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T Godefroy-Colburn

Publications and source records attributed to T Godefroy-Colburn.

At least 19 recordsLinked to original sources
PubMed1994

Mapping of the RNA-binding domain of the alfalfa mosaic virus movement protein.

In-frame contiguous deletions were created in the movement protein gene of alfalfa mosaic virus by site-directed mutagenesis. The mutated movement proteins were expressed in Escherichia coli, extracted and then purified by denaturing gel electrophoresis and then renatured. Their binding ability with RNA was assayed by electrophoretic retardation and u.v.-crosslinking. Results indicated that a domain included within amino acids 36 to 81 was necessary for RNA binding.

Alfalfa mosaic virus↗
PubMed1993

Effect of the alfalfa mosaic virus movement protein expressed in transgenic plants on the permeability of plasmodesmata.

Symplastic transport of different sized fluorescent probes has been assessed in leaf epidermal cells of transgenic Nicotiana plants expressing the movement protein (MP) of alfalfa mosaic virus (AMV). In both N. tabacum and N. benthamiana, the size exclusion limit (SEL) of plasmodesmata increased from M(r) 1000, which represents the commonly accepted limit, to over 4.4K. However, in control plants, movement of a 3K probe was seen in 11 to 22% of the injections, indicating that plasmodesmata may on occasion allow the passage of molecules larger than was previously thought. The increase of SEL due to the presence of the AMV MP, although significant, remains insufficient to permit the passage of viral particles and the possibility of other mechanisms involved in viral cell-to-cell spread is discussed.

Alfalfa mosaic virus↗
PubMed1992

Binding of RNA by the alfalfa mosaic virus movement protein is biphasic.

The movement protein of alfalfa mosaic virus was expressed in Escherichia coli and purified by cation exchange chromatography. The purified protein bound single-stranded RNA cooperatively in a biphasic manner. At protein saturation, RNA/protein complexes (designated 'primary complexes') were detected by a nitrocellulose-retention assay within 1 min of mixing, both at 4 and 22 degrees C. In contrast, an incubation of 30 min at 22 degrees C was necessary to obtain electrophoretically retarded complexes ('stabilized complexes'), containing a large number of protein molecules bound stably to each molecule of RNA. Stabilization did not take place at 4 degrees C. The rate of formation of the primary complexes was strongly dependent on protein concentration, and thus appeared limited by a bimolecular interaction. In contrast, the rate of stabilization was independent of protein concentration, suggesting that this process consisted of a rearrangement of the primary complexes without binding of additional protein molecules. In agreement with this suggestion, the amount of complexed RNA at equilibrium was the same when assayed by nitrocellulose retention and by electrophoretic retardation. The possibility that these peculiar kinetics could be caused by the presence of Tween 20 in the incubation media is discussed.

Cloning, Molecular↗
PubMed1992

Nucleic acid binding properties of the 92-kDa replicase subunit of alfalfa mosaic virus produced in yeast.

The 92-kDa non-structural protein of alfalfa mosaic virus (one of the replicase subunits) was synthesized by Saccharomyces cerevisiae transformed with a recombinant expression vector. The yeast-expressed protein had the immunological and size characteristics of the naturally made viral protein. It was partially purified and its nucleic acid binding properties were tested by gel-retardation electrophoresis and nitrocellulose adsorption. The protein interacted with single-stranded RNA, double-stranded RNA and double-stranded DNA in a salt-dependent manner, with a slight preference for RNA. These properties may be related to its putative function as a core RNA polymerase.

Blotting, Western↗
PubMed1992

Nucleic acid-binding properties of the alfalfa mosaic virus movement protein produced in yeast.

The movement protein of alfalfa mosaic virus (P3) was purified from yeasts transformed with an expression vector containing the P3 gene. Its nucleic acid-binding properties were tested by electrophoretic retardation, nitrocellulose retention, and RNA-protein cross-linking. The recombinant protein had a higher affinity for single-stranded RNA and DNA than for double-stranded nucleic acids. Each nucleic acid molecule bound several protein molecules without sequence specificity. The binding was 80% inhibited by 0.2 M NaCl. These properties are qualitatively similar, but not strictly identical, to those of two other viral movement proteins, the 30-kDa protein of tobacco mosaic virus and the gene I product of cauliflower mosaic virus.

Base Sequence↗
PubMed1992

An N-proximal sequence of the alfalfa mosaic virus movement protein is necessary for association with cell walls in transgenic plants.

We have made transgenic tobacco plants (Nicotiana tabacum, cv. Xanthi nc) expressing the movement protein (P3, 300 amino acids) of alfalfa mosaic virus (A1MV) and two N-terminally deleted proteins lacking respectively 12 and 77 amino acids of the P3 sequence (P3 delta[1-12] and P3 delta[1-77]). The same proteins were expressed in recombinant yeast. By subcellular fractionation, the full-length P3 protein expressed by transgenic plants was found to be associated with cell walls as well as with cytoplasmic particulate material, as was the wild type movement protein expressed by A1MV-infected tobacco plants. P3 delta[1-12] behaved similarly but P3 delta[1-77] was found only in the cytoplasm. It thus appears that a polypeptide domain located between amino acids 13 and 77 of the P3 sequence is necessary for association of the protein with cell walls.

Cloning, Molecular↗
PubMed1985

Translational discrimination between the four RNAs of alfalfa mosaic virus. A quantitative evaluation.

In an attempt to relate the translational characteristics of alfalfa mosaic virus (A1MV) RNAs to their structure [Ravelonandro et al. (1983) Nucleic Acids Res. 11, 2815-2826; Gehrke et al. (1983) Biochemistry 22, 5157-5164] we measured the relative affinities (discrimination ratios) of these RNAs for the initiation complex, in the wheat germ extract and in the nuclease-treated reticulocyte lysate, using a competition method designed by Brendler et al. [(1981) J. Biol. Chem. 256, 11747-11754]. As a prerequisite of this study we ascertained that the molecular mass distribution of the translation products was independent of RNA concentration in both translation systems. In the wheat germ extract the discrimination ratios are very similar for two strains of A1MV (S and B) which differ mainly by the presence (strain S) or absence (strain B) of a stable 5'-proximal hairpin. Hence this structure has no bearing on discrimination. Taking the affinity of RNA 3 as reference, the following orders of magnitude are found for the affinities of the different RNAs in the wheat germ: RNA 3, 1.0; RNA 1, 10; RNA 2, 60; RNA 4, 150. In the reticulocyte lysate the discrimination ratios are not significantly different from the wheat germ. Thus it seems that the mechanism of discrimination is essentially the same in the two translation systems, despite a difference in rate-limitation.

Animals↗
PubMed1985

Cap accessibility correlates with the initiation efficiency of alfalfa mosaic virus RNAs.

The rate of cap removal from the alfalfa mosaic virus (A1MV) RNAs with tobacco acid pyrophosphatase (TAP) depends on the RNA species. At 37 degrees C and in the absence of divalent cation, RNA 3 reacts more slowly than the other three, which are decapped at similar rates. In the presence of magnesium, at 25 degrees C, TAP also discriminates against RNA 1. Thus the order of reactivity with TAP largely mimics the hierarchy of initiation efficiencies of the A1MV RNAs (Godefroy-Colburn et al., preceding paper in this journal). Our interpretation of these findings is that cap accessibility is what limits the rate of reaction with initiation factors as well as with TAP. In this hypothesis, translational discrimination between naturally capped messages would be related to the rate of 'breathing' of their 5' ends.

Animals↗
PubMed1983

Structure of the 5'-terminal untranslated region of the genomic RNAs from two strains of alfalfa mosaic virus.

We report the sequences of the 5'-terminal regions of the 3 Alfalfa Mosaic Virus genomic RNAs for the Strasbourg strain (AlMV-S) and for a new isolate, AlMV-B; they are compared to similar data obtained by Koper-Zwarthoff et al. (Nucleic Acids Res. 1980, 8, 5635-5647) for strain 425. The structure of these leaders is highly conserved in RNAs 1 and 2. The length of the leader is 102, 100 and 101 nucleotides in RNA1 for strains S, B and 425 respectively; 55 and 56 in RNA2 for strains S and B respectively. In RNA3 however, there are important differences near the 5'-terminus between strain S and the other two: The total leader length is 258 nucleotides for strain S and 242 for strain B. The secondary structure models show a conserved hairpin near the 5'-end of each genomic RNA of AlMV-S. This hairpin is inexistent in RNA3 of the B and 425 strains. The degree of base-pairing increases with leader length. The initiator codon is located in a single stranded region in RNA2 whereas it is found in a hairpin stem in RNA 3.

Base Sequence↗
PubMed1981

The role of mRNA competition in regulating translation. III. Comparison of in vitro and in vivo results.

Competition of encephalomyocarditis virus, reovirus, and L-cell mRNAs for a message-discriminatory component was studied in vitro. The data were analyzed qualitatively to determine the relative initiation efficiencies among the various mRNAs. The effects of potassium chloride concentration, magnesium acetate concentration, and m7G methylation on mRNA competition in vitro were also studied. These results were correlated with translation rates in vivo for the same mRNAs, to determine if the sites of competition in vitro and in vivo are the same. It was found that under a particular set of magnesium acetate and potassium chloride concentrations, the order of mRNA initiation efficiencies was the same both in vivo and in vitro, suggesting that the same limiting message-discriminatory factor is regulating initiation rates in both cases. This can only be accomplished in a competitive situation when RNA is in molar excess relative to the discriminatory component.

Animals↗
PubMed1981

The role of mRNA competition in regulating translation. I. Demonstration of competition in vivo.

SC-1 cells infected with reovirus continue to synthesize host proteins at a nearly normal rate for at least 12 h postinfection. The relative translational efficiencies of reovirus and host mRNAs were determined at various times during this interval. Analysis was performed by determining the sensitivity of individual mRNA translation rates to inhibition by low doses of cycloheximide, by measuring polysome sizes, and by quantitatively comparing mRNA concentration with protein synthesis rates. Treatment of infected cells with low doses of cycloheximide actually stimulates the synthesis of some of the major reovirus protein, whereas host protein synthesis is inhibited. An analysis of the polysomes encoding two reovirus proteins (microNS and sigma 3; synthesis of both is stimulated by cycloheximide) shows that these proteins are synthesized on polysomes smaller than those producing host proteins of comparable size. Reovirus mRNAs accumulate to unusually high levels during the first 8 h of infection, accounting for 30% to 45% by weight of the total mRNA in the cell. We interpret these results as indicating that viral and host mRNAs must compete for a message-discriminatory component prior to their binding to the 40 S ribosomal complex and that this component is limiting in reovirus-infected cells. Moreover, a hierarchy must exist among mRNAs in terms of their affinity for this component, host mRNAs in general having greater affinities than reovirus mRNAs.

Animals↗
PubMed1981

The role of mRNA competition in regulating translation. II. Development of a quantitative in vitro assay.

Competition among excess mRNAs for a limiting, message-discriminatory initiation component (or set of components) has been studied in vitro. Procedures have been devised which provide maximum sensitivity with minimum potential for artifact. These procedures produce data that can be analyzed quantitatively to obtain numerical constants characteristic of a given mRNA's intrinsic initiation efficiency or, more precisely, competitive efficiency. A major advantage of this approach is that this information can be determined for individual mRNAs within a mixture of RNAs without the necessity of determining individual mRNA concentrations. The effects of potassium chloride concentration and magnesium acetate concentration on mRNA competitive efficiencies have been studied using these procedures.

Animals↗
PubMed1981

The role of mRNA competition in regulating translation. IV. Kinetic model.

A kinetic model of protein synthesis is presented, primarily designed to analyze the accompanying data (Brendler, T., Godefroy-Colburn, T., Carlill, R. D., and Thach, R. E. (1981) J. Biol. Chem. 256, 11747-11754; Walden, W. E., Godefroy-Colburn, T., and Thach, R. E. (1981) J. Biol. Chem. 256, 11739-11746). Our model treats initiation as a multistep process in which mRNA must bind to a "discriminatory factor" prior to its recognition by the native 40 S subunit. Interaction with the latter is followed by an irreversible rearrangement which yields the functional 40 S initiation complex capable of binding the 60 S ribosome with release of all the factors. Elongation is simply treated as a series of irreversible steps with a single rate constant. The model takes into account the recycling of ribosomal subunits, initiation factors, discriminatory factor, and message initiation site. We can thus mimic the simultaneous translation of several messages, each with its own concentration, size, binding constants, and rate constants. The only limit to the number of messages is the capacity of the computer (3 kilobytes of accessible memory is sufficient for 5 messages). Thus, we are able to evaluate quantitatively the effect of each parameter on the rate of synthesis of individual polypeptides, on polysome size, and on the repartition of message species between the untranslated and the polysomal pools. Several applications are considered: (i) competitive translation of alpha- and beta-globin in vitro (Kabat, D., and Chappel, M. R. (1977) J. Biol. Chem. 252, 2684-2690); (ii) determination of the relative affinities of reoviral messages for the discriminatory factor in vitro (Brendler, T., Godefroy-Colburn, T., Carlill, R. D., and Thach, R. E. (1981) J. Biol. Chem. 256, 11747-11754); (iii) effect of elongation inhibitors on the translation of reoviral and cellular messages in SC-1 fibroblasts (Walden, W. E., Godefroy-Colburn, T., and Thach, R. E. (1981) J. Biol. Chem. 256, 11739-11746); and (iv) effect of the growth state on the initiation efficiency of Vero cell messages (Lee, G. T.-Y., and Engelhardt, D. L. (1979) J. Mol. biol. 129, 221-233). In each case we find that the experimental data are consistent with the notion that mRNAs compete for a discriminatory factor independent of the ribosome. This factor has a high enough affinity for mRNAs to ensure nearly quantitative binding. When present in a limiting amount (with respect to the message pool but not necessarily with respect to the rest of the translation apparatus), the discriminatory factor selects against those messages for which its affinity is lowest, thereby modulating their initiation efficiency. When the factor is present in excess, on the other hand, all the messages are translated at maximum efficiency. This form of translational control could be remarkably efficient as an on-off switch for the synthesis of a few key proteins.

Animals↗
PubMed1979

Characterization of the proteins of intracisternal type A and extracellular oncornavirus-like particles produced by MOPC-460 myeloma cells.

The mouse plasmacytoma cell line, MOPC-460, produces both intracisternal and intracytoplasmic A-type particles when grown as a solid tumor. When these cells are grown either as an ascites tumor or in tissue culture, a third type of particle is produced extracellularly. This particle, the "myeloma-associated virus," is closely related to, and probably an alternate form of, the intracisternal A-type particle. The proteins present in these two types of particles were compared by tryptic peptide mapping. Both types of particles were found to contain essentially the same major proteins of 76,000 (p76), 68,000 to 70,000 (p68-70), and 45,000 (p45) daltons, in addition to varying amounts of smaller proteins. The relative proportions of all these proteins varied from preparation to preparation in an unpredictable way. The p45, p68, and p70 proteins all contained sequences found in p76, suggesting precursor-product relationships of p76 leads to p70 leads to p45 for solid tumor A-type particles and p76 leads to p68 leads to p45 for extracellular myeloma-associated virus. In addition, immune precipitation experiments have established that p76 contains at least some of the antigenic determinants characteristic of murine leukemia virus p30. This confirms earlier nucleic acid hybridization studies which indicated a moderate degree of relatedness between MOPC-460 A-type particles and several standard murine leukemia and sarcoma viruses. Taken together, our results provide evidence supporting the concept that MOPC-460 A-type particles may represent aberrant forms of C-type murine viruses.

Animals↗
PubMed1979

Characterization of infectious oncornaviruses from MOPC-460 plasmacytomas: their relation to A-type particles.

MOPC-460 mouse plasmacytoma cells produce intracellular A-type particles and extracellular oncornavirus-like particles ("myeloma-associated virus," abbreviated MAV). The genomes of these two particles are closely related. During attempts to establish infections with MOPC-460 extracellular particles, we isolated ecotropic and xenotropic infectious forms of murine leukemia virus. We have investigated the relation of these isolates to A-type particles and to MAV by nucleic acid hybridization. Using complementary DNA probes prepared from the two isolates, we found that these infectious murine leukemia viruses differ from A-type particles and from MAV. Moreover, we found that MAV is the predominant extracellular component: the ecotropic and xenotropic forms of murine leukemia virus were present at only low levels (less than 5%) in MAV preparations. Neither the SC-1 cells infected with ectropic murine leukemia virus nor the mink cells infected with xenotropic murine leukemia virus showed any A-type particles in their cytoplasm when examined by electron microscopy. Our inability to demonstrate infection by the A-type particle-related component, MAV, suggests that these may be defective.

Animals↗
PubMed1978

Specificity of interferon action in protein synthesis.

Inhibitors of elongation steps in protein synthesis such as cycloheximide and anisomycin mimic interferon treatment in that they specifically inhibit the synthesis of certain viral proteins. These specific effects are seen only at very low concentrations of the antibiotics, under conditions where host cellular protein synthesis, as well as cell viability, are not severely reduced. A qualitatively as well as quantitatively close correlation between the effects of the two types of agents has been established for encephalomyocarditis virus, vesicular stomatitis virus and murine leukemia virus protein synthesis. It is concluded that one of the primary mechanisms of interferon action may be a nonspecific retardation of one or more elongation steps, and that this may be sufficient to account for its effects on the replication of certain viruses such as encephalomyocarditis and vesicular stomatitis viruses.

Anisomycin↗
PubMed1975

Ribosomal subunit interaction as studied by light scattering. Evidence of different classes of ribosome preparations.

The Mg2+-dependent equilibrium of ribosomal subunits was studied by light scattering in the absence of any other factor of protein synthesis. The difference of intensity in the light scattered by dissociated 30S-50S couples and by 70S particles was used as a method to measure the percentage of association as a function of Mg2+ under conditions of nonperturbation of the equilibrium. We found that pressure-resistant (type A) and non-pressure-resistant (type B) ribosomes may be characterized by the different behavior of their association equilibrium curves. The thermodynamic parameters of this equilibrium and their comparison for the two classes of ribosomes were calculated from experiments at different temperatures.

Cell Fractionation↗