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P Couble

Publications and source records attributed to P Couble.

33 records · Page 2Linked to original sources
PubMed1990

Specific expression of a silk-encoding gene of Bombyx in the anterior salivary gland of Drosophila.

Successful expression of genes transferred into distantly related species in which genetic functions have been maintained through evolution has been reported previously. In the case of the silkmoth Bombyx mori and the fruitfly Drosophila melanogaster, both of which produce chorions (eggshells), Bombyx chorion genes are correctly expressed in Drosophila despite their estimated 240-Myr phylogenetic divergence. Here we report that, although Drosophila does not produce silk, mechanisms regulating transcription have been conserved between the salivary gland of the fruitfly and the silk gland of the silkmoth larva.

Amino Acid Sequence↗
PubMed1987

mRNA characterization of human fetal enamel matrix.

Preliminary characterization of the human enamel matrix at 16-18 weeks in utero was performed. Using an homogenizing buffer, the proteins were extracted and analysed by gel electrophoresis. Total cellular RNA was isolated and the cell-free mRNA translated. The major component was a 68,000 protein with an enamelin-like amino-acid composition. Other translation products included a 55,000 polypeptide and lower mol. wt components of 26,000, 22,000 and 20,000 size of amelogenin size. It is suggested that high mol. wt component in the enamelin range is the most prevalent at the early stage of human tooth development.

Amino Acids↗
PubMed1987

Developmental switches of sericin mRNA splicing in individual cells of Bombyx mori silkgland.

Four mRNA of 10.5, 9.0, 4.0, and 2.8 kb are made from the sericin Ser1 gene by alternative maturation of a unique mRNA precursor. By means of RNA blots and in situ hybridization, we investigated variations in the distribution of these mRNA during the last larval instar in different territories of the middle silkgland. Taken together, the results from these two techniques show that 150 out of the 266 cells of this region of the organ express the Ser1 gene, but accumulate distinct mature mRNA species. Of these 150 cells 42 are specialized in a processing pathway resulting in the production of the 2.8-kb Ser1 mRNA throughout the larval instar. The 108 others perform successively three distinct splicing pathways leading to a development-dependent accumulation of, respectively, the 4.0-, the 10.5-, and the 9.0-kb mRNA. This suggests the occurrence of two switches in the splicing capacities of these cells during the fifth instar. The middle silkgland cells also express another sericin gene (Ser2) which encodes two mRNA of 5.4 and 3.1 kb, also arising by differential splicing. At the beginning of development, all the middle silkgland cells express this gene but, as development proceeds, expression becomes restricted to only the anterior cells. The biological consequence of this topological and temporal regulation of the mode of expression of these two genes is the sequential secretion and layering of the different sericins around the silk thread.

Animals↗
PubMed1985

Structural organization of the P25 gene of Bombyx mori and comparative analysis of its 5' flanking DNA with that of the fibroin gene.

We have cloned a large portion of the P25 gene of Bombyx mori encoding the 25,000 dalton polypeptide which associates with fibroin to constitute the major silk protein. Its structure has been investigated by restriction mapping R-loop analysis, S1 nuclease protection experiments and nucleotide sequencing of the region spanning the 5' end of the gene and its flanking DNA. This has permitted a comparative sequence analysis of the DNA from the P25 and fibroin genes. The genes demonstrate no relatedness in their coding regions but they exhibit large blocks of sequence homology in their 5' flanking regions. In particular, the DNA upstream of the P25 gene possesses a sequence very similar to a region of fibroin 5' flanking DNA that is known to possess transcription modulation signals. The functional significance of these homologous regions is discussed with regard to the highly coordinated expression of these two genes.

Amino Acid Sequence↗
PubMed1984

Actin microfilaments and fibroin secretion in the silkgland cells of Bombyx mori. Effects of cytochalasin B.

Bombyx mori posterior silkgland cells exhibit an impressive microfilament apparatus located at the cellular apex. It consists of bundles of packed, long microfilaments of 50-70 A diameter running along circumferences delimiting the lumen of the gland, perpendicularly to the flow of luminal silk. Microfilaments are closely associated with microtubules of the cytoplasmic 'radial microtubule system'. Immunolabelling with purified antihuman actin antibodies was used to demonstrate their actin-like nature. Apical microfilaments are sensitive to cytochalasin B (CB) which selectively inhibits the secretion of fibroin. Following the removal of the drug, microfilaments recover their normal morphology and secretion resumes. The possible implication of contraction of microfilaments in the process of secretion is discussed.

Actins↗
PubMed1983

Developmental variations of a nonfibroin mRNA of Bombyx mori silkgland, encoding for a low-molecular-weight silk protein.

The characterization of a new silk protein mRNA (P25 mRNA) in posterior silkgland cells (PSG) and the developmental variations of its cell molecular concentration versus that of fibroin mRNA are described. A 80% pure P25 cDNA was obtained by class separation of total nonfibroin cDNA from PSG and used to identify the mRNA in blotted PSG mRNA as a single 1100 nucleotide long species. When purified from agarose gel and translated in a reticulocyte cell-free system, P25 mRNA yielded a 25-kD polypeptide (P25), identical to a 25-kD protein of the cocoon in terms of pI value and partial peptide mapping pattern. Moreover, this protein comigrated with an abundant polypeptide of the posterior silkgland (PSG) and of the middle silkgland (MSG). When tritiated leucine was injected in vivo, labeled P25 showed up in the PSG after a 2-hr pulse but appeared in the MSG only after 24 hr of labeling. Since MSG cells were found to be devoid of P25 mRNA, we concluded that P25 is exclusively synthesized in the PSG, that it accumulates in the MSG lumen and that it is spun out in the same way as fibroin. Specific probes were used to measure the concentrations of P25 mRNA and also fibroin mRNA in PSG total RNA by hybridization with an excess of cDNA. Both species are highly degraded in the few hours following the physiological arrest of feeding which precedes the fourth molting period. Their subsequent accumulation during the fifth intermolt is triggered by food uptake and proceeds in such a way that a constant 1:1 molar ratio is maintained during the period of silk secretion.

Animals↗
PubMed1981

[Gene expression in Bombyx mori silkgland cells during the last larval instar].

Poly (A)-containing RNA was isolated from the posterior silkgland of Bombyx mori just after the fourth molt, when the gland is not producing fibroin (stage V0), and at the middle of the fifth larval instar, when the fibroin is massively synthesized (stage V6). The hybridization kinetics of these mRNA with their complementary DNA (cDNA) were remarkably different. The mRNA from stage V0 consisted of two abundance classes, comprising 38 and 2 915 different species, respectively. At stage V6, fibroin mRNA (FmRNA) was separated from the rest of the poly (A)-containing RNA (F-mRNA) by centrifugation on sucrose gradients, and both preparations were analyzed separately. The kinetic complexity of FmRNA was very low as compared to its actual size. This result agrees with the existence of a short repetitive sequence accounting for 60 p. 100 of the molecule. Stage V6 F-mRNA was resolved into four classes containing 1, 20, 319 and about 2 600 species, respectively. We carried out cross-hybridization with each of the two cDNA classes from stage V0 and stage V6 F-mRNA. They demonstrated that almost all the sequences present at stage V0 were also present at stage V6, but that their abundance class distribution was modified. Our data show that the specialization of the silkgland cell for fibroin production is characterized by massive accumulation of FmRNA and another unknown species, and that stage variations of mRNA populations are related to relative quantitative variations rather than to qualitative ones.

Animals↗
PubMed1980

The programming of silk-gland development in Bombyx mori. I. Effects of experimental starvation on growth, silk production, and autolysis during the fifth larval instar studied by electron microscopy.

The cytological development of the silk gland has been studied by light and electron microscopy in silkworms experimentally starved at different periods of the natural feeding stage during the fifth instar. When newly molted animals are not provided with food, no sign of growth is observed. Starvation initiated early during the obligatory feeding period, stops cell growth and development of the organelles involved in protein synthesis and secretion, whereas it induces the appearance of organelles concerned with autolysis. These effects are reversible if starvation is not prolonged beyond two days. Starvation during the facultative feeding period, at the time of massive fibroin production, results in quantitative and qualitative modifications of organelles related to the decrease of fibroin production and the onset of autolysis. Rough endoplasmic reticulum, responsible for fibroin synthesis, forms transitory whorls. Fibroin transport via the Gjolgi apparatus and secretion of the protein into the gland lumen decrease parallel to fibroin synthesis, so that no fibroin storage can be detected in any organelle. After food deprivation, autophagosomes and secondary lysosomes rapidly develop in the cytopolasm, and if starvation continues portions opf the cytoplasm are sequestered and completely destroyed. If animals are refed, fibroin production is resumed and autolysis declines. These ultrastructural alterations of the silk gland during experimental starvation are very similar to those observed during the periods of physiological starvation (molt and cocoon spinning) and generally considered to be under hormonal control. Our results raise the question of the nature of interactions between alimentary and hormonal factors which control silk-gland development.

Animals↗
PubMed1979

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↗
PubMedDate not supplied

A single gene produces multiple sericin messenger RNAs in the silk gland of Bombyx mori.

The sericins are a family of major cocoon proteins specifically synthesized in the middle silk gland of the silkworm Bombyx mori. The 5' part of one sericin gene had been cloned and described by Okamoto et al. (1982, J. Biol. Chem. 257, 15192-15199). Using a differential screening procedure of Bombyx genomic libraries, we obtained the 3' part of this gene. We demonstrate that it consists of a single gene extending over 24 kb, present in two allelic forms in hybrid strains. This gene encodes for four mRNAs which result from a unique transcript by an alternative splicing mechanism. This explains, at least partially, the diversity of the sericins found in the cocoon.

Base Sequence↗