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PubMed2000

Germline transformation of the silkworm Bombyx mori L. using a piggyBac transposon-derived vector.

We have developed a system for stable germline transformation in the silkworm Bombyx mori L. using piggyBac, a transposon discovered in the lepidopteran Trichoplusia ni. The transformation constructs consist of the piggyBac inverted terminal repeats flanking a fusion of the B. mori cytoplasmic actin gene BmA3 promoter and the green fluorescent protein (GFP). A nonautonomous helper plasmid encodes the piggyBac transposase. The reporter gene construct was coinjected into preblastoderm eggs of two strains of B. mori. Approximately 2% of the individuals in the G1 broods expressed GFP. DNA analyses of GFP-positive G1 silkworms revealed that multiple independent insertions occurred frequently. The transgene was stably transferred to the next generation through normal Mendelian inheritance. The presence of the inverted terminal repeats of piggyBac and the characteristic TTAA sequence at the borders of all the analyzed inserts confirmed that transformation resulted from precise transposition events. This efficient method of stable gene transfer in a lepidopteran insect opens the way for promising basic research and biotechnological applications.

Actins↗
PubMed1995

Transovarian transmission of a foreign gene in the silkworm, Bombyx mori, by Autographa californica nuclear polyhedrosis virus.

We introduced a firefly luciferase gene, expressed under control of Drosophila heat shock protein gene promoter, into Autographa californica nuclear polyhedrosis virus (AcNPV). When the 5th instar larvae of the silkworm, Bombyx mori, were inoculated with the recombinant virus, luciferase activities were detected in the virus-infected larvae and pupae, and in the newly hatched larvae of the next generation. PCR amplification and Southern blot hybridization analysis demonstrated that the luciferase gene was transmitted through at least the F2 generation. In addition, the V-cathepsin gene, encoding a cysteine protease of AcNPV, was also detected in the DNA of all individuals of the F2 generation. These results show that AcNPV can be utilized as vector for the transovarian transmission of foreign genes in the silkworm.

Animals↗
PubMed1999

Structure and expression of tandemly duplicated xanthine dehydrogenase genes of the silkworm (Bombyx mori).

Xanthine dehydrogenase (XDH) is a molybdoenzyme which catalyses oxidation of xanthine and hypoxanthine to uric acid. We isolated genomic clones of silkworm (Bombyx mori) XDH genes (BmXDH1 and BmXDH2). The BmXDH2 gene is located upstream from the BmXDH1 gene and they show a tandemly duplicated structure. Both BmXDH genes were expressed in the fat body and Malpighian tubules, whereas only the BmXDH1 gene was expressed in the midgut. Phylogenetic analysis indicates that BmXDH gene duplication occurred after the divergence of the silkworm and dipteran species. Intron insertion site comparison shows that some introns were lost during insect XDH gene evolution.

Amino Acid Sequence↗
PubMed2000

Extrachromosomal transposition of the transposable element Minos occurs in embryos of the silkworm Bombyx mori.

To assess the ability of the transposable element Minos to act as a vector for genetic manipulation of the silkworm Bombyx mori, an extrachromosomal transposition assay based on three plasmids was performed. The three plasmids - helper, donor and target - were co-injected into preblastoderm embryos. Low molecular weight DNA was extracted from the embryos at the stage of blastokinesis and used to transform Escherichia coli. High frequency of transposition was observed in the presence of a helper plasmid possessing an intronless Minos transposase gene, whereas transposition did not occur in the presence of a helper plasmid with the intron-bearing transposase gene. Sequence analysis of the insertion sites showed that Minos always inserts into a TA dinucleotide. Although the insertions are distributed throughout the target gene, there was a preference for certain insertion sites. However, no consensus could be identified in the sequence flanking the target site. The results strongly suggest that the transposable element Minos has the potential to be used as a vector in the silkworm and probably in other lepidopteran insects.

Animals↗
PubMed2002

Nested retrotransposons on the W chromosome of the wild silkworm Bombyx mandarina.

The W chromosome of the silkworms Bombyx mori or B. mandarina is recombinationally isolated from the Z chromosome and the autosomes. We previously characterized a female-specific randomly amplified polymorphic DNA (RAPD), designated W-Yamato, derived from the W chromosome of the wild silkworm Bombyx mandarina. To further analyse the W chromosome of B. mandarina, we obtained a lambda phage clone that contains the W-Yamato RAPD sequence and sequenced the 16.7 kb DNA insert. We found that this DNA comprises a nested structure of at least seven elements: six retrotransposons and one transposable element-like sequence. The transposable element-like sequence is inserted into a micropia-like retrotransposon (Karate). The Karate and the non-long terminal repeat (non-LTR) retrotransposon BMC1 are inserted into a 412-like retrotransposon (Judo). Furthermore, this Judo, and two non-LTR retrotransposons (Kurosawa and Kendo) are inserted into a Pao-like retrotransposon (Yamato). These results indicate that the retrotransposons inserted into the W chromosome are not efficiently removed but accumulate gradually as strata without recombination.

Animals↗
PubMed2000

Molecular cloning of NADPH-cytochrome P450 oxidoreductase from silkworm eggs. Its involvement in 20-hydroxyecdysone biosynthesis during embryonic development.

Using RT-PCR, a cDNA fragment of NADPH-cytochrome P450 oxidoreductase from silkworm, Bombyx mori, was cloned from three-day-old nondiapause eggs. RACE was used to isolate the ends of the DNA. The full-length cDNA obtained was composed of 3471 bp with an open reading frame encoding a protein of 687 amino-acid residues with a relative molecular mass of 77 700. The protein, fused with glutathione S-transferase, was expressed in Escherichia coli and purified to homogeneity. The fused protein not only had NADPH-dependent cytochrome c-reducing activity, but also acted as an electron carrier from NADPH to bovine adrenal 21-hydroxylase P450 in the steroid hydroxylation reaction, confirming that the protein is the silkworm NADPH-cytochrome P450 oxidoreductase. Ecdysone 20-hydroxylase activity in the nondiapause egg microsomes increased until the fourth day after oviposition, and then decreased, little being detected on the ninth day. An antibody raised against the P450 reductase inhibited the ecdysone hydroxylation. Immunoblot analyses of the microsomes indicated that the P450 reductase protein appeared distinctly in the three-day-old nondiapause eggs and, in contrast to the developmental pattern of ecdysone hydroxylase activity, continued to increase as the embryos developed. These results suggest that ecdysone hydroxylation in the early stage of embryogenesis is dependent on the presence of both P450 reductase and ecdysone 20-hydroxylase P450, but its gradual reduction in the later stage may be due to the decrease in the level of ecdysone 20-hydroxylase P450.

Amino Acid Sequence↗
PubMed1997

Behaviors of nucleus, basal bodies and microtubules during eupyrene and apyrene spermiogenesis in the silkworm, Bombyx mori (Lepidoptera).

Observations with immunostaining for tubulin and electron microscopy revealed that silkworm eupyrene spermiogenesis was characterized by an attachment of the basal body to the nucleus except in the period of movement for unidirectional arrangement. In young eupyrene sperm, a microtubule basket caught the nucleus, which thereafter was transformed elliptically. Microtubules were also observed along the elongated acrosome and mitochondrial derivatives. During apyrene spermiogenesis, however, the basal body was not attached to the nucleus and approached the head cyst cell after the completion of unidirectional arrangement, leaving the round nucleus in the middle of the cell. The presence or absence of the phenomenon in which the basal body attaches to the nucleus seems to be essential in the course of diverse spermatogenesis of the silkworm.

Acrosome↗
PubMed1994

L-thyroxine (T4) elevates the free amino acid pool of haemolymph plasma of tasar silkworm, Antheraea mylitta drury (Lepidoptera: Saturniidae).

Concentration of free amino acids (FAA) in the haemolymph plasma of male and female tasar silkworm, A. mylitta, during fifth larval instar were determined by amino acid analyzer after administration of vertebrate thyroxine (T4). Twenty FAA have been identified in haemolymph plasma in both sexes, out of which ten were found to be predominating. Treatment with anabolic doses of thyroxine (0.5 and 1.0 micrograms/g) were able to enhance the titre of all the individual FAA which has been also reflected in the total concentrations. A specific pattern of variations in plasma FAA recorded during 5th larval instar, was not altered by the administered thyroxine. Our results thus indicate that T4 has a controlling influence on the FAA profiles of silkworm as observed in other biochemical parameters of the insect.

Aging↗
PubMed1996

Purification of a peptidoglycan recognition protein from hemolymph of the silkworm, Bombyx mori.

A method was developed for obtaining a homogeneous silkworm hemolymph protein (peptidoglycan recognition protein, PGRP) which has affinity for peptidoglycan and the ability to trigger the prophenoloxidase cascade upon its binding to peptidoglycan. The purified PGRP had a molecular mass of about 19 kDa and is composed of a single polypeptide with an isoelectric point of 6.5. It bound to peptidoglycan in the absence of divalent cation, whereas its binding to beta1,3-glucan and chitin was not detected. N-Acetyl-D-glucosaminyl-(beta1-4)-N-acetylmuramyl-L-alanyl-D-isogluta mine did not inhibit purified PGRP to bind insoluble peptidoglycan, but fragmented soluble peptidoglycan did. PGRP seemed to require peptidoglycan as a possible ligand to keep its glycan portion consisting of at least two or more of the repeating unit. PGRP did not have any detectable lysozyme activity, and its amino acid composition and amino-terminal sequence of 20 amino acid residues were shown to be different from those of silkworm lysozyme. PGRP seems to be a hitherto unknown protein. In the absence of PGRP, the prophenoloxidase cascade in the plasma fraction of hemolymph could not be triggered by peptidoglycan, indicating that some type of activity, capable of activating the cascade, is generated upon their binding. However, the exact nature of this activity is not yet known. The purified PGRP bound to peptidoglycan did not hydrolyze significantly any of the 26 commercially available peptidyl-7-amino-4-methylcoumarins, substrates for various proteases.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed1999

Prophenoloxidase-activating enzyme of the silkworm, Bombyx mori. Purification, characterization, and cDNA cloning.

Prophenoloxidase-activating enzyme (PPAE) was purified to homogeneity as judged by SDS-polyacrylamide gel electrophoresis from larval cuticles of the silkworm, Bombyx mori. The purified PPAE preparation was shown to be a mixture of the isozymes of PPAE (PPAE-I and PPAE-II), which were eluted at different retention times in reversed-phase high performance liquid chromatography. PPAE-I and PPAE-II seemed to be post translationally modified isozymes and/or allelic variants. Both PPAE isozymes were proteins composed of two polypeptides (heavy and light chains) that are linked by disulfide linkage(s) and glycosylated serine proteases. The results of cDNA cloning, peptide mapping, and amino acid sequencing of PPAE revealed that PPAE is synthesized as prepro-PPAE with 441 amino acid residues and is activated from pro-PPAE by cleavage of a peptide bond between Lys152 and Ile153. The homology search showed 36.9% identity of PPAE to easter, which is a serine protease involved in dorso-ventral pattern formation in the Drosophila embryo, and indicated the presence of two consecutive clip-like domains in the light chain. A single copy of the PPAE gene was suggested to be present in the silkworm genome. In the fifth instar larvae, PPAE transcripts were detected in the integument, hemocytes, and salivary glands but not in the fat body or mid gut. A polypeptide cross-reactive to mono-specific anti-PPAE/IgG was transiently detected in the extract of eggs between 1 and 3 h after they were laid.

Amino Acid Sequence↗
PubMed2000

The chitinase gene of the silkworm, Bombyx mori, contains a novel Tc-like transposable element.

We have determined the cDNA sequence and the genomic organization of the chitinase gene of the silkworm, Bombyx mori. The cDNA encodes 544 amino acids having 83% amino acid homology to the chitinase of the tobacoo hornworm, Manduca sexta. The total length of the gene is larger than 25 kilobase pairs, and it is separated into 11 exons. The intron-exon boundaries are all in accordance with the GT-AG rule. Also, the TATA box sequence was found in the 5' upstream region of the gene, and the gene is mapped on the seventh chromosome. A novel DNA type transposon that shows similarity to the Tc-like element was found in the third intron in some strains of B. mori; other strains, however, lack this element in the same intron. This element has long terminal inverted repeats, presumably encodes a transposase of about 340 amino acids with a DDE motif, and has an amino-terminal domain with a strong nuclear localization function. Seven other transposable elements with homologous but distinct sequences were isolated from the B. mori genome. Together with plaque hybridization results, our findings suggest that these novel elements exist in multiple copies constituting a new Tc-like transposable element family in the silkworm genome.

Amino Acid Sequence↗
PubMed2002

Malpighi, Swammerdam and the colourful silkworm: replication and visual representation in early modern science.

In 1669, Malpighi published the first systematic dissection of an insect. The manuscript of this work contains a striking water-colour of the silkworm, which is described here for the first time. On repeating Malpighi's pioneering investigation, Swammerdam found what he thought were a number of errors, but was hampered by Malpighi's failure to explain his techniques. This may explain Swammerdam's subsequent description of his methods. In 1675, as he was about to abandon his scientific researches for a life of religious contemplation, Swammerdam destroyed his manuscript on the silkworm, but not before sending the drawings to Malpighi. These figures, with their rich and unique use of colour, are studied here for the first time. The role played by Henry Oldenburg, secretary of the Royal Society, in encouraging contact between the two men is emphasized and the way this exchange reveals the development of some key features of modern science - replication and modern scientific illustration - is discussed.

Animals↗
PubMed1983

Possible growth factors for chickens in silkworm pupae meal.

Silkworm pupae were included in the diet of growing chickens, causing little effect at 50 g/kg but a marked depression in performance at 100 g/kg. Direct comparisons of diets containing silkworm pupae of different ages indicated a growth stimulating effect correlated with ecdysteroid activity.

Amino Acids↗
PubMed2002

Expression of human endostatin in larvae of silkworm (Bombyx mori) and in vitro activity assays.

Human endostatin is a novel antiangiogenic molecule, which can inhibit the proliferation and development of new blood vessels, and experimentally can cause nearly complete regression of established tumors. In this paper, the cDNA encoding human endostatin was cloned into a baculovirus shuttle vector pBacPAK8 and co-infected with linearized Bm-BacPAK6 DNA into and BmN cells. The recombinant virus was screened and identified by PCR, DNA and RNA dot hybridization, and ELISA assay. The recombinant endostatin was expressed in culture cells, and the larvae and pupa of silkworm by inoculation of recombinant virus. The biological activity assay showed that the expression product in larvae was over 150 microg/ml, about 50-fold higher than that expressed in cultured cells. SDS-PAGE and Western blotting analysis showed a pattern of molecular weight of about 20 kDa. The bio-activity of the protein product was determined by human umbilical vein endothelial cells (ECV304) proliferation test in vitro and the chick chorioallantoic membrane (CAM) vascular inhibition test. Endostatin showed significant inhibitory effect on endothelial cells in a dose-dependent manner. Silkworm-produced endostatin induced apoptosis of endothelial cells and also inhibited angiogenesis in the CAM assay. Combination regimen using angiostatin and endostatin showed more than additive effect in angiogenic inhibition and increasing apoptosis when compared with treatment with the individual antiangiogenic protein.

Angiostatins↗
PubMed2006

Sinusoidal magnetic fields and chawki (silkworm) rearing in sericulture.

Effects of sinusoidal magnetic fields on chawki silkworm rearing have been studied. The experiment was conducted using a multi X Bi silkworm hybrid, PM x CSR 2. Disease-free layings were reared from hatching to cocooning and by subjecting first and second instar to three magnetic field frequencies: 0.1, 1.0, and 10 Hz at 1500 nT, pp, for six days at six hours per day. Controls were maintained simultaneously. Larval durations for both young-stage chawki (I and II) and late stage (III, IV, V) were calculated in days and hours, including the feeding and moulting periods for I to IV instars, and the feeding period for V instar up to the time of spinning. The study revealed that the magnetic exposures reduced both feeding and moulting times with no adverse effects on larval growth. The substantial reductions in time, 33 hours in instars I and II, and 64 hours in total larval duration, could be commercially important in chawki rearing, saving time, leaf consumption, and labor expenses.

Agriculture↗
PubMed1953

Immunological studies on insect metamorphosis. I. Qualitative and quantitative description of the blood antigens of the Cecropia silkworm.

1. The cell-free blood of the Cecropia silkworm produces a maximum of nine bands of antigen-antibody precipitate when reacted in antiserum-agar tests with antisera prepared by injecting Cecropia extracts into rabbits. The blood antigens producing these bands of precipitate have the properties of proteins in that they are non-dialyzable, labile at 75 degrees C., and salted out by 75 per cent saturated ammonium sulfate. One antigen was identified as a carotenoid protein. 2. Six bands of precipitate were selected for further study. Absorption tests revealed that the blood, at all stages of metamorphosis, is capable of precipitating the antibodies which produce five of these bands. This result indicates that five of the six antigens are present in the blood throughout metamorphosis. The sixth antigen is undetectable in blood from fourth instar larvae, appears in the blood late in the fifth instar, persists during the pupal stage, and disappears again during adult development. 3. When blood samples from various stages of metamorphosis were tested in antiserum-agar tubes, the rates of advance of the six bands of precipitate underwent systematic change in close correlation with the morphological stage of the silkworm's metamorphosis. These changes are interpreted in terms of concentration changes of the corresponding blood antigens. The validity of this interpretation was tested in several ways, with the conclusion that the interpretation was generally acceptable for the system under consideration. 4. All six antigens appear to increase in concentration during the last larval instar and to decrease in concentration during the period of adult development. However, each antigen has its own characteristic pattern of concentration change which differs from those of the other five. In order to explain this diversity, we conclude that the physiological mechanisms which regulate the synthesis and utilization of the blood antigens control each antigen on an individual basis.

Animals↗
PubMed1956

Culture in vitro of tissue from the silkworm, Bombyx mori L.

1. Ovarian tissue from Bombyx mori L. larvae about to pupate was cultured in Trager's (1935) salt solution and 10 per cent hemolymph, with indifferent results. Improvement of cultures was sought by modifying the culture medium. 2. To reduce the activity of the tyrosinase, hemolymph for culture medium was heated for 5 minutes at 60 degrees C., and the coagulated protein removed. 3. A physiological solution was formulated containing cations and amino acids as they occur normally in silkworm hemolymph. In both hanging-drop and small tube cultures use of this medium brought about increased cell number, improved cell appearance, more rapid mitoses, and longer life of cultures. 4. To the solution formulated from analyses, tryptophan, cystine, cysteine, malate, fumarate, succinate, and alpha-ketoglutarate were added after testing individually, resulting in improved growth in cultures. 5. Use of a silkworm egg extract prepared 4 to 5 days after acid treatment produced an increase in cell number. 6. In small roller tube cultures, when the new medium was changed twice a week, the cells spread over the walls of the tube in 4 or 5 days (Figs. 8 and 9), rapid mitoses were observed after 2 weeks, and transparent active cells were present at 3 weeks. Subculturing was not attempted.

Animals↗
PubMed1956

The chemistry of insect hemolymph; organic components of the hemolymph of the silkworm, Bombyx mori, and two other species.

1. Hemolymph was collected for analysis from the silkworm, Bombyx mori, in a series of developmental stages ranging from the second molt to the late pupa. The mean pH of larval hemolymph after collection was found to be 6.45, that of pupal hemolymph, 6.57; in vivo values may be slightly lower. Total dry solids ranged from 5.4 to 10.6 per cent. Total protein ranged from 1.2 to 5.3 per cent, increasing rapidly during the fifth instar. 2. Free amino acids were separated chromatographically and estimated. Of 19 amino acids identified, amounting collectively to 823 to 1497 mg. per 100 ml., glutamine, histidine, and lysine generally occurred in greatest amount. Tryptophan was not detected, and cystine (or cysteine) was found in only one sample. The total free amino acids account for 35 to 55 per cent of the non-protein nitrogen of the plasma. 3. Free sugars, estimated semiquantitatively on chromatograms, comprise glucose, fructose, and sucrose in total amount ranging from about 5 to 40 mg. per 100 ml. Total acid-soluble, ultrafiltrable carbohydrate, estimated as glucose by the anthrone reaction, ranged from 166 to 635 mg. per 100 ml., indicating the presence of low molecular weight sugar derivatives. 4. Inorganic phosphate amounted to 5 to 15 mg. per 100 ml., and acid-soluble organic phosphate to 100 to 200 mg. per 100 ml. The latter fraction includes several substances, of which one was tentatively identified as glucose-6-phosphate and the remainder are as yet unidentified. 5. Single samples of hemolymph were also taken from larvae of the wax moth, Galleria mellonella, and the spruce sawfly, Diprion hercyniae. These contained even higher concentrations of solutes than the silkworm samples, but with a generally similar distribution. The proportions of the free amino acids were different in each species.

Amino Acids↗