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PubMed2003

Cloning, expression, and assembly of sericin-like protein.

Recombinant sericin proteins of different molecular masses (17.4, 31.9, and 46.5 kDa), based on the 38-amino acid repetitive motif of native sericin, were cloned, expressed, and purified. The recombinant sericin self-assembled during dialysis (starting concentration of 2.5 mg/ml) forming twisted fibers. Circular dichroism and Fourier transform infrared spectroscopy studies demonstrated protein conformational transitions occurred from random coil to beta-sheets during the dialysis. Congo red-stained recombinant sericin fibrils exhibited apple-green birefringence, indicating long-range order in the array of beta-sheets. Biosynthetic sericin has a high content of polar amino acids (e.g. > 40 mol % serine), leading to a beta-sheet conformation formed by hydrogen bonding via polar zipper interactions. Analysis of recombinant sericin sequence using Mandel-Gutfreund's (Mandel-Gutfreund, Y., and Gregoret, L. M. (2002) J. Mol. Biol. 323, 453-461) definition of polar and non-polar amino acids showed that the hydrophobicity pattern resembles the most frequent pattern of amyloidogenic proteins, polar amino acid aggregates (PPPPP). Many beta-proteins and peptides are designed to study amyloidogenesis using a polar/non-polar alternating pattern (PNPNPN). Sericin-like proteins or peptides provide an alternative model in terms of hydrophobicity pattern with which to explore questions related to beta-sheet formation and amyloidogenesis. The glue-like property of sericin is attributed to the hydrogen bonding between serine residues of sericin with serine residues in the fibroin structural components of silk fiber.

Amino Acid Motifs↗
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↗
PubMed2004

Bombyx Y-box protein BYB facilitates specific DNA interaction of various DNA binding proteins independently of the cold shock domain.

A new member of the Y-box protein family of the silkworm Bombyx mori (BYB) was co-purified with the fibroin gene enhancer-binding protein FMBP-1, and stimulated the binding of FMBP-1 to its cognate DNA element. However, the stimulatory effect was not specific to FMBP-1, BYB also enhancing the binding of mammalian transcription factors OTF2, SP1 and AP2 to their specific binding elements. Besides the above transcription regulatory factors, BYB facilitated the binding of basal transcription factor TBP, and enhanced transcription from the adenovirus 2 major late promoter in a reconstituted transcription system. Moreover, BYB stimulated the reactions of some restriction endonucleases under cold conditions. The C-terminal region of BYB was sufficient for these stimulatory effects, and the highly conserved cold shock domain (CSD) in the N-terminal region was dispensable. GST-pull down experiments showed that the C-terminal region could interact with DNA independently of the CSD. The above results suggest that the C-terminal region of BYB causes the active interaction of various DNA binding proteins with their targets. Such a function of the C-terminal region of BYB may partly explain the functional diversity of Y-box proteins.

Amino Acid Sequence↗
PubMed1977

The nucleotide sequence of a major glycine transfer RNA from the posterior silk gland of Bombyx mori L.

The nucleotide sequence of tRNA1Gly isolated from the posterior silk gland of Bombyx mori has been determined. This transfer RNA is present in high amounts in the posterior silk gland during the fifth larval instar. It has a GCC anticodon, capable of decoding a major glycine codon in the fibroin messenger RNA, GGU. Structural features of Bombyx tRNA1Gly and its homology to other eukaryotic glycine tRNAs are discussed.

Animals↗
PubMed1980

Hydrophobic affinity chromatography of nucleic acids and proteins.

5' tritylated oligonucleotides binding hydrophobically to low trityl cellulose/sepharose (< 15 microMTr/ml) retain their hydrogen-bonding specificities for complementary sequences. This, constitutes a novel mode of attaching affinity ligands to solid supports, is more convenient than existing methods, and proceeds with 100% yield. The salt, dielectric constant and temperature dependence of these non-covalently anchored ligands permits the isolation of a variety of RNAs including fibroin mRNA. Medium trityl sepharose (15-40 microM Tr/ml) has a high binding specificity for poly A and poly A containing mRNA, equivalent to dT cellulose. Most proteins, including nucleic acid enzymes, bind to these columns and retain enzymatic activity, thus mimicking enzymes attached covalently to solid phases. A number of in vivo counterparts to this hydrophobically determined specificity are noted, as are homologies to nitro-cellulose filters.

Affinity Labels↗
PubMed1981

Codon catalog usage is a genome strategy modulated for gene expressivity.

The nucleic acid sequence bank now contains 161 mRNAs, 43 new genes are added. One sequence, that of B. mori fibroin, is dropped due to uncertainty on the starting point for translation. Frequencies of all codons are given for each gene added and for each genome type in the total bank. A new series of correspondence analyses on codon use is presented, substantiating the genome hypothesis. Internal regulation of mRNA expression by different third base choices between quartet and duet codons is proposed for bacterial genes.

Amino Acid Sequence↗
PubMed1975

The biosynthesis of transfer RNA in insects. I. Increase of amino acid acceptor activity of specific tRNA's utilized for silk protein biosynthesis in the silk gland of Bombyx mori.

1) To detect the quantitative changes of amino acid acceptor activity of tRNA's from the posterior and middle silk glands of Bombyx mori at various ages, a relatively simple and rapid method was established using a mixture of radioactive amino acids in Chlorella hydrolysate. 2) The acceptor activities of silk gland tRNA for 15 amino acids tested seemed to be almost on the same level at the end of the 4th moult stage. During the 5th instar, however, characteristic increases were observed in glycine, alanine, and serine acceptor activities in both silk glands. 3) In the posterior silk gland, which produces fibroin, the acceptor activities for glycine and alanine increased more than that for serine. In the middle silk gland, which produces sericine, the acceptor activity for serine increased more than those for glycine and alanine. 4) In the light of observations on the increase of corresponding aminoacyl-tRNA synthetase activities in the silk glands, a functional adaptation of tRNA synthesis in the tissue is discussed.

Alanine-tRNA Ligase↗
PubMed1982

Differential synthesis rates of tRNA species in the silk gland of Bombyx mori are required to promote tRNA adaptation to silk messages.

In studying the mechanism of tRNA adaptation to silk fibroin and sericin mRNA translation, we investigated the accumulation rates of tRNA species rapidly labeled in vivo in the posterior and middle silk glands of the silkworm Bombyx mori during the last larval instar. We found that the rates of synthesis of total and individual tRNA species labeled with [3H]uridine during the growth and the secretion phases and separated by electrophoresis on polyacrylamide gels, are specific for each tRNA species and each tissue. We noted continuous variations of the tRNA levels designed to reflect changes in the population of mRNAs. The tRNA adaptation to silk mRNAs is completed roughly at the end of the growth phase and at the beginning of the secretion phase (day four of the Vth instar). Its stabilization during the secretion phase occurs by a reversal of the relative rates of predominant tRNA species, mainly tRNAGly. The sum total of the synthetic rates for each tRNA species corroborates their known adaptive distribution during the secretion phase of silk proteins. In addition, when coupled to the similarity of their turnover rates, observed previously, the data support a transcriptional control of tRNA genes.

Animals↗
PubMed1992

P25 gene regulation in Bombyx mori silk gland: two promoter-binding factors have distinct tissue and developmental specificities.

The gene encoding the silk protein P25 is expressed in the posterior silk gland of Bombyx mori with strict territorial and developmental specificities. The cis-acting regulatory elements previously located within the 441-bp 5' proximal sequence of the gene were examined for protein-binding capacities. We identified two factors, BMFA and SGFB, that lead to prominent band shifts and the target sites for which are included in a region homologous to the fibroin gene enhancer sequence. Analysis of the tissue-specific incidence of both factors showed that BMFA is ubiquitous, whereas SGFB is restricted to the silk gland cells. However, SGFB was found in both posterior and middle silk gland cells and therefore likely directs organ-specific, but not territory-specific, expression. Developmental studies throughout the fourth larval molt, at which the P25 gene status changes from derepressed to repressed, revealed that BMFA is reversibly modified at the transition from intermolt to molt. Indeed, the preexisting BMFA is replaced by a structurally related factor, BMFA', during the 2 h following head capsule apolysis. The exact temporal coincidence of this conversion with the onset of gene repression suggests that BMFA' is involved in transcription inactivation and likely results from a transduction process initiated by the hormonal change at molting.

Animals↗
PubMed1995

Exotic collagen gradients in the byssus of the mussel Mytilus edulis.

Byssal threads of the common mussel Mytilus edulis contain collagenous molecules from which two pepsin-resistant fragments have been isolated and characterized. These show a complementary distribution along the length of the thread, such that one predominates distally (Col-D) and the other proximally (Col-P). Both fragments contain three identical alpha-like chains with molecular masses of 50 kDa (Col-P) and 60 kDa (Col-D) and have typically collagenous amino acid compositions; for example, 35% glycine and almost 20% proline plus 4-trans-hydroxyproline. Hydroxylysine and 3-hydroxyproline were absent. Col-P sequences are also typical of collagen in consisting of tandem repeats of the triplet Gly-X-Y in which X and Y generally represent any amino acid. When proline occurs, it is hydroxylated to 4-trans-hydroxyproline only in the Y position. Seven instances where X is glycine have been detected in Col-P. Specific polyclonal anti-Col antibodies were used to isolate the precursors of Col-P and Col-D from the mussel foot. PreCol-P has a molecular mass of 95 kDa and contains 36% glycine but a lower imino acid content (13%). It has a complementary distribution with another precursor (preCol-D, 97 kDa) along the length of the foot. The two precursor compositions suggest resilin-like and silk-fibroin-like structures, respectively, in the noncollagenous domains of preCol-P and preCol-D. Immunogold labelling studies indicate that Col-P is associated with the coiled fibers of the inner core in the proximal portion of the thread, whereas Col-D is localized to the straight fiber bundles of the distal thread as well as to the outer core of the proximal thread.

Amino Acid Sequence↗
PubMed1998

Intracellular electrolyte levels and transport of secretory granules in exocrine gland cells

We demonstrate the intracellular transport of secretory granules of a silk protein, fibroin, from the Golgi region to the apical cytoplasm with special reference to microtubule organization, electrolyte concentrations and the acidic intragranular pH of normal and mutant posterior silk gland cells, using the techniques of electrophysiological microelectrode and microprobe analysis and of light and electron microscopic autoradiography. The silk gland cells of a recessive mutant making only flimsy cocoons were defective in the microtubule systems, did not stain with an anti-tubulin antibody in immunofluorescent microscopy, and accumulated intracellular granules in the apical and basal cytoplasm. The increase in intracellular calcium concentration and levels of chloride secretion were also reduced in the mutant cells. A carboxylic ionophore, monensin, which collapsed the granular H+ gradient, induced the transport of chloride and an increase in the intracellular calcium concentration, while it blocked the intracellular transport of granules from the Golgi region to the apical cytoplasm in normal cells. Thus, we conclude that the H+ gradient across the membrane of secretory granules is responsible for the intracellular transport of the secretory granules along the microtubule systems in silk gland cells, while Ca2+ is thought to be required for the exocytosis of the granules.

Journal Article↗
PubMed2000

Effects of applaud on the growth of silkworm (Lepidoptera: Bombycidae).

An experiment was carried out to evaluate the effect of the insecticide Applaud (buprofezin 25% WP) on the silkworm Bombyx mori (L.). This insecticide belongs to the class of insect growth regulators (IGR). The larvae were fed on leaves treated with 3 different concentrations (0.5, 1, 2 g/liter) of Applaud on the 1st d of each instar. Analysis of data with the Tukey-Kramer test at 1% significant level revealed that mortality and larval duration did not differ among the treatments. On the contrary, the larval weight, which was estimated just before mounting (procedure during which the mature larva climbing on a branch or other material to spin the cocoon), differed among the treatments. Also, cocoon weight, shell weight, and cocoon sericin and fibroin content were different among the treatments, except the shell cocoon ratio. Maximum weight was observed in the controls and minimum in the last instar treatments. Our data suggest that supplementation of Applaud through food to larvae does not affect their mortality rate. On the contrary, it affects larval growth and cocoon parameters.

Animals↗
PubMed1999

Transformation of Neomycin Resistance Gene (neo(R)) into Silkworm (Bombyx mori.L.).

Neomycin resistance gene (neo(R)) flanked by 5' and 3' regions of fibroin H-chain gene of silkworm (Bombyx mori.L.) was transferred into eggs of silkworm by gene gun in the early period of fertilization. The larvae were fed with an artificial diet containing neomycin in early 24 hours post transfettion, and some of them survived. The neo(R) encoding sequence in G(2) generation derived from the survivals was detected by Southern blotting. The results indicated that neo(R) could be used as a selective marker for studies on transgenic silkworm.

Journal Article↗
PubMed1986

Xylosylation and glucuronosylation reactions in rat liver Golgi apparatus and endoplasmic reticulum.

We have studied in rat liver the subcellular sites and topography of xylosylation and galactosylation reactions occurring in the biosynthesis of the D-glucuronic acid-galactose-galactose-D-xylose linkage region of proteoglycans and of glucuronosylation reactions involved in both glycosaminoglycan biosynthesis and bile acid and bilirubin conjugation. The specific translocation rate of UDP-xylose into sealed, "right-side-out" vesicles from the Golgi apparatus was 2-5-fold higher than into sealed right-side-out vesicles from the rough endoplasmic reticulum (RER). Using the above vesicle preparations, we only detected endogenous acceptors for xylosylation in the Golgi apparatus-rich fraction. The specific activity of xylosyltransferase (using silk fibroin as exogenous acceptor) was 50-100-fold higher in Golgi apparatus membranes than in those from the RER. Previous studies had shown that UDP-galactose is translocated solely into vesicles from the Golgi apparatus. In these studies, we found the specific activity of galactosyltransferase I to be 40-140-fold higher in membranes from the Golgi apparatus than in those from the RER. The specific translocation rate of UDP-D-glucuronic acid into vesicles from the Golgi apparatus was 10-fold higher than into those from the RER, whereas the specific activity of glucuronosyltransferase (using chondroitin nonasaccharide as exogenous acceptor) was 12-30-fold higher in Golgi apparatus membranes than in those from the RER. Together, the above results strongly suggest that, in rat liver, the biosynthesis of the above-described proteoglycan linkage region occurs in the Golgi apparatus. The specific activity of glucuronosyltransferase, using bile acids and bilirubin as exogenous acceptor, was 10-25-fold higher in RER membranes than those from the Golgi apparatus. This suggests that transport of UDP-D-glucuronic acid into the RER lumen is not required for such reactions.

Animals↗
PubMed1985

Mechanisms of chain initiation in the biosynthesis of connective tissue polysaccharides.

Carbohydrate-protein linkages of three types are found in the connective tissue proteoglycans; these linkages involve the following monosaccharide-amino acid pairs: xylose-serine; N-acetylglucosamine-asparagine; and N-acetylgalactosamine-threonine (or serine). The biosynthesis of carbohydrate groups containing linkages of the latter two types presumably occurs by the same pathways that have been well established for many glycoproteins, but details of these processes as they pertain to proteoglycans are not yet known. Initiation of polysaccharide chains linked by the xylose-serine linkage takes place by direct transfer of xylose from UDP-xylose to the hydroxyl groups of specific serine residues in the core proteins of the respective proteoglycans, and the xylosyltransferase catalyzing these reactions has been detected in the rough endoplasmic reticulum of embryonic chick chondrocytes. Although the completed or nascent core proteins are the natural substrates for xylose transfer in the intracellular assembly of proteoglycans, a survey of potential exogenous substrates has shown that small peptides containing alternating serine and glycine residues may also serve as acceptors in this reaction. Nevertheless, larger substrates are preferred, such as chondroitin sulfate proteoglycan, which has been deglycosylated by Smith degradation or HF treatment, or silk fibroin, which contains Ser-Gly pairs. In contrast to the sulfated polysaccharides, which are synthesized by carbohydrate transfer to protein in the endoplasmic reticulum and the Golgi apparatus, hyaluronic acid is formed in the plasma membrane by a different mechanism. The reaction by which chains are initiated is not yet known, but recent work by Prehm suggests that this process occurs either by transfer of the glucuronosyl component of UDP-glucuronic acid to UDP-N-acetylglucosamine or by the converse reaction, i.e., transfer of the N-acetylglucosaminyl unit of UDP-N-acetylglucosamine to UDP-glucuronic acid.

Acetylgalactosamine↗
PubMed1984

Glycyl- and alanyl-tRNA synthetases from Bombyx mori. Purification and properties.

Glycyl- and alanyl-tRNA synthetases have been purified from an extract of Bombyx mori posterior silk glands by a procedure that allows for the simultaneous isolation of both enzymes. Glycyl-tRNA synthetase is a dimer of Mr = 160,000 consisting of similar or identical subunits, whereas alanyl-tRNA synthetase is a monomer of Mr = 110,000 to 115,000. The abundance of these two enzymes in the posterior silk gland is consistent with the observed adaptation of this organ to the production of the silk protein, fibroin. The two enzymes are similar in oligomeric structure to the corresponding enzymes in Saccharomyces cerevisiae, but dissimilar from their counterparts in Escherichia coli.

Alanine-tRNA Ligase↗
PubMed1997

Determination of xylosyltransferase activity in serum with recombinant human bikunin as acceptor.

Xylosyltransferase (XT) is the chain-initiating enzyme of the biosynthesis of chondroitin sulfate. So far, XT activity has been detected by incorporation of [14C]xylose in chemically deglycosylated cartilage proteoglycan or silk fibroin. However, these acceptors allow no reliable determination in blood. We found that recombinant bikunin is an excellent acceptor for XT. The Michaelis-Menten constants for the xylosylation of silk, deglycosylated cartilage proteoglycans, and bikunin were 545, 155, and 0.9 micromol/L, respectively. With recombinant bikunin as acceptor, we developed a sensitive assay that allows a precise determination of XT activity in serum. We measured the serum XT activities of 500 blood donors and observed a considerable sex and age dependence. XT activities in men (0.77-1.50 mU/L) were approximately 30% higher than in women (0.58-1.20 mU/L) and reached a maximum in donors of approximately 40 years of age. During the menstrual cycle, serum XT activity showed a significant coincidence with the beta-estradiol concentration, and in the first trimester of pregnancy we observed a strong increase in serum XT activity.

Adolescent↗