Translation Of Rabbit Globin mRNA Research Articles

Inhibition of hepatitis C virus IRES-mediated translation by oligonucleotides

Two oligodeoxynucleotides (ODNs) were found to have a strong inhibition on the hepatitis C virus (HCV) internal ribosomal entry sites (IRES)-mediated translation but not the rabbit globin mRNA translation. Specific inhibition of those ODNs on HCV IRES-mediated translation was confirmed with heat treatment of ODNs in formic acid and dosage-dependent manners. Heat treatment of ODNs presented a decreasing inhibitory effect on HCV IRES-mediated translation. A dosage-dependent decrease of HCV IRES-mediated translation was observed with increasing amount of these ODNs in HeLa cell extracts. The minimal sequences of ODNs (A11) were identified as 5′-CGCGTTACG-3′ with the strongest inhibition of the HCV IRES-mediated translation. In a search for cellular factors, two cellular factors (p68 and p70) were identified to interact with ODNs A1 and A11, but not A5 (CT-oligo). This data showed new kinds of cellular proteins involved in HCV IRES-mediated translation. Further study of ODNs and these cellular proteins will provide important information for understanding the mechanistic basis and molecular regulation of HCV IRES-mediated translation.

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.

A c-myc antisense oligodeoxynucleotide inhibits entry into S phase but not progress from G0 to G1.

Initiation of T-lymphocyte proliferation by mitogen or antigen involves a cascade of gene activation events. Thus, by the time mitogen-activated T cells have reached the G1/S interface, many genes that are transcriptionally silent in G0, like the c-myc, IL-2, IL-2 receptor (IL-2R) and transferrin receptor (TfR) genes, have been transcriptionally activated. To understand the role of the individual genes in the activation process, one must be able to interfere specifically with the expression or function of each particular gene product. In this way, by blocking the IL-2R with an antibody, it has been demonstrated that IL-2/IL-2R interaction is required to induce TfR expression in activated T cells. When the function or expression of intracellular proteins is to be blocked, however, the need to introduce antibodies into the cytoplasm of viable cells, although possible, is a limiting factor. We have taken another approach, namely the exogenous addition to bulk cell cultures of small antisense oligomers. Sequence-specific antisense oligodeoxyribonucleotides have been reported to inhibit intracellular viral replication without interfering with cellular protein synthesis. Similarly, rabbit globin mRNA translation in a cell-free system and in rabbit reticulocytes has been inhibited by oligomers complementary to the globin mRNA initiation codon region. Recently, a pentadecadeoxyribonucleotide complementary to the initiation codon and four downstream codons of human c-myc mRNA was reported to inhibit the proliferation of the human leukaemic cell line HL-60 specifically. We report here that the same c-myc complementary oligonucleotide inhibits mitogen-induced c-myc protein expression in human T lymphocytes and prevents S phase entry. Interestingly, c-myc antisense treatment did not inhibit G0 to G1 traversal as assessed by morphologic blast transformation, transcriptional activation of the IL-2R and TfR genes, or induction of 3H-uridine incorporation.

Inhibition of rabbit globin mRNA translation by sequence-specific oligodeoxyribonucleotides.

Oligodeoxyribonucleotides 8-12 nucleotides in length whose sequences are complementary to the 5' end, the initiation codon regions, or the coding regions of rabbit globin mRNA were tested for their ability to inhibit translation in a rabbit reticulocyte lysate and in a wheat germ extract. The oligomers interact specifically with their target mRNAs as shown by their ability to serve as primers with reverse transcriptase. In the reticulocyte lysate, oligomers complementary to the 5' end or the initiation codon regions inhibit translation of both alpha- and beta-globin mRNA, whereas oligomers complementary to the coding regions have little or no effect. This suggests that reticulocyte ribosomes are able to displace the oligomers from the mRNA during the elongation but not the initiation step of translation. In the wheat germ system, translation was effectively inhibited by all oligomers regardless of their binding site on the message. In contrast to their behavior in the reticulocyte system, the oligomers inhibited alpha- and beta-globin synthesis in a specific manner. This observation suggests that control of alpha- and beta-globin mRNA translation is coordinated in the reticulocyte lysate system but not in the wheat germ extract. The results of our studies indicate that oligodeoxyribonucleotides may be useful probes for studying control of mRNA translation in cell-free systems.

Simplified in vitro system for study of eukaryotic mRNA translation by measuring di- and tripeptide formation.

An in vitro system for measurement of rabbit globin mRNA translation has been developed based on the formation of the NH2-terminal dipeptide, fMet-Val. The basic components include a partially purified initiation factor preparation from rabbit reticulocytes supplemented with eukaryotic initiation factor 4A, purified and formylated yeast Met-tRNAi, and rabbit liver or Escherichia coli Val-tRNA1Val. Picomole quantities of fMet-Val are synthesized, dependent on mRNA, and the dipeptide is readily assayed by a simple extraction procedure. In the presence of Leu-tRNA or His-tRNA, the tripeptides fMet-Val-Leu and fMet-Val-His are synthesized, corresponding to the NH2-terminal sequence of alpha- and beta-globin, respectively. Therefore, tripeptide synthesis provides a simple means to distinguish between the expression of the alpha- and beta-globin mRNA species.

Variable rate of polypeptide chain elongation in vitro. Effect of spermidine.

Translation of rabbit globin mRNA and tobacco mosaic virus mRNA was studied in a wheat germ system. After a short pulse with labeled methionine, the reaction products were separated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate and visualized by fluorography. Discrete bands corresponding to incomplete products were detected both with globin and tobacco mosaic virus RNA as messenger at a time when no release of peptides from tRNA could be detected. In the absence of spermidine certain intermediate products accumulated during the chase period. With increasing incubation time, the radioactivity of most bands was chased into higher-molecular-weight products. This change in size distribution was more pronounced when magnesium was partially replaced by spermidine. Evidence is presented that some of the incomplete products had in part been released from tRNA. No such release of polypeptides could be detected in experiments using the artificial messenger poly(U), indicating that non-specific peptidyl-tRNA hydrolases were not present in the system. The transient accumulation of discrete bands during the chase period indicates that in the wheat germ system polypeptide chain elongation occurs at discontinuous rates. The fact that spermidine increases the rate of elongation and the yield of full-length translation products may be due to its ability to facilitate the movement of the ribosomes beyond regions where the elongation is retarded.

Translation of rabbit globin mRNA upon injection into fused HeLa cells

Translation of rabbit globin mRNA upon injection into fused HeLa cells

Identification and nucleotide sequence of the sup8-e UGA-suppressor leucine tRNA from Schizosaccharomyces pombe

Using the translation of rabbit globin mRNA in wheat germ extracts as an assay for ochre and opal suppression, a UGA suppressor tRNA from Schizosaccharomyces pombre strain sup8-e was purified by column chromatography and two-dimensional gel electrophoresis. The purified tRNA can be aminoacylated with leucine by a crude aminoacyl-tRNA synthetase preparation from a wild type S. pombe strain, and has high activity in the suppressor assay. By a combination of post-labeling fingerprinting and rapid gel sequencing methods the nucleotide sequence of this suppressor tRNA was determined to be: pG-C-G-G-C-U-A-U-G-C-C-ac4C-G-A-G-D-G-G-D-G-D-A-A-G-G-G-m22G-G-C-A-G-A-psi-U-U*-C-A-m1G-C-C-C-U-G-C-U-G-U-U-G-U-A-A-A-A-C-G-m5C-G-A-G-A-G-T-psi-C-G-m1A-A-C-C-U-C-U-C-U-G-G-C-C-G-C-A-C-C-AOH. The anticodon sequence U*CA is complementary to the UGA codon. An interesting feature of the suppressor tRNA is an expanded anticodon loop of nine nucleotides owing to an A-C nonpair at the first anticodon stem position.

Evidence for translation of rabbit globin mRNA after liposome-mediated insertion into a human cell line.

THE ability to insert messenger RNA selectively into large numbers of differentiated eukaryotic cells and thereby alter the expression of the eukaryotic genome would provide a new and direct approach to the study of the molecular mechanisms involved in protein synthesis. Several procedures have been used to facilitate the cellular incorporation of administered RNA, including microinjection of RNA into Xenopus oocytes1–6 and into fully differentiated eukaryotic cells7,8, direct co-cultivation of cells with naked RNA (refs 9–12), and encapsulation of mRNA within erythrocyte ghosts followed by Sendai virus induced fusion of the ghosts to recipient cells in culture13. We describe here the use of liposomes for the selective insertion of functional mRNA into differentiated eukaryotic cells in vitro. We provide evidence which indicates that cultured human epithelial carcinoma cells (HEp-2) treated with liposomally encapsulated rabbit globin mRNA are stimulated to produce a globin-like protein.

Translation of rabbit globin mRNA introduced by liposomes into mouse lymphocytes.

SEVERAL interesting questions in developmental biology can be studied by the combination of messenger RNA from one kind of cell with the translational apparatus of another cell type. Such experiments can provide proof of the identity of a mRNA and can yield information regarding the species and cell-type specificities of translation systems. The first successful approach to this problem was that taken by Lane et al.1 who injected globin mRNA into frog oocytes. However, results obtained using the oocyte, which is an unspecialised cell, cannot necessarily be extended to the translational apparatus of more specialised cells. Many workers have tried to introduce informational macromolecules directly into differentiated cells, using a variety of methods2–6. Stacey et al. have translated duck globin mRNA after injection into HeLa cells7. We have demonstrated8,9 the entrapment of ribonucleic acids of different sizes (28S, 18S, 9S and 4S) in large unilamellar liposomes. Such entrapped macromolecules are protected from ribonucleases and can be isolated both intact and biologically active8. Moreover, fusion of these liposomes yields RNA-filled cells9. We present here evidence that rabbit reticulocyte 9S mRNA introduced into mouse spleen lymphocytes directs the synthesis of globin.

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.

Inhibition of initiation of protein synthesis by 7-methylguanosine-5'-monophosphate.

Translation of rabbit globin mRNA in a wheat germ protein-synthesizing system is inhibited by the nucleotide 7-methylguanosine-5'-monophosphate (m7G5'p) but not by other guanosine nucleotides without the 7-methyl group or with the phosphate in a different position. Translation of RNA of tobacco mosaic virus and poly(A) + HeLa RNA is also inhibited by m7G5'p. We show that m7G5'p prevents the association of mRNA with ribosomal subunits to form an initiation complex. We propose that m7G5'p interacts with a site on initiation factor(s) or ribosomes which is involved in mRNA recognition, presumably by binding to the 5'-terminal sequence m7G5'ppp. m7G5'p does not inhibit translation of poly(U) and RNA of satellite tobacco necrosis virus, which do not have the 5'-terminal sequence m7G5'ppp. In the case of RNA of satellite tobacco necrosis virus, some stimulation of its translation is consistently observed in the presence of m7G5'p; possible interpretations of this finding are discussed.

Differential translation of rabbit globin mRNA. Effect of concentrations of creatine phosphate, ATP and GTP

Rabbit reticulocyte lysate can synthesize the (Y and p globin chains in equal amounts [ 1 ] . There are many works concerning the effect of various factors on the synthetic ratio of ar to fl globin chains in rabbit reticulocyte lysate systems [l-6] . However, there is no publication that deals with the effect of concentrations of creatine phosphate(CP), ATP and GTP on the ratio of (Y to /3 chains synthesized. The present paper reports the changes in the cr and 0 chains synthesized after incubation of rabbit reticulocyte lysate with a different concentration of CP, ATP and GTP. The possibility that the ollp ratio is controlled by the concentration of ATP through the aminoacylation of tRNA was discussed.

Heparin as inhibitor of mammalian protein synthesis. II. Degree of sulfation; related sulfated mucopolysaccharides.

Abstract Heparin is a potent inhibitor of initiation of translation of rabbit globin mRNA in a Krebs ascites cell-free system, as well as being a potent anticoagulant. Progressive desulfation of heparin in the presence of acid for 1 h decreases heparin's anticoagulation activity by 50%, while incubation of heparin for longer than 8 h is necessary to cause 50% loss of heparin's activity as a protein-synthesis inhibitor. Partial resulfation of amino groups of inactive desulfated heparin restores heparin's protein-synthesis inhibitory ability almost completely, while negligible anticoagulant activity is restored. Proteoheparin is active as both a protein-synthesis inhibitor and an anticoagulant, while heparan sulfate lacks both activities.

Translation of duck-globin messenger RNA in a partially purified mammalian cell-free system.

Duck globin messenger RNA purified by three different procedures from duck immature erythrocyte polyribosomes was translated in a mammalian cell‐free system which is strongly dependent upon exogenous mRNA. The system consists of purified mouse liver ribosome sub‐units, partially purified rabbit reticulocyte initiation factors, and rat liver pH‐5 enzymes (containing elongation and termination factors, aminoacyl‐tRNA synthetases and tRNA). If offered alone, duck globin mRNA directed the synthesis of all known duck globin chains. The efficiency of duck globin synthesis was similar to that of rabbit globin if the two types were translated separately. If duck and rabbit globin mRNA were offered together each in saturating amounts in this system, rabbit globin synthesis occurred almost exclusively. Differences in the secondary structure between the two types of globin mRNA must be involved in this strongly discriminating translation in the competition experiments. Furthermore, the quantitative requirement of at least one initiation factor was found to be different for duck and rabbit globin mRNA translation. While there is no evidence that this factor is directly involved in mRNA binding, it seems likely that the difference in factor requirement is related to the preferential translation of rabbit globin mRNA in the competition experiments.

Translation of rabbit hemoglobin meessenger RNA in vitro with purified and partially purified components from brain or liver of different species.

Two mammalian systems have been developed for efficient in vitro translation of exogenous messenger RNA (rabbit globin mRNA). One system is completely derived from guinea pig brain, and the other is from liver of different species. Both systems consist of purified 60S and 40S ribosomal sununits, unseparated initiation factors partially purified by ammonium sulfate precipitation and DEAE-cellulose chromatography, and pH 5 enzyme fractions as sources of elongation and termination factors, aminoacyl-tRNA synthetases, and transfer RNA. Translation depends completely upon exogenous mRNA and initiation factors. Extraction of initiation factors from microsomes or ribosomes has been improved for these tissues by inclusion of Mg(++) ions in the 0.5 M KCl extraction solution. Both systems synthesize complete rabbit alpha- and beta-globin chains, but in variable ratios. The overall efficiencies of the two systems are about 60% (liver) and 40% (brain) of a comparable system with rabbit reticulocyte initiation factors.

Translation of rabbit globin mRNA.

IN certain conditions bacterial initiation factors can discriminate between different classes of mRNA, or the individual cistrons of bacteriophage RNA1–5. Whether messenger specificity is a general phenomenon in eukaryotes as well is controversial, but several reports support the idea that mRNAs can be translated in heterologous systems without the addition of homologous tissue-specific factors, suggesting a lack of specificity6,10,17. These results have been obtained by adding mRNA either to oocytes7 or to crude cell-free systems such as lysates or 30,000g supernatants (S-30). With more fractionated systems—separated ribosomes reconstituted with cell sap—the existence of messenger specific factors is supported11–13. We have already demonstrated that an S-30 derived from either mouse or rat liver can faithfully translate rabbit globin mRNA without the addition of reticulocyte factors and now report that fractionation of this S-30 using methods similar to those used by previous workers11–13 results in a system which will also translate globin mRNA. This fractionated system does not show a requirement for reticulocyte specific factors.