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Biomedical subjects

R E Thach

Publications and source records attributed to R E Thach.

At least 55 records · Page 3Linked to original sources
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↗
PubMed1981

Encephalomyocarditis viral RNA can be translated under conditions of poliovirus-induced translation shutoff in vivo.

Superinfection with poliovirus of HeLa cells already infected with encephalomyocarditis (EMC) virus does not inhibit translation of EMC viral mRNA, whereas residual host translation is completely inhibited. This result indicates that the cap recognition factors inactivated by poliovirus are not required for translation of EMC viral mRNA in vivo, in agreement with previous in vitro experiments. This raises the question of why EMC virus has evolved a capindependent translation mechanism.

Carrier Proteins↗
PubMed1981

Translational elongation rate changes in encephalomyocarditis virus-infected and interferon-treated cells.

Infection of mouse L cells with encephalomyocarditis virus results in a rapid inhibition of host protein synthesis before the synthesis of viral proteins. Although no alterations in initiation factor activities have been demonstrated in encephalomyocarditis virus-infected mouse cells, a defect in polypeptide chain elongation has been shown to occur in infected cell extracts. We investigated the significance of this elongation defect in the host shutoff phenomenon in vivo. Average polypeptide chain elongation rates were measured at various times after infection. Interferon was used as a reagent to separate temporarily the virus-induced alterations. Encephalomyocarditis virus infection of L cells was shown to lead to a progressive reduction in the elongation rate. Whereas interferon pretreatment delayed the decrease in elongation rate in a dose-dependent manner, it failed to alter the kinetics of host shutoff, suggesting that slowing of elongation steps played no significant role in this phenomenon. In addition, interferon pretreatment of either mock-infected or virus-infected cells led to no elongation defect that could be attributed to interferon action.

Animals↗
PubMed1980

Specificity of protein synthesis inhibitors in the inhibition of encephalomyocarditis virus replication.

Effect of protein synthesis inhibitors on encephalomyocarditis virus production in L-cells was studied. Inhibition of initiation by hypertonicity, harringtonine, or pactamycin decreased viral protein synthesis to a lesser extent than that of host. Virus yield was unaffected or actually enhanced by low concentrations of these inhibitors. On the contrary, the elongation inhibitors cycloheximide, anisomycin, and emetine, shown previously to inhibit viral protein synthesis preferentially, had a greater effect on virus yield than on overall protein synthesis. These results support our earlier proposal that the antiviral activity of cycloheximide derives from its specific effect on the rate of elongation of protein synthesis, and that elongation inhibitors in general may show varying degrees of specific antiviral activity.

Animals↗
PubMed1980

Shutoff of HeLa cell protein synthesis by encephalomyocarditis virus and poliovirus: a comparative study.

Previous experimental results have suggested that poliovirus and encephalomyocarditis (EMC) virus employ very different mechanisms for shutting off host protein synthesis. However, this conclusion is suspect, inasmuch as different cell types were used for the two viruses; hence the apparent mechanistic differences might be specific for cell type and not virus type. To test this possibility we compared shutoff mechanisms in poliovirus- and EMC virus-infected HeLa cells. Striking differences were seen: poliovirus-induced shutoff was much more rapid and extensive than that induced by EMC virus; relative translation rates of certain host proteins were inhibited to different extents by the two viruses; initiation factors prepared from poliovirus-infected cells were specifically defective for translation of capped mRNA's in vitro, whereas those from EMC virus-infected cells were not. These results indicate that EMC virus and poliovirus employ different mechanisms for the shutoff of HeLa cell protein synthesis. This conclusion is consistent with much earlier work and indicates that many differences previously reported are specific to virus type.

Dactinomycin↗
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

Comparison of initiation rates of encephalomyocarditis virus and host protein synthesis in infected cells.

The relative initiation rates for encephalomyocarditis virus mRNA and host mRNA's in infected cells were measured using two independent techniques. In both cases the results showed that viral mRNA initiates at a much higher rate than host mRNA'S. This difference was observed midway in the infectious cycle, well before virus-induced cytopathic effects (leakage of low-molecular-weight metabolites, failure to exclude trypan blue) were apparent. These results confirm that encephalomyocarditis viral mRNA is a more efficient initiator than host mRNA's in vivo, as has previously been demonstrated in in vitro experiments.

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↗
PubMed1976

Competition between cellular and viral mRNAs in vitro is regulated by a messenger discriminatory initiation factor.

Encephalomyocarditis viral RNA has previously been shown to outcompete host cellular mRNA has for translation in vitro in crude and fractionated protein synthesizing systems. In the present communication it is shown that the competition is regulated by an initiation factor or complex of factors, and not the 40S initiation complex per se. The factor primarily involved is the murine equivalent of a component present in a partially purified preparation of rabbit initiation factor M3. Both the murine and rabbit factors are clearly messenger discriminatory.

Animals↗
PubMed1976

Relationships between intracisternal type A and extracellular oncornavirus-like particles produced in murine MOPC-460 myeloma cells.

Oncornavirus-like particles of the "A" (both intracisternal and intracytoplasmic) and "B" or "C" (extracellular) types are produced by murine MOPC-460 myeloma cells. This communication describes a comparative study on tracisternal A and extracellular particles. Both types of particles contain an RNA-dependent DNA polymerase activity, traces of 35S and 70 S RNA in addition to larger amounts of degraded RNA, and proteins of approximately 76,000 and 45, 000 daltons. The 76,000-dalton proteins from intracisternal A and extracellular particles have the same cyanogen bromide peptides. Hybridization kinetic analysis indicates that the RNAs in the two particles are identical or very closely related and share partial homology with Moloney leukemia virus RNA. In contrast, the particles appear to have little or no relationship to murine mammary tumor virus as judged by several different criteria. Electron microscope studies indicate that the extracellular particles arise from the budding of core components through the plasma membrane. These results suggest that the intracisternal A and extracellular oncornavirus-like particles produced by MOPC-460 cells are closely related.

Animals↗
PubMed1975

Reverse transcriptase associated with A-type particles from murine myeloma cells.

Our findings may be summarized as follows: (a) a DNA polymerase is associated with murine type A particles, which is very similar to the reverse transcriptases found in true RNA tumor viruses; (b) the majority of the RNA isolated from A particles is of low molecular weight (probably due to degradation), byt appears to contain an amount of unique sequence information comparable to that found in other oncornavirus particles. These results suggest that A-type particles may be very similar, and perhaps related, to the two other classes (B and C) of oncornaviruses.

Base Sequence↗
PubMed1975

Characterization of DNA polymerase and RNA associated with A-type particles from murine myeloma cells.

The RNA-dependent DNA polymerase present in intracisternal A-type particles from mouse myeloma tumor cells has been studied. This polymerase can use either endogenous A particle RNA or an exogenous synthetic polynucleotide [poly (rA)] as a template. The DNA reaction product is small (4S-10S) and over 90% of it hybridizes to A particle RNA, whereas up to 50% of it hybridizes to murine sarcoma-leukemia virus RNAs. The RNA isolated from purified A particles is generally of low molecular weight (5S-15S) but contains small amount of 70S and 35S components. These results suggest that A-type particles may be related to C-type oncornaviruses.

Animals↗
PubMed1975

Identification of a viral protein involved in post-translational maturation of the encephalomyocarditis virus capsid precursor.

Translation of encephalomyocarditis virus RNA in a cell-free system from uninfected Krebs ascites cells results in the synthesis of a major polypeptide product with a molecular weight of approximately 112,000. In contrast, when the viral RNA is translated in a cell-free system from virus-infected cells, this polypeptide is absent and the largest polypeptide produced has a molecular weight of about 100,000. This latter polypeptide comigrates on sodium dodecyl sulfate-gels with in vivo virus capsid precursor A, and the two have identical patterns of CNBr-generated peptides. A polypeptide having a molecular weight of 12,500 is also a major translation product in the system from infected cells (but not from uninfected cells). This polypeptide appears to be generated by cleavage of the NH-2-terminal portion of the viral RNA-dependent polypeptides by a proteolytic activity present in the infected cell-free system. This proteolytic activity copurifies with the 23,000-molecular weight viral capsid protein gamma, found in infected cells, through chromatography on DEAE-cellulose and cellulose phosphate. This suggests that gamma is itself a proteolytic enzyme involved in maturation of the viral capsid precursor.

Animals↗