Annie Garel

Structure and organization of the Bombyx mori sericin 1 gene and of the sericins 1 deduced from the sequence of the Ser 1B cDNA

The sericin 1 primary transcript of the silkworm Bombyx mori is differentially spliced via a tissue- and developmentally-regulated process. From a middle silk gland cDNA library, we have elucidated the sequence of one of the four mRNAs, the 4.0 kb Ser1B mRNA. Determination of alternative or constitutive exons and intron-exon boundaries allowed us to establish the nine exon-eight intron structure of the Ser1 gene. From these and previous data, it was possible to deduce the sequence of the sericins 1 and to predict the secondary structure and physiochemical properties of the different regions of the proteins.

Fork head alternative binding drives stage-specific gene expression in the silk gland of Bombyx mori

Here, we identified the main transactivator of fhx, the gene encoding the silk protein fibrohexamerin in posterior silk gland cells (PSG), as the homeotic SGF1/fork head factor. The same factor also stimulates sericin-1, another silk protein encoding gene, in the middle silk gland cells. SGF1/fork head is present in the silk gland nuclei during the whole course of larval life, but its binding to the fhx promoter occurs at intermolt and not during molt, when fhx is respectively turned on and off. The alternative binding of the factor is associated with specific changes in the fhx chromatin topology in PSG cells. Taken together, our results show that stabilization of SGF1/fork head to its target sequence is critical to promote fhx transcription at each intermolt. We also found that fhx is characterized by a PSG-specific DNase I hypersensitive site in the first intron, present during molt and intermolt, i.e. independent of the transcriptional status of the gene. All these data suggest that differential chromatin accessibility and fork head activation are crucial in controlling the spatial and temporal regulation of the fhx gene in the posterior silk gland cells.

Complexity and diversity of polyadenylated mRNA in the silk gland of Bomyx mori: Changes related to fibroin production

Abstract Poly(A) containing RNA was isolated from the posterior silk gland of Bombyx mori at the end of the fourth molt when the gland does not produce fibroin (stage V 0 ) and at the middle of the fifth larval instar when fibroin synthesis is massive (stage V 6 ). The kinetics of hybridization of these mRNAs with their complementary DNA (cDNA) were markedly different. The mRNA from V 0 stage consists of two abundance classes comprising, respectively, 38 and 2915 different sequences. V 6 mRNA-cDNA hybridization could not be analyzed properly by standard calculations since we found that the messenger RNA coding for fibroin (FmRNA) was about eight times less transcribed than the other mRNAs. This low yield of reverse transcription was assumed to be due to the exceptional length of FmRNA (16,000 nucleotides) as compared to the average length of the other silk gland mRNAs (1400 nucleotides). Therefore we separated the FmRNA from the rest of polyadenylated RNA (F ⊝ mRNA) by sucrose gradients and analyzed the two preparations separately. The kinetic complexity of FmRNA was very low for its size and can be accounted for by a 66-nucleotide-long driving sequence. This result agrees with the existence of a short repetitive sequence known to represent 60% of the molecule. The V 6 F ⊝ mRNA preparation was resolved into four classes containing, respectively, 1, 20, 319, and approximately 2600 sequences. Cross hybridizations have been performed with each of the cDNA abundance classes from V 0 stage and V 6 F ⊝ mRNA. They demonstrated that almost all the sequences present at the V 0 stage are also detected at V 6 stage, but their distribution among the abundance classes was modified. The relative concentration of 26 species of the abundant class and 291 species of the rare class of the V 0 stage was increased at V 6 and therefore appears to be implicated in the building of the fibroin synthesizing machinery, whereas 12 mRNA species from the V 0 abundant class decreased in relative concentration and are probably subject to another mode of control. Finally a large number of mRNA species remained in a stable low concentration at both stages and may be attributed to housekeeping functions. Our data show that the spectacular silk gland cell adaptation to fibroin production is characterized by massive accumulation of FmRNA and of an unknown mRNA species, and that stage variations in mRNA populations are related to relative quantitative variations rather than qualitative differences.

Comparison between histones FV and F2a2 of chicken erythrocyte: II. Interaction with homologous DNA

The conformation and stability of artificial complexes between chicken erythrocyte DNA and homologous histones FV and F2a2 was studied by circular dichroism (CD) and thermal denaturation followed by both absorbance and CD measurements. The complexes are made after a stepwise potassium fluoride gradient dialysis without urea and studied at low ionic strength (10-minus 3 M). 1) No structural changes of the DNA can be detected up to r equals 0.2 with FV and r equals 0.6 for F2a2. With FV at higher values of r the CD spectrum is altered, indicating the organization of DNA and histones in some kind of aggregate. 2) The conformation of histone molecules inside the complexes is not related to the ionic strength of the medium but to an effective ionic environment close to 0.1 M. This ionic strength would also correspond to the melting temperature of histone-covered DNA. 3) From the analysis of the absorbance melting profile the length of DNA covered with an histone molecule can be estimated. A good agreement is found between the negative charge of this piece of DNA and the net positive charge of the histone. 4) Since the CD transition at 227 nm occurs before the second absorbance transition at 280 nm, the DNA is stabilized no longer by native histone but partially or fully denatured histones. The helical regions of the histone molecule are not involved in the binding process, which appears to be almost purely coulombian and most likely related to some structural fit between the pattern of negative charges in the DNA helix and that of positive charges along the peptide chain.

Histones d'érythrocytes de poulets: III. Mise en évidence de l'hétérogénéité du fragment N-Terminal de l'histone spécifique

Summary After splitting the molecule of chicken erythrocyte specific histone (Fv) with cyanogen bromide we have isolated three fractions, by chromatography on a carboxy-methyl-cellulose column eluted with an ionic strength gradient, buffered at pH 4,3 : 2 N-terminal fractions A1 and A2 and the C-terminal fraction B. The results of amino-acid analysis and N-terminal determination of the tryptic peptides of the two N-terminal fractions are in complete agreement with the microheterogeneity which has been recently suggested by Greenaway and Murray : at amino-acid no 15, a glutamyl residue in the fraction A1, and an arginyl residue in the fraction A2 have been found.