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M Buisson

Publications and source records attributed to M Buisson.

At least 73 records · Page 4Linked to original sources
PubMed1989

The Epstein-Barr virus (EBV) early protein EB2 is a posttranscriptional activator expressed under the control of EBV transcription factors EB1 and R.

From the cloning and characterization of cDNAs, we found that the Epstein-Barr virus (EBV) open reading frame (ORF) BMLF1-BSLF2 coding for the early protein EB2 is present in several mRNAs generated by alternative splicing and expressed in the leftward direction from two promoters PM and PM1. The PM promoter controls the expression of two abundant mRNA species of 1.9 and 2 kilobases (kb), whereas the PM1 promoter controls the expression of at least three mRNAs 3.6, 4.0, and 4.4 kb long. The PM promoter probably overlaps with the PS promoter which controls the transcription of a 3.6-kb mRNA expressed in the rightward direction and containing the ORF BSRF1. Although it increases the amount of chloramphenicol acetyltransferase enzyme expressed from the chimeric pMCAT gene, EB2 is not a promiscuous trans-activator of gene expression and does not positively regulate its own expression from promoter PM. The EB2 activation is not promoter dependent but could possibly act by stabilizing mRNAs and increasing their translation. The PM promoter is, however, activated by the two EBV transcription trans-acting factors, EB1 and R, encoded by the EBV ORFs BZLF1 and BRLF1, respectively. EB1 activates the PM promoter from a consensus AP-1 binding site, and R activates the PM promoter from an enhancer.

Base Sequence↗
PubMed1989

The Epstein-Barr virus (EBV) early promoter DR contains a cis-acting element responsive to the EBV transactivator EB1 and an enhancer with constitutive and inducible activities.

The Epstein-Barr Virus (EBV) DR promoter controlled the expression of the PstI repeat region IR4. This promoter was activated by the EBV trans-acting factor EB1, mainly at the transcriptional level, and the activation was mediated by the TATA box and two cis-acting regulatory regions, one proximal to the TATA box and one distal to the TATA box. The distal region had enhancer properties. In HeLa cells, it activated transcription from the herpes simplex virus type 1 thymidine kinase promoter linked to the chloramphenicol acetyltransferase gene when located in inverted orientation upstream of the thymidine kinase promoter or downstream of the chloramphenicol acetyltransferase gene coding sequence. This enhancer also activated transcription from the simian virus 40 early upstream regulatory elements. These results indicate that the DR These results indicate that the DR enhancer can constitutively activate heterologous promoters in HeLa cells. However, the DR enhancer was not active in EBV genome-negative B cell lines, but it became active when these cells were infected by EBV and when the expression of the EBV early genes was induced by EB1. This suggests that an EBV early gene product induces the DR enhancer activity. The DR promoter TATA box-proximal cis-acting regulatory element contained EB1-responsive sequences.

Enhancer Elements, Genetic↗
PubMed1982

Carbodiimide-induced protein--RNA crosslinking in mammalian ribosomal subunits.

RNA--protein interactions in the 60 S subunits of rat liver ribosomes were studied using 1-ethyl-3-dimethylaminopropyl carbodiimide (EDC) under conditions which neither changed the sedimentation coefficient of subunits nor the intactness of their rRNA. EDC induced RNA--protein and protein--protein crosslinkings. Proteins crosslinked to 28 S RNA were identified by two-dimensional gel electrophoreses as L17, L19, L23a and L37a, shown to react with 28 S RNA when using a low dose of UV radiation. Attempts have also been made to use EDC for the studies of RNA--protein interactions in 40 S ribosomal subunits.

Carbodiimides↗
PubMed1981

Photoincorporation of puromycin into rat liver ribosomes and subunits.

[3H]Puromycin was covalently incorporated into rat liver ribosomes and isolated 40S and 60S subunits on irradiation at 254 nm. A study of the concentration dependence of this photolytic incorporation suggested that it arose from specific sites on isolated subunits but also from unspecific ones in the case of ribosomes, these sites being probably located on contaminant nonribosomal proteins. Puromycin was incorporated simultaneously into ribosomal proteins and rRNAs. The results from simultaneous one-dimensional and two-dimensional gel electrophoreses showed a small distribution of label among ribosomal proteins in 60S subunits and in 80S ribosomes, L10 being the most radioactive protein. Some antibiotics, which act on the peptidyltransferase center (amicetin and gougerotin), and also tetracycline competed with this labeling. Therefore, it was concluded that puromycin interaction with protein L10 occurred most likely at a functional site. In the case of free 40S subunits, labeling distribution among proteins was much wider. The possibility that proteins S3 and perhaps S23-24, which were significantly labeled in crude ribosomes too, also belong to a specific site interacting with puromycin is discussed.

Animals↗
PubMed1980

tRNA binding stabilizes rat liver 60 S ribosomal subunits during treatment with LiCl.

We have shown recently that, in the absence of mRNA, 1 molecule of nonacylated tRNA binds to the large ribosomal subunit of rat liver with a high affinity constant (Buisson, M., Reboud, A.M., Dubost, S., and Reboud, J. P. (1979) Biochem. Biophys. Res. Commun. 90,634-640). In this paper, free and tRNA-bound 60 S subunits were treated with increasing concentrations of LiCl to obtain information on tRNA binding site. The rationale for using deacylated tRNA was that it is assumed to bind to the peptidyl donor site. We observed that tRNA has a strong protective effect on subunit modifications produced by LiCl: tRNA prevents subunit inactivation as measured by puromycin reaction and polyphenylalanine synthesis and it shifts the Li+/Mg2+ ratio value needed to reach 50% inactivation, from 60 to 250; it also prevents ribosomal protein and 5 S RNA release and large sedimentation changes of subunits, induced by LiCl. To explain the mechanism of 60 S subunit stabilization by tRNA, two hypotheses are considered: stabilization can be consequent on direct interaction of tRNA with specific proteins, or on maintenance on subunits of essential cations which are otherwise displaced by Li+, or both.

Animals↗
PubMed1980

Photoinduced protein-RNA cross-linking in mammalian 80-S ribosomes.

RNA-protein interactions in the 80-S rat liver ribosomes were studied by measuring cross-linking of proteins to rRNAs induced by ultraviolet radiation, as already reported for free 40-S and 60-S subunits. Our results are compatible with the model in which most of the ribosomal proteins are accessible to rRNAs in the native conformational state of the ribosomes. Subunit association in 80-S ribosomes does not seem to induce modifications in protein-RNA interactions as measured by this irradiation technique. However, two proteins, S9 and S13, appeared to be significantly less cross-linked with RNA in ribosomes than in free subunits. Ribosomes which had been frozen and thawed several times were highly sensitive to ultraviolet radiation. Such treatment in the cold chiefly modified their 60-S subunit moiety.

Animals↗
PubMed1979

Spot position of rat liver ribosomal proteins by four different two-dimensional electrophoreses in polyacrylamide gel.

Separation of the proteins from rat liver 40S and 60S ribosomal subunits and polysomes was done in four different two-dimensional polyacrylamide gel electrophoresis systems. The first dimension was run at acidic or basic pH, the second dimension either with sodium dodecyl sulphate or at acidic pH in 18% acrylamide. The position of each individual protein of both subunits and polysomes was determined in each system. This identification resulted from a new method avoiding any pervious purification of individual proteins. The new "proposed uniform nomenclature for mammalian ribosomal proteins" (McConkey et al. in press) was used for numbering the proteins in the four systems.

Animals↗
PubMed1979

Photo-induced protein-RNA cross-linking in mammalian 60-S ribosomal subunits.

Rat liver 60-S ribosomal subunits were submitted to increasing doses of radiation (253.7 nm), at 4 degrees C and 25 degrees C, as previously reported fro 40-S subunits. The existence of protein-RNA cross-linking was demonstrated by two methods. The first consisted in the separation of protein-RNA complex; the second was indirect, and took into account alteration either in the electrophoretic mobility of cross-linked proteins or the separability of 28-S RNA in a 4 M urea/3 M LiCl buffer. The peptide synthetase activity and the sedimentation characteristics of the particles irradiated at 4 degrees C were well preserved, but at 25 degrees C the large subunits were progressively inactivated and unfolded for doses higher than 2 x 10(18) quanta. The dose-dependent variations of protein cross-linkage determined by two-dimensional gel electrophoresis allowed us to distinguish those proteins which reacted at the lowest doses with a first-order reaction from those which cross-linked to RNA after a subtle modification of the subunit structure. At 25 degrees C, all proteins became low-dose reactive. The curve obtained for 28-S RNA cross-linkage was similar to that of the total protein moiety, while those obtained fro the 5-S and 5.8-S RNA (which were parallel) suggest a lower reactivity of these RNAs. As a general rule, proteins from the large subunits were more reactive to RNA than those from the small subunits. This could indicate differences in the organisation of the two subunits.

Animals↗
PubMed1978

Photo-induced protein-RNA cross-linking in mammalian 40-S ribosomal subunits.

RNA-protein interaction in the 40-S subunits of rat liver ribosomes were studied by measuring cross-linking of proteins to RNA induced by ultraviolet radiation. Under conditions which caused neither extensive degradation of the 40-S subunits (or 18-S RNA) nor biological inactivation, the total staining intensity of the proteins extracted from irradiated subunits was considerably reduced on the two-dimensional electrophoregrams. Convincing evidence was obtained that cross-linking of the proteins to 18-S RNA was the predominant reaction. The cross-linking extent of the individual proteins was studied as a function of the radiation dose. At 4 degree C, 13--15 proteins were found to cross-linked to RNA even at low doses of quanta. They generally correspond to proteins which have been previously shown to react poorly on the ribosomes with various chemical reagents. At 25 degree C, all the proteins became cross-linked to RNA using the same radiation doses.

Kinetics↗
PubMed1977

Change of protein reactivity in mammalian ribosomal subunits as a function of temperature.

Reductive methylation of rat liver ribosomal subunits was carried out at different temperatures and the reaction was followed as a function of time. The percentage of protein lysine residues which were methylated reached a definite plateau for each temperature, and was considerably increased by heating (four times at 40 degrees C). This increase was not observed when free ribosomal proteins were heated under the same conditions. Ribosomal subunits kept their biological activity (elongation steps of protein synthesis) even when methylated to a high extent. Half of the subunit activity was still found when 67% of the lysine residues were methylated. Proteins were divided into different classes on the basis of their alkylation in response to temperature. Those which reacted poorly within subunits even at high temperature should interact directly with rRNA. The results suggest that the protein reactivity modifications induced by heating (up to 37 degrees C), which were often totally or partially reversible, reflect a conformational change of the proteins themselves within the subunits. Besides protein unfolding, higher temperatures produced structural modification and inactivation of the subunits, which were restrained by the presence of aminoacyl tRNA.

Animals↗
PubMed1977

Reductive alkylation of mammalian ribosomes.

40- and 60-S ribosomal subunits and 80-S ribosomes from rat liver were highly labelled by reductive methylation using formaldehyde and sodium boro-[3H] hydride, under conditions which did not decrease their activity in poly-U-directed polyphenylalanine synthesis. Dissociation of the monosomes, subunits dimers, and polysomes into free subunits was observed after methylation. Free proteins labelled after extraction from the ribosomal subunits incorporated 7 times more radioactivity than when labelled in the subunits. Proteins extracted from methylated subunits and ribosomes were analyzed by two-dimensional gel electrophoresis, and the radioactivity of each protein was compared to that of the same free protein. A classification of the proteins was established according to their accessibility to the reagents in the subunits and the ribosomes.

Alanine↗
PubMed1976

Specific ribonucleoprotein fragments from 40-S ribosomal subunits.

Well-defined ribonucleoprotein fragments, resulting from the action of endogenous nuclease on 40-S subunits, were able to be separated when using high concentrations of LiCl. The ribonucleoproteins obtained sedimented at 12, 17 S, 23 S and 30 S and contained 8 S, 12 S and 17 S RNA, respectively, associated with a few proteins. The proteins extracted from the fragments were [3H] labeled by reductive methylation and their molar proportion was determined. The smallest fragment (12, 17 S) contained only three proteins, S8, S9 and S24. The 23-S and 30-S materials contained some proteins in common, S15, S19, S22, S25; S16 was found mainly in 30 S. Two proteins, S26 and "protein y" were found mainly in 23 S material. Thus, these results can give information on the relative location of certain proteins in the 40-S subunits.

Alkylation↗
PubMed1975

Study of mammalian ribosomal protein reactivity in situ. I. - Effect of 2-methoxy-5-nitrotropone on 40S and 60S subunits.

Liver ribosomes and subunits were reacted with increasing concentrations of 2-methoxy-5-nitrotropone. At low reagent concentrations (0.3 mM), the molar uptake by 60S subunits was more efficient than the uptake by 40S subunits, and the amount of reagent bound to 80S ribosomes was less than that bound to both free subunits considered together. At higher reagent concentrations, the molar uptake of both subunits was equivalent. Subunits and ribosomes remained fully active when reacted with up to 0.3 mM and 1 mM of the reagent, respectively. With 2 mM of the reagent, both subunits were half inactivated, although their sedimentation characteristics were unaltered. The reactivity of each ribosomal protein was assessed by two-dimensional gel electrophoresis and quantitative measurement of the unmodified proteins. From these results, considered together with the uptake characteristics and the inactivation curves, a number of tentative conclusions about ribosome topography can be drawn. The over-all sensitivity of the 60S subunits to the reagent is higher than that of the 40S subunits. Both subunits undergo a conformational change when they combine to form 80S ribosomes. Proteins S18, S20, S28 and L5, L9, L11, L15, L16, L25, L29, L30, L31, L34, L37 have NH2 groups exposed in native subunits. These groups are not essential for subunit function.

Animals↗
PubMed1975

Study of mammalian ribosomal protein reactivity in situ. II. - Effect of glutaraldehyde and salts.

Results concerning ribosomal protein sensitivity to glutaraldehyde were compared to protein depletion studies using LiCl centrifugation. The relative degree of reactivity of the different proteins was determined by two-dimensional acrylamide gel electrophoresis, and the activity of the reacted subunits was measured. The results obtained mostly confirmed the studies of methoxynitrotropone reactivity reported earlier. For example, L16, L25, L29, L30, L31, S18, S20 appeared to be definitely exposed to both NH2-reagents and LiCl. Some interesting points emerged from this study regarding protein topography in both subunits: (1) with few exceptions, almost all ribosomal proteins were accessible to the surrounding medium; (2) the sensitivity of the 40S proteins to the three reagents used was lower than was that of the 60S proteins; (3) the reactivities of the subunit components changed when subunits were associated: L8 was more reactive with glutaraldehyde in 60S subunits than in 80S ribosomes. In contrast, S14, S15 and S19 were more exposed in ribosomes than in the 40S subunits.

Aldehydes↗