Enhancement of interferon mRNA levels in butyric acid-treated Namalwa cells.
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Burkitt Lymphoma↗
Biomedical subjects
J Morser
Publications and source records attributed to J Morser.
Effects of sodium butyrate on a human lymphoblastoid cell line (Namalwa) and its interferon production.
Treatment of lymphoblastoid cells with sodium butyrate before infection with Sendai virus increases the subsequent yield of interferon and of its messenger RNA. This treatment caused almost complete inhibition of cellular DNA synthesis and consequently cell division, and some inhibition of cellular RNA and protein synthesis. It also caused increased acetylation of histones and alterations in the plasma membrane. There was no decrease in cell viability or change in cell morphology but the cells were smaller than untreated cells and were all in the G1 phase. Sodium butyrate treatment had no effect on the course of Sendai virus infections. The observed increase in interferon yields after butyrate treatment did not correlate with its effects on DNA synthesis, size, cell division and histone acetylation, but did with its effects on RNA and protein synthesis.
Acetylation↗
Post-translational events in the production of human lymphoblastoid interferon.
Newly synthesized interferon and its mRNA were membrane-associated in a human lymphoblastoid cell (Namalwa) that had been induced with Sendai virus. Treatment with zinc, which acts as an inhibitor of proteolytic cleavage, prevented interferon production. When cytoskeletal function was disrupted by adding both colchicine and cytochalasin B to the induced Namalwa cells, secretion of interferon was inhibited. It is concluded that after translation of the interferon mRNA, the newly synthesized interferon polypeptide is discharged into the lumen of the endoplasmic reticulum, and undergoes a proteolytic cleavage before it is secreted by a process involving the cytoskeleton.
Burkitt Lymphoma↗
The Xenopus oocyte as a surrogate secretory system. The specificity of protein export.
Combining messenger RNA from one kind of secretory cell with the cytoplasm of another such cell can reveal the nature and specificity of protein export mechanisms. We show that messenger RNAs from secretory cells of chickens, rats, mice, frogs, guinea-pigs, locusts and barley plants, when injected into Xenopus oocytes, direct the synthesis and export of proteins. Chicken ovalbumin, Xenopus albumin, mouse thyroid-stimulating hormone, locust vitellin and guinea-pig milk proteins were identified using specific antibodies, whilst chicken lysozyme and ovomucoid, rat albumin, Xenopus vitellogenin and rat seminal vesicle basic proteins were identified provisionally from their molecular weights. Certain endogenous proteins are sequestered and secreted although most oocyte proteins are not exported. Similarly the major polyoma viral protein and the simian virus 40 and polyoma tumour antigens are retained within the oocyte. Radioactive proteins exported by oocytes programmed with chicken oviduct or Xenopus liver RNA are not re-exported in detectable amounts when injected into fresh oocytes, nor is there secretion of chicken oviduct or guinea-pig mammary gland primary translation products prepared using wheat germ extracts. Thus the export of secretory proteins from oocytes cannot be explained by leakage and may require a cotranslational event. The secretory system of the oocyte is neither cell-type nor species-specific yet is highly selective. We suggest that the oocyte can be used as a general surrogate system for the study of gene expression, from transcription through translation to the final subcellular or extracellular destination of the processed protein.
Animals↗
Interferon and double-stranded RNA.
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Interferons↗
Export of proteins from oocytes of Xenopus laevis.
When human lymphoblastoid mRNA was microinjected into X. laevis oocytes, titers of interferon rapidly reached a maximum inside the oocyte while accumulation of interferon continued in the incubation medium for at least 45 hr. If interferon protein was injected into oocytes it was rapidly inactivated. Significantly, newly synthesized interferon but not injected interferon was found to be membrane-associated. Further experiments involving the co-injection of mRNAs coding for secretory proteins (guinea pig milk proteins and human interferon) and nonsecretory proteins (rabbit globin) revealed that only the secretory proteins were exported from the oocyte. Moreover, different proteins were exported at different rates. A distinct subclass of newly synthesized oocyte proteins of unknown function also accumulated in the incubation medium. Since the information encoded in the messenger RNAs of secretory proteins is sufficient to specify synthesis, compartmentation and secretion of these proteins, the oocyte may provide a complete system for the analysis of the secretory process.
Animals↗
The effect of inhibitors of glycosylation on interferon production in human lymphoblastoid cells.
Interferon production was inhibited in the Namalwa line of human lymphoblastoid cells by treatment with 2-deoxy-D-glucose or D-glucosamine. D-Glucosamine also inhibited protein synthesis and the cells were no longer viable, whereas 2-deoxy-D-glucose allowed protein and RNA synthesis to continue at control rates, and the cells remained fully viable. It is concluded that a glycosylation step is essential for production of lymphoblastoid interferon.
Burkitt Lymphoma↗
The toxic effect of double-stranded RNA for interferon-treated cells: evidence for a heterogeneous cellular response and the role of the cell nucleus.
When L929 cells were treated with interferon and subsequently with poly(rI).poly(rC), there was a pronounced toxic effect. Most of the cells lysed, but some survived and grew at the same rate as control cells to yield cells which were as sensitive to the effects of interferon and poly(rI).poly(rC) as the original population. The proportion of surviving cells did not vary with either the cell cycle or the cell density. The treated cells produced interferon and some of the interferon was produced by the resistant cells. Cells which had been X-irradiated before treatment with interferon and poly(rI).poly(rC) behaved similarly so cell division was not necessary for the development of toxicity. The toxic effect also developed when cells were enucleated with the aid of cytochalas in B after treatment with interferon, but not if they were enucleated before treatment. It is concluded that the nucleus is essential for interferon to exert its effect on the cells, but not for the development of cytotoxicity after the addition of poly(rI).poly(rC).
Animals↗
The toxic effect of double-stranded RNA for interferon-treated L cells: studies on membrane transport and with cell-free systems.
We have tested two possible explanations for the toxic effects observed in L cells treated with interferon followed by poly(rI).poly(rC), namely (1) that the toxicity was preceded by and due to a change in membrane permeability which could be monitored by measurement of ion fluxes, or (2) that the toxicity was due to the action of the inhibitors of protein synthesis which are known to be formed when extracts from interferon-treated cells are treated with poly(rI).poly(rC) in vitro. Neither explanation was found to be correct.
Animals↗
Characterization of interferon messenger RNA from human lymphoblastoid cells.
After treatment with Sendai virus, Namalwa cells form large amounts of interferon. RNA extracted from treated whole cells or from their polysomes was injected into Xenopus laevis oocytes and the interferon formed was titrated. The results show that the amount of interferon mRNA was maximal by 9 h after treatment of the cells with Sendai virus and then declined. Sucrose gradient centrifugation of the mRNA gave substantial purification and showed that its size was 12 S.
Animals↗
The effect of 5-bromodeoxyuridine on interferon production in human cells.
5-Bromodeoxyuridine (BrdUrd) increased interferon production by the Namalwa line of human lymphoblastoid cells treated with Sendai virus, but inhibited their growth. Thymidine, which also inhibited cell growth had no effect on interferon production, so that growth inhibition per se was not the cause of the stimulation. BrdUrd was incorporated into cellular DNA; 5-chlorodeoxyuridine and 5-iododeoxyuridine (which are also incorporated) increased the interferon yield, but 5-fluorodeoxyuridine (which is not incorporated) did not. Thymidine reduced both the incorporation of BrdUrd and its stimulatory effect on interferon production. Deoxycytidine (which prevents the cytotoxic effects of BrdUrd) had no effect on the stimulation. BrdUrd also stimulated interferon production in response to poly(rI) . poly(rC) in growing human diploid fibroblasts but not in SV40 virus-transformed human cells. Since BrdUrd was incorporated into the DNA of all these cells, we concluded that incorporation is necessary, but not sufficient for the stimulation of interferon formation.
Bromodeoxyuridine↗
Differences in sialic acid content of human interferons.
Human leucocyte, lymphoblastoid and fibroblast interferons were separately treated with sialidase and the effect of this on their isoelectric focusing was examined using a system in which full dissociation of complexes occurred. Both leucocyte and lymphoblastoid interferons showed a single form with an isoelectric point which was unaltered by treatment with sialidase. In contrast, fibroblast interferon showed three forms which were reduced to one by treatment with the enzyme.
Fibroblasts↗
Simultaneous translation of structural and nonstructural proteins from Semliki-forest-virus RNA in two eukaryotic systems in vitro.
The Semliki Forest virus genome, 42-S RNA, and the virus-specific intracellular 26-S RNA were translated in two cell-free protein-synthesising systems, the wheat germ extract, and a partially purified system from mammalian tissues. The 26-S RNA directed the synthesis of structual proteins only, as revealed by tryptic peptide mapping. About 75--80% of the radioactivity in the products comigrated with capsid and about 4--8% with envelope protein peptides. All the capsid peptides and the full-sized capsid protein were found in the products in vitro, no complete envelope protein was formed and fewer than half of the envelope peptides were detected. This result is consistent with reports that there is only one initiation site for the translation of virus structural proteins, and that the capsid protein is N-terminal in the polyprotein followed by envelope proteins. The systems programmed with 42-S RNA yielded virtually the same structural peptides. However, the bulk of the radioactivity was in peptides which did not comigrate with the structural ones. These peptides were mostly associated with relatively small-sized products. This shows that Semliki Forest virus 42-S RNA has at least two initiation sites, one for the structural proteins and the other(s) for the nonstructural proteins.
Cell Line↗
Initiation of translation directed by 42S and 26S RNAs from Semliki Forest virus in vitro.
The proteins synthesized in vitro in response to 42S and 26S RNAs from Semliki Forest virus were labeled with formyl-[35S]methionine from initiator tRNA. One protein which comigrated with viral capsid protein was labeled under the direction of 26S RNA, and only one labeled peptide was detected after digestion with trypsin. Further digestion with pronase gave rise to the dipeptide fMet-AsN. Several labeled polypeptides were found in the 42S RNA directed product and these had molecular weights of up to 150,000. However, tryptic digestion of the product yielded only one formylmethionyl-labeled peptide, which had a different mobility from that directed by the 26S RNA. Further digestion with pronase gave a single dipeptide, fMet-Ala. This indicates that nonstructural proteins as large as 150,000 daltons are probably synthesized from one initiation site on the 42S RNA. Translation starting from the internal initiation site on the 42S RNA, which is equivalent to that on the 26S RNA, could not be detected under the conditions used. Internal initiation sites which are similarly inactive have also been detected in other viral RNAs (e.g., brome mosaic virus, tobacco mosaic virus, and polyoma 19S RNA) and this suggests that, although eukaryotic mRNAs can contain more than one initiation site for protein synthesis, only the site nearer the 5' terminus is active in vitro.
Cell-Free System↗
Translation of Semliki forest virus 42S and 26S RNAs in a cell-free system derived from Escherichia coli.
The SFV 42S RNA and the intracellular 26S RNA have been translated in a prokaryotic cell-free system, the E. coli S30. About half of the [35S]methionine-labelled products directed by both RNAs had molecular weights larger than 20,000 on polyacrylamide gels. Both products contained tryptic peptides which comigrated with all the capsid and envelope protein-derived peptides. The most striking difference between the prokaryotic and eukaryotic systems lay in the translation of the 42 S RNA: The "42S RNA-specific nonstructural" peptides, which predominate in the eukaryotic systems, were apparently absent from the product translated by the prokaryotic system.
Cell-Free System↗
Translation of Semliki forest virus 42S and 26S RNAs in two eukaryotic cell-free protein synthesising systems.
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Cell-Free System↗
Polysomes and initiation complexes in vitro induced by Semliki Forest virus 42S and 26S RNA.
Semliki Forest virus 42S and 26S RNA induce the formation of polysomes when translated in vitro in cell-free systems. After analysis on sucrose gradients the polysomal structures, containing prelabelled RNA and nascent peptide chains labelled with [35S]methionine, had sedimentation values from 100 to 200S in the case of 26S RNA and from 150 to over 250S with 42S RNA. After incubation in the presence of inhibitors of elongation 26S RNA was found in initiation complexes sedimenting at about 80S. Under identical conditions 42S RNA had a heterogenous sedimentation pattern, being attached to complexes sedimenting even faster than 250S. These structures, however, fulfilled the criteria of initiation complexes suggesting that there were more than one ribosome attached to each 42S RNA under conditions where 26S RNA bound only one.
Cell-Free System↗