Jean-Claude Prudhomme

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.

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.

Cloning and characterization of the highly polymorphic Ser2 gene of Bombyx mori.

Three alleles of the sericin (Ser) 2-encoding gene (Ser2), called L, C and mC, were isolated from a Bombyx mori genomic library, and two related ones, called mCL and Cv, were also characterized in B. mori European strains. The Ser2 gene gives rise to two middle silk gland mRNAs by differential splicing. The size of a short mRNA (3.1 kb) is constant, but the length of a longer one ranges from 5 to 6.4 kb depending on the Ser2 allele. These length variations probably result from unequal recombinations in a region which contains about 30 well conserved 45-bp repeats coding for a Ser-like peptide. Furthermore, the L allele (and probably the mCL one) contains a 4.4-kb retrotransposon, resembling the copia-like ones of Drosophila.

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.

The expression of five middle silk gland specific genes is territorially regulated during the larval development of Bombyx mori

Abstract By differential screening of a Bombyx mori genomic library, three genes specifically expressed in the middle silk gland ( MSGS 3, 4 and 5 ) have been isolated. They code respectively for a 3500, 2950 and 450 nt long mRNA. By probing Northern blotted RNA, we investigated their topological distribution during the last larval instar. The results parallel those previously observed for the Ser 1 and Ser 2 genes. As Ser 1, MSGS 3 is expressed in a strictly limited middle silk gland (MSG) area, including the middle and posterior subparts, whereas MSGS 5 expression, as the Ser 2 one, precociously occurs in the whole MSG, but is progressively restricted to its anterior subpart. The MSGS 4 mRNA is detected only in the MSG anterior subpart. This parallelism was confirmed by probing dot-blotted MSG RNA from fourth to mid-fifth larval instar, which shows also that the maximal relative accumulation of the MSGS 5 and Ser 2 RNAs occurs earlier than that of the MSGS 3 and Ser 1 ones. The topographical and temporal regulation of these five genes recalls that of the genes governing pigment accumulation, suggesting that the former are under the same hormonal control as the latter.

The biosynthesis of fibroin: III. Involvement of the Golgi apparatus in transport and secretion—An electronmicroscope autoradiographic study☆

Abstract The intracellular transport of fibroin and its secretion have been studied in the silk gland of Bombyx mori incubated in vitro. Sites of synthesis and transport were determined radioautographically by pulse-chase labelling with tritium-labelled glycine. The label, initially located in the rough endoplasmic reticulum (RER) is found in the complexes and the fibroin globules of the Golgi apparatus before it accumulates in the cell-lumen border and further on reaches the compact cylinder of silk in the lumen. Study of the relative distribution of the radioactivity in the gland has allowed us to use a compartment model, developed in the appendix, to calculate the mean residence times of a molecule in the RER and the Golgi apparatus. They were found to be respectively 35 and 13 min. The involvement of the Golgi apparatus in the transport of secreted protein is discussed by comparing our results with current knowledge on fibrous protein transport and secretion.