The Saccharomyces cerevisiae translation initiation factor Tif3 and its mammalian homologue, eIF-4B, have RNA annealing activity.

The Saccharomyces cerevisiae TIF3 gene encodes the yeast homologue of mammalian translation initiation factor eIF-4B. We have added six histidine residues to the C-terminus of Tif3 protein (Tif3-His6p) and purified the tagged protein by affinity chromatography. Tif3-His6p stimulates translation and mRNA binding to ribosomes in a Tif3-dependent in vitro system. Furthermore, it binds to single-stranded RNA and catalyses the annealing of partially complementary RNA strands in vitro. In parallel experiments, RNA annealing activity could also be demonstrated for mammalian eIF-4B. A role for Tif3/eIF-4B and RNA annealing activity in the scanning process is proposed.     

Transformation of chicory and expression of the bacterial uidA and nptll genes in the transgenic regenerants

Transgenic chicory plants were obtained from different explantsco-cultured with Agrobacterium tumefaciens. Among tap-root,leaf and cotyledonary tissues, etiolated cotyledons showed thegreatest competence for transformation. The Agrobacterium strainsused contained either pGSGLUC1 or pTDE4 as a vector which carryboth the neomycin phosphotransferase II gene (nptll) for kanamycinresistance and ß-glucuronidase gene (uidA) under thecontrol of different promoters. Transformation was confirmedby NPTII enzymatic assay, histochemical analysis of GUS activityand DNA hybridization. Transgenic plants expressed both markergenes in root and shoot tissues. In leaves, GUS activity wasexpressed in all tissue types, whatever the nature of the promoter.Nevertheless, variable heterogeneous patterns of expressionwere observed in the different root tissues. Differential expression of the GUS fusions controlled by thedual TR or the CaMV 35S promoters are discussed. Key words: Chicory, genetic transformation, GUS activity, kanamycin resistance     

Induction of alternative oxidase synthesis by herbicides inhibiting branched-chain amino acid synthesis

Sycamore suspension cells ( Acer pseudoplatanus L.) were incubated in the presence of sulfonylurea and imidazolinone herbicides. These inhibitors of acetolactate synthase (ALS), a key enzyme of branched-chain amino acid synthesis, triggered a dramatic induction of the alternative oxidase (AOX). AOX activity increased in treated cells, eventually exceeding cytochrome (cyt) pathway activity. This induction of AOX activity was correlated with the accumulation of a 35 kDa AOX protein in isolated mitochondria, detected by Western blotting with a monoclonal antibody against Sauromatum guttatum AOX. It was preceded by the accumulation of putative 1.6 kb AOX mRNA, detected using an Aox cDNA probe from soybean. The metabolic perturbations induced by the herbicides rather than the herbicide molecules themselves were responsible for this induction of AOX. However, α-oxobutyrate (one of the substrates of ALS) and its transamination product, α-aminobutyrate, which accumulated after herbicide treatment, were not involved. The inhibition of branched-chain amino acid synthesis was probably somehow responsible for the AOX induction since: (i) a mixture of those amino acids (leucine, isoleucine, valine) prevented AOX induction by ALS inhibitors; (ii) the herbicide Hoe 704, a potent inhibitor of acetolactate reducto-isomerase (the enzyme following ALS in the branched-chain amino acid pathway), also triggered AOX induction.     

Calmodulin during development and metamorphosis in urodelan amphibians

Calmodulin isolated and purified to homogeneity from young larvae is very similar to that obtained from adult Pleurodeles waltlii and these proteins are almost identical to previously described vertebrate calmodulins. During P. waltlii development, an increase in total individual calmodulin content is observed after the heart beating stage. In dorsal axial muscle, calmodulin level which is very high at the beginning of larval life (premetamorphosis) decreases strikingly in the first part of prometamorphosis. Such an evolution is observed in Ambystoma mexicanum too. Then, a significant increase occurs during metamorphosis. In contrast, calmodulin level in P. waltlii cardiac ventricular muscle increases continuously from hatching to the end of metamorphic climax. Thyroxine treatment which promotes precocious metamorphosis in P. waltlii and experimental metamorphosis in neotenic A. mexicanum, induces a rapid and significant increase in muscle calmodulin concentration.     

A fetal skeletal muscle actin mRNA in the mouse and its identity with cardiac actin mRNA

We compare a recombinant cDNA plasmid (pAF81) complementary to a fetal skeletal muscle actin mRNA with a plasmid (pAM91) complementary to the actin mRNA expressed in adult skeletal muscle. The two mRNAs are significantly diverged in silent nucleotide positions; they are coexpressed in fetal skeletal muscle, and in differentiating muscle cell cultures their accumulation begins coordinately. The sequence of pAF81 shows that the amino acid sequence of mouse fetal skeletal muscle actin is almost identical to that of adult bovine cardiac actin. Hybridization of pAF81 to RNA from different mouse tissues shows that fetal skeletal muscle actin mRNA is very homologous or identical to fetal and adult cardiac actin mRNA. Only one gene homologous to pAF81 is detected on blots of restricted mouse DNA. We conclude that this gene must be expressed both in fetal skeletal muscle and in fetal heart. Whereas mRNA transcribed from this gene is the major actin mRNA species in adult heart, it is present in low amounts, if at all, in adult skeletal muscle.     

The multiple conformations of a cyclic disaccharide: DI-β-d-glucopyranose 1,6′:1′,6-dianhydride hexaacetate

The conformational behaviour of a cyclic disaccharide, di-β-d-glucopyranose 1,6′:1′,6-dianhydride hexaacetate, has been investigated. Because this molecule can exist only with the glucose rings in the unusual flexible forms, such conformational parameters as pseudorotation phase-angles have been used. Within a given number of approximations, the conformational space available for the whole system can be explored by considering only one two-dimensional map. Detailed investigations have shown that three stable conformations may be proposed. Among these, two correspond to minima found in the solid state. In one form, the six-membered rings adopt a boat conformation, whereas a skew conformation is found for the other form. However, these two conformations cannot be considered to be unique models of the conformation in solution; they both produce sets of proton-proton coupling-constants inconsistent with observed n.m.r.-spectroscopic results. At least the third form, having the six-membered rings in skew conformations, has to be taken into account. Deviations from coupling constants-molecular conformation relationships are thought to originate from ring strain.