Mapping and recombination analysis of two moth colour mutations, Black moth and Wild wing spot, in the silkworm Bombyx mori.

Many lepidopteran insects exhibit body colour variations, where the high phenotypic diversity observed in the wings and bodies of adults provides opportunities for studying adaptive morphological evolution. In the silkworm Bombyx mori, two genes responsible for moth colour mutation, Bm and Ws, have been mapped to 0.0 and 14.7 cM of the B. mori genetic linkage group 17; however, these genes have not been identified at the molecular level. We performed positional cloning of both genes to elucidate the molecular mechanisms that underlie the moth wing- and body-colour patterns in B. mori. We successfully narrowed down Bm and Ws to ~2-Mb-long and 100-kb-long regions on the same scaffold Bm_scaf33. Gene prediction analysis of this region identified 77 candidate genes in the Bm region, whereas there were no candidate genes in the Ws region. Fluorescence in-situ hybridisation analysis in Bm mutant detected chromosome inversion, which explains why there are no recombination in the corresponding region. The comparative genomic analysis demonstrated that the candidate regions of both genes shared synteny with a region associated with wing- and body-colour variations in other lepidopteran species including Biston betularia and Heliconius butterflies. These results suggest that the genes responsible for wing and body colour in B. mori may be associated with similar genes in other Lepidoptera.

Identification and functional analysis of a Masculinizer orthologue in Trilocha varians (Lepidoptera: Bombycidae).

We recently showed that the Masculinizer gene (Masc) plays a primary role in sex determination in the lepidopteran model insect Bombyx mori. However, it remains unknown whether this Masc protein-dependent sex determination system is conserved amongst lepidopteran insects or within the family Bombycidae. Here we cloned and characterized a Masc homologue (TvMasc) in Trilocha varians (Lepidoptera: Bombycidae), a species closely related to B. mori. To elucidate the role of TvMasc in the sex determination cascade of T. varians, TvMasc expression was knocked down in early embryos by the injection of small interfering RNAs (siRNAs) that targeted TvMasc mRNAs. Both female- and male-type splice variants of Tvdsx, a doublesex (dsx) homologue in T. varians were observed in control siRNA-injected embryos. By contrast, only female-type splice variants were observed in TvMasc siRNA-injected embryos. These results indicate that the TvMasc protein directly or indirectly regulates the splicing patterns of Tvdsx. Furthermore, we found that male-type splice variants of B. mori dsx (Bmdsx) were produced in TvMasc-overexpressing BmN4 cells. The mRNA level of B. mori Imp, a gene whose product induces male-specific Bmdsx splicing also increased. These results suggest that Masc genes play similar roles in the sex-determination cascade in Bombycidae.

A homolog of the human Hermansky-Pudluck syndrome-5 (HPS5) gene is responsible for the oa larval translucent mutants in the silkworm, Bombyx mori.

Normally, many granules containing uric acid accumulate in the larval integument of the silkworm, Bombyx mori. These uric acid granules cause the wild-type larval integument to be white or opaque, and the absence of these granules results in a translucent integument. Although about 30 B. mori loci governing larval translucency have been mapped, most have not been molecularly identified yet. Here, based on a structural analysis of a deletion of chromosome 14 that included the oa (aojyuku translucent) locus, we concluded that the BmHPS5 encoding a Bombyx homolog of the HPS5 subunit of biogenesis of lysosome-related organelles complex-2 is the candidate for the oa locus. Nucleotide sequence analyses of cDNAs and genomic DNAs in three mutant strains, each of which were homozygous for the respective allele of the oa locus (oa, oa ( 2 ), and oa ( v )), revealed that each mutant strain has a frame shift or a premature stop codon (caused by deletion or nonsense mutation, respectively) in the BmHPS5 gene. Our findings indicate that some genes that cause the translucent phenotype in Bombyx, some HPS-associated genes in humans, and some genes that cause mutant eye color phenotypes in Drosophila are homologous and participate in an evolutionarily conserved mechanism that leads to biogenesis of lysosome-related organelles.

Identification and characterization of the fusion transcript, composed of the apterous homolog and a putative protein phosphatase gene, generated by 1.5-Mb interstitial deletion in the vestigial (Vg) mutant of Bombyx mori.

The vestigial (Vg) mutant is a Z-linked mutant that causes vestigial wings in the silkworm, Bombyx mori. We have previously reported a 1.5-Mb interstitial deletion on the Z chromosome bearing the Vg mutation (Z(Vg) chromosome). In this study, we found that exons 3-8 of a gene named Bmptp-Z encoding a putative tyrosine-specific protein phosphatase are deleted by the 1.5-Mb interstitial deletion. We found that a gene encoding the Bombyx homolog of Drosophila Apterous (BmAp-A) protein is located 4.5 kb downstream of the distal breakpoint of the 1.5-Mb interstitial deletion. Moreover, an in-frame fusion transcript composed of the 5' part of Bmptp-Z and the 3' part of Bmap-A is generated specific to the Z(Vg) chromosome. Effects of the in-frame fusion transcript on the vestigial phenotype are discussed.

Interspecies linkage analysis of mo, a Bombyx mori locus associated with mosaicism and gynandromorphism.

The mo (hereditary mosaic) mutation is one of the most famous and interesting mutations of the silkworm, Bombyx mori. Females homozygous for mo generate mosaic and gynandromorphic offspring due to non-elimination of polar bodies and subsequent double fertilization events, irrespective of the genotype of the mated males. Although mo was first reported in 1927, the locus has not been mapped to linkage groups, as the mutation is unstable and appears to be sensitive to genetic background. In this study, linkage analysis of mo was performed using PCR-based markers on single nucleotide polymorphism linkage maps. Bombyx mandarina was used as the mating partner for the B. mori mo strain, as it is easier to identify polymorphic markers between B. mori and B. mandarina than within B. mori strains. Surprisingly, we identified two homozygous linkage groups (LGs) in all of the 12 B(1) (first backcross generation) moths that had deposited mosaic eggs. It was revealed that +( mo ) is located on the M chromosome of B. mandarina, which corresponds to two linkage groups of B. mori, LG 14 and 27. Based on further linkage analysis using B. mori as a mating partner, mo was mapped to LG 14. Additionally, we found that mo activity could be modified by a gene(s) on LG 17.

Transgenic analysis of the BmBLOS2 gene that governs the translucency of the larval integument of the silkworm, Bombyx mori.

The larval integument of the silkworm, Bombyx mori, is opaque because urate granules accumulate in the epidermis. Although the biosynthetic pathway of uric acid is well studied, little is known about how uric acid accumulates as urate granules in epidermal cells. In the distinct oily (od) mutant silkworm, the larval integument is translucent because of the inability to construct urate granules. Recently, we have found that the od mutant has a genomic deletion in the B. mori homologue of the human biogenesis of lysosome-related organelles complex1, subunit 2 (BLOS2) gene (BmBLOS2). Here, we performed a molecular and functional characterization of BmBLOS2. Northern blot analysis showed that BmBLOS2 was ubiquitously expressed in various tissues. We analysed the structure of a newly isolated mutant (od(B) ) allelic to od and found a premature stop codon in the coding sequence of BmBLOS2 in this new mutation. Moreover, the translucent phenotype was rescued by the germ-line transformation of the wild-type BmBLOS2 allele into the od mutant. Our results suggest that BmBLOS2 is responsible for the od mutant phenotype and plays a crucial role in biogenesis of urate granules in the larval epidermis of the silkworm. The relationships amongst Hermansky-Pudlak syndrome (HPS) genes in mammals, granule group genes in Drosophila and translucent mutant genes in B. mori are discussed.

Functional characterization of chitinase from Cydia pomonella granulovirus.

Baculovirus chitinases (V-CHIAs) play a crucial role in the terminal liquefaction of virus-infected larvae after death. Although v-chiAs from nucleopolyhedroviruses (NPVs) have been well characterized, little is known about v-chiAs from granuloviruses (GVs). We characterized the v-chiA of Cydia pomonella GV (CpGV) by constructing a recombinant Bombyx mori NPV (BmNPV) in which BmNPV v-chiA was replaced by CpGV v-chiA (103CpGV virus). CpGV v-chiA encoded an approximately 70-kDa chitinase with an exo-type substrate preference. CpGV V-CHIA lacked a C-terminal KDEL endoplasmic reticulum retention motif and was suggested to be a secretory protein. Terminal host liquefaction of B. mori larvae and proper folding of BmNPV-encoded cysteine protease (BmNPV V-CATH) were observed following infection with 103CpGV, indicating that CpGV v-chiA is able to compensate for the absence of its BmNPV counterpart. Our data suggest that the molecular interaction between V-CHIA and V-CATH may be conserved across a broad range of lepidopteran GVs and NPVs.

Reduced cysteine protease activity of the hemolymph of Bombyx mori larvae infected with fp25K-inactivated bombyx mori nucleopolyhedrovirus results in the reduced postmortem host degradation.

We previously reported that the FP25K gene (fp25K) product of the baculovirus Bombyx mori nucleopolyhedrovirus (BmNPV) is involved in postmortem host degradation. Here, we investigated the mechanism by which reduced postmortem host degradation is caused after the infection of fp25K-mutated BmNPVs. Firstly, we investigated gene expression levels of vcath and chiA both of which products are involved in postmortem host degradation. We found that transcriptional levels of these genes infp25K-mutated BmNPV-infected BmN cells were comparable to those in cells infected with wild-type (wt) BmNPV. Next, we examined the cysteine protease activity in fp25K-mutated BmNPV-infected BmN cells. Although the cysteine protease activity in BmN cells infected with fp25K-mutated BmNPVs was comparable to that of wt BmNPV-infected cells, the released activity in the culture medium is dramatically reduced in that of cells infected with fp25K mutants. We also found that the cysteine protease activity in the hemolymph of fp25K-mutated BmNPV-infected B. mori larvae is drastically reduced compared to that of wt BmNPV-infected larvae. These results show that the release of cysteine protease into the hemolymph of B. mori larvae infected with fp25K-mutated BmNPVs is reduced and, as a consequence, postmortem host degradation of infected insects is lessened.

Molecular monitoring of bleomycin-induced pulmonary fibrosis by cDNA microarray-based gene expression profiling.

Pulmonary fibrosis is a progressive disorder whose molecular pathology is poorly understood. Here we developed an in-house cDNA microarray ("lung chip") originating from a lung-normalized cDNA library. By using this lung chip, we analyzed global gene expression in a murine model of bleomycin-induced fibrosis and selected 82 genes that differed by more than twofold intensity in at least one pairwise comparison with controls. Cluster analysis of these selected genes showed that the expression of genes associated with inflammation reached maximum levels at 5 days after bleomycin administration, while genes involved in the development of fibrosis increased gradually up to 14 days after bleomycin treatment. These changes in gene expression signature were well correlated with observed histopathological changes. The results show that microarray analysis of animal disease models is a powerful approach to understanding the gene expression programs that underlie these disorders.

Genome medicine promised by microarray technology.

The human genome project has now been completed, which markedly changes the way to analyze gene functions. Recently developed DNA microarray technologies enable us to explore genome-wide gene expression in the diseased tissues. In this review, we introduce the principles and applications of microarray technologies (such as DNA, tissue and cell microarrays) to molecular diagnostics, drug target discovery and validation of drug effects.

Genomic analysis of a mouse model of immunoglobulin A nephropathy reveals an enhanced PDGF-EDG5 cascade.

The molecular mechanism of immunoglobulin A nephropathy (IgAN), the most common primary renal glomerular disease worldwide, is unknown. HIGA (high serum IgA) mouse is a valid model of IgAN showing almost all of the pathological features, including mesangial cell proliferation. Here we elucidate a pattern of gene expression associated with IgAN by analyzing the diseased kidneys on cDNA microarrays. In particular, we showed an enhanced expression of several genes regulating the cell cycle and proliferation, including growth factors and their receptors, as well as endothelial differentiation gene-5 (EDG5), a receptor for sphingosine 1-phosphate (SPP). One of the growth factors, platelet-derived growth factor (PDGF) induces a marked upregulation of EDG5 in proliferative mesangial cells, and promotes cell proliferation synergistically with SPP. The genomic approach allows us to identify families of genes involved in a process, and can indicate that enhanced PDGF-EDG5 signaling plays an important role in the progression of IgAN.

Functional genomic research of alpha 1-adrenoceptors.

The Human Genome Project is now almost completed, and we are about to move into the post-genome sequence era of functional genomics. The advent of genome science has markedly changed the way life science research including pharmacological study is conducted; thus, systematic and integrated 'genome-wide' survey is feasible. The stream of 'Genome-->Transcriptome--> Proteomics' is logical and, in each aspect, approaches for functional genomics are now pursued at a high pace. We have recently developed a standardized technical platform (in various levels, such as transcription, cell and whole animal levels, etc.), and applied these techniques to the study of functional genomics of G-protein-coupled receptors, particularly alpha1-adrenoceptors as a model. Combining the genome information and technology, future pharmacological studies would become the genome-based search and research.

Identification of novel residues involved in nuclear localization of a baculovirus polyhedrin protein.

A baculovirus polyhedrin protein has proven to possess a nuclear localization signal (NLS) sequence and a domain required for supramolecular assembly. Here we investigated five Bombyx mori nucleopolyhedrovirus (BmNPV) mutants that did not produce polyhedra. Two of five mutants were generated during routine baculoviral expression vector screening, and three were isolated by treatment with the mutagen 5-bromo-2'-deoxyuridine (BrdU). Marker rescue mapping and nucleotide sequence analysis showed that mutations in the polyhedrin gene caused the altered phenotype of these mutants. Biochemical fractionation indicated that cells infected with these mutants exhibited polyhedrin protein in both the nucleus and the cytoplasm. Electron microscopic observation revealed that polyhedrin produced by these mutants ocurred in both the nucleus and the cytoplasm, but did not form a crystalline lattice. Despite the incompleteness of polyhedrin nuclear localization, the NLSs of the five mutants were unchanged, although some of the mutations occurred within residues just outside of the domain reported to be required for polyhedron assembly (4). This result suggests that (a) the polyhedrin NLS directs polyhedrin to the nucleus, but the efficiency of this localization is regulated by regions other than the NLS (probably, polyhedrin conformation and its association with the nucleus are also involved), and (b) formation of a crystalline lattice may also be determined by several domains within polyhedrin.

Molecular characterization of baculovirus Bombyx mori nucleopolyhedrovirus polyhedron mutants.

Four newly isolated and two previously isolated polyhedron mutants of Bombyx mori nucleopolyhedrovirus (BmNPV) were studied. Two polyhedron deficient mutants, #126 and #136, produced small uncrystallized particles of polyhedrin in the nuclei and cytoplasm of infected cells. Mutant #211 produced a large number of variably sized polyhedra in the nucleus and #220 produced a few large cuboidal polyhedra in the nucleus. Mutant #24 and #128 were previously isolated BmNPV mutants. Mutant #24 could not produce polyhedrin mRNA and polyhedra produced by mutant #128 lacked oral infectivity. Nucleotide sequence analysis indicated that five mutants (#126, #136, #211, #220 and #128) had amino acid substitutions in polyhedrin and mutant #24 had a point mutation only in the promoter region of the polyhedrin gene. Cotransfection experiments showed that the altered phenotypes were due to the mutations found in the polyhedrin gene regions. In mutants #126 and #136, amino acid sequences of the nuclear localization signal of polyhedrin were identical to those of wild-type BmNPV, suggesting that this sequence was necessary but not sufficient for nuclear localization of polyhedrin. Electron microscopic observation revealed that fewer occluded virions were contained in polyhedra of #128 and #220.

Characterization of the 25K FP gene of the baculovirus Bombyx mori nucleopolyhedrovirus: implications for post-mortem host degradation.

Mutagenesis experiments on the baculovirus Bombyx mori nucleopolyhedrovirus (BmNPV) using 5-bromo-2'-deoxyuridine generated five mutants with a 'few polyhedra' (FP) phenotype. Sequence analysis of the 25K gene homologue of the BmNPV FP mutants revealed nucleotide substitutions in the coding region. Rescue experiments indicated that the FP phenotype of the BmNPV mutants resulted from mutations in the 25K coding region. Effects of infection by these FP mutants were analysed following injection of the viruses into silkworm (B. mori) larvae. Compared to infection with wild-type virus, infection with each FP mutant resulted in reduced host degradation (liquefaction). The degree to which liquefaction was blocked corresponded to the degree of functional disruption of the 25K gene product and to the extent to which polyhedron production was reduced. Electron microscopy revealed that (1) polyhedron production was reduced, (2) very few virions were occluded and those that were lacked envelopes, and (3) the basal lamina of fat-body tissue was not destroyed by infection and accumulations of virions occurred along the membrane. Typical NPV-induced liquefaction was observed following infection with a polyhedrin deletion mutant, indicating that host degradation was not related to polyhedron production. These results suggest that (1) the 25K gene product is involved in the host degradation process caused by virus infection and (2) the FP phenotype is an indirect result of disruption of the 25K gene; activation or suppression of a specific host or viral gene related to tissue degradation and polyhedron formation may be involved.