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PubMed2005

Possible effect of 30K proteins in embryonic development of silkworm Bombyx mori.

The silkworm Bombyx mori possesses a 30K protein family of 3x10(4) Da, the biological functions of which have not been fully identified. The relationship between the 30K protein family and the embryonic development of temperature sensitive sex-linked mutant strain of silkworm was investigated by two dimensional polyacrylamide gel electrophoresis (2D-PAGE) and Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The results show that protein spots 1-5 of the 30K protein family, mainly existing in normal strain, are possibly related to embryonic development. The early consumption of a 30K protein named 6G1-30K-1 and the accumulation of 30K proteins named 6G1-30K-3 and 6G1-30K-4 are likely caused by the destruction of physiological balance in normal embryonic development, which may lead to lower hatchability of the temperature sensitive strain. The results suggest that reasonable metabolism of 30K proteins is a prerequisite for the embryo's normal development.

Animals↗

PubMed2006

Structural analysis of fibroin heavy chain signal peptide of silkworm Bombyx mori.

To study the minimal length required for the secretion of recombinant proteins and silk proteins in posterior silk gland, the signal peptide (SP) of the fibroin heavy chain (FibH) of silkworm Bombyx mori was systematically shortened from the C-terminal. Its effect on the secretion of protein was observed using enhanced green fluorescent protein (EGFP) as a reporter. Secretion of EGFP fusion proteins was examined under fluorescence microscope. FibH SPs with lengths of 20, 18, 16 and 12 a.a. can direct the secretion of the reporter, yet those with lengths of 11, 10, 9, 8 and 1 a.a. can not. When the FibH SP was shortened to 12 a.a., the secretion efficiency was decreased slightly and cleavage occurred within EGFP. When 16 a.a. of the FibH SP were used, the secretion of fusion protein was normal and the cleavage site was between the Gly-Ser linker and Met, the starting amino acid of EGFP. These findings are applicable for the expression of foreign proteins in silkworm silk gland. The cleavage site of the SP is discussed and compared with the predictive results of the SignalP 3.0 online prediction program.

Animals↗

PubMed2004

A draft sequence for the genome of the domesticated silkworm (Bombyx mori).

We report a draft sequence for the genome of the domesticated silkworm (Bombyx mori), covering 90.9% of all known silkworm genes. Our estimated gene count is 18,510, which exceeds the 13,379 genes reported for Drosophila melanogaster. Comparative analyses to fruitfly, mosquito, spider, and butterfly reveal both similarities and differences in gene content.

Algorithms↗

PubMed1967

Antibiotics in the laboratory-rearing of cecropia silkworms.

A mixture of aureomycin and kanamycin prevents a fatal intestinal infection that usually occurs in all cecropia silkworms reared in the laboratory. Thus, for the first time, laboratory experimentation with these larvae and with those of other wild silkworms is practical.

Animals↗

PubMed1977

2-Deoxy-alpha-ecdysone from ovaries and eggs of the silkworm, Bombyx mori.

From ovaries dissected from developing and emerged adults of the silkworm, Bombyx mori, two substances having high molting hormone activity were isolated. One of these was identified as 2-deoxy-alpha-ecdysone by means of high-pressure liquid chromatography and mass spectrometry. Although this compound had previously been isolated from the fern Blechnum minus and postulated to be the precursor of alpha-ecdysone, it had not been obtained from insect material. The compound is also contained in the form of a conjugate in ovaries as well as in diapausing silkworm embryos.

Animals↗

PubMed1983

Dissolution and Degradation of Bacillus thuringiensis delta-Endotoxin by Gut Juice Protease of the Silkworm Bombyx mori.

The dissolution and degradation of dagger-endotoxin (crystal) of Bacillus thuringiensis subsp. kurstaki strain HD-1 were investigated. Crystals were dissolved in 0.1 M phosphate-carbonate-NaOH buffer at pH > 12. Swelling of crystals occurred in the buffer between pH 10 and 11, and crystals dissolved in the same buffer supplemented with gut juice protease of the silkworm Bombyx mori. The proteolytic dissolution of crystals occurred after a time lag of several minutes in 0.1 M carbonate-NaOH buffer, pH 10.2. The time lag was not observed when crystals were suspended in the buffer for 30 min before the addition of protease. After the dissolution of the crystals and further degradation of the solubilized protein, the appearance of a toxic protein with a molecular weight of 59,000, designated P-59, was observed. Lower-molecular-weight peptides (less than 40,000) showed no toxicity to the silkworm larvae on feeding. Digestion of the 120,000-dalton subunit of the crystal by gut juice protease also produced P-59. These observations suggest the occurrence of a similar process in vivo, i.e., the swelling of crystals due to the alkalinity of gut juice and the production of P-59, dependent on the hydrolysis of swollen crystals by gut juice protease.

Journal Article↗

PubMed1994

Silk gland-specific tRNA(Ala) genes interact more weakly than constitutive tRNA(Ala) genes with silkworm TFIIIB and polymerase III fractions.

Constitutive and silk gland-specific tRNA(Ala) genes from silkworms have very different transcriptional properties in vitro. Typically, the constitutive type, which encodes tRNA(AlaC), directs transcription much more efficiently than does the silk gland-specific type, which encodes tRNA(AlaSG). We think that the inefficiency of the tRNA(AlaCG) gene underlies its capacity to be turned off in non-silk gland cells. An economical model is that the tRNA(AlaSG) promoter interacts poorly, relative to the tRNA(AlaC) promoter, with one or more components of the basal transcription machinery. As a consequence, the tRNA(AlaSG) gene directs the formation of fewer transcription complexes or of complexes with reduced cycling ability. Here we show that the difference in the number of active transcription complexes accounts for the difference in tRNA(AlaC) and tRNA(AlaSG) transcription rates. To determine whether a particular component of the silkworm transcription machinery is responsible for reduced complex formation on the tRNA(AlaSG) gene, we measured competition by templates for defined fractions of this machinery. We find that the tRNA(AlaSG) gene is greatly impaired, in comparison with the tRNA(AlaC) gene, in competition for either TFIIIB or RNA polymerase III. Competition for each of these fractions is also strongly influenced by the nature of the 5' flanking sequence, the promoter element responsible for the distinctive transcriptional properties of tRNA(AlaSG) and tRNA(AlaC) genes. These results suggest that differential interaction with TFIIIB or RNA polymerase III is a critical functional distinction between these genes.

Animals↗

PubMed1994

tRNA(IleIAU) (TFIIIR) plays an indirect role in silkworm class III transcription in vitro and inhibits low-frequency DNA cleavage.

tRNA(IleIAU) provides an activity, originally called TFIIIR, necessary to reconstitute transcription by silkworm RNA polymerase III in vitro from partially purified components. Here we report studies on the role of tRNA(IleIAU) in in vitro transcription. We show that tRNA(IleIAU) does not act positively but, rather, is required to prevent the action of a transcriptional inhibitor. We also show that the presence of tRNA(IleIAU) in transcription reaction mixtures prevents low-frequency DNA cleavage by the TFIIIB fraction. Studies on the mechanism of transcriptional inhibition suggest that this DNA cleavage could cause transcriptional inhibition through trans-inactivation of transcription machinery. The ability to block DNA cleavage, like the ability to facilitate transcription, is highly specific to silkworm tRNA(IleIAU).

Animals↗

PubMed1996

Silkworm TFIIIB binds both constitutive and silk gland-specific tRNA Ala promoters but protects only the constitutive promoter from DNase I cleavage.

We have identified a complex between TFIIIB and the upstream promoter of silkworm tRNA Ala genes that is detectable by gel retardation and DNase I footprinting. Formation of this complex depends on the integrity of previously identified upstream promoter elements and on the presence of other silkworm transcription factors, either TFIIID or a fraction that contains both TFIIIC and TFIIID. We have used this complex to compare the interactions of TFIIIB with two kinds of tRNA Ala genes whose different in vitro transcription properties are conferred by the upstream segments of their promoters. These are the tRNA C Ala genes, which are transcribed constitutively, and the tRNA SG Ala genes, which are transcribed only in the silk gland. We find that TFIIIB binds tRNA SG Ala genes with lower affinity than it binds tRNA C Ala genes. In addition, the TFIIIB complex formed on tRNA SG Ala genes differ qualitatively from those formed on tRNA C Ala genes. Both the transcriptional activity of tRNA SG Ala complexes and the ability of the complexes to protect upstream DNA from DNase I digestion are reduced.

Animals↗

PubMed1982

Silkworm 5S RNA and alanine tRNA genes share highly conserved 5' flanking and coding sequences.

A fragment of Bombyx mori genomic DNA containing one tRNA2Ala gene and one 5S RNA gene has been used to compare the structural features of silkworm 5S RNA and tRNA genes. The nucleotide sequences of both genes and of the primary transcripts produced from them in homologous in vitro transcription systems have been determined. Comparison of the sequences of these two genes with that of another previously analyzed B. mori tRNA2Ala gene reveals common oligonucleotides which may be important transcriptional signals. The oligonucleotides TA(C)TAT, AATTTT, and TTC are located approximately (+/- 1 nucleotide) 29, 19, and 3 nucleotides, respectively, before the transcription initiation sites of the two tRNA2Ala genes and the one 5S RNA gene we have analyzed. The sequence GGGCGTAG(C)TCAG lies within the coding regions of all three genes. The functional significance of these sequences is suggested by their location within regions required for the transcription of silkworm alanine tRNA genes in vitro.

5' Flanking Region↗

PubMed2003

Identification of ISSR markers associated with productivity traits in silkworm, Bombyx moni L.

Bombyx mori L., commonly recognised around the world as the mulberry silkworm, is characterized by a wide variability in yield and developmental traits, which have been proven through conventional genetic analysis to be of polygenic nature. A large number of morpho-biochemical traits and RFLP and RAPD markers are mapped on different linkage groups, but to this point very little attention has been given to unravelling the genetics of yield traits. To address this issue, polymorphic profiles of 147 markers generated with 12 ISSR primers on the genomic DNA of 20 silkworm stocks of diverse yield status were subjected to multiple regression and discriminant function analyses (DFA). This led to the identification of eight markers generated by six primers, which demonstrated high beta-coefficient indices of -0.451 to -0.940. Furthermore, a significant difference between the yield traits for stocks with and without the specific marker could also be established. The inheritance pattern of one marker, L13800bp, identified at the first step of selection of markers through stepwise regression analyses for five yield parameters is discussed in the context of applying multiple regression analysis for establishing association, if not linkage, between a group of DNA markers and a particular yield trait of polygenic nature and using such markers in molecular marker-assisted breeding programs.

Analysis of Variance↗

PubMed2006

Linkage and mapping analyses of the densonucleosis non-susceptible gene nsd-Z in the silkworm Bombyx mori using SSR markers.

In the silkworm Bombyx mori, non-susceptibility to the Zhenjiang (China) strain of the densonucleosis virus (DNV-Z) is controlled by the recessive gene nsd-Z (non-susceptible to DNV-Z), which is located on chromosome 15. Owing to a lack of crossing over in females, reciprocal backcrossed F1 (BC1) progeny were used for linkage analysis and mapping of the nsd-Z gene using silkworm strains Js and L10, which are classified as being highly susceptible and non-susceptible to DNV-Z, respectively. BC1 larvae were inoculated with the DNV-Z virus at the first instar, and DNA was extracted from the individual surviving pupae and analyzed for simple sequence repeat (SSR) markers. The nsd-Z gene was found to be linked to 7 SSR markers, as all the surviving larvae in the BC1female (F1female x L10male) showed the homozygous profile of strain L10, and the sick larvae in the BC1female (F1female x L10male) showed the heterozygous profile of Js x L10 F1 hybrids. Using a reciprocal BC1male (L101female x F1male) cross, we constructed a linkage map of 80.6 cM, with nsd-Z mapped at 30 cM and the closest SSR marker at a distance of 4.4 cM.

Animals↗

PubMed2005

The genetics and genomics of the silkworm, Bombyx mori.

We review progress in applying molecular genetic and genomic technologies to studies in the domesticated silkworm, Bombyx mori, highlighting its use as a model for Lepidoptera, and in sericulture and biotechnology. Dense molecular linkage maps are being integrated with classical linkage maps for positional cloning and marker-assisted selection. Classical mutations have been identified by a candidate gene approach. Cytogenetic and sequence analyses show that the W chromosome is composed largely of nested full-length long terminal repeat retrotransposons. Z-chromosome-linked sequences show a lack of dosage compensation. The downstream sex differentiation mechanism has been studied via the silkworm homolog of doublesex. Expressed sequence tagged databases have been used to discover Lepidoptera-specific genes, provide evidence for horizontal gene transfer, and construct microarrays. Physical maps using large-fragment bacterial artificial chromosome libraries have been constructed, and whole-genome shotgun sequencing is underway. Germline transformation and transient expression systems are well established and available for functional studies, high-level protein expression, and gene silencing via RNA interference.

Animals↗

PubMed2004

Distribution of circadian clock-related proteins in the cephalic nervous system of the silkworm, Bombyx mori.

In the circadian timing systems, input pathways transmit information on the diurnal environmental changes to a core oscillator that generates signals relayed to the body periphery by output pathways. Cryptochrome (CRY) protein participates in the light perception; period (PER), Cycle (CYC), and Doubletime (DBT) proteins drive the core oscillator; and arylalkylamines are crucial for the clock output in vertebrates. Using antibodies to CRY, PER, CYC, DBT, and arylalkylamine N-acetyltransferase (aaNAT), the authors examined neuronal architecture of the circadian system in the cephalic ganglia of adult silkworms. The antibodies reacted in the cytoplasm, never in the nuclei, of specific neurons. A cluster of 4 large Ia(1) neurons in each dorsolateral protocerebrum, a pair of cells in the frontal ganglion, and nerve fibers in the corpora cardiaca and corpora allata were stained with all antibodies. The intensity of PER staining in the Ia(1) cells and in 2 to 4 adjacent small cells oscillated, being maximal late in subjective day and minimal in early night. No other oscillations were detected in any cell and with any antibody. Six small cells in close vicinity to the Ia(1) neurons coexpressed CYC-like and DBT-like, and 4 to 5 of them also coexpressed aaNATlike immunoreactivity; the PER- and CRY-like antigens were each present in separate groups of 4 cells. The CYC- and aaNAT-like antigens were further colocalized in small groups of neurons in the pars intercerebralis, at the venter of the optic tract, and in the subesophageal ganglion. Remaining antibodies reacted with similarly positioned cells in the pars intercerebralis, and the DBT antibody also reacted with the cells in the subesophageal ganglion, but antigen colocalizations were not proven. The results imply that key components of the silkworm circadian system reside in the Ia(1) neurons and that additional, hierarchically arranged oscillators contribute to overt pacemaking. The retrocerebral neurohemal organs seem to serve as outlets transmitting central neural oscillations to the hemolymph. The frontal ganglion may play an autonomous function in circadian regulations. The colocalization of aaNAT- and CYC-like antigens suggests that the enzyme is functionally linked to CYC as in vertebrates and that arylalkylamines are involved in the insect output pathway.

ARNTL Transcription Factors↗

PubMed2006

Identification and analysis of YELLOW protein family genes in the silkworm, Bombyx mori.

BACKGROUND: The major royal jelly proteins/yellow (MRJP/YELLOW) family possesses several physiological and chemical functions in the development of Apis mellifera and Drosophila melanogaster. Each protein of the family has a conserved domain named MRJP. However, there is no report of MRJP/YELLOW family proteins in the Lepidoptera. RESULTS: Using the YELLOW protein sequence in Drosophila melanogaster to BLAST silkworm EST database, we found a gene family composed of seven members with a conserved MRJP domain each and named it YELLOW protein family of Bombyx mori. We completed the cDNA sequences with RACE method. The protein of each member possesses a MRJP domain and a putative cleavable signal peptide consisting of a hydrophobic sequence. In view of genetic evolution, the whole Bm YELLOW protein family composes a monophyletic group, which is distinctly separate from Drosophila melanogaster and Apis mellifera. We then showed the tissue expression profiles of Bm YELLOW protein family genes by RT-PCR. CONCLUSION: A Bombyx mori YELLOW protein family is found to be composed of at least seven members. The low homogeneity and unique pattern of gene expression by each member among the family ensure us to prophesy that the members of Bm YELLOW protein family would play some important physiological functions in silkworm development.

Amino Acid Sequence↗

PubMed1993

Purification and characterization of a lectin-like substance from silkworm faeces.

To obtain biologically active substances that are useful in the study of cell structures and functions, we examined several biological activities of an extract of silkworm faeces with hot buffer. We detected a lectin-like substance in the extract and purified it. When the extract of silkworm faeces was added to a culture of quail myoblasts transformed with temperature-sensitive Rous sarcoma virus (QM-RSV cells), the plated cells which were cultured at 35.5 degrees C (the permissive temperature for RSV that suppresses myogenic differentiation) became detached from the dishes and the cells in suspension aggregated. However, when the same amount of extract was added to plated QM-RSV cells cultured at 41 degrees C, which is a nonpermissive temperature for RSV, the cells did not become detached. Other kinds of plated cells examined also did not become detached. Unlike plated cells, nonplated cells of all kinds aggregate in suspension upon the addition of the extract. This active substance was purified by monitoring its induction of cell aggregation. The purified substance was found to be a lectin-like glycoprotein with an apparent molecular mass of about 60 kDa. Further studies showed that the binding sites of this glycoprotein are sugar chains on the cell surface and that mannose is an epitope.

Animals↗

PubMed1989

Role of cholesterol 10-methyl group and effect of "extra" 14-methyl group on silkworm growth and development.

In order to establish the functional importance of the 10-methyl group of cholesterol and the planarity of the steroid ring, silkworms (Bombyx mori) were reared on an artificial diet containing 19-norcholesterol (1), 14 alpha-methylcholesterol (3) or 19,19-difluorocholesterol (2). The former two sterols (1 and 3) only partially satisfied the silkworm sterol requirement; growth and development were seriously retarded. The fluorinated sterol (2) was much more deleterious and was totally inadequate in meeting the sterol requirement.

Animals↗

PubMed1990

Transient expression of chimeric CAT genes injected into early embryos of the domesticated silkworm Bombyx mori.

In order to establish a transient expression system for genes introduced into early embryos of the silkworm, Bombyx mori, we tested various promoters ligated with CAT reporter genes. The embryos into which we injected supercoiled plasmid DNA of pFb(-860/+10)CAT containing the Bombyx fibroin promoter region ligated to the CAT gene showed a reasonably high CAT activity beginning around 30 h after oviposition. This high activity was observed only when the plasmid was injected before termination of the early nuclear cleavage stage, which was about 8 h after oviposition, but not after this stage. This means that the expression of injected DNA is closely related to the presence of cleavage nuclei in early embryos. Promoters originating from insect genes, like the Bombyx sericin-1 gene, Drosophila hsp70 and Drosophila copia LTR, functioned as strong promoters in the embryos. On the contrary, promoters from mammalian virus genes, such as the SV40 early and Moloney murine leukemia virus LTR genes, functioned as weak promoters. Moreover, linearized DNAs showed no or weak activity of expression in embryos. From these results, we conclude that the silkworm embryo transient expression system is a useful tool for studying the mechanism of regulation of insect genes.

Animals↗