmN cells. In addition, the expression levels of the very late AcMNPV polyhedrin and p10 genes were dramatically reduced in BmN cells, which presumably led to the inability of AcMNPV to form polyhedral inclusion bodies in BmN cells. Nearly all of the 136 genes of BmNPV appeared to be expressed in BmN cells, however, BmNPV gene expression was dramatically reduced in Sf-9 cells inoculated with BmNPV. Experiments in which BmNPV DNAs were transfected to Sf-9 cells suggested that this dramatic reduction in gene expression was not the result of poor attachment, penetration or uncoating of the BmNPV virion into Sf-9 cells. In conclusion, we established a system to monitor global gene expression patterns during baculovirus infection in permissive and nonpermissive cell lines. This system was used to identify global trends in the transcription of baculovirus genes during productive and nonproductive infection.

Animals↗
PubMed2004

The genome sequence of silkworm, Bombyx mori.

We performed threefold shotgun sequencing of the silkworm (Bombyx mori) genome to obtain a draft sequence and establish a basic resource for comprehensive genome analysis. By using the newly developed RAMEN assembler, the sequence data derived from whole-genome shotgun (WGS) sequencing were assembled into 49,345 scaffolds that span a total length of 514 Mb including gaps and 387 Mb without gaps. Because the genome size of the silkworm is estimated to be 530 Mb, almost 97% of the genome has been organized in scaffolds, of which 75% has been sequenced. By carrying out a BLAST search for 50 characteristic Bombyx genes and 11,202 non-redundant expressed sequence tags (ESTs) in a Bombyx EST database against the WGS sequence data, we evaluated the validity of the sequence for elucidating the majority of silkworm genes. Analysis of the WGS data revealed that the silkworm genome contains many repetitive sequences with an average length of <500 bp. These repetitive sequences appear to have been derived from truncated transposons, which are interspersed at 2.5- to 3-kb intervals throughout the genome. This pattern suggests that silkworm may have an active mechanism that promotes removal of transposons from the genome. We also found evidence for insertions of mitochondrial DNA fragments at 9 sites. A search for Bombyx orthologs to Drosophila genes controlling sex determination in the WGS data revealed 11 Bombyx genes and suggested that the sex-determining systems differ profoundly between the two species.

Animals↗
PubMed2004

Microarray analysis of gene expression profiles in wing discs of Bombyx mori during pupal ecdysis.

Wing discs of holometabolous insects undergo dramatic morphological changes during metamorphosis, a process that is controlled by the actions of hundreds of gene products. Using cDNA microarrays constructed from 5086 ESTs, we monitored the gene expression profiles in wing discs of Bombyx mori at 13 time points during pupal ecdysis (day-4 fifth instar larvae to day-0 pupae). Of the 5086 ESTs on the microarrays, 2998 ESTs had significant signals in more than half of the experiments. Of the 2998 ESTs, genes represented by 683 ESTs showed significant perturbations during pupal ecdysis. Genes previously known to be induced during metamorphosis were identified, including E75, Urbain, Chitinases, and cuticle proteins. The expressions of genes represented by 59 ESTs induced at the beginning of wandering contained genes predicted to be involved in protein degradation, amino acid metabolism, and amino acid transport. The expressions of genes represented by 147 ESTs induced after the ecdysteroid peak had a role in cuticle synthesis, pigmentation, ion transport, protein transport, and transcription regulation. The expressions of genes represented by 85 ESTs repressed after the ecdysteroid peak were predicted to be involved in nucleotide and nucleic acid metabolism and cell cycle. This indicates the involvement of several biological processes in wing disc development during metamorphosis.

Animals↗
PubMed2004

Characterization of the baculovirus Bombyx mori nucleopolyhedrovirus gene homologous to the mammalian FGF gene family.

We characterized a gene of the baculovirus Bombyx mori nucleopolyhedrovirus (BmNPV) homologous to the mammalian fibroblast growth factor (FGF) family. We termed it vfgf, and examined its transcription and the properties of the gene product (vFGF). RT-PCR analysis showed that vfgf is one of the baculovirus early genes, although there are no consensus sequences of the baculovirus early gene promoters. 5'-RACE analysis revealed that its transcription started at 10 nucleotides upstream of the translation start codon. vFGF has a hydrophobic amino terminus (approximately 16 amino acids), which is a typical signal sequence. As expected, vFGF was efficiently secreted from BmNPV-infected BmN cells. Because possible glycosylation sites are found at positions 44 (Asn) and 171 (Asn), we examined whether BmNPV vFGF is glycosylated or not. Cleavage of recombinant vFGF with PNGase F revealed that BmNPV vFGF was glycosylated. We also found that secretion of vFGF is completely blocked by the treatment of Tunicamycin, which blocks N-linked glycosylation. This is the first report to characterize a virus-encoded FGF.

Amino Acid Sequence↗
PubMed2003

The construction of an EST database for Bombyx mori and its application.

To build a foundation for the complete genome analysis of Bombyx mori, we have constructed an EST database. Because gene expression patterns deeply depend on tissues as well as developmental stages, we analyzed many cDNA libraries prepared from various tissues and different developmental stages to cover the entire set of Bombyx genes. So far, the Bombyx EST database contains 35,000 ESTs from 36 cDNA libraries, which are grouped into approximately 11,000 nonredundant ESTs with the average length of 1.25 kb. The comparison with FlyBase suggests that the present EST database, SilkBase, covers >55% of all genes of Bombyx. The fraction of library-specific ESTs in each cDNA library indicates that we have not yet reached saturation, showing the validity of our strategy for constructing an EST database to cover all genes. To tackle the coming saturation problem, we have checked two methods, subtraction and normalization, to increase coverage and decrease the number of housekeeping genes, resulting in a 5-11% increase of library-specific ESTs. The identification of a number of genes and comprehensive cloning of gene families have already emerged from the SilkBase search. Direct links of SilkBase with FlyBase and WormBase provide ready identification of candidate Lepidoptera-specific genes.

Amino Acid Sequence↗
PubMed2003

W-derived BAC probes as a new tool for identification of the W chromosome and its aberrations in Bombyx mori.

We isolated four W chromosome-derived bacterial artificial chromosome (W-BAC) clones from Bombyx mori BAC libraries by the polymerase chain reaction and used them as probes for fluorescence in situ hybridization (FISH) on chromosome preparations from B. mori females. All four W-BAC probes surprisingly highlighted the whole wild-type W sex chromosome and also identified the entire original W-chromosomal region in W chromosome-autosome translocation mutants. This is the first successful identification of a single chromosome by means of BAC-FISH in species with holokinetic chromosomes. Genomic in situ hybridization (GISH) by using female-derived genomic probes highlighted the W chromosome in a similar chromosome-painting manner. Besides the W, hybridization signals of W-BAC probes also occurred in telomeric and/or subtelomeric regions of the autosomes. These signals coincided well with those of female genomic probes except one additional GISH signal that was observed in a large heterochromatin block of one autosome pair. Our results support the opinion that the B. mori W chromosome accumulated transposable elements and other repetitive sequences that also occur, but scattered, elsewhere in the respective genome.

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PubMed2003

Analysis of the biological functions of a doublesex homologue in Bombyx mori.

We have previously reported that Bmdsx, a homologue of the sex-determining gene doublesex ( dsx), was sex-specifically expressed in various tissues of the silkworm. The primary transcript of Bmdsx is alternatively spliced in males and females to yield sex-specific mRNAs that encode male-specific (BmDSXM) and female-specific (BmDSXF) polypeptides. In the studies reported here, we expressed BmDSXF in males from a ubiquitous promoter and examined its regulatory activities. We show that BmDSXF functions as a positive regulator of the hexameric storage protein termed SP1 and vitellogenin genes that are predominantly expressed in females. We also show that expression of Bmdsx(F) in males results in the repression of the pheromone-binding protein gene that is preferentially expressed in males. Gel-mobility shift assays demonstrated that BmDSX proteins bind to the sequence (ACATTGT) between -95 and -89 nt relative to the transcriptional initiation site of the vitellogenin gene. These results strongly suggest that Bmdsx is a final regulatory gene in the hierarchy of regulatory genes controlling the expression of female-specific protein in Bombyx mori.

Animals↗
PubMed2003

Isolation and comparison of different ecdysone-responsive cuticle protein genes in wing discs of Bombyx mori.

Microarray analysis was used to isolate an ecdysone up-regulated cuticle protein gene from wing discs of Bombyx mori. Transcripts of isolated cDNAs were identified by Northern blot analysis. Expression of the BMWCP10 gene was observed during the W0-W3 stages with the strongest signal being at the W2 stage. In contrast, expression of the BMWCP2 gene was observed at the W3and P0 stages. Expression of BMWCP10 was identified after exposure to 20E in vitro, while that of BMWCP2 was identified after exposure to 20E followed by its removal. Induction of BMWCP10 by 20E was observed in 30 min and was not inhibited by cycloheximide. Expression of BMWCP2 was observed in wing discs cultured for more than 18 h in a hormone-free medium after 20E removal. At least 4 h exposure to 20E was required before removal for induction of BMWCP2. Induction of BMWCP2 required protein synthesis. Thus, different ecdysone-responsive cuticle protein genes in wing discs of Bombyx mori were isolated.

Amino Acid Sequence↗
PubMed2003

A Bombyx mori gene, BmChi-h, encodes a protein homologous to bacterial and baculovirus chitinases.

We have cloned and characterized a novel chitinase gene (BmChi-h) from the silkworm, Bombyx mori. BmChi-h cDNA has an open reading frame of 1,665 nucleotides, encoding a protein of 555 amino acid residues. The predicted protein shared extensive similarities with bacterial and baculovirus chitinases in both amino acid sequences (73% identity with Serratia marcescens chiA and 63% with Autographa californica nucleopolyhedrovirus chiA) and domain architectures. BmChi-h was a single-copy gene and located on chromosome 7. The expression of BmChi-h mRNA was observed in a stage- and tissue-specific manner that was almost identical to that of another chitinase gene previously cloned from B. mori. We further determined the overall genomic organization of BmChi-h. There was no intron in the ORF of BmChi-h. However, BmChi-h was transcribed from three promoters, which generated three isoforms in the 5'-UTR of the transcript. Phylogenetic analysis suggested that ancestral species of B. mori acquired the chitinase gene from a bacterium or an ancestral baculovirus via horizontal gene transfer.

Amino Acid Sequence↗
PubMed2003

Cloning of Cyc (Bmal1) homolog in Bombyx mori: structural analysis and tissue specific distributions.

Cycle (Bmal1) is one of the circadian clock genes and the key regulator of the circadian system in many organisms, encoding a bHLH-PAS transcription factor. In the present study, we cloned cycle homolog (BmCyc) in Bombyx mori. We performed polymerase chain reaction with degenerated primers deduced from the conserved amino acid sequences of mammalian BMAL1 and Drosophila CYCLE. Then the partial clone obtained was used as a probe for screening a cDNA library constructed from pupal brains of B. mori. BmCyc is 5703 nucleotides long and encodes 700 amino acid residues. The BmCYC has bHLH, PAS A and PAS B domains, and the sequence identities for these domains are 85, 60 and 50%, to Drosophila CYCLE (dCYC) and 69, 58 and 50%, to human BMAL1 (hBMAL1), respectively. The deduced amino acid sequence of BmCYC is 37% identical to that of dCYC and 28% to hBMAL1. Northern blot analysis demonstrates that BmCyc gene was expressed in all the tissues tested, which were head, fat body, silk glands, and midgut. Also no significant day, night time-specific difference in expression of BmCyc gene in the head was detected.

Amino Acid Sequence↗
PubMed2003

Detachment analysis of the translocated W chromosome shows that the female-specific randomly amplified polymorphic DNA (RAPD) marker, female-218, is derived from the second chromosome fragment region of the translocated W chromosome of the sex-limited p(B) silkworm (Bombyx mori ) strain.

The sex chromosomes of the silkworm, Bombyx mori, are designated ZW for the female and ZZ for the male. We previously characterized a female-specific randomly amplified polymorphic DNA (RAPD) marker, designated Female-218, from the translocation-bearing W chromosomes. These W chromosomes contain a region of the second chromosome, which carries visible larval markers of the p loci. We used strain TWPB in which female larvae have black skin due to the p(B) gene (T(W;2)p(B), +p/+p) while male larvae have whitish skin (+p/+p). To determine whether the Female-218 RAPD marker is derived from the "W region" or a "second chromosome fragment", we induced a detachment of the translocated W chromosome, T(W;2)p(B), by treating the eggs with hot water at an early developmental stage. After hot water treatment, we obtained 27 white female larvae out of 4850 female larvae. The Female-218 RAPD marker was not amplified in 26 out of 27 white female larvae, and was amplified from one white female larva. Moreover, we obtained 11 black male larvae out of 5377 male larvae. Eight out of 11 black male larvae became adult moths, and the Female-218 RAPD marker was amplified from all eight male moths. Examination of the genetic relationship between the Female-218 RAPD marker and the second chromosome fragment of the translocated W chromosome strongly indicates that the Female-218 RAPD marker is amplified from the region of second chromosome fragment of the T(W;2)p(B) chromosome.

Animals↗
PubMedDate not supplied

Role of the male BmDSX protein in the sexual differentiation of Bombyx mori.

The sex determination pathway is different between Drosophila melanogaster and Bombyx mori in the initial signal. Here we show evidence that the sex determination pathway in B. mori is similar to that of D. melanogaster at the level of the terminal regulator, doublesex (dsx), which is essential for the proper differentiation of the sexually dimorphic somatic features of D. melanogaster. In B. mori, a homolog of dsx (Bmdsx) is expressed in various tissues, and its primary transcript is alternatively spliced in males and females to yield sex-specific mRNAs that encode male-specific (BmDSXM) and female-specific (BmDSXF) polypeptides. In the studies reported here, transgenic silkworms carrying a construct with a Bmdsx male cDNA placed under the control of either an hsp70 promoter or a Bombyx actin3 promoter were generated by piggyBac-mediated germline transformation. Ectopic expression of the male cDNA in females resulted in abnormal differentiation of certain female-specific genital organs and caused partial male differentiation in female genitalia. Transgenic analysis also revealed that the expression of BmDSXM in females caused repression of the female-specifically expressed gene, the vitellogenin gene, and also resulted in activation of the pheromone-binding protein gene that is dominantly expressed in males. These results provide evidence that the role of BmDSXM includes the activation of some aspects of male differentiation as well as the repression of female differentiation. Taken together with our previous data on the function of BmDSXF, we can conclude that Bmdsx is a double-switch gene at the final step in the sex-determination cascade of B. mori.

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