Protein S1 counteracts the inhibitory effect of the extended Shine-Dalgarno sequence on translation

There are two major components of Escherichia coli ribosomes directly involved in selection and binding of mRNA during initiation of protein synthesis-the highly conserved 3' end of 16S rRNA (aSD) complementary to the Shine-Dalgarno (SD) domain of mRNA, and the ribosomal protein S1. A contribution of the SD-aSD and S1-mRNA interactions to translation yield in vivo has been evaluated in a genetic system developed to compare efficiencies of various ribosome-binding sites (RBS) in driving beta-galactosidase synthesis from the single-copy (chromosomal) lacZ gene. The in vivo experiments have been supplemented by in vitro toeprinting and gel-mobility shift assays. A shortening of a potential SD-aSD duplex from 10 to 8 and to 6 bp increased the beta-galactosidase yield (four- and sixfold, respectively) suggesting that an extended SD-aSD duplex adversely affects translation, most likely due to its redundant stability causing ribosome stalling at the initiation step. Translation yields were significantly increased upon insertion of the A/U-rich S1 binding targets upstream of the SD region, but the longest SD remained relatively less efficient. In contrast to complete 30S ribosomes, the S1-depleted 30S particles have been able to form an extended SD-aSD duplex, but not the true ternary initiation complex. Taken together, the in vivo and in vitro data allow us to conclude that S1 plays two roles in translation initiation: It forms an essential part of the mRNA-binding track even when mRNA bears a long SD sequence, and through the binding to the 5' untranslated region, it can ensure a substantial enhancing effect on translation.     

Identification of a flunixin metabolite in camel by gas chromatography–mass spectrometry

A flunixin metabolite, a hydroxylated product, has been identified in camel urine and plasma samples using gas chromatography–mass spectrometry (GC–MS) and GC–MS–MS in the electron impact and chemical ionization modes. Its major fragmentation pattern has been verified by GC–MS–MS in daughter ion and parent ion scan modes. The method could detect flunixin and its metabolite in camel urine after a single intravenous dose of 2.2 mg of flunixin/kg body weight for 96 and 48 h, respectively, which increases the reliability of antidoping control analysis.     

Interaction of glucagon with sphingomyelins

In the presence of either egg or bovine brain sphingomyelin, the spectral properties of glucagon undergo changes which are similar to those which occur in the presence of synthetic phosphatidylcholines. The fluorescence emission spectra are blue shifted about 10 nm in the presence of lipid and the peptide acquires an increased helical content, determined by circular dichroism. As with phosphatidylcholines, the changes in spectral properties do not occur above the phase transition temperature of the glucagon-lipid mixture. Freeze-fracture electron microscopy indicates that glucagon forms an ellipsoidal complex with bovine brain sphingomyelin, similar to the glucagon-dimyristoylphosphatidylcholine complex. However, the sphingomyelin complexes break down to vesicular structures both above and below the region of the phase transition. These results indicate that the dissociation of glucagon from the lipid at higher temperatures results from changes in the phase of the lipid rather than from a thermal denaturation of glucagon. The effect of glucagon on the phase transition behaviour of palmitoyl sphingosine phosphorylcholine was measured by differential scanning calorimetry. The major effect of glucagon on both this lipid and on dimyristoylphosphatidylcholine is to broaden the phase transition and to shift it to higher temperatures. Similar results are obtained for the effects of glucagon on an equimolar mixture of dimyristoylphosphatidylcholine and palmitoyl sphingosine phosphorylcholine. Glucagon is able to solubilize mixtures of bovine brain sphingomyelin with either dimyristoylphosphatidylcholine or egg lecithin. The lipid composition of the solubilized material is similar to that of the starting lipid film. These results together with those from the differential scanning calorimetry on the synthetic mixtures indicate that glucagon can bind to sphingomyelin-phosphatidylcholine mixtures and that it does not induce extensive lateral phase separation between the components. The maximal stability of the glucagon-lipid complex at the phase transition of the lipids indicates that the glucagon-lipid interaction is highly dependent on the structural organization of the lipid.     

Ca(2+) current activity decreases during meiotic progression in bovine oocytes

By using the whole cell voltage-clamptechnique, we studied changes in plasma membrane permeability atdifferent meiotic stages of bovine oocytes. Follicular oocytes werematured in vitro and activated by Ca²⁺ ionophore. Oocytesat germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphaseI (MI), metaphase II (MII), and meiosis exit were used forelectrophysiological recording. By clamping the oocytes at 30 mV, wefound that the L-type voltage-dependent Ca²⁺ channels wereactive at the GV stage and that their activity decreased after the GVBDstage. Furthermore, the resting potential decreased from the GV to theMI stage and increased again at MII. A significant decrease of thesteady-state conductance occurred from the GV to the MI stage, followedby a sharp increase at the MII stage. With the addition of organicL-type Ca²⁺ channel blockers (nifedipine and verapamil), weinhibited the Ca²⁺ currents. However, only in the case ofverapamil was there a decrease of in vitro maturation efficiency. Ourresults suggest that, in addition to the cumulus-oocyte junctions, theplasma membrane channels provide another mode of Ca²⁺ entryinto bovine oocytes during meiosis.

     

GATA4, 5 and 6 mediate TGFbeta maintenance of endodermal gene expression in Xenopus embryos

The individual contributions of the three vertebrate GATA factors to endoderm formation have been unclear. Here we detail the early expression of GATA4, 5 and 6 in presumptive endoderm in Xenopus embryos and their induction of endodermal markers in presumptive ectoderm. Induction of HNF3beta by all three GATA factors was abolished when protein synthesis was inhibited, showing that these inductions are indirect. In contrast, whereas induction of Sox17alpha and HNF1beta by GATA4 and 5 was substantially reduced when protein synthesis was inhibited, induction by GATA6 was minimally affected, suggesting that GATA6 is a direct activator of these early endodermal genes. GATA4 induced GATA6 expression in the same assay and antisense morpholino oligonucleotides (MOs), designed to knock down translation of GATA6, blocked induction of Sox17alpha and HNF1beta by GATA4, suggesting that GATA4 induces these genes via GATA6 in this assay. All three GATA factors were induced by activin, although GATA4 and 6 required lower concentrations. GATA MOs inhibited Sox17alpha and HNF1beta induction by activin at low and high concentrations in the order: GATA6>GATA4>GATA5. Together with the timing of their expression and the effects of GATA MOs in vivo, these observations identify GATA6 as the predominant GATA factor in the maintenance of endodermal gene expression by TGFbeta signaling in gastrulating embryos. In addition, examination of gene expression and morphology in later embryos, revealed GATA5 and 6 as the most critical for the development of the gut and the liver.     

Non-canonical mechanism for translational control in bacteria: synthesis of ribosomal protein S1

Translation initiation region (TIR) of the rpsA mRNA encoding ribosomal protein S1 is one of the most efficient in Escherichia coli despite the absence of a canonical Shine-Dalgarno-element. Its high efficiency is under strong negative autogenous control, a puzzling phenomenon as S1 has no strict sequence specificity. To define sequence and structural elements responsible for translational efficiency and autoregulation of the rpsA mRNA, a series of rpsA'-'lacZ chromosomal fusions bearing various mutations in the rpsA TIR was created and tested for beta-galactosidase activity in the absence and presence of excess S1. These in vivo results, as well as data obtained by in vitro techniques and phylogenetic comparison, allow us to propose a model for the structural and functional organization of the rpsA TIR specific for proteobacteria related to E.coli. According to the model, the high efficiency of translation initiation is provided by a specific fold of the rpsA leader forming a non-contiguous ribosome entry site, which is destroyed upon binding of free S1 when it acts as an autogenous repressor.