Jean-Pierre Cecchini

Peroxidases associated with lentil root ribosomes

Abstract Three peroxidases are associated with the ribosomes of lentil roots. During cellular fractionation, the three haemoproteins are only present on these particles and not in the other fractions. The three peroxidases were isolated and obtained in a highly purified form. Their spectral characteristics and their molecular weights were determined. One is slightly acidic at pH 6·8, whereas the other two are slightly basic at this same pH. The incorporation of a 14 C-amino acid into these haemoproteins has enabled us to show that the basic peroxidases turn over rapidly whereas the acid peroxidase is very stable. Treatment of the roots with IAA considerably stimulates the de novo biosynthesis of the two basic peroxidases but not that of the acid peroxidase.

Stage and tissue-specific expression of a collagen gene during Drosophila melanogaster development☆

Based on data from developmental RNA profiles and in situ hybridization, we report a direct examination of the expression of one collagen gene (Dcg1) during drosophila melanogaster life cycle. These studies show, for the first time, that the expression of a collagen gene is both differential and tissue-specific during the course of development. Moreover, they demonstrate that the connective tissues in Drosophila do contain a collagen type synthesized by mesodermal tissues. Indeed the accumulation of Dcg1 transcripts was located mainly within the second instar fat bodies, the third instar lymph glands, and over adepithelial cells associated with third instar imaginal discs. In addition, these results seem to confirm the interpretation that wandering hemocytes released by the lymph glands could contribute in extracellular matrix composition in some tissues in the larva.

Ribosomal cistrons in higher plant cells. I. A definitive scheme for the maturation pathway of the primary transcriptional product of ribosomal cistrons in Acer pseudoplatanus L. cells.

1. Four precursor ribosomal RNAs have been previously characterized in cultured sycamore cells (Acer pseudoplatanus L.). The present paper reports a study of the two smallest ones. 2. (Me-3H)-labelling of the precursor and mature rRNAs has been submitted to kinetic analysis. From pulse-chase experiments it is concluded that the 27S and 20S precursor rRNAs behave like intermediate molecules in the maturation of the 42S and 36S precursor rRNAs into the 26S and 17S components. They are direct precursors to the cytoplasmic and mature rRNAs. 3. Base-composition analyses have been performed on carefully purified molecules. They show that the 27S and 20S precursor rRNAs arise from a single cleavage of the 36S precursor rRNA. In addition, they strongly suggest that the 27S and 20S precursor rRNAs can be taken as specific precursors to the 26S and 17S rRNAs, respectively. These results are reinforced by DNA - RNA hybridization experiments, the measured values being in good agreement with the theoretical ones expected from a specific relationship between the four smallest molecules. Base-compositions of the conserved and non-conserved nucleotide sequences of the primary transcriptional product have been estimated or calculated. 4. On the basis of the present results and those previously reported, a scheme for the biosynthesis of ribosomal RNA in sycamore cells is proposed.

Contribution a l'etude d'acides ribonucleiques a marquage rapide de la racine de lentille: Isolement et caracterisation, action d'une hormone sur leur synthese et sur celle de peroxydases

Abstract Chromatography on methylated albumin columns of RNAs from Lentil roots, shows the presence of rapidly labelled RNAs tenaciously bound onto the column and which cannot be eluted by high NaCl molarities. Elution at 35°, with sodium dodecyl sulfate, gives two peaks (α and β). At 70°, a third elution peak (γ) is obtained. For short labelling periods, RNAs of the α peak are uridine rich (UMP/AMP approx. 1.2), heterodisperse and may have an important percentage of high-molecular-weight species. RNAs from the β peak are adenine rich (UMP/AMP approx. 0.4). Their modal sedimentation constant is about 12 S. The mean life of the uridine-rich RNAs of the α peak is lower than 1 h; that of the RNAs from the β peak is higher than 2 h. RNAs from α peak are essentially, or exclusively, of nuclear origin. On the other hand, polysomic messenger RNAs are present in the β peak. A close similitude exists between uridine-rich RNAs described in animal cells and the nuclear rapidly labelled RNAs of Lentil roots contained in the α peak. Short treatment (45 min) of Lentil roots with indoleacetic acid increases the amount of nuclear uridine-rich RNAs. A prolonged treatment (5 h) causes an increase in various rapidly labelled RNAs, probably polysomic messenger RNAs. Treatment of excised roots with indoleacetic acid leads, after a lag period of about 5 h, to the synthesis of two peroxydases associated with the ribosomes. The mean life of their messenger RNAs is about 2.5 h.

DCg1 αIV collagen chain of Drosophila melanogaster is synthesized during embryonic organogenesis by mesenchymal cells and is deposited in muscle basement membranes

Abstract We report the purification and characterization of three sequence-specific polyclonal antibodies raised against specific portions of the Drosophila αIV collagen chain produced from the gene DCg1. These antibodies were used for immunolocalization experiments on tissue sections from embryonic organogenesis stages (13–17) and first larval stages. This analysis was paralleled by in situ hybridization experiments with a labeled fragment of the gene DCg1. We demonstrated that, by late embryogenesis, the DCg1 αIV chain was synthesized by individual mesoblasts and deposited in basement membranes of skeletal and visceral muscles. These sites of αIV collagen deposition were the same, by first and second instars, but the protein was then synthesized by fat body cells. Our results were reminiscent of those obtained for vertebrate in vitro myogenesis, they suggested, moreover, a tissue-specific composition of basement membranes in Drosophila melanogaster .

Ribosomal cistrons in higher plant cells: II. Sequence homology between the two mature rRNAs of sycamore cells and intracistronic reiteration. A DNA · rRNA hybridization study

1. Uniformly labelled rRNA of sycamore cells has been annealed with homologous DNA. The fractions of DNA complementary to the 17S, or 26S, or 17S + 26S rRNAs are found to be 0.19%, 0.15% and 0.23%. They are not in the ratio of the molecular weight values (0.8, 1.2 and 2 - 10(6), respectively for the 17S, 26S and 17S + 26S rRNAs). This result is compatible with the large hybridization competition observed between the two rRNAs (53 and 72%) and with the shift-down of saturation curves when DNA is presaturated with unlabelled rRNA before the incubation with the other labelled rRNA. 2. Under the selected experimental procedure, the DNA - rRNA hybrids formed appear to be specific. Since there is an equal number of structural genes for the 17S and 26S rRNAs, these results mean the occurrence of a great sequence homology, strictly restricted to the two rRNAs. Homologous and specific sequences have been estimated to 0.1 and 0.7, or 0.85 and 0.35 million daltons, respectively in the 17S or 26S structural genes. 3. From the calculated lengths of homologous sequences, an intracistronic reiteration of some ribosomal sequences can be deduced. This internal reiteration is directly evidenced by the complex pattern of DNA - rRNA annealing curves. As demonstrated by base-composition analysis, the internal reiteration is heterogeneous and concerns both the homologous and specific sequences. In addition, the DNA saturation values allow the calculation of 4000 copies for the ribosomal cistron in the whole sycamore genome.

Effect of ligand binding and of pH change on the accessibility of thiol residues of fructose-1,6-bisphosphatase from spinach chloroplast

Sulfhydryl groups of reduced fructose-1,6-bisphosphatase (d-fructose-1,6-bisphosphatase 1-phosphohydrolase, EC 3.1.3.11) from spinach chloroplast have been classified in several sets of different accessibility according to their rate of reaction with dithiobis(nitrobenzoic acid). The oxidation by this reagent results from the linkage of vicinal thiols into disulfide bridges. The thiol groups involved in the activation-deactivation process have been characterized by analyzing the effects of a stepwise oxidation on the catalytic activity of the reduced enzyme. The target of the redox activation process is constituted by two disulfide bridges as indicated by the fact that the four resulting thiols are essential residues for the activity. Mg2+ or substrate fixation modifies the enzyme conformation and the accessibility of the strategic sulfhydryls. O-Methylfructofuranoside 1,6-bisphosphate, a non-reactive substrate analog, induces the same conformational transition as the one induced by the substrate. The simultaneous fixation of Mg2+ and the substrate analog maintains the reduced enzyme in a specific conformation which is very sensitive to pH variations. At pH 7 the four strategic thiol groups are deeply embedded in the protein but are directly exposed to oxidation at pH 8.

Partial base-methylation and other structural differences in the 17 S ribosomal RNA of sycamore cells during growth in cell culture

Sycamore (Acer pseudoplatanus L.) cytoplasmic rRNA was investigated in rapidly dividing cells, cells starting mitosis after the lag phase of growth (4 days) induced by deconditioning of the culture medium and also in growth-arrested cells from 10 day-old cultures deprived of exogenous auxin (i.e. exponential, early exponential and 2,4-dichlorophenoxyacetic acid (2,4-D)-deprived cultures). rRNA was extracted and purified from mixed 14C-labelled exponential cultures and 3H-labelled early exponential cultures. A 14C-labelled exponential culture and a 3H-labelled 2,4-D-deprived culture were analyzed in the same way. The 17 S rRNA molecules from both early exponential and 2,4-D-deprived cultures displayed a lower electrophoretic mobility on polyacrylamide gels than those from exponential cultures. Alkaline and acid hydrolysates of purified 17 S rRNA labelled on the phosphate groups or the methyl groups were analyzed on ion-exchange resins. There was no change in the extent of ribose methylation of the molecule from the three different cultures. However, the base methylation of the 17 S rRNA was decreased in early exponential cultures and in 2,4-D-deprived cultures. Part of the molecules synthesized in early exponential cultures specifically lacked 7-methylguanine, N6-methyladenine and N6,N6-dimethyladenine. The possible significance of these changes in the 17 S rRNA were discussed.