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PubMed1968

Double-stranded Ribonucleic Acid from Cytoplasmic Polyhedrosis Virus of the Silkworm.

Ribonucleic acid (RNA) was extracted by phenol treatment from cytoplasmic polyhedrosis virus isolated from the midgut of infected silkworms. This RNA appears as threads when precipitated in alcohol. Two components having different sedimentation constants were observed. The molecular weight of the RNA preparation obtained by sedimentation coefficient (weight-averaged) and intrinsic viscosity was about 2 x 10(6) to 3 x 10(6). It was one-half to one-third the size of the calculated molecular weight for an entire RNA molecule in a virion. Electron micrographs of this RNA preparation showed two peaks in the distribution of contour length, at 0.4 and 1.3 mum, which would correspond to molecular weights of 10(6) and 3 x 10(6), respectively. The extracted RNA seemed to split into segments at a preferential breaking point. This RNA was soluble in concentrated salt solution, differing from single stranded high-molecular-weight RNA. The base composition of this RNA was complementary in the ratios of adenosine to uridine and guanosine to cytosine. It contained 43% guanosine plus cytosine. Based on its filamentous appearance by electron microscopy, typical pattern of optical rotatory dispersion and circular dichroism, sharp transition of the optical properties on heating, great hyperchromicity on degradation, nonreactivity with formaldehyde, and resistance to ribonucleases, it is concluded that this RNA is double-stranded and has regular base pairings of guanosine-cytosine and adenosine-uridine.

Journal Article↗
PubMed1971

Separation and structure of components of nuclear polyhedrosis virus of the silkworm.

Morphology of structural components of nuclear polyhedrosis virus (NPV) particles of the silkworm (Bombyx mori Linné) was studied by electron microscope using negative staining. NPV particles isolated from polyhedra could be separated into five structural components by centrifugation in sucrose density gradients. The lowest band (band I) was found to consist of thick rod-shaped particles (330 by 80 nm) with knobby surfaces and with occasional protrusion at one end. The second band from the bottom (band II) was shown to consist mainly of slender rod-shaped particles (360 by 60 nm), in which internal structures were visible as a dense mass. Regular striations were also seen on the surface of these particles. By treatment with mercaptoethanol, these particles were drastically damaged, and in some cases the internal substances were partially released, producing empty inner membranes of various degrees of disintegration. In bands III and IV, both empty spherical and empty rod-shaped membranes were present. Band III was rich in empty spherical membranes which were shown to be the outer membranes of thick rod-shaped particles. The empty rod-shaped membranes, the inner membranes, were mainly located in band IV and have cross striations on the surface. It is remarkable that the uppermost band (band V) consisted purely of small spherical particles, somewhat heterogeneous in size and shape (around 20 to 25 nm in diameter), indicating the particles to be the degradation product of the virus particles. Similar particles could also be observed within the empty inner membranes.

Animals↗
PubMed1993

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

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

Animals↗
PubMed1995

Structural analysis of TRAS1, a novel family of telomeric repeat-associated retrotransposons in the silkworm, Bombyx mori.

We characterized TRAS1, a retrotransposable element which was inserted into the telomeric repetitive sequence (CCTAA)n of the silkworm, Bombyx mori. The complete sequence of TRAS1, a stretch of 7.8 kb with a poly(A) tract at the 3' end, was determined. No long terminal repeat (LTR) was found at the termini of the element. TRAS1 contains gag- and pol-like open reading frames (ORFs) which are similar to those of non-LTR retrotransposons. The two ORFs overlap but are one nucleotide out of frame (+1 frameshift). Most of the approximately 250 copies of TRAS1 elements in the genome were highly conserved in the structure. Chromosomal in situ hybridization showed that TRAS1 elements are clustered at the telomeres of Bombyx chromosomes. A phylogenetic analysis using the amino acid sequence of the reverse transcriptase domain within the pol-like ORF revealed that TRAS1 falls into one lineage with R1, which is a family of non-LTR retrotransposons inserted into the same site within the 28S ribosomal DNA unit in most insects. TRAS1 may have been derived from R1 and changed the target specificity so that TRAS1 inserts into the telomeric repetitive sequence (CCTAA)n. Southern hybridization and Bal 31 exonuclease analyses showed that TRAS1 elements are clustered proximal to the terminal long tract of (CCTAA)n. TRAS1 is a novel family of non-LTR retrotransposons which are inserted into the telomeric repetitive sequences as target sites.

Amino Acid Sequence↗
PubMed2000

TATA-Binding protein-TATA interaction is a key determinant of differential transcription of silkworm constitutive and silk gland-specific tRNA(Ala) genes.

We have investigated the contribution of specific TATA-binding protein (TBP)-TATA interactions to the promoter activity of a constitutively expressed silkworm tRNA(C)(Ala) gene and have also asked whether the lack of similar interactions accounts for the low promoter activity of a silk gland-specific tRNA(SG)(Ala) gene. We compared TBP binding, TFIIIB-promoter complex stability (measured by heparin resistance), and in vitro transcriptional activity in a series of mutant tRNA(C)(Ala) promoters and found that specific TBP-TATA contacts are important for TFIIIB-promoter interaction and for transcriptional activity. Although the wild-type tRNA(C)(Ala) promoter contains two functional TBP binding sequences that overlap, the tRNA(SG)(Ala) promoter lacks any TBP binding site in the corresponding region. This feature appears to account for the inefficiency of the tRNA(SG)(Ala) promoter since provision of either of the wild-type TATA sequences derived from the tRNA(C)(Ala) promoter confers robust transcriptional activity. Transcriptional impairment of the wild-type tRNA(SG)(Ala) gene is not due to reduced incorporation of TBP into transcription complexes since both the tRNA(C)(Ala) and tRNA(SG)(Ala) promoters form transcription complexes that contain the same amount of TBP. Thus, the deleterious consequences of the lack of appropriate TBP-TATA contacts in the tRNA(SG)(Ala) promoter must come from failure to incorporate some other essential transcription factor(s) or to stabilize the complete complex in an active conformation.

Animals↗
PubMed1988

Silk gland-specific tRNA(Ala) genes are tightly clustered in the silkworm genome.

To understand the basis for tissue-specific production and accumulation of alanine tRNA in silkworms, we have examined the organization of the genes that code for silk gland-specific and constitutive alanine tRNAs. We have found that all of the silk gland-specific tRNA(Ala) genes (approximately 20) appear to be tightly clustered at a single locus in the Bombyx genome. These genes are arranged in tandem at intervals of approximately 150 base pairs. In contrast to the arrangement of the silk gland-specific tRNA(Ala) genes, most of the 20 to 30 constitutive tRNA(Ala) genes are dispersed in the genome. Silk gland-specific tRNA(Ala) genes are not amplified or grossly rearranged in the silk gland. Thus it is likely that differential transcription, rather than changes in gene number or structure, accounts for the tissue-specific accumulation of tRNA(Ala).

Animals↗
PubMed2005

Integration of molecular and classical linkage groups of the silkworm, Bombyx mori (n = 28).

Previously published linkage groups (LGs) composed of molecular markers were assigned to classical LGs in the silkworm, Bombyx mori (n = 28). Four markers from the classical linkage map, og, w-1, Lp, and Pfl, were assigned to the molecular linkage maps using sequence tagged sites. In addition, linkage analysis was carried out using BF1 progeny between wild-type and mutant stocks carrying morphological phenotypic markers. As a result, the counterparts for 26 of 28 molecular LGs were identified with their counterparts of the classical LGs. Two visible markers, Sel and Xan, representing different classical LGs, were found to be linked.

Animals↗
PubMed1999

Geographic dimorphism of the wild silkworm, Bombyx mandarina, in the chromosome number and the occurrence of a retroposon-like insertion in the arylphorin gene.

Individuals of the wild silkworm, Bombyx mandarina, collected in South Korea (Taegu City) and Japan (Tsushima Islands and Fukuoka City) had the chromosome number of 2n = 54, while those collected in China (Hangzhou City) had the chromosome number of 2n = 56. Analysis by PCR (polymerase chain reaction) showed that the 66-bp-long retroposon-like insertion known in the arylphorin gene was present in the B. mandarina specimens with 2n = 54, but not in those with 2n = 56. Thus, dimorphism in the chromosome number coincided with the occurrence of the insertion. It is likely that the boundary dividing the two geographic B. mandarina populations lies somewhere in the northern part of the Korean Peninsula.

Animals↗
PubMed2005

Retrotransposable elements on the W chromosome of the silkworm, Bombyx mori.

The sex chromosomes of the silkworm, Bombyxmori, are designated ZW(XY) for females and ZZ(XX) for males. The W chromosome of B. mori does not recombine with the Z chromosome and autosomes and no genes for morphological characters have been mapped to the W chromosome as yet. Furthermore, femaleness is determined by the presence of a single W chromosome, regardless of the number of autosomes or Z chromosomes. To understand these interesting features of the W chromosome, it is necessary to analyze the W chromosome at the molecular biology level. Initially to isolate DNA sequences specific for the W chromosome as randomly amplified polymorphic DNA (RAPD) markers, we compared the genomic DNAs between males and females by PCR with arbitrary 10-mer primers. To the present, we have identified 12 W-specific RAPD markers, and with the exception of one RAPD marker, all of the deduced amino acid sequences of these W-specific RAPD markers show similarity to previously reported amino acid sequences of retrotransposable elements from various organisms. After constructing a genomic DNA lambda phage library of B. mori we obtained two lambda phage clones, one containing the W-Kabuki RAPD sequence and one containing the W-Samurai RAPD sequence and found that these DNA sequences comprised nested structures of many retrotransposable elements. To further analyze the W chromosome, we obtained 14 W-specific bacterial artificial chromosome (BAC) clones from three BAC libraries and subjected these clones to shotgun sequencing. The resulting assembly of sequences did not produce a single contiguous sequence due to the presence of many retrotransposable elements. Therefore, we coupled PCR with shotgun sequencing. Through these analyses, we found that many long terminal repeat (LTR) and non-LTR retrotransposons, retroposons, DNA transposons and their derivatives, have accumulated on the W chromosome as strata. These results strongly indicate that retrotransposable elements are the main structural component of the W chromosome.

Animals↗
PubMed1997

Detection of peptidoglycan and endotoxin in dialysate, using silkworm larvae plasma and limulus amebocyte lysate methods.

Silkworm larvae plasma (SLP) reagent is activated by peptidoglycan (PG), a fragment of both the gram-positive and gram-negative bacterial cell wall, as well as beta-glucan (BG), a component of fungi. It is possible to measure contamination of gram-positive bacteria quantitatively by combining the conventional limulus amebocyte lysate (LAL) and PG measurement methods. Therefore, a more highly accurate analysis of dialysate can be made using both SLP and LAL methods to detect endotoxin (ET) and/or PG contamination. We studied the effects of contaminated dialysate on human peripheral blood mononuclear cells (PBMC) by producing various cytokines in vitro. Muramyl dipeptide (MDP) was used as the biologically active minimum constituent of PG. A total of 54 dialysate samples were obtained under sterile conditions from 4 sites: (1) reverse osmosis water unit; (2) proportioning unit; (3) multiple dialysate preparation console, and (4) personal dialysate preparation console, at 9 dialysis facilities. To detect bacterial contamination, the samples were measured with LAL(C), LAL(G) and SLP methods. PBMC were collected from 10 healthy controls and from 10 hemodialysis patients and cultured for 24 h with ET, MDP, ET + MDP and contaminated dialysate. IL-1 receptor antagonist (IL-1Ra), IL-1 beta and TNF-alpha in the culture medium supernatants were measured using the ELISA method. PG was not detected in dialysate from sites 1 or 2. However, dialysate from the inlet of the dialyzer at the bedside monitor of the central supply and personal console showed 4.1 +/- 6.1 ng/ml for site 3 (in 7 of 18 samples) and 3.3 +/- 4.6 ng/ml for site 4 (in 3 of 18 samples). Contamination by PG alone and complex contamination by PG and ET were also detected. Furthermore, IL-1Ra, IL-1 beta and TNF-alpha production by PBMC increased in accordance with the concentrations of MDP. Cytokine production was enhanced 5-10 times more where MDP and ET coexisted than where either MDP or ET existed alone, showing the synergic effects of MDP and ET. Based on these results, there is a high possibility that PG may also be a pyrogen in the dialysate prior to this study. ET had been considered the only pyrogen in dialysate. Therefore, it is essential to recognize the existence of both ET and PG in investigating dialysate contamination.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed2006

Casein kinases I of the silkworm, Bombyx mori: their possible roles in circadian timing and developmental determination.

Doubletime (DBT), a homolog of casein kinase Iepsilon (CKIepsilon), is an essential circadian clock component and developmental regulator in Drosophila melanogaster. The authors cloned a dbt homolog from the silkworm, Bombyx mori(Bmdbt), and examined its spatial and temporal expression in comparison to a CKI[alpha] homolog (BmCKIalpha). Four Bmdbt splice variants and 2 BmCKIalpha splice variants were detected, and their expression patterns varied in different tissues. The level of Bmdbt transcript in the brain was constant under LD 12:12 while those of BmCKIalpha transcripts fluctuated with a decrease at ZT12. In situ hybridization showed presumably identical distribution of dbt, CKIalpha, and per transcripts in the putative clock neurons of the head ganglia, as well as in the retina, where CKI-and PER-like immunoreactivities were colocalized, suggesting a possible involvement of both CKIs in the B. mori circadian system. Signals were detected at 4 Ia(1) neurons in each dorsolateral protocerebrum, 6 to 8 cells in the pars intercerebralis, about 6 cells in the suboesophageal ganglion, 2 neurons in the frontal ganglion, and most of the photoreceptors. All these cells contained dbt, CKIalpha, and per antisense transcripts. The Northern analysis of dbtand CKIalpha transcripts at different developmental stages showed that both genes were expressed at relatively high levels during early embryogenesis and in the ovary. The levels of CKIalpha transcripts were also high in the late larval stages until the mid-fifth instar and then suddenly disappeared before larval-pupal ecdysis. In contrast, the transcriptional activity of both genes was low in diapausing eggs.

Alternative Splicing↗
PubMed1981

Intracellular transport and secretion of fibroin in the posterior silk gland of the silkworm Bombyx mori.

Intracellular transport and secretion of fibroin in the posterior silk gland cells of the silkworm, Bombyx mori, were investigated in relation to the radial microtubule and circular microtubule-microfilament systems of the cells. The silk glands were pulse-labelled for 3 min with [3H]glycine in vitro and then chased in media containing excess cold glycine and in some cases antimitotic reagents (colchicine or vinblastine) or cytochalasin (B or D), and the flow of the label in the glands was investigated by radioautography. It was revealed that the label initially located over the rough endoplasmic reticulum subsequently moves to the Golgi bodies to be condensed there. The secretory granules of fibroin or fibroin globules thus formed are transported via the radial microtubule system to the apical cytoplasm to be secreted there under some regulation by the circular microtubule-microfilament system. In the presence of colchicine or vinblastine, the secretion of fibroin was suppressed an marked accumulation of fibroin globules in the Golgi regions was observed, while in the presence of cytochalasin B or D the secretion was accelerated and extensive invagination of the luminal surface, which was probably due to the serial exocytosis of fibroin globules, was observed. These results suggest that the radial microtubule system and the circular microtubule-microfilament system are responsible for intracellular transport of fibroin globules from Golgi bodies to the apical cytoplasm and secretion by exocytosis at the luminal surface, respectively.

Animals↗
PubMed2000

Sniffing by a silkworm moth: wing fanning enhances air penetration through and pheromone interception by antennae.

Many organisms increase the air or water flow adjacent to olfactory surfaces when exposed to appropriate chemical stimuli; such 'sniffing' samples fluid from a specific region and can increase the rate of interception of odorant molecules. We used hot-wire anemometry, high-speed videography and flow visualization to study air flow near the feathery olfactory antennae of male silkworm moths (Bombyx mori L.). When exposed to conspecific female sex pheromone, male B. mori flap their wings through a stroke angle of 90-110 degrees at approximately 40 Hz without flying. This behavior generates an unsteady flow of air (mean speed 0.3-0.4 m s(-1)) towards the antennae from the front of the male. A pulse of peak air speed occurs at each wing upstroke. The Womersley number (characterizing the damping of pulsatile flow through the gaps between the sensory hairs on the antennae) is less than 1; hence, pulses of faster air (at 40 Hz) should move between sensory hairs. Calculation of flow through arrays of cylinders suggest that this wing fanning can increase the rate of interception of pheromone by the sensory hairs on the antennae by at least an order of magnitude beyond that in still air. Although wing fanning produces air flow relative to the antennae that is approximately 15 times faster than that generated by walking at top speed (0.023 m s(-1)), air flow through the gaps between the sensory hairs is approximately 560 times faster because a dramatic increase in the leakiness of the feathery antennae to air flow occurs at the air velocities produced by fanning.

Air↗
PubMed2002

Molecular cloning and function of ecdysis-triggering hormones in the silkworm Bombyx mori.

Inka cells of the epitracheal endocrine system produce peptide hormones involved in the regulation of insect ecdysis. In the silkworm Bombyx mori, injection of Inka cell extract into pharate larvae, pupae or adults activates the ecdysis behavioural sequence. In the present study, we report the identification of three peptides in these extracts, pre-ecdysis-triggering hormone (PETH), ecdysis-triggering hormone (ETH) and ETH-associated peptide (ETH-AP), which are encoded by the same cDNA precursor. Strong immunoreactivity associated with each peptide in Inka cells prior to ecdysis disappears during each ecdysis, indicating complete release of these peptides. Injection of either PETH or ETH alone is sufficient to elicit the entire ecdysis behavioural sequence through the direct action on abdominal ganglia; cephalic and thoracic ganglia are not required for the transition from pre-ecdysis to ecdysis behaviour. Our in vitro data provide evidence that these peptides control the entire ecdysis behavioural sequence through activation of specific circuits in the nervous system. Ecdysis of intact larvae is associated with the central release of eclosion hormone (EH) and elevation of cyclic 3',5'-guanosine monophosphate (cGMP) in the ventral nerve cord. However, injection of ETH into isolated abdomens induces cGMP elevation and ecdysis behaviour without a detectable release of EH, suggesting that an additional central factor(s) may be involved in the activation of this process. Our findings provide the first detailed account of the natural and hormonally induced behavioural sequence preceding larval, pupal and adult ecdyses of B. mori and highlight significant differences in the neuro-endocrine activation of pre-ecdysis and ecdysis behaviours compared with the related moth, Manduca sexta.

Amino Acid Sequence↗
PubMed1996

Suppression of enveloped virus production with a substance from silkworm faeces.

In the course of investigating effective biological active substances, we detected a substance in an extract of silkworm faeces that markedly suppresses viral production. The extract, prepared with hot phosphate-buffered saline and purified with ammonium sulfate precipitation, inhibited HVJ (Sendai virus), HSV (herpes simplex virus type-1), and HIV (human immunodeficiency virus type-1), but not poliovirus, suggesting that it is effective on enveloped virus production but not on non-enveloped ones. In the case of HVJ, indirect immunofluorescent staining using anti-HVJ antibody and Northern blotting analysis showed that, while viral adsorption and entry into the host cells were not affected, the synthesis of viral specific gene was inhibited by pretreatment of the virions with the extract. The extract affected more effectively aged virion, which losses membrane function as barrier and its envelope is leaky, than young virion that maintains barrier function. The active substance was partially purified by gel filtration after treatment of the extract with 1 N NaOH solution. From analysis with SDS-PAGE (SDS-polyacrylamide gel electrophoresis), protein bands were detected with molecular masses of about 25 kDa and near 14 kDa, while sugars were also detected with lectin blotting.

Animals↗
PubMed1998

Biological activities of a novel lectin derived from silkworm faeces: characteristic changes of mouse peritoneal macrophages by the lectin.

A novel lectin derived from silkworm faeces, named NUE, activates phagocytosis of mouse peritoneal macrophages. At this time, significant morphological changes of the cells take place. NUE-treated macrophages formed projection-like neurocytes within 12 hr of treatment, and appeared somewhat flat in shape with the activation of cell growth. Associated with the morphological changes, actin was organized in dot-like structures corresponding to cell-substratum contact sites in NUE-treated macrophages. Vinculin, a cytoskeletal protein involved in microfilament-membrane interaction, formed doughnut-like rings matching the actin-dots, called "podosomes". Furthermore, observation by interference reflection microscopy showed that NUE-treated macrophages adhered more strongly to the substratum at local areas. According to these changes, more proteins associated with cell-substratum contact sites became to detergent-resistant. It was shown that NUE changed adhesive form of mouse peritoneal macrophages structurally and qualitatively.

Animals↗
PubMed1997

Antiviral substance from silkworm faeces: purification and its chemical characterization.

In a previous paper, we reported that an extract of silkworm faeces has a marked antiviral activity on enveloped viruses, but not on a non-enveloped virus, and we showed that it inhibits the synthesis of a viral specific gene of HVJ (Sendai virus) without affecting the viral adsorption and entry into the host cell. In this paper, we accomplished the purification of an antiviral active substance by extraction of a hydrophobic substance and thin layer chromatography. The active substance was found to be a chlorophyll-like substance with a molecular mass of about 530. This substance shows clear antiviral activity against HVJ, HSV (herpes simplex virus type-1), and HIV (human immunodeficiency virus type-1). Its antiviral activity was dependent on light irradiation and temperature. Furthermore, it also possesses a strong hemolytic activity under light.

Animals↗
PubMed2002

Recombinant human chymase produced by silkworm-baculovirus expression system: its application for a chymase detection kit.

Human chymase is a mast cell-derived serine proteinase, which is a non-angiotensin converting enzyme angiotensin II-generating enzyme. It appears to participate in various diseases, but it is unclear whether chymase plays major roles in physiological and pathophysiological functions in vivo. To obtain information on the physiological and pathophysiological functions of chymase and to search for diseases in which chymase participates, in the present study, we aimed at producing recombinant human chymase in large quantities and at developing an ELISA system using anti-human chymase antibodies. A recombinant human chymase was produced by a silkworm-baculovirus expression system. The recombinant chymase in active form was efficiently purified from larval hemolymph using cation-exchange and heparin column chromatography. This recombinant enzyme was enzymatically identical with native human chymase. On the other hand, the stability of the recombinant enzyme in cultured medium for mammalian cells at 37 degrees C was very high as compared with the stability of the native enzyme; 20% of the activity was maintained 120 h after addition of medium. These results indicated that the recombinant enzyme could also utilize in vitro and in vivo assay systems. We obtained several anti-chymase monoclonal antibodies by using the recombinant human chymase as antigen. These antibodies were used to construct an ELISA system for measuring the chymase concentration in blood. As a result of preliminary examination using this ELISA system, it was shown that the chymase concentration in each serum from hypertensive patients is significantly higher than in normal serum. The ELISA system will be applicable for clinical diagnosis and in vivo evaluation systems for chymase-targeting drugs.

Animals↗