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PubMed2000

Production of canine IFN-gamma in silkworm by recombinant baculovirus and characterization of the product.

Canine interferon-gamma (CaIFN-gamma) cDNA was expressed in silkworm by infecting recombinant baculovirus. Western blot analyses revealed that the resulting preparation contained various CaIFN-gamma protein molecules. Genetic engineering of CaIFN-gamma to remove potential glycosylation sites resulted in reduced components of the CaIFN-gamma, suggesting that one cause of this heterogeneity was glycosylation. Nonglycosylated CaIFN-gamma produced in silkworm still had several components that were deleted at the carboxy-terminal end. The major component was the CaIFN-gamma protein truncated 17 or 16 carboxy-terminal amino acid residues. CaIFN-gamma showed antiviral activity, class II major histocompatibility complex (MHC) expression-enhancing activity, and antiproliferation activity on tumor cells.

Animals↗
PubMed2003

Targeted gene expression using the GAL4/UAS system in the silkworm Bombyx mori.

The silkworm Bombyx mori is one of the most well-studied insects in terms of both genetics and physiology and is recognized as the model lepidopteran insect. To develop an efficient system for analyzing gene function in the silkworm, we investigated the feasibility of using the GAL4/UAS system in conjunction with piggyBac vector-mediated germ-line transformation for targeted gene expression. To drive the GAL4 gene, we used two endogenous promoters that originated from the B. mori actin A3 (BmA3) and fibroin light-chain (FiL) genes and the artificial promoter 3xP3. GFP was used as the reporter. In initial tests of the function of the GAL4/UAS system, we generated transgenic animals that carried the UAS-GFP construct plus either BmA3-GAL4 or 3xP3-GAL4. GFP fluorescence was observed in the tissues of GFP-positive animals, in which both promoters drove GAL4 gene expression. Animals that possessed only the GAL4 gene or UAS-GFP construct did not show GFP fluorescence. In addition, as a further test of the ability of the GAL4/UAS system to drive tissue-specific expression we constructed FiL-GAL4 lines with 3xP3-CFP as the transformation marker. FiL-GAL4 x UAS-GFP crosses showed GFP expression in the posterior silk gland, in which the endogenous FiL gene is normally expressed. These results show that the GAL4/UAS system is applicable to B. mori and emphasize the potential of this system for controlled analyses of B. mori gene function.

Animals↗
PubMed2005

The Bmdsx transgene including trimmed introns is sex-specifically spliced in tissues of the silkworm, Bombyx mori.

Bmdsx is an orthologue of the sex-determining gene doublesex (dsx) and known to be sex-specifically expressed in various tissues of the silkworm, Bombyx mori. Its pre-mRNA is sex-specifically spliced and encodes female-specific or male-specific polypeptides. The open reading frame of Bmdsx consists of 5 exons, of which exons 3 and 4 are female-specific and its pre-mRNA was known to undergo default processing to generate the female-type mRNA. Previous reports have shown that the mechanism of splicing of the doublesex gene is different in Drosophila melanogaster and Bombyx mori. However, intron 4 is so long that it is difficult to identify the intronic cis-element(s) required for male-specific splicing of Bmdsx pre-mRNA using Bmdsx minigenes whose introns are shortened in various manners. As a first step toward discovery of the cis-element, the Bmdsx mini gene, which consisted of exon 1 and 5 and internally shortened introns 2 to 4, was constructed, and transgenic silkworms expressing this construct were generated. Bmdsx pre-mRNA transcribed derived from transgene was sex-specifically spliced. This result shows that the mini gene contained the information necessary for the correct regulation of alternative splicing.

Alternative Splicing↗
PubMed1993

Transcription of a silkworm tRNA(cAla) gene is directed by two AT-rich upstream sequence elements.

A region within 35 nucleotides upstream of the transcription initiation site of a variety of silkworm Class III templates is absolutely required for transcription in vitro. To determine whether the activity of this region can be attributed to a particular sequence element, we systematically replaced 4-5 bp segments of the region upstream of a silkworm tRNA(cAla) gene. We show that replacement of either of two AT-rich blocks markedly impairs promoter function, whereas replacement of other sequences has little or no effect. Additional mutants were constructed to test whether base composition or sequence is important for function of the AT blocks. We find that some sequences are more effective than others, but that various AT-rich sequences can direct transcription at a high level. Possible mechanisms by which such elements could act are discussed.

Animals↗
PubMed1997

A new family of site-specific retrotransposons, SART1, is inserted into telomeric repeats of the silkworm, Bombyx mori.

The telomeres of the silkworm, Bombyx mori, consist of pentanucleotide repeats (TTAGG)n . We previously characterized the non-LTR element TRAS1, which terminates with oligo (A) in a head to tail orientation at the exact position (between A and C) of the (CCTAA) n repeats. Here we characterized another family of telomere-specific non-LTR retrotransposon named SART1. The SART1 family was inserted at another site of the (TTAGG) n in a reverse orientation from that of TRAS1. The complete unit of SART1, 6.7 kb in length with a poly (A) stretch, contains two open reading frames encoding putative gag and pol products, overlapping by 54 bp in the -1 reading frame. Most of the 600 SART1 copies in the silkworm haploid genome are completely conserved in structure without 5'truncation. All SART1 sequences analyzed were inserted at the same position (between T and A) within the (TTAGG) n repeats. Fluorescence in situ hybridization showed that many of the SART1 copies were localized in the chromosomal ends. A phylogenetic tree showed that the SART1, TRAS1 and two other site-specific elements, R1 and RT, which insert into 28S ribosomal RNA genes in insects, belong to the same group. Based on the orientation for the chromosomal insertion and structural similarities, these elements could be further classified into two subgroups, R1/TRAS1 and RT/SART1, suggesting that the target specificity of the two telomere-associated elements was changed independently.

Animals↗
PubMed1999

Transcription analysis of the telomeric repeat-specific retrotransposons TRAS1 and SART1 of the silkworm Bombyx mori.

The telomeres of the silkworm Bombyx mori consist of (TTAGG)n repeats and harbor a large number of sequence-specific non-LTR retrotransposons such as TRAS1 and SART1. In order to ascertain if TRAS1 and SART1 are transcribed in vivo and if there is a novel transcription mechanism peculiar to the sequence-specific retrotransposons, we studied their transcription. We detected transcripts of TRAS1 and SART1 by northern hybridization in many tissues and the BmN4 cell line of the silkworm. 5'-Rapid amplification of cDNA ends analysis showed that transcription of both elements was initiated precisely from their own 5'-ends and that most of their genomic copies contained these initiation sites. TRAS1 contained an internal promoter and positively regulating elements in the +1/+581 nucleotides in its 2432 bp 5'-untranslated region (UTR). We could not, however, detect any promoter activity in the SART1 5'-UTR. This difference may be related to the fact that only TRAS1 contained an initiator-like element at its 5'-end. Placing 1-52 units of the telomeric repeat (TTAGG)n upstream of TRAS1 reduced transcription 5-fold. The evidence suggests that most of the TRAS1 genomic copies within the telomeric repeats are weakly transcribed in vivo.

5' Untranslated Regions↗
PubMed1987

Interaction of silkworm larval hemolymph antitrypsin and bovine trypsin.

Antitrypsin isolated from silkworm larval hemolymph can inhibit bovine trypsin. The apparent molar ratio of silkworm antitrypsin (sw-AT) to trypsin at extrapolated null trypsin activity was determined to be 1.3 by titration of trypsin with sw-AT. The undissociability of the complex between sw-AT and trypsin on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was confirmed by immunoblotting and fluorescence labeling techniques. Chemical analysis of the complex elucidated that it contained equimolar sw-AT and trypsin. Densitometric analysis of electrophoretic patterns obtained during the titration of trypsin by sw-AT suggested the presence of a suicide product formed from sw-AT. This was the reason why excess sw-AT was needed for complete inhibition of trypsin. In the complex, the sw-AT molecule was cut at one site but the fragments produced were still joined together. Trypsin in the complex was released by treatment at pH 10.0, and it was deduced that the complex formation involved acyl-bond formation between sw-AT and trypsin. The sw-AT component obtained from the alkali-treated complex possessed two kinds of NH2-terminal amino acid sequences. Non-covalent forces may bind the two fragments of sw-AT, which could be separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Amino-terminal amino acid sequence analysis of the large fragment gave a sequence identical with that of intact sw-AT. This indicated that the reactive site of sw-AT with trypsin was located at the COOH-terminal region of the molecule. These characteristics resemble those of inhibitors belonging to the serpin family.

Amino Acid Sequence↗
PubMed1989

Membrane anchors of alkaline phosphatase and trehalase associated with the plasma membrane of larval midgut epithelial cells of the silkworm, Bombyx mori.

The larval midgut epithelial cell of the silkworm, Bombyx mori, has two forms of alkaline phosphatase and trehalase, soluble and membrane-bound. Alkaline phosphatase and trehalase of the latter form are found in the brush border membrane and the basolateral membrane, respectively. In this work we studied the membrane anchors of these membrane-bound enzymes. Alkaline phosphatase was solubilized by phosphatidyl-inositol-specific phospholipase C, but not by papain. Conversely, trehalase was released from the membrane by papain, but not by phosphatidylinositol-specific phospholipase C. Both enzymes were solubilized in an amphiphilic form with 0.5% Triton X-100 plus 0.5% sodium deoxycholate (pH 7.0). The detergent-solubilized alkaline phosphatase and trehalase were converted to hydrophilic form on incubation with phosphatidylinositol-specific phospholipase C and papain, respectively. The effects of papain on solubilization and conversion of trehalase were completely inhibited by leupeptin. These results suggest that, in the silkworm larvae, alkaline phosphatase is anchored in the brush-border membrane via a glycosyl-phosphatidylinositol, while trehalase is associated with the basolateral membrane through a hydrophobic segment of the polypeptide.

Alkaline Phosphatase↗
PubMed1994

Expression and characterization of human bone morphogenetic protein-2 in silkworm larvae infected with recombinant Bombyx mori nuclear polyhedrosis virus.

Recombinant human bone morphogenetic protein-2 (rhBMP-2) was expressed in silkworm larvae, and a milligram quantity of the protein was purified and characterized. The expressed rhBMP-2 was biologically active in terms of induction of alkaline phosphatase activity in MC3T3-E1 cells and ectopic bone formation in mice. On SDS-polyacrylamide gel electrophoretic analysis, the purified protein showed a 16 kDa band under reducing conditions and a 30 kDa band under non-reducing conditions. The silkworm-expressed rhBMP-2 was glycosylated and susceptible to endo-beta-N-acetylglucosaminidase F (endo F) and endo H, but resistant to endo D. Deglycosylated rhBMP-2 treated with endo F retained its biological activity. These results suggest that rhBMP-2 exists as a dimer and disulfide bond(s) are responsible for the dimerization. Moreover, sugar chains have no direct effect on the biological activity of the protein. The availability of a quite large amount of rhBMP-2 has allowed us to study the biological function of this interesting factor in detail.

3T3 Cells↗
PubMed1985

Isoprenoid enzyme systems of silkworm. II. Formation of the juvenile hormone skeletons by farnesyl pyrophosphate synthetase II.

Comparative substrate specificities of farnesyl pyrophosphate synthetases I and II purified from larvae of silkworm, Bombyx mori, were studied by use of the possible biosynthetic intermediates of juvenile hormones in the insect. In the presence of Mn2+ ions farnesyl pyrophosphate synthetase II showed higher activity than synthetase I and the corresponding enzyme from pig liver with the following substrate homologues: (Z)-3-methyl-2-pentenyl-, 3-ethyl-3-butenyl-, (2E,6Z)-3,7-dimethyl-2,6-nonadienyl-, and (2E,6Z)-3-ethyl-7-methyl-2,6-nonadienyl pyrophosphate. When (Z)-3-methyl-2-pentenyl-, 3-ethyl-3-butenyl-, and isopentenyl pyrophosphate were mixed and incubated with farnesyl pyrophosphate synthetase II, (2E,6E,10Z)-3,11-dimethyl-7-ethyl-2,6,10-tridecatrienyl-, (2E,6E,10Z)-3,7,11-trimethyl-2,6,10-tridecatrienyl, and a trace amount of (2E,6E,10Z)-3,7-diethyl-11-methyl-2,6,10-tridecatrienyl pyrophosphate, whose carbon skeletons were the same as those of juvenile hormone I, II, and O, respectively, were formed. (Z)-3-Methyl-2-pentenyl pyrophosphate was produced from 3-ethyl-3-butenyl pyrophosphate as a single product by the action of silkworm isopentenyl pyrophosphate isomerase, though the enzyme activity was much lower with this substrate than with the usual substrate, isopentenyl pyrophosphate.

Animals↗
PubMed1992

Induction of a novel protein kinase in pupae of the silkworm Bombyx mori after infection with nuclear polyhedrosis virus.

Protein kinases induced by Bombyx mori nuclear polyhedrosis virus in pupae of the silkworm B. mori were examined by activity gel analysis using phosvitin as a protein substrate. The method involved PAGE of the soluble fraction from pupae under native conditions and in the presence of SDS, followed by in situ renaturation of proteins and recovery of protein kinase activity in the intact gel. A novel protein kinase able to phosphorylate phosvitin was detected in the infected pupae from 2 days post-infection. This enzyme was not present in uninfected silkworms at any stage of the pupal period. The novel kinase activity was found by SDS-PAGE to be associated with a single polypeptide with an apparent M(r) of 50K. However, on electrophoresis under native conditions its activity was associated with a set of polypeptides with similar but not identical electrophoretic mobilities. Microheterogeneity of the catalytically active polypeptides suggests that the virus-induced protein kinase undergoes post-translational modification during the course of infection.

Animals↗
PubMed2004

Molecular diversity and phylogenetic analysis of mariner-like transposons in the genome of the silkworm Bombyx mori.

Genome-wide screening of mariner-like elements (MLEs) in the silkworm Bombyx mori has revealed the presence of five different types of MLEs (Bmmar1, Bmmar2, Bmmar3, Bmmar4 and Bmmar5). We isolated and characterized sixty copies of the MLEs representing the five Bmmar types. Their nucleotide sequences, nucleotide compositions, deduced transposase sequences, codon preferences, and the copy numbers showed extensive variations. Phylogenetic analysis of the sequences revealed that Bmmar1, Bmmar2, and Bmmar3 have been in the B. mori genome for a long time, while Bmmar4 is probably a recent invader of the genome. Because of the long-term association of Bmmar1 and Bmmar2 with the genome, highly mutated miniature Bmmar1 and Bmmar2 are widespread in the genome, and the footprints of these elements are also present in different silkworm genes. However, miniature copies of Bmmar4 were not detected. This recently acquired element has very few mutations. None of the characterized copies had functional transposase open reading frames. They essentially exist as fossils in the genome.

Amino Acid Sequence↗
PubMed2005

Partial deletions of the W chromosome due to reciprocal translocation in the silkworm Bombyx mori.

In the silkworm, Bombyx mori (female, ZW; male, ZZ), femaleness is determined by the presence of a single W chromosome, irrespective of the number of autosomes or Z chromosomes. The W chromosome is devoid of functional genes, except the putative female-determining gene (Fem). However, there are strains in which chromosomal fragments containing autosomal markers have been translocated on to W. In this study, we analysed the W chromosomal regions of the Zebra-W strain (T(W;3)Ze chromosome) and the Black-egg-W strain (T(W;10)+(w-2) chromosome) at the molecular level. Initially, we undertook a project to identify W-specific RAPD markers, in addition to the three already established W-specific RAPD markers (W-Kabuki, W-Samurai and W-Kamikaze). Following the screening of 3648 arbitrary 10-mer primers, we obtained nine W-specific RAPD marker sequences (W-Bonsai, W-Mikan, W-Musashi, W-Rikishi, W-Sakura, W-Sasuke, W-Yukemuri-L, W-Yukemuri-S and BMC1-Kabuki), almost all of which contained the border regions of retrotransposons, namely portions of nested retrotransposons. We confirmed the presence of eleven out of twelve W-specific RAPD markers in the normal W chromosomes of twenty-five silkworm strains maintained in Japan. These results indicate that the W chromosomes of the strains in Japan are almost identical in type. The Zebra-W strain (T(W;3)Ze chromosome) lacked the W-Samurai and W-Mikan RAPD markers and the Black-egg-W strain (T(W;10)+(w-2) chromosome) lacked the W-Mikan RAPD marker. These results strongly indicate that the regions containing the W-Samurai and W-Mikan RAPD markers or the W-Mikan RAPD marker were deleted in the T(W;3)Ze and T(W;10)+(w-2) chromosomes, respectively, due to reciprocal translocation between the W chromosome and the autosome. This deletion apparently does not affect the expression of Fem; therefore, this deleted region of the W chromosome does not contain the putative Fem gene.

Animals↗
PubMed1990

Purification and characterization of a cysteine proteinase from silkworm eggs.

Eggs of the silkworm, Bombyx mori, contain a high level of a proteinase which is most active in acidic pH region. The proteinase was purified from an extract of eggs by a six-step procedure which included conventional chromatographic fractionations. The molecular mass of the proteinase was estimated to be 350 kDa by gel filtration and 47 kDa by electrophoresis on sodium dodecyl sulfate/polyacrylamide gels, suggesting an octameric structure. The amino acid composition was found to resemble that of mammalian lysosomal cysteine proteinases, in particular cathepsin L. The NH2-terminal 10-residue sequence is Val-Gln-Phe-Phe-Asp-Leu-Val-Lys-Glu-Glu-. The enzyme appears to be a member of the class of cysteine proteinases since it was strongly inhibited by sulfhydryl-reactive compounds and N-[N-(1,3-trans-carboxyoxiran-2-carbonyl)-L-leucyl]-agmatine (E-64). The enzyme hydrolyzed various protein substrates, such as hemoglobin, vitellogenin, vitellin, and lipophorin, with maximal activity around pH 3-3.5. The specificity of the cleavage sites in the oxidized B chain of insulin was rather well defined and there was high affinity for hydrophobic residues at the P2 and P3 positions. The cysteine proteinase is thought to be involved in protein degradation during embryonic development of silkworm eggs.

Amino Acid Sequence↗
PubMed1993

Molecular cloning of silkworm (Bombyx mori) antichymotrypsin. A new member of the serpin superfamily of proteins from insects.

The cDNA of silkworm (Bombyx mori) antichymotrypsin (sw-Achy) was cloned from larval fat body and its nucleotide sequence was determined. The deduced amino acid sequence of mature sw-Achy begins with Phe1 and ends with Phe384, with a preceding 16-amino-acid signal peptide. The amino-acid sequence similarities of sw-Achy with the serine-proteinase inhibitors (serpins) silkworm antitrypsin, tobacco hornworm alaserpin, human alpha-1-antitrypsin and human alpha-1-antichymotrypsin were 29.6%, 30.3%, 26.1%, and 25.0%, respectively. The highly conserved amino acids in other serpins are also conserved in sw-Achy. sw-Achy is thought to be a new member of the serpin family. Multiple alignment of sw-Achy with 23 other kinds of serpin by the progressive method produced a phylogenetic tree in which all four insect serpins are grouped separately within one branch. The reactive site of sw-Achy with alpha-chymotrypsin was identified as Thr343-Ser344 by direct amino-acid sequence analysis of cleaved and purified protein.

Amino Acid Sequence↗
PubMed1997

Lipopolysaccharide-binding proteins and their involvement in the bacterial clearance from the hemolymph of the silkworm Bombyx mori.

Proteins having the ability to bind to Escherichia coli K12W3110 (rough (R) mutant) were isolated and purified by affinity precipitation from the larval hemolymph of the silkworm Bombyx mori. These proteins were found to consist of two components with molecular masses of 43 kDa and 40 kDa by SDS/PAGE. They bound to all E. coli R mutants (Ra, Rb1, Rc, Rd1 and Re) and Salmonella minnesota R mutants. However, they did not bind to smooth types of the above bacteria. They bound to both lipopolysaccharide(LPS)-coated and lipid-A-coated microtiter plates and have similar dissociation constants for LPS and lipid A. This indicates that the binding proteins recognize the lipid A portion of LPS and thus, we have named these proteins BmLBP (B. mori LPS-binding proteins). We also found that BmLBP participated in the clearance of E. coli cells injected into the body cavity of the silkworm.

Acute-Phase Proteins↗
PubMed1996

Detection of peptidoglycan and beta-glucan with silkworm larvae plasma test.

A method to detect peptidoglycan and (1-->3) beta-D-glucan with silkworm larvae plasma (SLP) derived from the hemolymph of the silkworm, Bombyx mori was developed. SLP contains all of the factors of the pro-phenol oxidase cascade, an important self-defense mechanism of insects. Peptidoglycan or (1-->3)-beta-D-glucan initiates the cascade, in which pro-phenol oxidase is finally activated to phenol oxidase. The phenol oxidase activity was colorimetrically or visually detected with 3,4-dihydroxyphenylalanine as a substrate. SLP displayed high reactivity with peptidoglycan and polysaccharides containing 1,3-beta-glucosidic linkages, but not with endotoxins. SLP is useful for the detection of microbial contamination because peptidoglycan and (1-->3)-beta-D-glucan are cell wall components of bacteria and fungi, respectively.

Animals↗
PubMed2000

Detection of peptidoglycan in human plasma using the silkworm larvae plasma test.

Silkworm larvae plasma (SLP) reagent, which is prepared from the body fluid of the silkworm, reacts with peptidoglycan (PG), a fragment of both the Gram-positive and Gram-negative bacterial cell wall, as well as with beta-glucan, a component of fungi. We developed a quantitative method for the detection of PG in human plasma from cases with bacterial infection using the SLP reagent. Tested in this way, the SLP method showed 86.2% sensitivity, 90.6% specificity, 89.3% positive predictive value, and 88.5% efficiency. The SLP method provides a valuable tool for the diagnosis of systemic infection using patients' blood.

Animals↗