Fine mapping of Green a, Ga, on chromosome 27 in Bombyx mori.

Some strains of silkworms produce green cocoons of varying intensities. This results from quantitative and qualitative differences in flavonoid pigments, which are influenced by the environment and genetic background. We discovered that the appearance of a faint green cocoon is regulated by a gene (G27) located on chromosome 27. Through mating experiments, we found that G27 is identical to an essential flavonoid cocoon gene, Ga. This locus has not been previously described. Furthermore, we narrowed down the Ga region to 438 kbp using molecular markers. Within this region, several predicted genes for sugar transporters form a cluster structure, suggesting that Ga is among them.

Large scale full-length cDNA sequencing reveals a unique genomic landscape in a lepidopteran model insect, Bombyx mori.

The establishment of a complete genomic sequence of silkworm, the model species of Lepidoptera, laid a foundation for its functional genomics. A more complete annotation of the genome will benefit functional and comparative studies and accelerate extensive industrial applications for this insect. To realize these goals, we embarked upon a large-scale full-length cDNA collection from 21 full-length cDNA libraries derived from 14 tissues of the domesticated silkworm and performed full sequencing by primer walking for 11,104 full-length cDNAs. The large average intron size was 1904 bp, resulting from a high accumulation of transposons. Using gene models predicted by GLEAN and published mRNAs, we identified 16,823 gene loci on the silkworm genome assembly. Orthology analysis of 153 species, including 11 insects, revealed that among three Lepidoptera including Monarch and Heliconius butterflies, the 403 largest silkworm-specific genes were composed mainly of protective immunity, hormone-related, and characteristic structural proteins. Analysis of testis-/ovary-specific genes revealed distinctive features of sexual dimorphism, including depletion of ovary-specific genes on the Z chromosome in contrast to an enrichment of testis-specific genes. More than 40% of genes expressed in specific tissues mapped in tissue-specific chromosomal clusters. The newly obtained FL-cDNA sequences enabled us to annotate the genome of this lepidopteran model insect more accurately, enhancing genomic and functional studies of Lepidoptera and comparative analyses with other insect orders, and yielding new insights into the evolution and organization of lepidopteran-specific genes.

KONAGAbase: a genomic and transcriptomic database for the diamondback moth, Plutella xylostella.

BACKGROUND: The diamondback moth (DBM), Plutella xylostella, is one of the most harmful insect pests for crucifer crops worldwide. DBM has rapidly evolved high resistance to most conventional insecticides such as pyrethroids, organophosphates, fipronil, spinosad, Bacillus thuringiensis, and diamides. Therefore, it is important to develop genomic and transcriptomic DBM resources for analysis of genes related to insecticide resistance, both to clarify the mechanism of resistance of DBM and to facilitate the development of insecticides with a novel mode of action for more effective and environmentally less harmful insecticide rotation. To contribute to this goal, we developed KONAGAbase, a genomic and transcriptomic database for DBM (KONAGA is the Japanese word for DBM). DESCRIPTION: KONAGAbase provides (1) transcriptomic sequences of 37,340 ESTs/mRNAs and 147,370 RNA-seq contigs which were clustered and assembled into 84,570 unigenes (30,695 contigs, 50,548 pseudo singletons, and 3,327 singletons); and (2) genomic sequences of 88,530 WGS contigs with 246,244 degenerate contigs and 106,455 singletons from which 6,310 de novo identified repeat sequences and 34,890 predicted gene-coding sequences were extracted. The unigenes and predicted gene-coding sequences were clustered and 32,800 representative sequences were extracted as a comprehensive putative gene set. These sequences were annotated with BLAST descriptions, Gene Ontology (GO) terms, and Pfam descriptions, respectively. KONAGAbase contains rich graphical user interface (GUI)-based web interfaces for easy and efficient searching, browsing, and downloading sequences and annotation data. Five useful search interfaces consisting of BLAST search, keyword search, BLAST result-based search, GO tree-based search, and genome browser are provided. KONAGAbase is publicly available from our website (http://dbm.dna.affrc.go.jp/px/) through standard web browsers. CONCLUSIONS: KONAGAbase provides DBM comprehensive transcriptomic and draft genomic sequences with useful annotation information with easy-to-use web interfaces, which helps researchers to efficiently search for target sequences such as insect resistance-related genes. KONAGAbase will be continuously updated and additional genomic/transcriptomic resources and analysis tools will be provided for further efficient analysis of the mechanism of insecticide resistance and the development of effective insecticides with a novel mode of action for DBM.

CD36 homolog divergence is responsible for the selectivity of carotenoid species migration to the silk gland of the silkworm Bombyx mori.

Dietary carotenoids are absorbed in the intestine and delivered to various tissues by circulating lipoproteins; however, the mechanism underlying selective delivery of different carotenoid species to individual tissues remains elusive. The products of the Yellow cocoon (C) gene and the Flesh (F) gene of the silkworm Bombyx mori determine the selectivity for transport of lutein and ß-carotene, respectively, to the silk gland. We previously showed that the C gene encodes Cameo2, a CD36 family member, which is thought to function as a transmembrane lipoprotein receptor. Here, we elucidated the molecular identity of the F gene product by positional cloning, as SCRB15, a paralog of Cameo2 with 26% amino acid identity. In the F mutant, SCRB15 mRNA structure was severely disrupted, due to a 1.4 kb genomic insertion in a coding exon. Transgenic expression of SCRB15 in the middle silk gland using the binary GAL4-UAS expression system enhanced selective ß-carotene uptake by the middle silk gland, while transgenic expression of Cameo2 enhanced selective lutein uptake under the same GAL4 driver. Our findings indicate that divergence of genes in the CD36 family determines the selectivity of carotenoid species uptake by silk gland tissue and that CD36-homologous proteins can discriminate among carotenoid species.

Single amino acid mutation in an ATP-binding cassette transporter gene causes resistance to Bt toxin Cry1Ab in the silkworm, Bombyx mori.

Bt toxins derived from the arthropod bacterial pathogen Bacillus thuringiensis are widely used for insect control as insecticides or in transgenic crops. Bt resistance has been found in field populations of several lepidopteran pests and in laboratory strains selected with Bt toxin. Widespread planting of crops expressing Bt toxins has raised concerns about the potential increase of resistance mutations in targeted insects. By using Bombyx mori as a model, we identified a candidate gene for a recessive form of resistance to Cry1Ab toxin on chromosome 15 by positional cloning. BGIBMGA007792-93, which encodes an ATP-binding cassette transporter similar to human multidrug resistance protein 4 and orthologous to genes associated with recessive resistance to Cry1Ac in Heliothis virescens and two other lepidopteran species, was expressed in the midgut. Sequences of 10 susceptible and seven resistant silkworm strains revealed a common tyrosine insertion in an outer loop of the predicted transmembrane structure of resistant alleles. We confirmed the role of this ATP-binding cassette transporter gene in Bt resistance by converting a resistant silkworm strain into a susceptible one by using germline transformation. This study represents a direct demonstration of Bt resistance gene function in insects with the use of transgenesis.

Identification of the Bombyx red egg gene reveals involvement of a novel transporter family gene in late steps of the insect ommochrome biosynthesis pathway.

Ommochromes are one of the major pigments involved in coloration of eggs, eyes, and body surface of insects. However, the molecular mechanisms of the final steps of ommochrome pigment synthesis have been largely unknown. The eggs of the silkworm Bombyx mori contain a mixture of ommochrome pigments, and exhibit a brownish lilac color. The recessive homozygous of egg and eye color mutant, red egg (re), whose eggs display a pale orange color instead of normal dark coloration, has been long suggested to have a defect in the biosynthesis of the final ommochrome pigments. Here, we identify the gene responsible for the re locus by positional cloning, mutant analysis, and RNAi experiments. In the re mutants, we found that a 541-bp transposable element is inserted into the ORF of BGIBMGA003497-1 (Bm-re) encoding a novel member of a major facilitator superfamily transporter, causing disruption of the splicing of exon 9, resulting in two aberrant transcripts with frameshifts yielding nonfunctional proteins lacking the C-terminal transmembrane domains. Bm-re function in pigmentation was confirmed by embryonic RNAi experiments. Homologs of the Bm-re gene were found in all insect genomes sequenced at present, except for 12 sequenced Drosophila genomes, which seemed to correlate with the previous studies that have demonstrated that eye ommochrome composition is different from other insects in several Dipterans. Knockdown of the Bm-re homolog by RNAi in the red flour beetle Tribolium castaneum caused adult compound eye coloration defects, indicating a conserved role in ommochrome pigment biosynthesis at least among holometabolous insects.

Positional cloning of silkworm white egg 2 (w-2) locus shows functional conservation and diversification of ABC transporters for pigmentation in insects.

The white, scarlet and brown genes of Drosophila melanogaster encode three half-type ATP-binding cassette (ABC) transporters. In Drosophila, precursors of ommochromes and pteridines are transported by White/Scarlet and White/Brown heterodimers, respectively. The white egg 2 (w-2) mutant of the silkworm, Bombyx mori, has white eggs and eyes because of lack of ommochrome granules in the serosa and eyes. Here, we report that the silkworm w-2 locus encodes an ortholog of Drosophila scarlet. Our results indicate that Bombyx Scarlet forms a heterodimer with Bombyx White to transport ommochrome precursors, suggesting that formation of a White/Scarlet heterodimer and its involvement in the transport of ommochrome precursors are evolutionarily ancient and widely conserved traits in insects. Contrary to dipteran insects, white and scarlet were juxtaposed in a head-to-tail orientation in the silkworm genome, suggesting that the origin of white and scarlet was a tandem duplication of their ancestral transporter gene. In Bombyx, White is also essential for the transport of uric acid in larval epidermis. However, our results suggest that a Bombyx White/Scarlet heterodimer is not involved in this process. Our results emphasize the functional conservation and diversification of half-type ABC transporter families in insects, which may contribute to their extremely diverse color patterns.

Repression of tyrosine hydroxylase is responsible for the sex-linked chocolate mutation of the silkworm, Bombyx mori.

Pigmentation patterning has long interested biologists, integrating topics in ecology, development, genetics, and physiology. Wild-type neonatal larvae of the silkworm, Bombyx mori, are completely black. By contrast, the epidermis and head of larvae of the homozygous recessive sex-linked chocolate (sch) mutant are reddish brown. When incubated at 30 degrees C, mutants with the sch allele fail to hatch; moreover, homozygous mutants carrying the allele sch lethal (sch(l)) do not hatch even at room temperature (25 degrees C). By positional cloning, we narrowed a region containing sch to 239,622 bp on chromosome 1 using 4,501 backcross (BC1) individuals. Based on expression analyses, the best sch candidate gene was shown to be tyrosine hydroxylase (BmTh). BmTh coding sequences were identical among sch, sch(l), and wild-type. However, in sch the approximately 70-kb sequence was replaced with approximately 4.6 kb of a Tc1-mariner type transposon located approximately 6 kb upstream of BmTh, and in sch(l), a large fragment of an L1Bm retrotransposon was inserted just in front of the transcription start site of BmTh. In both cases, we observed a drastic reduction of BmTh expression. Use of RNAi with BmTh prevented pigmentation and hatching, and feeding of a tyrosine hydroxylase inhibitor also suppressed larval pigmentation in the wild-type strain, pnd(+) and in a pS (black-striped) heterozygote. Feeding L-dopa to sch neonate larvae rescued the mutant phenotype from chocolate to black. Our results indicate the BmTh gene is responsible for the sch mutation, which plays an important role in melanin synthesis producing neonatal larval color.

A CD36-related transmembrane protein is coordinated with an intracellular lipid-binding protein in selective carotenoid transport for cocoon coloration.

The transport pathway of specific dietary carotenoids from the midgut lumen to the silk gland in the silkworm, Bombyx mori, is a model system for selective carotenoid transport because several genetic mutants with defects in parts of this pathway have been identified that manifest altered cocoon pigmentation. In the wild-type silkworm, which has both genes, Yellow blood (Y) and Yellow cocoon (C), lutein is transferred selectively from the hemolymph lipoprotein to the silk gland cells where it is accumulated into the cocoon. The Y gene encodes an intracellular carotenoid-binding protein (CBP) containing a lipid-binding domain known as the steroidogenic acute regulatory protein-related lipid transfer domain. Positional cloning and transgenic rescue experiments revealed that the C gene encodes Cameo2, a transmembrane protein gene belonging to the CD36 family genes, some of which, such as the mammalian SR-BI and the fruit fly ninaD, are reported as lipoprotein receptors or implicated in carotenoid transport for visual system. In C mutant larvae, Cameo2 expression was strongly repressed in the silk gland in a specific manner, resulting in colorless silk glands and white cocoons. The developmental profile of Cameo2 expression, CBP expression, and lutein pigmentation in the silk gland of the yellow cocoon strain were correlated. We hypothesize that selective delivery of lutein to specific tissue requires the combination of two components: 1) CBP as a carotenoid transporter in cytosol and 2) Cameo2 as a transmembrane receptor on the surface of the cells.

yellow and ebony are the responsible genes for the larval color mutants of the silkworm Bombyx mori.

Many larval color mutants have been obtained in the silkworm Bombyx mori. Mapping of melanin-synthesis genes on the Bombyx linkage map revealed that yellow and ebony genes were located near the chocolate (ch) and sooty (so) loci, respectively. In the ch mutants, body color of neonate larvae and the body markings of elder instar larvae are reddish brown instead of normal black. Mutations at the so locus produce smoky larvae and black pupae. F(2) linkage analyses showed that sequence polymorphisms of yellow and ebony genes perfectly cosegregated with the ch and so mutant phenotypes, respectively. Both yellow and ebony were expressed in the epidermis during the molting period when cuticular pigmentation occurred. The spatial expression pattern of yellow transcripts coincided with the larval black markings. In the ch mutants, nonsense mutations of the yellow gene were detected, whereas large deletions of the ebony ORF were detected in the so mutants. These results indicate that yellow and ebony are the responsible genes for the ch and so loci, respectively. Our findings suggest that Yellow promotes melanization, whereas Ebony inhibits melanization in Lepidoptera and that melanin-synthesis enzymes play a critical role in the lepidopteran larval color pattern.

A BAC-based integrated linkage map of the silkworm Bombyx mori.

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.

End-sequencing and characterization of silkworm (Bombyx mori) bacterial artificial chromosome libraries.

BACKGROUND: We performed large-scale bacterial artificial chromosome (BAC) end-sequencing of two BAC libraries (an EcoRI- and a BamHI-digested library) and conducted an in silico analysis to characterize the obtained sequence data, to make them a useful resource for genomic research on the silkworm (Bombyx mori). RESULTS: More than 94000 BAC end sequences (BESs), comprising more than 55 Mbp and covering about 10.4% of the silkworm genome, were sequenced. Repeat-sequence analysis with known repeat sequences indicated that the long interspersed nuclear elements (LINEs) were abundant in BamHI BESs, whereas DNA-type elements were abundant in EcoRI BESs. Repeat-sequence analysis revealed that the abundance of LINEs might be due to a GC bias of the restriction sites and that the GC content of silkworm LINEs was higher than that of mammalian LINEs. In a BLAST-based sequence analysis of the BESs against two available whole-genome shotgun sequence data sets, more than 70% of the BESs had a BLAST hit with an identity of > or = 99%. About 14% of EcoRI BESs and about 8% of BamHI BESs were paired-end clones with unique sequences at both ends. Cluster analysis of the BESs clarified the proportion of BESs containing protein-coding regions. CONCLUSION: As a result of this characterization, the identified BESs will be a valuable resource for genomic research on Bombyx mori, for example, as a base for construction of a BAC-based physical map. The use of multiple complementary BAC libraries constructed with different restriction enzymes also makes the BESs a more valuable genomic resource. The GenBank accession numbers of the obtained end sequences are DE283657-DE378560.

Construction of a single nucleotide polymorphism linkage map for the silkworm, Bombyx mori, based on bacterial artificial chromosome end sequences.

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 x an F1 (p50T x 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.

Phosphorylation of clock protein PER1 regulates its circadian degradation in normal human fibroblasts.

Recent advances suggest that the molecular components of the circadian clock generate a self-sustaining transcriptional-translational feedback loop with a period of approx. 24 h. The precise expression profiles of human clock genes and their products have not been elucidated. We cloned human clock genes, including per1, per2, per3, cry2 and clock, and evaluated their circadian mRNA expression profiles in WI-38 fibroblasts stimulated with serum. Transcripts of hPer1, hPer2, hPer3, hBMAL1 and hCry2 (where h is human) underwent circadian oscillation. Serum-stimulation also caused daily oscillations of hPER1 protein and the apparent molecular mass of hPER1 changed. Inhibitor studies indicated that the CKI (casein kinase I) family, including CKIepsilon and CKIdelta, phosphorylated hPER1 and increased the apparent molecular mass of hPER1. The inhibition of hPER1 phosphorylation by CKI-7 [ N -(2-aminoethyl)-5-chloro-isoquinoline-8-sulphonamide], a CKI inhibitor, disturbed hPER1 degradation, delayed the nuclear entry of hPER1 and allowed it to persist for longer in the nucleus. Furthermore, proteasome inhibitors specifically blocked hPER1 degradation. However leptomycin B, an inhibitor of nuclear export, did not alter the degradation state of hPER1 protein. These findings indicate that circadian hPER1 degradation through a proteasomal pathway can be regulated through phosphorylation by CKI, but not by subcellular localization.