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The adaptation of the silkgland cell to the production of fibroin in Bombyx mori L.

At the end of the larval life, the posterior silk gland of Bombyx mori is highly specialized in the biosynthesis of a specific protein : silk fibroin. The successive steps of fibroin production : amino supply, synthesis and secretion are described. Their analysis shows that fibroin synthesis is important enough to orient the overall cellular activities. Thus, the terminal differentiation of the posterior silk gland cell corresponds to the cells adaptation to the production of fibroin. Cytological and biochemical studies of the silk gland development show that specialization occurs discontinuously; the fourth molt, when fibroin is no more produced, is a phase of regression of the cellular adaptation whereas cell differentiation proceeds during the growth phase of the following fifth intermolt. After the spinning of the cocoon, the cells are lysed and disappear entirely at the nymphal stage. Biometrical analysis of silk production of different Bombyx strains in relation with the development of the proteosynthesis apparatus leads to the conclusion that the specific messenger RNA content determines the amount of synthetized fibroin. At maximum secretion, the mRNAF recruits almost all the cell ribosomes. The variations of the size of the proteosynthesis machinery are sufficient to explain the differences of productivity of the various silkworm strains. Different experimental factors affect silk production. Topical applications of juvenile hormone induce an increase of the RNA content and a consequent rise of the amount of secreted protein. In contrast, starvation reduces the silk production by acting at both transcriptonal and translational levels. Current researches on this system are devoted to the study of the differential gene expression, with particular interest to the regulation of the transcription of the specific fibroin messenger RNA.

Amino Acids

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

Cell junctions in the cyst envelope in the silkworm testis, Bombyx mori Linné.

The cell junctions of the cyst envelope in the testes of Bombyx mori were examined by electron miscroscopy utilizing a thin-sectioning technique following conventional fixation, tannic acid fixation and lanthanum tracer study, and also using a freeze-fracture technique. There are three kinds of junctions; septate junctions, gap junctions and tight junctions. Septate junctions are of the pleated type. Gap junctions are characterized by four electron-dense lines and three electron-lucent lines in the reduced intercellular spaces seen by thin-sectioning. They are of the "E" type, having clusters of intramembraneous particles on the E-fracture face. The most striking finding is the frequent presence of tight junctions on the fracture planes, while focally fused outer leaflets of the junctional unit membranes are rarely detected on thin-sectioned preparations. Tight junctions are characterized by branching zigzag ridges on the P-fracture face and complementary grooves on the E-fracture face. It is proposed that tight junctions are new morphological evidence of blood-germ cell barrier in an insect.

Animals

Oxygen permeability of the chorion in relation to diapause termination in Bombyx eggs.

Oxygen permeability of the chorion of the silkworm, Bombyx mori, was measured in relation to embryonic diapause. It did not change appreciably when the eggs were freed from diapause by being kept under long chilling. This finding suggests that the increase in oxygen permeability of the chorion is not a pre-requisite for the termination of the diapause and resynthesis of glycogen from 2 polyols.

Animals

Induction of dominant lethals with ethyl methane-sulfonate in male germ cells of mulberry silkwork, Bombyx mori l.

Sensitivity of male germ cells in the mulberry silkworm, Bombyx mori L., to ethyl methanesulfonate (EMS) was determined by treating newly emerged 5th- instar larvae, and 2-day- and 7-day-old pupae with 3 concentrations, 0.05, 0.1 and 0.15%, of the mutagen. The frequency of dominant-lethal mutations induced by EMS treatment was used as the parameter for the study. Spermatids and spermatozoa were markedly sensitive to EMS. Statistical analysis confirmed that differences in respect of percentage of egg hatch among the 3 different treatments as well as the interactions between the 3 factors, e.g. stages, hatchability and EMS treatment, were highly significant.

Animals

Transcription of a cloned Bombyx mori tRNA2Ala gene: nucleotide sequence of the tRNA precursor and its processing in vitro.

We have analyzed the transcription of a cloned silkworm tRNA2Ala gene in germinal vesicle extracts of X. laevis oocytes. The primary transcript was sequenced; it is 98 nucleotides long, beginning with a 5' triphosphate nucleotide and ending in a 3' oligouridine stretch. After transcription for long periods of time, enzymes in the frog extract also process the tRNA2Ala precursor to remove extra 5' and 3' nucleotides and to add a CCA end. The twenty-two extra nucleotides at the 3' end of this precursor are recovered as an intact fragment, implicating a new site of endoribonuclease cleavage in eucaryotic tRNA processing. This enzyme activity has also been demonstrated by reincubation of isolated pre-tRNA2Ala with a germinal vesicle extract. The products of in vitro cleavage are the same as those seen in the transcription reactions. The tRNA2Ala precursor molecules are made faithfully in the system with as few as 6 bp of Bombyx morti DNA upstream of the transcription initiation site of the tRNA2Ala gene. This result narrows down the minimal amount of DNA adjacent to the 5' end of a eucaryotic tRNA gene needed to support proper initiation by RNA polymerase III.

Animals

The genes for 18S, 5.8S and 28S ribosomal RNA of Bombyx mori are organized into tandem repeats of uniform length.

The organization of the multiple genes for 18S, 5.8S and 28S rRNA in the genome of the silkworm, Bombyx mori was determined by restriction endonuclease digestion and Southern blot hybridization. The ribosomal genes (rDNA) are tandemly reiterated, with a uniform repeat length of 6.9 . 10(6) daltons. Each rDNA repeat has a single site for EcoRI, HindIII, HpaI and SmaI and each of these sites has been mapped with respect to the others and to the rRNA genes; each repeat consists of a transcribed region (6 . 10(6)daltons) containing the 18S, 5.8S and 28S rRNA genes (5' leads to 3') and also a small non-transcribed spacer (approximately 10(6) daltons). Complete rDNA repeats were cloned using the vector RSF2124 and grown in Escherichia coli. Characterization of the rDNA plasmids confirmed the conclusions from studies of the total rDNA. The organization of B. mori rDNA is similar to that of other eukaryotes, except for the absence of heterogeneity in the rDNA repeat length; thus, there is neither variation in the length of the non-transcribed spacer nor the presence of inserts in a detectable portion of the rDNA. The utility of this map, and particularly of the rDNA plasmids, for detailed studies of rRNA transcription and processing is discussed.

Animals

Characterization of an alpha-like DNA polymerase from Bombyx mori silkglands.

A soluble DNA polymerase has been purified near to homogeneity from Bombyx mori silkglands. The following characteristics were observed: high molecular weight (about 150 000 - 220 00); optimum pH about 8; inhibition by high salt concentrations, sulfhydryl-group blocking agents and polyamines; absence of nuclease activity; preference for magnesium as required divalent cation with all the efficient template-primers tested; and clear template-primer specificity, the purified enzyme being able to copy primed - polydeoxyribonucleotide templates [activated DNA, poly(dA).oligo(dT), poly(dA).oligo(rU)] but not polyribonucleotide chains [poly(rA).oligo(dT), poly(rA).oligo(rU)] in the presence of either Mg++ or MN++. Believed to represent the bulk of silkgland DNA polymerase activity, the purified soluble enzyme most resembles vertebrate DNA polymerases alpha when it is compared to other eukaryotic DNA polymerases as yet characterized.

Animals

Occurrence of an active regulatory mechanism of protein synthesis during starvation and refeeding in Bombyx mori fat body.

The origin of the amino acids which participate in protein synthesis at the recovery from starvation have been determined in the fat body from Bombyx mori larvae. Endogeneous amino acids have been labelled with [3H] leucine and ingested ones with [14C] leucine, allowing their discrimination in the organism. 22 minutes after refeeding, proteosynthetic activity of the fat body, estimated by the polysome level, is increased 2.5 fold. Endogeneous leucine represents more than 90 p. cent of the leucine present in nascent polypeptides. Free leucine pools of the fat body and of hemolymph increase, mainly through the release of endogeneous leucine. It is therefore concluded that refeeding with amino acids induces the production of a signal or critical factor, responsible for the increase in proteosynthetic activity in the fat body.

Adipose Tissue, Brown

Effect of starvation on tRNA synthesis, amino acid pool, tRNA charging levels and aminoacyl-tRNA synthetase activities in the posterior silk gland of Bombyx mori L.

Changes in the translational machinery components of the Bombyx mori posterior silk gland were analysed during starvation and refeeding and compared to the regularly fed larvae. During starvation, tRNA and ribosomal RNA synthesis are stopped. The amounts of different RNA classes and of the different tRNA species slow down at the same rate. Thus various tRNA show similar half-lifes and the preexisting tRNA adaptation to fibroin mRNA translation persists during starvation. Similarly, the tRNA/rRNA ratio is constant during starvation and refeeding (12 tRNA molecules for one ribosome) as in silk glands of control animals. Aminoacyl-tRNA synthetases and tRNA charging levels are decreased during starvation. The maximal tRNA charging level obtained during maximal protein synthesis in control animals is regained after 24 h refeeding of starved larvae. Changes observed in the free amino acid pool are not similar from one amino acid to another and levels reached after starvation do not differ strongly from the controls. Our results suggest that the production of translation apparatus components is coordinated and adjusted to the protein synthesis activity. Whether this coordination occurs in the silk gland is discussed on the basis of the "metabolic regulation", primarily described in prokaryotes and Yeast. Transfer RNA charging levels seem to play a key role in the process of regulation and could be implicated in the mechanism of tRNA adaptation if this phenomenon results as expected from a transcriptional control.

Amino Acids

Studies on tRNA adaptation, tRNA turnover, precursor tRNA and tRNA gene distribution in Bombyx mori by using two-dimensional polyacrylamide gel electrophoresis.

Eighteen out of twenty amino acids have been used for identifying tRNAs from the silkworm Bombyx mori L. fractionated on two-dimensional polyacrylamide gel electrophoresis. 43 spots out of 53 have been identified. This mapping confirms previous results and brings new answers to some questions on the regulation of tRNA biosynthesis. 1. In addition to quantitative adaptation of tRNAs to the composition of silk proteins (fibroin from the posterior silk gland, sericin from the middle part) and of iso-tRNAs from posterior silk gland to the major codons of fibroin mRNA, we also observe adaptation of tRNA from various tissues to the average amino acid content of proteins from fat body, gut, gonads and carcass of the silkworm. 2. In the silk gland, turnover rates of several tRNA species are similar. The selective accumulation of tRNAs needed for decoding fibroin and sericin mRNAs which takes place during the Vth larval instar, cannot be explained by the occurrence of a preferential degradation of some tRNA species. 3. Under given conditions for incubating silk glands, it is possible to obtain an accumulation of precursor tRNA species, which are enriched in pre-tRNAAla and pre-tRNAGly in the posterior silk gland and pre-tRNASer in the middle part. 4. The distribution of tRNA genes is not random. tRNA genes for glycine, alanine and serine are prominent. Selective transcription of batteries of iso-tRNA genes could explain our data.

Animals

Juvenile hormone modifies larvae and silk gland development in Bombyx mori.

Topical application of a Juvenile Hormone Analogue (ZR 515) during the first half of the first half of the last (fifth) larval instar of Bombyx mori induces a prolongation of this instar and increases larvae weight and silk secretion. Later treatment can lead to an extra larval molt. Moreover, the hormone analogue stops either the development or the secretory activity of the silk gland, depending on the period of treatments. When Juvenile Hormone is administered at the beginning of the fifth instar, silk gland growth as well as RNA and protein syntheses are first inhibited and later resumed and amplified. Finaly silk production increases up to 150 per cent of controls. One of the primarly effects of treatments seems to be RNA synthesis. Thus Juvenile Hormone analogue application could be a new tool for studying RNA transcription and/or processing.

Animals

Quantitative data on the Bombyx mori L. silkworm: a review.

This paper summarizes a variety of quantitative data on the silkworm Bombyx mori, collected in the literature, to help building models on silk gland differentiation. The properites of the silk gland and their changes especially during the last larval instar have been reviewed (size, DNA, RNA amino acids, enzymes). The components of the silk (fibroin and sericin) are also studied (molecular weight, composition). Thus translation and transcription rates have been estimated. The relevant data on the fat body and the haemolymph are also given, as well as some characteristics of the oocyte/egg system.

Animals

Identification of precursor molecules to individual tRNA species from Bombyx mori.

Short-labeled 4.5S RNA molecules isolated from the posterior silk gland of Bombyx mori can be separated by two-dimensional polyacrylamide gel electrophoresis into many discrete species, some of which are radiochemically pure by the criteria of RNA fingerprinting. One region of the gel contains two precursor RNAs, one to each of the known alanine transfer RNAs. Each precursor tRNAAla molecule contains all of the internal oligonucleotides present in the corresponding tRNAAla species plus new 5'-and 3'-terminal sequences. Precursor molecules to tRNA1Gly (which differ from each other in size) are contained in two other gel regions, and a fourth region contains a precursor to tRNA2Gly. Both of the transcription initiator purine tetraphosphate nucleosides are present in unfractionated tRNA precursor mixtures, with pppA- predominating over pppG-. Minor nucleotides are also present in B. mori tRNA precursors. No polycistronic tRNA gene transcripts were observed.

Alanine

Homology of the 3' terminal sequences of the 18S rRNA of Bombyx mori and the 16S rRNA of Escherchia coli.

The terminal 220 base pairs (bp) of the gene for 18S rRNA and 18 bp of the adjoining spacer rDNA of the silkworm Bombyx mori have been sequenced. Comparison with the sequence of the 16S rRNA gene of Escherichia coli has shown that a region including 45 bp of the B. mori sequence at the 3' end is remarkably homologous with the 3' terminal E. coli sequence. Other homologies occur in the terminal regions of the 18S and 16S rRNAs, including a perfectly conserved stretch of 13 bp within a longer homology located 150--200 bp from the 3' termini. These homologies are the most extensive so far reported between prokaryotic and eukaryotic genomic DNA.

Animals

Isolation and characterization of malic enzyme from the pupa of Bombyx mori.

Malic enzyme, which requires NADP+ as a coenzyme, was isolated and purified from pupae of the silkworm, Bombyx mori. The purified enzyme appeared homogeneous and had a molecular weight of 195,000 on polyacrylamide gel electrophoresis. The optimum pH for the oxidative decarboxylation of malate, measured in terms of the increase of NADPH (MH activity) and CO2 (MC activity), was pH 7.5, while that for the decarboxylation of oxaloacetate measured in terms of the increase of CO2 (OC activity) was pH 4.6. Several differences between MH and OC activity were investigated.

Ammonium Sulfate

Purification and some properties of a specific nuclease which cleaves transfer RNA precursors from the posterior silk gland of Bombyx mori.

A specific endonuclease involved in the processing of tRNA precursors was isolated and partially purified from the posterior silk gland of Bombyx mori, and designated as RNase P.Bmo. This enzyme was shown to catalyze the conversion of 4.5 S precursor RNA to 4.1 S RNA by trimming the 5'-additional segment from the precursor RNA. RNase P.Bmo required divalent cations, Mg2+ or Mn2+. In the presence of these divalent cations, K+ or NH4+ activated the RNase P.Bmo reaction. Optimum pH was observed around 8.0. Ribosomal RNA's and mature tRNA from the silk gland were not cleaved by RNase P.Bmo. A 4.5 S precursor RNA fraction containing formycin, an adenosine analog, was less susceptible to RNase P.Bmo than the normal one. These results indicate that RNase P.Bmo has a high substrate specificity. An additional nuclease(s) was isolated. This activity was assumed to remove the extra 3'-segment of the 4.5 S precursor RNA.

Ammonium Chloride