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Bombyx mori densovirus type 2 (BmDNV-2), a parvo-like virus, replicates only in midgut columnar cells and causes fatal disease. The resistance expressed in some silkworm strains against the virus is determined by a single gene, nsd-2 , which is characterized as nonsusceptibility irrespective of the viral dose. However, the responsible gene has been unknown. We isolated the nsd-2 gene by positional cloning. The virus resistance is caused by a 6-kb deletion in the ORF of a gene encoding a 12-pass transmembrane protein, a member of an amino acid transporter family, and expressed only in midgut. Germ-line transformation with a wild-type transgene expressed in the midgut restores susceptibility, showing that the defective membrane protein is responsible for resistance. Cumulatively, our data show that the membrane protein is a functional receptor for BmDNV-2. This is a previously undescribed report of positional cloning of a mutant gene in Bombyx and isolation of an absolute virus resistance gene in insects.

Despite more than 40 years of intense study, essential features of the silkmoth chorion (eggshell) are still not fully understood. To determine the precise structure of the chorion locus, we performed extensive EST analysis, constructed a bacterial artificial chromosome (BAC) contig and obtained a continuous genomic sequence of 871,711 base pairs. We annotated 127 chorion genes in two segments interrupted by a 164 kb region with 5 non-chorion genes, orthologs of which were on chorion bearing scaffolds in 4 ditrysian families. Detailed transcriptome analysis revealed expression throughout choriogenesis of most chorion genes originally categorized as “middle” and evidence for diverse regulatory mechanisms including cis-elements, alternative splicing and promoter utilization and antisense RNA. Phylogenetic analysis revealed multigene family associations and faster evolution of early chorion genes and transcriptionally active pseudogenes. Proteomics analysis identified 99 chorion proteins in the eggshell and micropyle localization of 1 early and 6 Hc chorion proteins.

A bacterial artificial chromosome (BAC) contig was constructed by chromosome walking, starting from the Hox genes of the silkworm, Bombyx mori. Bombyx orthologues of the labial (lab) and zerknult (zen) genes were newly identified. The size of the BAC contig containing the Hox gene cluster-except the lab and Hox 2 genes-was estimated to be more than 2 Mb. The Bombyx Hox cluster was mapped to linkage group (LG) 6. The lab gene was mapped on the same LG, but far apart from the cluster. Fluorescence in situ hybridization analysis confirmed that the major Hox gene cluster and lab were at different locations on the same chromosome in B. mori.

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.

Tubulin is one of the most widespread classes of multiprotein families and is well known to construct microtubules with two different subunits, alpha- and beta-tubulin. In the course of genome analysis of Bombyx mori, we have constructed an EST database by large-scale sequencing of clones that were randomly selected from cDNA libraries of various tissues and organs belonging to different developmental stages. Using this EST database, we have identified four types of beta-tubulin gene and three types of alpha-tubulin gene. Based on the analysis of deduced amino acid sequences, we have determined the phylogenetic relationships of tubulins between Bombyx and Drosophila melanogaster as well as two other moth species, suggesting that each tubulin is classified into at least three distinct subfamilies: a ubiquitously expressed one, a developmentally regulated one and a testis specific one.

The new gene, DCRR1, from the proximal part of the Down's syndrome critical region (DCR) was identified by the GRAIL analysis of the 97-kb nucleotide sequence of two P1 DNAs and the cDNA for DCRR1 gene was cloned. A 7.36-kb cDNA encodes the imcompleted open reading frame composed of 1941 amino acid residues (220.2 kDa). The deduced amino acid sequence contains the conserved domain for protein phosphatases at the N-terminus. The domain encoding the rod-like tail of a myosin heavy chain was also found near the C-terminal region besides the signature for an actin binding protein, profilin, suggesting its possible role as a microtuble-associated protein. Two different sizes (7.9 and 9.0 kb) of mRNAs were detected in the poly(A)+ RNA from abundant tissues by the Northern analysis. The smaller transcript was only transcribed at a high level in the testis. The imbalance of the DCRR1 gene dosage may contibute to the pathogenesis of Down's syndrome.

We have developed a linkage map for the silkworm Bombyx mori based on single nucleotide polymorphisms (SNPs) between strains p50T and C108T initially found on regions corresponding to the end sequences of bacterial artificial chromosome (BAC) clones. Using 190 segregants from a backcross of a p50T female × an F1 (p50T × C108T) male, we analyzed segregation patterns of 534 SNPs between p50T and C108T, detected among 3840 PCR amplicons, each associated with a p50T BAC end sequence. This enabled us to construct a linkage map composed of 534 SNP markers spanning 1305 cM in total length distributed over the expected 28 linkage groups. Of the 534 BACs whose ends harbored the SNPs used to construct the linkage map, 89 were associated with 107 different ESTs. Since each of the SNP markers is directly linked to a specific genomic BAC clone and to whole-genome sequence data, and some of them are also linked to EST data, the SNP linkage map will be a powerful tool for investigating silkworm genome properties, mutation mapping, and map-based cloning of genes of industrial and agricultural interest.

The cDNA and genomic structure of a putative lipophorin receptor from the silkworm, Bombyx mori (BmLpR), indicated the presence of four isoforms, designated LpR1, LpR2, LpR3, and LpR4. The deduced amino acid sequence of each isoform showed five functional domains that are homologous to vertebrate very low density lipoprotein receptor (VLDLR). All four isoforms seem to have originated from a single gene by alternative splicing and were differentially expressed in a tissue- and stage-specific manner. BmLpR1 harbored an additional 27 amino acids in the O-linked sugar domain, resulting in an extra exon. The silkworm BmLpR gene consisted of 16 exons separated by 15 introns spanning >122 kb and was at least three times larger than the human VLDLR gene. Surprisingly, one of the isoforms, LpR4, was expressed specifically in the brain and central nervous system. Additionally, it had a unique cytoplasmic tail, leading to the proposition that it represents a new candidate LpR for possible brain-related function(s). This is the first report on the genomic characterization of an arthropod lipoprotein receptor gene and the identification of a brain-specific receptor variant from a core member of the low density lipoprotein receptor family in invertebrates.

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 ≈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.

This report presents the first hsp90 complete cDNA sequences from two Lepidoptera. The Bombyx mori full sequence was reconstituted from 15 partial cDNA clones belonging to expressed sequence tag libraries obtained from different tissues or cultured cells, thus showing the ubiquitous expression of the hsp90 gene. The Spodoptera frugiperda cDNA was isolated as a full-length clone from a cDNA library established from the Sf9 cell line. Both cDNAs are highly homologous and display the classical amino acid (aa) stretches representing the HSP90 signature. They potentially encode a 716 aa (B. mori) and a 717 aa (S. frugiperda) protein, with a calculated molecular mass of 83 kDa similar to the Drosophila homologous protein. We show that, unlike the vertebrates, hsp90 is a unique gene in both S. frupiperda and B. mori genomes. Sequencing of the corresponding genomic region shows that, contrary to the dipteran homologous gene, the lepidopteran hsp90 gene does not display any intron. Phylogenetic analysis based on the two lepidopteran and 23 other HSP90 aa sequences displays a high consistency with known phylogeny at both high and low taxonomic levels. Transcriptional analysis performed in S. frugiperda shows that the induction of the hsp90 gene only occurs 14 degrees C above physiological growth conditions (42 degrees C).

In order to analyze the mutation sites related to abnormal induction of sister chromatid exchanges (SCEs) in the RNA polymerase II largest subunit (RpII LS) gene of the Chinese hamster CHO-K1 cell mutant, we have completely sequenced the whole region of the RpII LS cDNAs obtained from normal and mutant cells. By comparing both sequences, a mutation that results in an amino acid (aa) change in the RpII LS gene was found. This aa change was Pro ( C CC) to Ser ( T CC) at position 1006. Multiple alignment for aa sequences of RpII LS from various species revealed that this Pro residue was highly conserved throughout the eukaryotes. Considering the differences in physico-chemical properties between Pro and Ser residues, the Pro→Ser substitution may alter the RpII LS structure.

An integrated map of the Bombyx mori genome has been constructed using 361.1 Mb of BAC contigs and singletons together with a genetic map containing 1689 independent genes and synteny among Apis, Tribolium, and Bombyx was examined., Background In 2004, draft sequences of the model lepidopteran Bombyx mori were reported using whole-genome shotgun sequencing. Because of relatively shallow genome coverage, the silkworm genome remains fragmented, hampering annotation and comparative genome studies. For a more complete genome analysis, we developed extended scaffolds combining physical maps with improved genetic maps. Results We mapped 1,755 single nucleotide polymorphism (SNP) markers from bacterial artificial chromosome (BAC) end sequences onto 28 linkage groups using a recombining male backcross population, yielding an average inter-SNP distance of 0.81 cM (about 270 kilobases). We constructed 6,221 contigs by fingerprinting clones from three BAC libraries digested with different restriction enzymes, and assigned a total of 724 single copy genes to them by BLAST (basic local alignment search tool) search of the BAC end sequences and high-density BAC filter hybridization using expressed sequence tags as probes. We assigned 964 additional expressed sequence tags to linkage groups by restriction fragment length polymorphism analysis of a nonrecombining female backcross population. Altogether, 361.1 megabases of BAC contigs and singletons were integrated with a map containing 1,688 independent genes. A test of synteny using Oxford grid analysis with more than 500 silkworm genes revealed six versus 20 silkworm linkage groups containing eight or more orthologs of Apis versus Tribolium, respectively. Conclusion The integrated map contains approximately 10% of predicted silkworm genes and has an estimated 76% genome coverage by BACs. This provides a new resource for improved assembly of whole-genome shotgun data, gene annotation and positional cloning, and will serve as a platform for comparative genomics and gene discovery in Lepidoptera and other insects.