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At least 109 records · Page 6Linked to original sources
PubMed2004

Use of RNAi technology to confer enhanced resistance to BmNPV on transgenic silkworms.

dsRNA is a powerful tool for gene-specific silencing in plants and animals. In this study, we examined the use of gene silencing in generating transgenic silkworms resistant to the Bombyx mori nucleopolyhedrovirus (BmNPV). Using a transposon piggyBac system, we first generated BmN cells (rBmN-lef1), which carried artificial genes designed for expressing dsRNAs with sequences of the essential viral gene lef-1. NPV DNA microarray analysis revealed that the accumulation of lef-1 mRNA was successfully inhibited in rBmN-lef1 infected with BmNPV. The virus titer in the culture medium of rBmN-lef1 at 48 hr post-infection (h.p.i.) was 50% of that of the control cells. Moderate BmNPV-resistance caused by transgenesis of the artificial dsRNA-expressing gene was confirmed in the transgenic silkworms. Virus production was reduced in transgenic silkworms relative to controls up to 96 hrs after viral inoculation. Although complete protection was not achieved and the transgenic larvae ultimately died, this is the first report to show the use of RNAi in confering enhanced viral resistance on transgenic animals.

Animals↗
PubMed2005

Silk gland specific secretory expression of egfp gene in silkworm Bombyx mori with rAcMNPV system.

To evaluate the possibility of establishing an in vivo baculovirus expression system in a silk gland specific secretory way, the recombinant Autographa californica nucleopolyhedrovirus (AcserpegfpDeltaEGT) carrying the reporter gene egfp downstream of silkworm ser1 promoter and signal peptide coding sequence was generated. The purified recombinant baculovirus AcserpegfpDeltaEGT was injected into the haemocoel of newly ecdysed 5thHendekl) instar silkworm larvae at the amount of 10(6) pfu per larva. At 5 days post injection, green fluorescence derived from EGFP could be observed with fluorescent microscope in only the silk gland but not other tissues after dissection of the silkworm. By making an opening on the silk gland wall, green fluorescence could be observed in the outflow of silk gland indicating the secretion of EGFP and the effectiveness of ser1 signal peptide. Western blotting assay confirmed that EGFP exists in the water-soluble part of cocoon silk too. We also established a simple protocol to purify EGFP from the secreted silk proteins.

Animals↗
PubMed1985

The reactive site of silkworm hemolymph antichymotrypsin is located at the COOH-terminal region of the molecule.

The complex of silkworm larval hemolymph antichymotrypsin (Mr = 43,000) and C-chain of bovine alpha-chymotrypsin was obtained. This complex showed two NH2-terminal amino acid sequences identical to those of intact silkworm antichymotrypsin and C-chain of alpha-chymotrypsin, respectively. Alkali treatment of the complex brought about its dissociation and the separated inhibitor component (Mr = 36,000) had an NH2-terminal amino acid sequence identical to that of intact silkworm antichymotrypsin. These results suggest that the reactive site of this inhibitor is located at the COOH-terminal region of the molecule and that the nature of association of this inhibitor and alpha-chymotrypsin is an acyl-bond.

Amino Acid Sequence↗
PubMed1986

Transposable genetic element found in the 5'-flanking region of the fibroin H-chain gene in a genomic clone from the silkworm Bombyx mori.

A transposable genetic element was found in the 5'-flanking region of the fibroin H-chain gene in one of the genomic clones from the silkworm Bombyx mori. This element, named K-1.4, is about 1 X 4 X 10(3) base-pairs long, contains an open reading frame of only 225 base-pairs and has inverted repeats of 12 base-pairs at both ends. Duplication of three base-pairs seems to have occurred when this element was integrated into the silkworm genome. About 15 copies of K-1.4 are present per haploid genome of various silkworm strains. Genomic loci of some of these elements are different among different strains or even among individual offspring of the same parents. K-1.4 is present also in the genome of Bombyx mandarina. The K-1.4-related sequences are present in some species belonging to the family Saturniidae.

Animals↗
PubMedDate not supplied

High-level expression and characterization of hepatitis B virus surface antigen in silkworm using a baculovirus vector.

The Bombyx mori nuclear polyhedrosis virus (BmNPV) in the silkworm was used successfully for mass production of biologically active foreign genes under the control of the polyhedrin promoter. This system was adapted for the production of large amounts of hepatitis B virus surface antigens (HBsAg). The DNA fragments coded for the middle protein, which is composed of the S protein with the pre-S2 region, were cloned, the signal protein gene of beta-IFN was added, and both were inserted into a cloning vector. After co-transfection with wild-type BmNPV, stable recombinant viruses were isolated by the limiting dilution method. Infected silkworm larvae with the recombinants expressed HBsAg at high levels (400-600 micrograms/ml). These products, consisting of two polypeptides with molecular weights of approximately 25,000 (p25) and glycosylated P25 (GP28), were purified as assembled 22-nm particles. We demonstrated that HBsAg from silkworms consists of S protein with 7 amino acids of Pre-S2.

Amino Acid Sequence↗
PubMed1995

Molecular characterization of the gene encoding the precursor protein of diapause hormone and pheromone biosynthesis activating neuropeptide (DH-PBAN) of the silkworm, Bombyx mori and its distribution in some insects.

The diapause hormone is a 24 amino acid peptide amide which induces embryonic diapause of the silkworm, Bombyx mori. Diapause hormone, pheromone biosynthesis activating neuropeptide, and other three neuropeptides of FXPRL amide peptide family have been shown to be generated from a polyprotein precursor which is encoded by a single mRNA. We have cloned the genomic sequence encoding the precursor protein of diapause hormone-pheromone biosynthesis activating neuropeptide (DH-PBAN) by using a DH-PBAN cDNA as a probe, and analyzed its structure. The gene comprised six exons interspersed by five introns. The diapause hormone sequence along with a signal sequence was encoded in the first and second exons, and the pheromone biosynthesis activating neuropeptide was in the fourth and fifth exons. The major transcription initiation site of the gene was localized at 25 bp upstream from the translation start site. A single copy of this gene was present in a haploid genome. The 5'-upstream region of the gene contained a sequence similar to the ecdysone responsive element of Drosophila hsp 23 gene, and five decanucleotide motifs, which shared the homeodomain binding core sequence, TAAT. Genomic Southern analysis on DNA from some insect species other than the silkworm showed positive bands which hybridized with DH-PBAN cDNA of the silkworm. Thus, the DH-PBAN-like gene seems to be widely distributed in insects.

Amino Acid Sequence↗
PubMed1994

Molecular characterization of ovary trehalase of the silkworm, Bombyx mori and its transcriptional activation by diapause hormone.

We have isolated a cDNA encoding ovary trehalase of the silkworm, Bombyx mori. Sequence analyses revealed that the isolated cDNA contains 3143 nucleotides and comprises 579 amino acids, including a cleavable signal sequence and five potential N-glycosylation sites. Northern blot analysis showed a 3.0 kb transcript in developing ovaries carrying membrane-bound trehalase. A single copy of trehalase gene was present in the haploid genome of the silkworm. The effect of diapause hormone on the accumulation of trehalase mRNA was examined on developing ovaries in in vivo and in vitro conditions. The synthetic diapause hormone brought about a 6-fold increase in trehalase mRNA content in ovaries 4 h after injection. The similar increase was found in ovaries which were incubated in vitro with diapause hormone. Coincubation of ovaries with diapause hormone and actinomycin D could not increase the mRNA level in ovaries, and maintained a basal level which was found in ovaries incubated without diapause hormone. These results indicate that diapause hormone stimulates transcription of the trehalase gene in developing ovaries of the silkworm.

Amino Acid Sequence↗
PubMedDate not supplied

Oral immunization with Escherichia coli K-12 of the fifth instar larvae of the silkworm, Bombyx mori, reared on an artificial diet under completely aseptic conditions.

1. Effect of oral administration of live or formalin-treated Escherichia coli (E. coli) K-12 to the fifth instar, days 1 and 3 larvae of the silkworm, Bombyx mori, on induction of antibacterial activity in the haemolymph was investigated using the silkworms reared on an artificial diet under completely aseptic conditions. 2. When live E. coli was administered to the male day 1 larvae, low but significant antibacterial activity of 3.8 mm was detectable in the haemolymph of one individual at 48 hr after immunization. The proportion of the larvae to express antibacterial activity increased thereafter and at 120 hr after immunization, all three individuals showed antibacterial activity. In day 3 male larvae, activity was detectable at 48 and 72 hr after immunization. 3. When formalin-treated E. coli was orally administered to days 1 and 3 male larvae, no activity was detectable at any time post-immunization. 4. In the second experiment, when day 1 larvae, females and males were orally immunized with live E. coli, only females showed antibacterial activity in the haemolymph, beginning from 24 hr after immunization and up to 96 hr. 5. Removal of an antibiotic, chloramphenicol, from ingredients of an artificial diet was required for induction of antibacterial activity with oral administration of live E. coli. 6. When live E. coli that grows at pH 9.0 was selected and used for oral immunization, antibacterial activity was induced both in females and males at 72 hr after immunization and the activity was observed at 96 hr. 7. These results suggest that establishment of oral immunization with live E. coli in the silkworm larvae requires multiplication of E. coli in the midgut lumen and possibly its colonization on the luminal surface.

Administration, Oral↗
PubMed2026

Integrated transcriptomic and metabolomic analysis of fluoride tolerance-related pathways and differentially expressed genes in silkworm strain XSKD.

XueSong KD (XSKD) silkworm strain exhibits prominent fluoride tolerance, yet the underlying molecular mechanisms of fluoride tolerance remains unclear. In the present study, fourth-instar pre-molting XSKD silkworms were used as experimental materials for integrated transcriptomic and untargeted metabolomic analyses. In total, 572 differentially expressed genes and 90 differential metabolites were screened. GO enrichment and KEGG enrichment based on the hypergeometric distribution model revealed that 13-Hydroxy-9Z,11E-octadecadienoic acid (13-(S)-HODE) acts as the core differential metabolite, which is significantly enriched in the linoleic acid metabolism pathway. Within this pathway, LOC101737302 and CYP338A1 display opposite expression trends and show correlations with pathway metabolites. Based on multi-omics data, this study preliminarily characterizes the lipid metabolic response under fluoride stress, providing omics dataset support for further in-depth exploration of the molecular mechanism of fluoride tolerance in silkworms.

Animals↗
PubMed2004

In vivo DNA double-strand breaks enhance gene targeting in cultured silkworm cells.

Alteration of genomic information through homologous recombination (HR) is a powerful tool for reverse genetics in bacteria, yeast, and mice. The low frequency of HR is, however, a major obstacle to achieve efficient gene targeting. In this study, we have developed an assay system for investigating the frequency of gene targeting in cultured silkworm cells using a firefly luciferase gene as a reporter. The introduction of a DNA double-strand break (DSB) either in the chromosomal target locus or in the targeting construct drastically increased the frequency of gene targeting. Interestingly, the inhibition of poly(ADP-ribose) polymerase (PARP), a protein known to play an important role in overall suppression of the HR pathway, stimulated the targeting efficiency, whereas the overexpression of two silkworm RecA homologs, BmRad51 and BmDmc1, had no effect. The presently devised assay system may serve as a useful tool to improve the gene targeting efficiency in the silkworm (Bombyx mori).

Animals↗
PubMed2006

Identification and characterization of the telomerase reverse transcriptase of Bombyx mori (silkworm) and Tribolium castaneum (flour beetle).

Chromosomal ends of most eukaryotes are composed of simple telomeric repeats. Arthropod telomeres are generally constituted by TTAGG pentanucleotide repeats; however, some insect species including Drosophila melanogaster do not have telomeric repeats. In contrast, the domestic silkworm Bombyx mori contains TTAGG-type telomeric repeats, but the telomerase activity has not been detected in all investigated tissues. To search for a cause of unusual telomere structure in insects, we here identified telomerase reverse transcriptase (TERT) subunit from the domestic silkworm B. mori and the flour beetle Tribolium castaneum. This is the first report of telomerase genes from arthropods. The domestic silkworm TERT gene (BmoTERT) and the flour beetle TERT gene (TcasTERT) both did not have the N-terminal GQ motif. Comparison between cDNA and genomic DNA of BmoTERT revealed that it includes no introns. BmoTERT contains five ATG codons in its 5'UTR, which could reduce the translation of BmoTERT proteins. Also, Northern hybridization indicated that BmoTERT is transcribed at a very low level. These unique features of BmoTERT possibly explain the undetectable Bombyx telomerase activity.

5' Untranslated Regions↗
PubMed2005

Survey of long terminal repeat retrotransposons of domesticated silkworm (Bombyx mori).

Long terminal retrotransposons are major components of eukaryotic transposable elements. We have surveyed the long terminal repeats (LTR) retrotransposons of domesticated silkworm (Bombyx mori) by mining the data produced by Bombyx mori Genome Sequencing Project. At least 29 separate families of LTR retrotransposons are identified in this survey, comprising of 11.8% of the complete sequence. Families of domesticated silkworm LTR retrotransposons can be mainly classified into three groups: gypsy-like, copia-like, Pao-Bel. Fourteen families identified consist of gypsy-like elements, four families consist of copia-like elements and seven families consist of Pao-Bel elements. In addition to the three groups of LTR retrotransposons, two families of unusual non-coding elements are identified in the genome of this species. Further phylogenetic analysis of RT domain indicates that the elements of B.mori show high diversity and can form different clades in each group. An analysis of sequence variation from different families reveals distinct patterns of variation for the elements belonging to three groups. The analysis of the domesticated silkworm LTR retrotransposons should assist in our understanding of the roles of retroelement in lepidopteron insect genome evolution.

Animals↗
PubMed2005

Juvenile hormone diol kinase, a calcium-binding protein with kinase activity, from the silkworm, Bombyx mori.

Juvenile hormone (JH) diol kinase (JHDK) is an important enzyme involved in the JH degradation pathway. Bombyx mori (Bommo)-JHDK cDNA (637bp) contains an open reading frame encoding a 183-amino acid protein, which reveals a high degree of identity to the two previously reported JHDKs. JHDK is similar to GTP-binding proteins with three conserved sequence elements involved in purine nucleotide binding, contains eight alpha-helices and three EF-hand motifs, and resembles the three-dimensional model of 2SCP and some other calcium-binding proteins. The Bommo-JHDK gene has only a single copy in the silkworm haploid genome, contains only one exon, and its 5'-upstream sequence does not have a JH response element. Although Bommo-JHDK is highly expressed in the gut of the silkworm, its mRNA expression remains at a constant level during larval development suggesting this enzyme is constitutive and not regulated by JH, at least at the transcriptional level. Recombinant Bommo-JHDK catalyzed the conversion of 10S-JH diol into JH diol phosphate, confirming its enzymatic function. Recombinant enzyme formed a dimer and had biochemical characteristics similar to other JHDKs. Bommo-JHDK, a calcium-binding protein with kinase activity, provides unique insights on how JH levels are regulated in the silkworm.

Amino Acid Sequence↗
PubMed2006

A transgenic silkworm expressing the immune-inducible cecropin B-GFP reporter gene.

To analyze cecropin B promoter (P-CecB) activity in vivo, we constructed transgenic silkworms that expressed EGFP under the control of P-CecB using the piggyBac transposable element. Genomic Southern blot analysis of the G1 and G2 generations indicated the stable insertion of EGFP in the genome. Injection of Escherichia coli cells into the larvae strongly induced EGFP expression in the fat bodies and all five hemocyte cell types. Northern blot analysis indicated that the expression kinetics of EGFP in the fat bodies following bacterial injection were correlated with that of endogenous CecB. Flow cytometric analysis of the hemocytes revealed that EGFP expression was increased by bacteria, but not by yeast. Our results indicate that the features of EGFP expression in the transgenic silkworm are equivalent to those of endogenous CecB and that P-CecB activation can be monitored by EGFP expression using transgenic silkworms.

Animals↗
PubMed2004

Expression of human VEGF165 in silkworm (Bombyx mori L.) by using a recombinant baculovirus and its bioactivity assay.

Silkworm larva has a lot of advantages as a "biofactory" to produce recombinant protein. A recombinant baculovirus, carrying cDNA encoding the 165 amino-acid long isoform of human vascular endothelial growth factor (VEGF) was successfully constructed for the large-scale production of this protein using silkworms as an in vivo host. The fifth-instar silkworm larvae were inoculated with the recombinant virus. Time-course expression analysis indicated that the expression level was highest at around 80 h post-infection and the recombinant protein was found mainly in the haemolymph. Therefore, the hemolymph was collected from the infected larvae and the recombinant protein was purified by using Nickel affinity chromatography under native condition. The expression level was estimated to be as high as approximately 426 microg per larva. Furthermore, the recombinant protein was characterized and was found biologically active in inducing endothelial cell proliferation in vitro.

Amino Acid Sequence↗
PubMed2005

Oral administration of a cholera toxin B subunit-insulin fusion protein produced in silkworm protects against autoimmune diabetes.

The oral administration of disease-specific autoantigens can induce oral immune tolerance and prevent or delay the onset of autoimmune disease symptoms. Here, we describe the construction of an edible vaccine consisting of a fusion protein composed of cholera toxin B subunit (CTB) and insulin that is produced in silkworm larvae at levels of up to 0.3 mg/ml of hemolymph. The silkworm bioreactor produced this fusion protein vaccine as the pentameric CTB-insulin form, which retained the GM1-ganglioside binding affinity and the native antigenicity of CTB and insulin. Non-obese diabetic mice fed hemolymph containing microgram quantities of the CTB-insulin fusion protein showed a prominent reduction in pancreatic islet inflammation and a delay in the development of symptoms of clinical diabetes. These results demonstrate that the silkworm bioreactor is a feasible production and delivery system for an oral protein vaccine designed to develop immunological tolerance against T-cell-mediated autoimmune diabetes by regulatory T-cell induction.

Administration, Oral↗
PubMed2006

Suppression of diabetes in non-obese diabetic (NOD) mice by oral administration of a cholera toxin B subunit-insulin B chain fusion protein vaccine produced in silkworm.

Oral tolerance has been applied successfully as a potential therapeutic strategy for preventing and treating autoimmune diseases including type 1 diabetes. In this paper we constructed an edible vaccine consisting of a fusion protein composed of cholera toxin B subunit (CTB) and insulin B chain (InsB) that was produced in silkworm larvae. The silkworm larvae produced this fusion protein at levels of up to 0.97mg/ml of hemolymph as the pentameric CTB-InsB form, which retained the GM1-ganglioside binding affinity and the native antigenicity of CTB. Non-obese diabetic mice fed hemolymph containing microgram quantities of the CTB-InsB fusion protein showed a prominent reduction in pancreatic islet inflammation and a delay in the development of diabetic symptoms. This study demonstrates that silkworm-produced CTB-InsB fusion protein can be used as an ideal oral protein vaccine for induction of immunological tolerance against autoimmune diabetes.

Administration, Oral↗
PubMed2003

Inhibition of apoptosis by recombinant 30K protein originating from silkworm hemolymph.

In a previous study, we reported that silkworm hemolymph inhibits apoptosis and that the anti-apoptotic component in silkworm hemolymph is a 30K protein. In this study, the 30K protein encoded by 30Kc6 was expressed in Escherichia coli. The recombinant 30K protein was expressed as an inclusion body, and the inclusion body was separated and refolded by affinity column chromatography using a 6xHis tag. We demonstrated that apoptosis is inhibited by supplementing the culture medium with this purified recombinant 30K protein. The recombinant 30K protein inhibited the virus- or chemical-induced apoptosis in human cells as well as insect cells. Apoptosis-inhibitory activity of recombinant 30K protein was comparable to that of whole silkworm hemolymph. The recombinant 30K protein can be effectively used to minimize cell death and consequently increase the productivity by extending the production time of host cells in commercial animal cell culture.

Amino Acid Sequence↗