Haruhiko Fujiwara

Identification of a pentanucleotide telomeric sequence, (TTAGG)n, in the silkworm Bombyx mori and in other insects.

A pentanucleotide repetitive sequence, (TTAGG)n, has been isolated from a silkworm genomic library, using cross-hybridization with a (TTNGGG)5 sequence, which is conserved among most eukaryotic telomeres. Both fluorescent in situ hybridization and Bal 31 exonuclease experiments revealed major clusters of (TTAGG)n at the telomeres of all Bombyx chromosomes. To determine the evolutionary origin of this sequence, two types of telomeric sequence, (TTAGG)5 and a hexanucleotide repetitive sequence, (TTAGGG)4, which is conserved mainly among vertebrate and several invertebrate telomeres so far examined, were hybridized to DNAs from a wide variety of eukaryotic species under highly stringent hybridization conditions. The (TTAGGG)5 oligonucleotide hybridized to genomic DNAs from vertebrates and several nonvertebrate species, as has been reported so far, but not to any DNAs from insects. On the other hand, the Bombyx type of telomere sequence, (TTAGG)n, hybridized to DNAs from 8 of 11 orders of insect species tested but not to vertebrate DNAs, suggesting that this TTAGG repetitive sequence is conserved widely among insects.

Structural basis for telomerase catalytic subunit TERT binding to RNA template and telomeric DNA

Telomerase is a specialized DNA polymerase that extends the 3′ ends of eukaryotic linear chromosomes, a process required for genomic stability and cell viability. Here we present the crystal structure of the active Tribolium castaneum telomerase catalytic subunit, TERT, bound to an RNA-DNA hairpin designed to resemble the putative RNA-templating region and telomeric DNA. The RNA-DNA hybrid adopts a helical structure, docked in the interior cavity of the TERT ring. Contacts between the RNA template and motifs 2 and B′ position the solvent-accessible RNA bases close to the enzyme active site for nucleotide binding and selectivity. Nucleic acid binding induces rigid TERT conformational changes to form a tight catalytic complex. Overall, TERT–RNA template and TERT–telomeric DNA associations are remarkably similar to those observed for retroviral reverse transcriptases, suggesting common mechanistic aspects of DNA replication between the two families of enzymes.

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. F2 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.

Melanin-synthesis enzymes coregulate stage-specific larval cuticular markings in the swallowtail butterfly, Papilio xuthus

Like the adult wing, butterfly larvae are unique in their coloring. However, the molecular mechanisms underlying the formation of insect larval color patterns are largely unknown. The larva of the swallowtail butterfly Papilio xuthus changes its color pattern markedly during the 4th ecdysis. We investigated its cuticular color pattern, which is thought to be composed of melanin and related pigments derived from tyrosine. We cloned three enzymes involved in the melanin-synthesis pathway in P. xuthus: tyrosine hydroxylase (TH), dopa decarboxylase (DDC), and ebony. Whole-mount in situ hybridization showed that the expression of both TH and DDC is strongly correlated with the black markings. ebony is strongly expressed only in the reddish-brown area. The expression pattern of each enzyme coincides with the cuticular color pattern of the subsequent instar. We also investigated the uptake of melanin precursors into cultured integument. Inhibition of either TH or DDC activity prevents in vitro pigmentation completely. Addition of dopamine to integuments in the presence of TH inhibitor causes overall darkening without specific markings. From these results, specific larval cuticular color patterns are regulated by stage-specific colocalization of enzymes in epidermal cells rather than by the differential uptake of melanin precursors into individual epidermal cells. Epidermal cells expressing TH and DDC, but not ebony, produce the black cuticle, and epidermal cells expressing TH, DDC, and ebony produce the reddish-brown cuticle.

A genetic mechanism for female-limited Batesian mimicry in Papilio butterfly

In Batesian mimicry, animals avoid predation by resembling distasteful models. In the swallowtail butterfly Papilio polytes, only mimetic-form females resemble the unpalatable butterfly Pachliopta aristolochiae. A recent report showed that a single gene, doublesex (dsx), controls this mimicry; however, the detailed molecular mechanisms remain unclear. Here we determined two whole-genome sequences of P. polytes and a related species, Papilio xuthus, identifying a single ∼130-kb autosomal inversion, including dsx, between mimetic (H-type) and non-mimetic (h-type) chromosomes in P. polytes. This inversion is associated with the mimicry-related locus H, as identified by linkage mapping. Knockdown experiments demonstrated that female-specific dsx isoforms expressed from the inverted H allele (dsx(H)) induce mimetic coloration patterns and simultaneously repress non-mimetic patterns. In contrast, dsx(h) does not alter mimetic patterns. We propose that dsx(H) switches the coloration of predetermined wing patterns and that female-limited polymorphism is tightly maintained by chromosomal inversion.

Transplantation of target site specificity by swapping the endonuclease domains of two LINEs

Long interspersed elements (LINEs) are ubiquitous genomic elements in higher eukaryotes. Here we develop a novel assay to analyze in vivo LINE retrotransposition using the telomeric repeat‐specific elements SART1 and TRAS1. We demonstrate by PCR that silkworm SART1, which is expressed from a recombinant baculovirus, transposes in Sf9 cells into the chromosomal (TTAGG)n sequences, at the same specific nucleotide position as in the silkworm genome. Thus authentic retrotransposition by complete reverse transcription of the entire RNA transcription unit and occasional 5′ truncation is observed. The retrotransposition requires conserved domains in both open reading frames (ORFs), including the ORF1 cysteine– histidine motifs. In contrast to human L1, recognition of the 3′ untranslated region sequence is crucial for SART1 retrotransposition, which results in efficient trans‐complementation. Swapping the endonuclease domain from TRAS1 into SART1 converts insertion specificity to that of TRAS1. Thus the primary determinant of in vivo target selection is the endonuclease domain, suggesting that modified LINEs could be used as gene therapy vectors, which deliver only genes of interest but not retrotransposons themselves in trans to specific genomic locations.

Telomere-specific non-LTR retrotransposons and telomere maintenance in the silkworm, Bombyx mori

Most insects have telomeres that consist of pentanucleotide (TTAGG) telomeric repeats, which are synthesized by telomerase. However, all species in Diptera so far examined and several species in other orders of insect have lost the (TTAGG)n repeats, suggesting that some of them recruit telomerase-independent telomere maintenance. The silkworm, Bombyx mori, retains the TTAGG motifs in the chromosomal ends but expresses quite a low level of telomerase activity in all stages of various tissues. Just proximal to a 6–8-kb stretch of the TTAGG repeats in B. mori, more than 1000 copies of non-LTR retrotransposons, designated TRAS and SART families, occur among the telomeric repeats and accumulate. TRAS and SART are abundantly transcribed and actively retrotransposed into TTAGG telomeric repeats in a highly sequence-specific manner. They have three possible mechanisms to ensure specific integration into the telomeric repeats. This article focuses on the telomere structure and telomere-specific non-LTR retrotransposons in B. mori and discusses the mechanisms for telomere maintenance in this insect.

Expression of ecdysteroid-regulated genes is reduced specifically in the wing discs of the wing-deficient mutant (fl) of Bombyx mori

Abstract The wing-deficient mutant, flügellos (fl), of the silkworm, lacks four wings in the pupa and the adult. Previous studies have suggested that the fl wing discs lose responsiveness to ecdysteroid during metamorphosis. To test this hypothesis at the molecular level we compared the expression of 12 genes when the wing discs from the wild-type (WT) and fl larvae were cultured in the presence or absence of 20-hydroxyecdysone (20E). Most of the genes tested here, ecdysteroid-inducible (EcR-A, -B1,and E75) and noninducible genes (actin A3, β-tubulin, apterous (ap), USP, and BHR38) were normally expressed in the fl wing discs. However, the amounts of mRNAs of two ecdysteroid-inducible genes, BHR3 (early-late gene; Bombyx homologue to DHR3 and MHR3) and Urbain (wing-specific late gene), were reduced to about 50% and 20% of WT in the cultured fl wing discs, respectively. We analyzed developmental profiles of these mRNAs during metamorphosis. They also demonstrated decreased BHR3 and Urbain mRNA 2 days after the onset of wandering. This reduction in transcription of BHR3 in the fl mutant was observed only in the wing disc, not in the testis and fatbody. These results imply that the aberrant expression of the fl gene affects the downstream pathway of ecdysteroid signaling specifically in the wing discs and thus leads to a deficiency in wing formation.

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.

Caterpillar color patterns are determined by a two-phase melanin gene prepatterning process: new evidence from tan and laccase2

SUMMARY The larval color patterns in Lepidoptera exhibit splendid diversity, and identifying the genes responsible for pigment distribution is essential to understanding color‐pattern evolution. The swallowtail butterfly, Papilio xuthus, is a good candidate for analyzing marking‐associated genes because its body markings change dramatically at the final molt. Moreover, the silkworm Bombyx mori is most suitable for identification of lab‐generated color mutants because genome information and many color mutants are available. Here, we analyzed the expression pattern of 10 melanin‐related genes in P. xuthus, and analyzed whether these genes were responsible for Bombyx larval color mutants. We found that seven genes correlated strongly with the stage‐specific larval cuticular markings of P. xuthus, suggesting that, compared with Drosophila, more genes showed marking specificity in lepidopteran larvae. We newly found that the expression of both tan and laccase2 is strongly correlated with the larval black markings in both P. xuthus and B. mori. The results of F2 linkage analysis and mutant analysis strongly suggest that tan is the responsible gene for Bombyx larval color mutant rouge, and that tan is important in emphasizing black markings of lepidopteran larvae. Detailed comparison of temporal and spatial expression patterns showed that larval cuticular markings were regulated at two different phases. Marking‐specific expression of oxidizing enzymes preceded the marking‐specific expression of melanin synthesis enzymes at mRNA level, which is the reverse of the melanin synthesis step.