Pierre Remy

Xl erg: Expression Pattern and Overexpression During Development Plead for a Role in Endothelial Cell Differentiation

The ets gene family encodes transcription factors related to the proto‐oncogene c‐ets‐1 and involved in cell proliferation, differentiation, and oncogenic transformation. We have characterized the Xenopus homologue of the human erg gene, an ets‐related‐gene, and its expression has been examined throughout early embryonic development. Xl erg encodes at least two proteins, resulting from alternative splicing events. The transcripts are restricted to the forming endocardium, the endothelial cells of the blood vessels and to the neural crest‐derived mesenchyme cells of the pharyngial arches. When Xl ERG is expressed ectopically in Xenopus embryos by microinjection of synthetic mRNA, multiple developmental defects are observed. Dorsally injected embryos have their AP axis shortened and present severe defects in eye and somite morphogenesis. Ventrally injected embryos show a posteriorization of the cells having received the message together with ectopic endothelial cell differentiation as revealed by the accumulation of X‐msr transcripts. In both cases, accumulation of erythrocytes in structures not connected with the blood circulatory system can be observed. Our data suggest that Xl erg may be involved in cell motility and in the development of the circulatory system. Dev Dyn 1999;216:420–433. ©1999 Wiley‐Liss, Inc.

A study of the interaction of Escherichia coli elongation factor-Tu with aminoacyl-tRNAs by partial digestion with cobra venom ribonuclease

Abstract The hydrolysis of several aminoacylated transfer RNAs, by double-strand-specific ribonuclease from Naja oxiana was studied. The sensitivity to this enzyme of Phe-tRNAPhe, Glu-tRNAGlu and Met-tRNAmMet from Escherichia coli and Phe-tRNAPhe from yeast was examined, both in the free state and complexed to E. coli elongation factor Tu. The hydrolysis patterns in the isolated state were similar for all aminoacylated tRNAs except Glu-tRNA2Glu, which exhibited striking differences probably arising from the existence of several subpopulations of tRNA2Glu. When engaged in a ternary complex with EF-Tu and GTP, the aminoacyl-tRNAs were efficiently protected in the amino acid acceptor and TΨC helices, showing that the interaction with EF-Tu primarily takes place at the -C-C-A end and at the amino acid acceptor and TΨC helices. In all cases an increased reactivity of the anticodon stem was observed in the complexed tRNA, possibly resulting from a conformational change in this region of the tRNAs.

Quantitative study of the ionic interactions between yeast tRNAVal and tRNAPhe and their cognate aminoacyl-tRNA ligases

A general characteristic of the interactions between the tRNAs and the aminoacyl-tRNA ligases is that they are lowered by ionic strength. One explanation is that ionic strength weakens ionic interactions existing between phosphate groups of the tRNA and positive charges of the enzyme. A quantitative study based on this assumption was undertaken by Loftfield [l] . Using the Debye-Hiickel law, he found 2 to 4 charges interacting with the same number of positive ones. Since the Debye-Htickel law is valid only for small ions, the validity of these conclusions is questionable. A second explanation for the salt effect was proposed by Yarus [2] who postulated that the interactions between tRNA and enzyme required a conformational change and that this change was inhibited by salt. We report here a study of the affinity of tRNAVa’ and tRNAPhe for their cognate aminoacyl-tRNA ligases as a function of ionic strength under conditions where conformational changes can be considered as negligeable. Our results agree with the model of interaction between nucleic acids and protein developed by Daune [3-51. Our data are consistent with the existence of 5-6 phosphate groups interacting with the same number of positive charges on the enzyme. The participation of ionic interactions in the binding of the tRNA by its cognate aminoacyl-tRNA ligase would be about 40 to 60% of the total energy of interaction under usual conditions. This model is also sufficient to explain the increase in tRNA affinity for its aminoacyl-tRNA ligase in the presence of methanol

Accumulation of dinucleoside polyphosphates in Saccharomyces cerevisiae under stress conditions. High levels are associated with cell death

Adenosine tetraphosphonucleosides (Ap4X) were measured in Saccharomyces cerevisiae by a coupled phosphodiesterase-luciferase assay. After exposure of the cells to cadmium or to hyperthermic treatment (46 degrees C) a marked increase of the cellular pool from 0.08 microM (base level) to 4 microM or higher was observed. The accumulation of Ap4X to high levels is associated with irreversible processes leading to cell death.

Yeast phenylalanyl-tRNA synthetase: Properties of the sulfhydryl groups; evidence for -SH requirement in tRNA acylation

In spite of the particular importance of aminoacyl-tRNA synthetases, little is known about the role of specific aminoacid residues in the catalytic activity [ 1,2]. Among the aminoacid residues the most studied were the sulfhydryl groups. In most cases, modification of thiol groups resulted in the disappearence of the acylation activity of the enzymes, while the ATP-pyrophosphate exchange activity was little or not affected [3-71. We report here the properties of the thiol groups of phenylalanyl-tRNA synthetase (PRS) and their participation in the activity of the enzyme. Two -SH groups per molecule of enzyme were found to be necessary for the acylation of tRNA, but not for phenylalanine activation. 2-mercaptoethanol was shown to inhibit the heterologous acylation of yeast tRNAV”l by yeast PRS, while the homologous acylation of yeast tRNAPhe was unaffected. The possible existence of a thioesteracylenzyme intermediate in tRNA acylation, as proposed by MC Elroy et al. [S] , will be discussed.

Identification of interaction partners for two closely-related members of the ETS protein family, FLI and ERG

Fli and erg are two members of the ETS gene family that encodes transcription factors related to the c-ets-1 proto-oncogene. The products of the ETS genes act as transcriptional effectors in cell proliferation, differentiation, and oncogenic transformation. FLI and ERG, two closely-related proteins, bind, as do all the ETS proteins characterized so far, to DNA sequences with an invariable central GGA core flanked by preferred nucleotides. Nevertheless, promoter-specific responses to FLI or ERG may be driven by mechanisms involving multicomponent complexes. Using a yeast two-hybrid screen, we have identified several proteins that physically interact with either FLI or ERG proteins used as bait. The Xenopus developmentally implicated Xvent-2 and Xvent-2B proteins, and the Xenopus splicing factor RNP-C/U1C physically interact with Xl-FLI and Xl-ERG, both in the yeast two-hybrid system and in vitro. We also report the potential interaction of FLI and ERG with Sox-D, a stabilizing protein that may modulate their transcriptional activity. Furthermore, the possible involvement of the transcriptional effectors FLI and ERG in mRNA processing, hematopoiesis or in the control of angiogenesis is suggested through possible interactions with, respectively, RNA binding proteins and hnRNPs, a repressor of the hematopoietic pathway (SAP18), and the HAF protein.

Dinucleoside tetraphosphate variations in cultured tumor cells during their cell cycle and growth

Asynchronous and synchronized cultures of A549 and HTC cells were used to detect possible, cell cycle or cell density specific variations in the intracellular pools of dinucleoside tetraphosphates (Ap4X). No important variations of the nucleotide pools were observed during cell growth. When HTC cells were released from mitotic arrest, a decrease by a factor of N3 Ap4X and ATP levels was observed when the cells entered the G1 phase. This decrease is essentially due to cell doubling. When A549 cells were released from an arrest at the G1/S boundary, the nucleotide pool size increased slightly during the G2 phase just before mitosis. This result is in agreement with both earlier data from our laboratory and the observed decrease in Ap4X pool after release from mitotic-arrested HTC cells. These results suggest that the Ap4X and ATP pools are only subjected to very small variations during the cell cycle, essentially in the G2 phase and after mitosis.

A study of the interaction of Escherichia coli initiation factor IF2 with formylmethionyl-tRNAMetf by partial digestion with cobra venom ribonuclease

The translation initiation factor IF-2 of prokaryotic organisms functions in the correct formation of the initiation complex fMet-tRNAfMet:mRNA:30 S ribosomal subunit ] 11. Similar to the role of the elongation factor EF-Tu in the elongation step, IF-2 can be regarded as an a~noacyl-tRNA carrier protein which ensures the correct binding of the first amino acid, formylmethionine, in the ribosomal peptidyl transferase centre. However, the binding constant of IF-2 to fMettRNA is too low to allow isolation of the complex. We have studied the interaction of IF-2 with the initiator tRNA by measuring the effect of the protein on the spontaneous hydrolysis of the aminoacyl ester bond [2]. We showed that IF-2 specifically protected the formylated form of the initiator tRNA, ~dicating that IF-2 interacts with the amino acid acceptor region of the initiator tRNA. Furthermore, we showed that this interaction was independent of GTP. To obtain more detailed information about the structural regions of the initiator tRNA involved in the binding to IF-2, we have employed the method of specific protection by IF-2 against ribonuclease digestion of jet-tRNA.

Analogues de nucléosides polyphosphates: V. Action de l'adf́nosine-5′-hypophosphate sur l'adénylate kinase et la pyruvate kinase. Préparation de l'adénosine-5′-hypophosphophosphate, analogue structural de l'ATP

Abstract Analogues of nucleosides polyphosphates. V. Action of adenosine 5′-hypophosphate on adenylate kinase and on pyruvate kinase. Preparation of adenosine 5′-hypophosphophosphate, a structural analogue of ATP The action of adenosine 5′-hypophosphate, a structural analogue of ADP, was studied in two enzymic reactions: the first one catalyzed by adenylate kinase, involving the scission of the phosphoanhydride bond of ADP, the second one, catalyzed by pyruvate kinase, giving rise to the phosphorylation of ADP. In the first reaction, adenosine 5′-hypophosphate is a competitive inhibitor of the reaction towards ADP; in the second, adenosine 5′-hypophosphate acts as a substrate which is phosphorylated into adenosine 5′-hypophosphophosphate, a new analogue of ATP in which the phosphoanhydride bond between the α and β phosphorus of ATP is replaced by a direct P-P bond. But the reaction can only be detected with high concentrations of the enzyme. In the presence of ADP, adenosine 5′-hypophosphate acts as a competitive inhibitor. Under our experimental conditions, the Km of adenylate kinase for ADP was found to be 5 · 10−4 M and the Ki for adenosine 5′-hypophosphate 0.27 · 10−4 M. In the reaction catalyzed by pyruvate kinase, the Km for ADP was found to be 2 · 10−4 M and for adenosine 5′-hypophosphate, 1.7 · 10−2 M. In the presence of ADP, the Ki measured for adenosine 5′-hypophosphate was 1.8 · 10−2 M.

Analogues de nucléosides polyphosphates: I. Synthèse de l'adénosine 5′-phosphohypophosphate

Abstract Analogues of nucleosides polyphosphates. I. The synthesis of adenose 5′-phosphohypophosphate A new analogue of ATP, adenosine 5′-phosphohypophosphate has been synthetized by anionic exchange between the diphenylphosphate group of P 1 -adenysyl-5′, P 2 -diphenylpyrophosphate and hypophosphoric acid. This analogue has been isolated after column or paper chromatography and has been characterized by physical and chemical analysis.