mRNA In Cell-free System Research Articles

Posttranscriptional Events in the Expression of Myelin Protein Genesa

A number of posttranscriptional events may be involved in regulating the expression of the myelin protein genes. One such event in the expression of the myelin basic protein (MBP) gene is the translocation of MBP mRNAs from oligodendrocyte cell bodies to their processes. This translocation can be observed in vivo and in primary mixed glial cell cultures. In jimpy brains the translocation of MBP mRNA appears to be disrupted, so that most of the mRNA remains associated with cell bodies. This apparent failure of translocation may account for the lack of incorporation of newly synthesized MBP into jimpy myelin. In quaking myelin, where MBP assembly is also defective, translocation appears to be normal, suggesting that incorporation of MBP into the membrane also is regulated posttranslationally. We have identified a number of the structural features of MBP mRNAs that influence the efficiencies with which they are translated and may be involved in regulating the levels of individual MBP produced. We also found that glucocorticoids stimulate the translation of MBP and PLP mRNAs and inhibit the translation of CNP mRNA in cell-free systems. Our results suggest that this pattern of translational regulation may be physiologically meaningful, especially during maturation of myelin. The mechanism by which the steroids modulate translation of these messages appears to be novel. Analysis of the effect of steroids on cRNAs produced from engineered MBP cDNA constructs has permitted the identification of a nine nucleotide element involved in this steroid modulation within the 5' untranslated region of the MBP mRNA.

Effect of soman intoxication on the organization of rat brain ribosomes and the translational activity of mRNA in a cell-free system.

The effect of soman on rat brain ribosomes organization and translational activity of mRNA in cell-free system was studied in rats exposed to 1.3 LD50 soman (120 micrograms/kg body weight) and in rats repeatedly injected with 0.4 LD50 soman (35 micrograms/kg). Fifteen minutes after the injection of 1.3 LD50 soman the heavy polyribosomal fraction from rat brain was found to be enriched and translational activity of mRNA was enhanced. In rats administered five injections of 0.4 LD50 soman at 24-h intervals, the low density ribosomes appeared as the predominant fraction whereas the activity of mRNA in all cell-free system was significantly impaired. It is concluded that soman intoxication expresses a stimulatory or inhibitory effect on the processes of protein synthesis in the rat brain, depending on the dose schedule of soman administration.

Comparative inhibition of rabbit globin mRNA translation by modified antisense oligodeoxynucleotides

We have studied the translation of rabbit globin mRNA in cell free systems (reticulocyte lysate and wheat germ extract) and in microinjected Xenopus oocytes in the presence of anti-sense oligodeoxynucleotides. Results obtained with the unmodified all-oxygen compounds were compared with those obtained when phosphorothioate or alpha-DNA was used. In the wheat germ system a 17-mer sequence targeted to the coding region of beta-globin mRNA was specifically inhibitory when either the unmodified phosphodiester oligonucleotide or its phosphorothioate analogue were used. In contrast no effect was observed with the alpha-oligomer. These results were ascribed to the fact that phosphorothioate oligomers elicit an RNase-H activity comparable to the all-oxygen congeners, while alpha-DNA/mRNA hybrids were a poor substrate. Microinjected Xenopus oocytes followed a similar pattern. The phosphorothioate oligomer was more efficient to prevent translation than the unmodified 17-mer. Inhibition of beta-globin synthesis was observed in the nanomolar concentration range. This result can be ascribed to the nuclease resistance of phosphorothioates as compared to natural phosphodiester linkages, alpha-oligomers were devoid of any inhibitory effect up to 30 microM. Phosphorothioate oligodeoxyribonucleotides were shown to be non-specific inhibitors of protein translation, at concentrations in the micromolar range, in both cell-free systems and oocytes. Non-specific inhibition of translation was dependent on the length of the phosphorothioate oligomer. These non-specific effects were not observed with the unmodified or the alpha-oligonucleotides.

Translation of vaccinia virus and cellular mRNA in cell-free systems prepared from uninfected and vaccinia virus infected L929 cells.

Cell-free translation systems were prepared from uninfected and vaccinia infected (3 and 5 hours post-infection) L929 cells. The systems were made mRNA dependent in order to translate exogenous mRNA mixtures. The overall rate of protein synthesis was similar in the three translation systems. However, one-dimensional electrophoresis showed that the systems differed in terms of the translation efficiency for individual mRNAs. This could be demonstrated with each of the following mRNA mixtures: early vaccinia mRNA synthesized by vaccinia cores in vitro, mRNA isolated from polysomes of vaccinia infected HeLa cells ("late" vaccinia mRNA) and cytoplasmic ascites mRNA. When the above mentioned groups of mRNAs were allowed to compete for translation in the cell-free systems and their products were analyzed on one-dimensional gels, the following order of translational efficiency was observed: the most prominent species of vaccinia early mRNA (other species could not be judged) were translated better than some late vaccinia mRNA species which in turn were slightly more efficiently translated than cellular mRNAs.

Expression of vaccinia virus early mRNA in Ehrlich ascites tumor cells. 1. Translation of cellular and viral early mRNA in cell-free systems from uninfected and virus-infected cells at the early stage.

Translation of cellular and early vaccinia RNA in nuclease-treated lysates, derived from uninfected and vaccinia-virus-infected cells at the early stage, has been investigated. When using limiting amounts of RNA no discrimination of translation was observed in the infected cells lysates; this conclusion was confirmed by sensitive RNA competition experiments for translation in vitro and also when using two different fractionated systems for protein synthesis in vitro. This absence of detectable discrimination in vitro was established both by comparing incorporation of [35S]methionine into proteins and by analysis of the products thus synthesized by sodium dodecylsulfate gel electrophoresis. However, a modification of the translational machinery from vaccinia-virus-infected cells did occur since the only the ribosomal salt wash derived from infected cells was able to reverse the inhibition of protein synthesis in vitro resulting from excess RNA (control or early). This property of vaccinia-virus-infected cell lysates may result from the synthesis l machinery from vaccinia-virus-infected cells did occur since the only the ribosomal salt wash derived from infected cells was able to reverse the inhibition of protein synthesis in vitro resulting from excess RNA (control or early). This property of vaccinia-virus-infected cell lysates may result from the synthesis l machinery from vaccinia-virus-infected cells did occur since the only the ribosomal salt wash derived from infected cells was able to reverse the inhibition of protein synthesis in vitro resulting from excess RNA (control or early). This property of vaccinia-virus-infected cell lysates may result from the synthesis of an early protein involved in translation or from a better recovery of translational factors from the infected cells, as suggested in the accompanying paper.

Interferon Heterogeneity Resulting from Differences in Glycosylation

35S methionine-labeled interferon synthesized in mouse C-243 induced by NDV in the presence and absence of glycosylation was analyzed after immunoprecipitation by gel electrophoresis. Interferons produced in the induced cells show large size heterogeneity in the region of 24,000-35,000-daltons. Interferon synthesized in the tunicamycin treated induced cells show two major bands of 15,000 and 18,000 daltons, which synthesis was also coded for by the interferon mRNA in cell-free system. The results indicate that the size heterogeneity of mouse interferons is due to the variability in the carbohydrate moiety of interferon polypeptides (alpha, beta).

Quantitation of human globin chain synthesis by cellulose acetate electrophoresis

We have developed a method for the separation and quantitation of human α-, β-, and γ-globins utilizing cellulose acetate electrophoresis. The relative rates of synthesis of globin chains in reticulocytes in peripheral blood is determined by: (i) incubating intact cells with [ 35S]methionine; (ii) preparing globin from the hemolysates; (iii) performing electrophoresis of the globin on cellulose acetate strips; and (iv) autoradiography or direct determination of the radioactivity incorporated into each globin chain. The method is simple and rapid, requires only small amounts of hemolysate (30 μg of globin), and provides excellent resolution and reproducible quantitation of α-, β A-, β S-, and γ-globin chains for up to 24 peripheral blood samples at one time. Measurements by this method in patients with thalassemia variants and sickle-cell disorders correlate well with analysis of the same samples by carboxymethyl cellulose chromatography. This methodology may permit more widespread analysis of globin synthesis in the thalassemia syndromes and may also be useful in the analysis of globins synthesized from human globin mRNA in cell-free systems.

The influence of rRNA and tRNA on the translation of avian globin mRNA in cell-free systems of protein synthesis

Cell-free synthesis of globin chains in the presence of globin mRNA in an Ehrlich ascites-cell-free system is further stimulated by addition of 18- and 28-S rRNA but not of 4-S tRNA and 5-S rRNA. This stimulation can not be observed in the wheat germ cell-free system. When 125I-labelled globin mRNA was incubated in the two systems we have found after 60 min a 75% decrease of trichloroacetic acid precipitable polynucleotides in the ascites but only a 20% decrease in the wheat germ system. The RNAase action on mRNA can be reduced by the addition of 18- and 28-S rRNA but not by 5-S rRNA and 4-S tRNA. We suggest that the stimulating effect of the two rRNA species in the ascites cell-free system is due to a higher activity of a specific RNAase in this system and a competitive protection of mRNA from RNAase action.

Translational recognition of the 5'-terminal 7-methylguanosine of globin messenger RNA as a function of ionic strength

The translation of rabbit globin mRNA in cell-free systems derived from either wheat germ or rabbit reticulocyte was studied in the presence of various analogues of the methylated 5' terminus (cap) as a function of ionic strength. Inhibition by these analogues was strongly enhanced by increasing concentrations of KCl, K(OAc), Na(OAc), or NH4(OAc). At appropriate concentrations of K(OAc), both cell-free systems were equally sensitive to inhibition by m7GTP. At 50 mM K(OAc), the reticulocyte system was not sensitive to m7GMP or m7GTP, but at higher concentrations up to 200 mM K(OAc), both nucleotides caused strong inhibition. The compound in m7G5'ppp5'Am was inhibitory at all concentrations of K(OAc) ranging from 50 to 200 mM, although more strongly so at the higher concentrations. Over the same range of nucleotide concentrations, the compounds GMP, GTP, and G5'ppp5'Am were not inhibitors. The mobility on sodium dodecyl sulfate-polyacrylamide electrophoresis of the translation product was that of globin at all K(OAc) concentrations in the presence of m7GTP. Globin mRNA from which the terminal m7GTP group had been removed by chemical treatment (periodate-cyclohexylamine-alkaline phosphatase) or enzymatic treatment (tobacco acid pyrophosphatase-alkaline phosphatase) was translated less efficiently than untreated globin mRNA at higher K(OAc) concentrations, but retained appreciable activity at low K(OAc) concentrations.

Translation of natural mRNA in cell-free systems from a polyamine-requiring mutant of [formula omitted

Cell-free extracts of polyamine-deficient and supplemented bacteria were used to study the translation of MS 2 bacteriophage RNA. The decreased synthetic activity observed with preparations of starved cells depends on a deficient initiation of polypeptide synthesis, as indicated by a lower capacity for the binding of formyl-methionyl-tRNA to ribosomes, induced by the trinucleotide AUG. The analysis of polypeptides formed showed that extracts from polyamine-depleted and supplemented cells are both able to synthesize complete protein chains.

Isolation and in vitro translation of delta-crystallin mRNA from embryonic chick lens fibers.

Of the protein synthesized and accumulated during differentiation of embryonic chick lens fibers, 70-80% is the tissue specific protein delta-crystallin. We have isolated and partially characterized the total cytoplasmic mRNA from purified lens fibers of 15-day-old embryos as an initial step toward understanding the regulated expression of delta-crystallin during development. Each lens fiber mass contained an average of 10 mug of cytoplasmic RNA; approximately 0.1 mug per fiber mass was recovered in the mRNA fraction by oligo(dT)-cellulose chromatography. The mRNA electrophoresed primarily as a single peak on a polyacrylamide-agarose gel with an apparent molecular weight of about 9 x 10(5) estimated by comparison with 28S and 18S rRNA markers. Of the protein synthesized in response to the mRNA in cell-free systems derived from Krebs II ascites tumor cells or rabbit reticulocytes, 70-80% comigrated with delta-crystallin on sodium dodecyl sulfatepolyacrylamide-agarose gels. Comparison of the tryptic peptides of delta-crystallin with those of the in vitro products from both heterologous systems established that the lens fiber mRNA contained delta-crystallin mRNA, and that no other functional mRNAs were present in detectable quantities. Thus, the specialization of protein synthesis in embryonic chick lens fibers apparently results from an accumulation of delta-crystallin mRNA in the cytoplasm.

Properties of the ribosomal salt wash from unfertilized and fertilized sea urchin eggs and its effect on natural mRNA translation

The acrylamide gel pattern of a ribosomal salt-wash fraction (Fraction RSW) prepared from unfertilized sea urchin eggs was compared with the pattern of a similar preparation from fertilized eggs. Significant differences were found indicating that as early as 2 min after fertilization a marked change occurs at the ribosomal level. Fraction RSW from unfertilized eggs is highly inhibitory on the translation of endogenous mRNA in cell-free systems prepared either from sea urchin or from rabbit reticulocytes. By contrast, Fraction RSW from fertilized eggs, at least at the concentration used in these experiments, does not significantly affect the endogenous protein synthesis. Furthermore, it was shown that Fraction RSW prepared from unfertilized eggs inhibits protein synthesis by interfering with the elongation process. The possibility of a simultaneous inhibitory effect at the initiation step cannot be ruled out.

Transfer of the genetic information carried by rabbit Hb mRNA onto guinea pig and allelic rabbit reticulocyte ribosomes

Summary Two different mRNAs from reticulocytes of rabbits with allelic hemoglobins, i.e. Hb α TIV Val and Hb α TIV Val-Leu, have been purified. The mRNAs were active for more than two hours when ribosomal KCl extract was added to the incubation medium, which makes it possible to obtain a de novo synthesis of the protein. Under these conditions we provide evidence for the translation of both mRNAs by studying the incorporation of radioactive amino-acids into the peptides specific of these hemoglobins. The translation of the rabbit hemoglobin mRNA has been studied in cell-free systems of two different types of reticulocytes. We have observed the translation of rabbit reticulocyte mRNA in a guinea-pig cell-free system, and the translation of Hb α TIV Val-Leu rabbit reticulocyte mRNA in a cell-free system containing ribosomes from Hb α TIV Val rabbit reticulocytes. This synthesis increased in proportion to the amount of exogenous mRNA added to the incubation medium.