A novel system for separation and purification of egg pigments from the nondiapause red-egg mutant Re-nd in the silkworm,Bombyx mori

Re-nd, which was induced from the wild-type C108 by the chemical mutagen N-methane-N-methylnitrourea, is a nondiapause red-egg mutant of silkworm Bombyx mori. The special significance of the Re-nd mutant is that it is an independent dominant mutant. The aim of this study was to establish the type of pigment responsible for the red coloration in the Re-nd mutant eggs in silkworm. We compared the eggs of Re-nd mutants with those of the other B. mori egg color strains and confirmed that the Re-nd mutant is the only strain with red color and red pigment granules in nondiapause, showing this mutant belongs to the pigmentation in the serosa. We speculated that the red substance, which contributed to the bright red pigmentation for nondiapause eggs of the Re-nd mutant, could potentially be a novel pigment according to its solubility, optimum absorption peak, and oxidation–reduction reaction. Moreover, we have successfully constituted the system for enrichment, extraction, and purification of the red substance responsible for the Re-nd mutant, providing a new method for the separation and purification of other known and unknown pigments or substances.     

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.     

The expression analysis of silk gland-enriched intermediate-size non-coding RNAs in silkworm Bombyx mori

Small non-protein coding RNAs （ncRNAs） play important roles in development, stress response and other cellular processes. Silkworm is an important model for studies on insect genetics and control of Lepidopterous pests. We have previously identified 189 novel intermediate-size ncRNAs in silkworm Bombyx mori, including 40 ncRNAs that showed altered expression in different developmental stages. Here we characterized the functions of these 40 ncRNAs by measuring their expressions in six tissues of the fifth instar larvae using Northern blot and real-time polymerase chain reaction assays. We identified nine ncRNAs （four small nucleolar RNAs and five unclassified ncRNAs） that were enriched in silk gland, including four ncRNAs that showed silk gland-specific expression. We further showed that three of nine silk gland-enriched ncRNAs were predominantly expressed in the anterior silk gland, whereas another three ncRNAs were highly accumulated in the posterior silk gland, suggesting that they may play different roles in fibroin synthesis. Furthermore, an unclassified ncRNA, Bm- 152, exhibited converse expression pattem with its antisense host gene gartenzwerg in diverse tissues, and might regulate the expression of gartenzwerg through RNA-protein complex. In addition, two silk gland-enriched ncRNAs Bm-102 and Bm-159 can be found in histone modification complex, which indicated that they might play roles through epigenetic modifications. Taken together, we provided the first expression and preliminary functional analysis of silk gland-enriched ncRNAs, which will help understand the molecular mechanism of silk gland-development and fibroin synthesis.     

Characterization and expression of the pseudorabies virus (NYJ strain) glycoproteins in Bombyx mori cells and larvae

To characterize the NYJ strain of pseudorabies virus (PRV; Alphaherpesvirus of swine) isolated from the serum of an infected swine in Korea, the nucleotide sequence of three major glycoproteins (gB, gC, and gD) was analyzed. The expression of most potent immunogenic glycoprotein (gD) was also investigated using a Bombyx mori nucleopolyhedrovirus (BmNPV) expression system. The length of the glycoprotein genes corresponding to gB, gC, and gD of the NYJ strain were 2751 bp, 1443 bp, and 1203, respectively, and their identity ranged from 94.2% to 99.8% when compared with other strains. Phylogenetic analyses using these sequences showed that the NYJ strain forms a distinct branch with high bootstrap support. A novel transfer vector (pBmKSK4) was engineered with the polyhedrin promoter of BmNPV and a 6xHis tag to express glycoprotein gD in Bm5 cells and silkworm, B. mori, larvae. The immunogenicity of recombinant gD was demonstrated by its specific detection in both Bm5 cells and silkworm larvae by porcine anti-PRV antibody. The results of this study have implications both for the taxonomy of Korean PRV strains and vaccine development.     

Discovering genes responsible for silk synthesis in Bombyx mori by piggyBac‐based random insertional mutagenesis

Silkworm mutants are valuable resources for both transgenic breeding and gene discovery. PiggyBac-based random insertional mutagenesis has been widely used in gene functional studies. In order to discover genes involved in silk synthesis, a piggyBac-based random insertional library was constructed using Bombyx mori, and the mutants with abnormal cocoon were particularly screened. By this means, a “thin cocoon” mutant was identified. This mutant revealed thinner cocoon shell and shorter posterior silk gland (PSG) compared with the wild type. The messenger RNA (mRNA) levels of all the three fibroin genes, including Fib-H, Fib-L and P25, were significantly down-regulated in the PSG of mutants. Four piggyBac insertion sites were identified in Aquaporin (AQP), Longitudinals lacking protein-like {Lola), Glutamyl aminopeptidase-like (GluAP) and Loc101744460. The mRNA levels of all the four genes were significantly altered in the silk gland of mutants. In particular, the mRNA amount of AQP, a gene responsible for the regulation of osmotic pressure, decreased dramatically immediately prior to the spinning stage in the anterior silk gland of mutants. The identification of the genes disrupted in the “thin cocoon” mutant in this study provided useful information for understanding silk production and transgenic breeding of silkworms in the future.     

D‐3‐phosphoglycerate dehydrogenase from the silkworm Bombyx mori: Identification,functional characterization,and expression

D‐3‐phosphoglycerate dehydrogenase (PHGDH) is a key enzyme involved in the synthesis of l ‐serine. Despite the high serine content in silk proteins and the crucial role of PHGDH in serine biosynthesis, PHGDH has not been described in silkworms to date. Here, we identified PHGDH in the silkworm Bombyx mori and evaluated its biochemical properties. On the basis of the amino acid sequence and phylogenetic tree, this PHGDH has been categorized as a new type and designated as bmPHGDH. The recombinant bmPHGDH was overexpressed and purified to homogeneity. Kinetic studies revealed that PHGDH uses NADH as a coenzyme to reduce phosphohydroxypyruvate. High expression levels of bmphgdh messenger RNA (mRNA) were observed in the middle part of the silk gland and midgut in a standard strain of silkworm. Moreover, a sericin‐deficient silkworm strain displayed reduced expression of bmphgdh mRNA. These findings indicate that bmPHGDH might play a crucial role in the provision of l ‐serine in the larva of B. mori.     

Expression of the Japanese oak silkworm Antheraea yamamai fibroin gene in the domesticated silkworm Bombyx mori

Abstract To understand the evolutionary conservation of the gene expression mechanism and secretion machinery between Antheraea and Bombyx fibroins, we introduced the genomic A. yamamai fibroin gene into the domesticated silkworm, B. mori. The spliced A. yamamai fibroin mRNA appeared only in the posterior region of the silk gland of the transgenic silkworm, suggesting that the functions of the fibroin promoter region and the splicing machinery are conserved between these two species. The A. yamamai fibroin protein was detected in the lumen of the silk gland of the transgenic silkworm, albeit at lower levels compared with the B. mori‐type fibroin. We found a strong degeneration of the posterior region of the silk gland of the transgenic silkworm. As a result, the cocoon shell weight was much lower in the transgenic silkworm than in the non‐transgenic line. These results indicate that the promoter function and splicing machinery are well conserved between A. yamamai and B. mori but that the secretion mechanism of fibroin is diversified between the two.     

Molecular characterization and expression analysis of BmNOX in two strains of Bombyx mori with contrasting viral resistance phenotype

We recently documented the identification of a 26.5 kDa protein named BmNox in the gut fluid of Nistari strain of Bombyx mori, which possessed antiviral activity against BmNPV in vitro. In this report, we report the characterization of the full‐length gene encoding BmNOX and the levels of expression of this gene in select tissues of silkworm larvae from a BmNPV‐susceptible and a BmNPV‐resistant strain to the defense capability in Bombyx mori larvae challenged with BmNPV. We also evaluated the BmNox expression in various stages of larval life of a resistant and a susceptible strain of Bombyx mori selected from among a panel of strains of silkworm. Nistari, a multivoltine strain of silkworm, expressed BmNOX during all five larval stages, and were highly resistant to BmNPV infection. In sharp contrast, CSR₂, a bivoltine strain, showed weaker expression of BmNOX in the anterior midgut in larval life and was highly susceptible to BmNPV infection. BmNOX is a secretory protein with dual expression in gut fluid and mid gut tissue. BmNOX is expressed heavily in the posterior mid gut, with weaker expression in the fore‐ and mid‐gut regions. © 2010 Wiley Periodicals, Inc.     

Identification of a PP2A gene in Bombyx mori with antiviral function against B. mori nucleopolyhedrovirus

Ser/Thr protein phosphatase 2A (PP2A) is one of the type 2 protein phosphatases, which is required for many intracellular physiological processes and pathogen infection. However, the function of PP2A is unclear in silkworm, Bombyx mori. Here, we cloned and identified BmPP2A, a PP2A gene from B. mori, which has two HEAT domains and a high similarity to PP2A from other organisms. Our results showed that BmPP2A is localized in the cytoplasm and highly expressed in silkworm epidermis and midgut, and that Bombyx mori nucleopolyhedrovirus (BmNPV) infection induces down‐regulation of BmPP2A expression. Furthermore, up‐regulation of BmPP2A via overexpression significantly inhibited BmNPV multiplication. In contrast, down‐regulation of BmPP2A via RNA interference and okadaic acid (a PP2A inhibitor) treatment allowed robust BmNPV replication. This is the first report of PP2A having an antiviral effect in silkworm and provides insights into the function of BmPP2A, a potential anti‐BmNPV mechanism, and a possible target for the breeding of silkworm‐resistant strains.     

Evidence for a relationship between the Bombyx mori middle repetitive Bm1 sequence family and U1 snRNA

Several genomic library equivalents of Bombyx mori were constructed in the EMBL-4 lambda derivative. The genomic bank was screened with purified Bombyx mori U1 RNA and twenty positive clones for the U1 gene were isolated. Three U1-related sequences were subcloned and sequenced. Two of the sequences are U1 pseudogenes while a third sequence represents a member of the Bm1 family of repetitive elements of B.mori with significant sequence similarity to U1 small nuclear RNA. The U1-related Bm1 element exhibits 82% sequence similarity with the Bm1 consensus sequence and, under less stringent computer comparison parameters, 60% similarity with a composite B.mori/Drosophila melanogaster U1 gene. The Bm1 family consensus sequence exhibits 53% sequence similarity with the composite U1 gene. The two pseudogenes possess highly conserved sequences with the B.mori U1 gene only for the first 101 nucleotides. These findings are indicative of at least two different categories of U1-related sequences in B.mori, one with a possible evolutionary relationship to the Bm1 family of repetitive elements and the other representing characteristic processed pseudogenes with retroposon mode of dispersion and target selection for the TTTA hotspot. In addition, the U1-related Bm1 element may demonstrate for the first time that a family of retroposons is ultimately derived from a U snRNA.This article is dedicated in memory of Ms. Deborah Lampert who helped so much in the preparation of this paper.     

Functional analysis of a miRNA‐like small RNA derived from Bombyx mori cytoplasmic polyhedrosis virus

Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) is a major pathogen of the economic insect silkworm, Bombyx mori. Virus‐encoded microRNAs (miRNAs) have been proven to play important roles in host–pathogen interactions. In this study we identified a BmCPV‐derived miRNA‐like 21 nt small RNA, BmCPV‐miR‐1, from the small RNA deep sequencing of BmCPV‐infected silkworm larvae by stem‐loop quantitative real‐time PCR (qPCR) and investigated its functions with qPCR and lentiviral expression systems. Bombyx mori inhibitor of apoptosis protein (BmIAP) gene was predicted by both target prediction software miRanda and Targetscan to be one of its target genes with a binding site for BmCPV‐miR‐1 at the 5′ untranslated region. It was found that the expression of BmCPV‐miR‐1 and its target gene BmIAP were both up‐regulated in BmCPV‐infected larvae. At the same time, it was confirmed that BmCPV‐miR‐1 could up‐regulate the expression of BmIAP gene in HEK293T cells with lentiviral expression systems and in BmN cells by transfecting mimics. Furthermore, BmCPV‐miR‐1 mimics could up‐regulate the expression level of BmIAP gene in midgut and fat body in the silkworm. In the midgut of BmCPV‐infected larvae, BmCPV‐miR‐1 mimics could be further up‐regulated and inhibitors could lower the virus‐mediated expression of BmIAP gene. With the viral genomic RNA segments S1 and S10 as indicators, BmCPV‐miR‐1 mimics could up‐regulate and inhibitors down‐regulate their replication in the infected silkworm. These results implied that BmCPV‐miR‐1 could inhibit cell apoptosis in the infected silkworm through up‐regulating BmIAP expression, providing the virus with a better cell circumstance for its replication.     

The possible evolutionary significance of repeat elements near and within an early chorion gene in the late chorion locus of Bombyx mori

Summary Three -type early chorion gene copies (6F76.1, 6F76.2, and 6F76.3) are dispersed in the late region of chorion locus Chl-2. Detailed analysis of the 5-flanking region and the intron of 6176.1 shows that they contain sequences that are homologous to Bombyx mori Bm l repeat elements. Interestingly, the Bm l -type segment of the intron is interrupted by the insertion of a sequence that shows significant similarities with part of an intron of B. mori and Bombyx mandarina fibroin genes, and with part of the 3-flanking region of B. mori prothoracicotropic hormone and tRNA-Glu genes; this sequence may represent a new repetitive, possibly transposable, element of B. mori. Following the Bm1-homologous sequence of the 6176.1 5-flanking region and preceding the gene promoter region, a short DNA segment shows sequence motifs that are also present in the ErA.1 promoter region. The occurrence of these sequences near one end or within the Bm1 repeat element is suggestive of complex sequence transfer events. Comparative analysis of known B. mori chorion -gene promoters and of Bm1 repeat elements suggests, with marginal statistical significance, that these two sets of sequences contain common elements.Offprint requests to: G. Rodakis     

Molecular structure of a novel gypsy-Ty3-like retrotransposon (Kabuki) and nested retrotransposable elements on the W chromosome of the silkworm Bombyx mori

We previously characterized a female-specific randomly amplified polymorphic DNA (RAPD), designated W-Kabuki, derived from the W chromosome of the silkworm, Bombyx mori. To further analyze the W chromosome of B. mori, we obtained a lambda phage clone which contains the W-Kabuki RAPD sequence and sequenced the 18.1-kb DNA insert. We found that this DNA comprises a nested structure of at least seven elements; three retrotransposons, two retroposons, one functionally unknown insertion, and one Bombyx repetitive sequence. The non-LTR retrotransposon BMC1, the retroposon Bm1, a functionally unknown inserted DNA (FUI), and a copia-like LTR retrotransposon (Yokozuna) are themselves inserted into a novel gypsy-Ty3-like LTR retrotransposon, named Kabuki. Furthermore, this Kabuki element is itself inserted into another copy of Bm1. The BMC1 and Yokozuna elements inserted in the Kabuki sequence are intact. Moreover, the Kabuki element is largely intact. These results suggest that many retrotransposable elements have accumulated on the W chromosome, and these elements are expected to evolve more slowly than those on other chromosomes. Received: 7 October 1999 / Accepted: 14 April 2000     

Expression pattern and tissue localization of the class B scavenger receptor BmSCRBQ4 in Bombyx mori

Class B scavenger receptors (SR‐Bs) are cell surface glycoproteins involved in various physiological processes in vivo, including the transport and metabolism of lipids, binding and phagocytosis of xenobiotics, and signaling. But little information is available about silkworm SR‐Bs; it is necessary to study these SR‐Bs for revealing their function. In this study, we cloned the full‐length coding sequence of BmSCRBQ4, a SR‐B gene from the silkworm Bombyx mori L. We found that the BmSCRBQ4 gene consists of nine exons and eight introns, with an open reading frame of 1371 bp encoding 456 amino acids. Gene expression studies determined that BmSCRBQ4 messenger RNA (mRNA) was expressed in unfertilized eggs, during embryonic development and throughout the majority of the larval period. Expression of mRNA was detected in the mid gut, middle silk gland, posterior silk gland, head, integumentum, fat body, testes and the ovaries of the larval B. mori Dazao strain, as well as in the silkworm cell lines BmN and BmE. Protein expression studies found BmSCRBQ4 protein was expressed only in the testes, fat body and middle silk gland of larvae, as well as in the silkworm cell lines BmN and BmE. The BmSCRBQ4 protein showed variability in banding patterns in different tissues and cells when analyzed by Western blotting. Immunohistochemical staining showed that the BmSCRBQ4 protein localizes to the constitutive membranes or cellular membranes of these tissues. These results indicated that BmSCRBQ4 gene may play some physiologically relevant roles at the cell surface in each tissue.     

Expression of EGFP driven by BmNPV orf4 promoter, a novel immediate early promoter

Bombyx mori nucleopolyhedrovirus (BmNPV) orf4 has been shown to be expressed at very early stage of Bm-NPV infection cycle. In this study, using transient expression experiment, we demonstrated for the first time that orf4 promoter is an immediate early promoter, indicating that orf4 may play a role in the immediate-early stage of BmNPV infection. Moreover, with the recently developed Bac-to-Bac/BmNPV baculovirus expression system and a modified pFast-Bac1 whose polyhedrin promoter was replaced with orf4 promoter, a recombinant bacmid baculovirus expressing enhanced green fluorescent protein (EGFP) under the control of orf4 promoter in Bombyx mori (Bm) cells was successfully constructed. The result not only showed that the polyhedrin promoter can be replaced easily with other promoters to direct the expression of foreign genes by using this novel system but also laid the foundation for the rescue experiment of orf4 deletion mutant.     

Distribution of Bm1, a SINE type transposable element, in cecropin B genes of the silkworm, Bombyx mori

In order to investigate the distribution of Bm1, a SINE type transposable element, in cecropin B genes of the silkworm, Bombyx mori, a genomic library was first screened with cecropin B cDNA as a probe. Eighteen out of 275 positive clones were selected at random and SalI digestion patterns were compared. Ten clones which showed different patterns were further analyzed by Southern blotting using a cecropin B cDNA fragment encoding exon 1, exon 2 or the entire coding region as probes. The same SalI digested genomic clones were hybridized with a Bm1 probe. Comparison of positive patterns hybridized with the Bm1 probe to those hybridized with the cecropin B probes indicated that all cecropin B-fragments except one fragment had Bm1. The exceptional fragment contained exon 2 only. The results indicate that Bm1 is widely distributed in cecropin B genes.