Synthesis of reovirus ribonucleic acid in L cells

Kudo, Hajime (The Wistar Institute of Anatomy and Biology, Philadelphia, Pa.), and A. F. Graham. Synthesis of reovirus ribonucleic acid in L cells. J. Bacteriol. 90:936-945. 1965.-There is no inhibition of protein or deoxyribonucleic acid (DNA) synthesis in L cells infected with reovirus until the time that new virus starts to form about 8 hr after infection. At this time, both protein synthesis and DNA synthesis commence to be inhibited. Neither the synthesis of ribosomal ribonucleic acid (RNA) nor that of the rapidly labeled RNA of the cell nucleus is inhibited before 10 hr after infection. Actinomycin at a concentration of 0.5 mug/ml does not inhibit the formation of reovirus, although higher concentrations of the antibiotic do so. Pulse-labeling experiments with uridine-C(14) carried out in the presence of 0.5 mug/ml of actinomycin show that, at 6 to 8 hr after infection, two species of virus-specific RNA begin to form and increase in quantity as time goes on. One species is sensitive to ribonuclease action and the other is very resistant. The latter RNA is probably double-stranded viral progeny RNA, and it constitutes approximately 40% of the RNA formed up to 16 hr after infection. The function of the ribonuclease-sensitive RNA is not yet known. Synthesis of both species of RNA is inhibited by 5 mug/ml of actinomycin added at early times after infection. Added 6 to 8 hr after infection, when virus-specific RNA has already commenced to form, 5 mug/ml of actinomycin no longer inhibit the formation of either species of RNA.     

Influence of Macromolecular Biosynthesis on Cellular Autolysis in Streptococcus faecalis

The addition of several different antibiotics to growing cultures of Streptococcus faecalis, ATCC 9790, was found to inhibit autolysis of cells in sodium phosphate buffer. When added to exponential-phase cultures, mitomycin C (0.4 mug/ml) or phenethyl alcohol (3 mg/ml) inhibited deoxyribonucleic acid synthesis, but did not appreciably affect the rate of cellular autolysis. Addition of chloramphenicol (10 mug/ml), tetracycline (0.5 mug/ml), puromycin (25 mug/ml), or 5-azacytidine (5 mug/ml) to exponential-phase cultures inhibited protein synthesis and profoundly decreased the rate of cellular autolysis. Actinomycin D (0.075 mug/ml) and rifampin (0.01 mug/ml), both inhibitors of ribonucleic acid (RNA) synthesis, also reduced the rate of cellular autolysis. However, the inhibitory effect of actinomycin D and rifampin on cellular autolysis was more closely correlated with their concomitant secondary inhibition of protein synthesis than with the more severe inhibition of RNA synthesis. The dose-dependent inhibition of protein synthesis by 5-azacytidine was quickly diluted out of a growing culture. Reversal of inhibition was accompanied by a disproportionately rapid increase in the ability of cells to autolyze. Thus, inhibition of the ability of cells to autolyze can be most closely related to inhibition of protein synthesis. Furthermore, the rapidity of the response of cellular autolysis to inhibitors of protein synthesis suggests that regulation is exerted at the level of autolytic enzyme activity and not enzyme synthesis.     

Polyadenylate sequences of human rhinovirus and poliovirus RNA and cordycepin sensitivity of virus replication.

The polyadenylate [poly(A)] content of the genome RNA of human rhinovirus type 14 (HRV-14) is nearly twice as large as that of the genome RNA of poliovirus type 2. The poly(A) content of viral RNA was determined to be the RNase-resistant fraction of 32P-labeled viral RNA extracted from purified virions. Polyacrylamide gel electrophoresis indicated that the poly(A) sequences of HRV-14 are more heterogenous and on an average larger than those of poliovirus RNA. On the basis of susceptibility to micrococcal polynucleotide phosphorylase the rhinovirus genome terminates in poly(A). Replication of both viruses is almost totally inhibited by cordycepin at 50 mug/ml. At lower concentrations, rhinovirus replication is more sensitive to cordycepin than poliovirus replication. Addition of cordycepin (75 mug/ml) to infected culture prior to or during viral RNA replication results in more or less complete inhibition of virus-specific RNA synthesis. The results do not indicate that cordycepin sensitivity of either virus is due to preferential inhibition of viral poly(A) synthesis by this antibiotic.     

Mode of Action of Myxin on Escherichia coli

The effect of the new antibiotic, myxin, on the syntheses of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein in Escherichia coli (strains B and 15T(-)) was examined. Within 7 min of the addition of myxin at 5 mug/ml, the synthesis of new bacterial DNA was almost completely inhibited. This was followed by an extensive degradation of the pre-existing DNA to an acid-soluble form. All of the evidence indicated that the primary effect of the antibiotic was on cellular DNA. The synthesis of RNA was completely inhibited after 15 min of exposure to myxin (5 mug/ml), and the synthesis of protein was markedly reduced after 30 min. There was no measurable breakdown of either RNA or protein in the myxin-treated cells. A marked stimulation of (14)C-uracil incorporation was found in the presence of myxin in 15T(-) cells only. This did not result from an increased rate of RNA synthesis but was due to an increase in the proportion of exogenous uracil, relative to endogenous uracil, incorporated into cellular RNA. This probably reflected a partial inhibition of the biosynthesis of uridine monophosphate from orotate. At 4.5 mug of myxin per ml and with 0.8 x 10(8) cells per ml, 50% of the antibiotic was reduced in 15 min from the biologically active oxidized form to the biologically inactive state. Under these conditions, a maximum of 0.6% (27 mumug/ml) of the myxin was retained in the cells.     

Interferon Action on Parental Semliki Forest Virus Ribonucleic Acid

Actinomycin D-treated chick fibroblasts were infected with purified (32)P-labeled Semliki forest virus, and ribonucleic acid (RNA) was extracted after 1 or 2 hr. Within 1 hr, viral RNA forms sedimenting in sucrose gradients at 42S, 30S, and 16S were present. The 42S form corresponded to the RNA of the virion. The 16S form appeared to be a double-stranded template for the formation of new viral RNA, since nascent RNA was associated with it and the molecule could be heat-denatured and subsequently reannealed by slow cooling. Interferon treatment before infection, or puromycin (50 mug/ml) or cycloheximide (200 mug/ml) added at the time of virus infection, had no effect on the formation of the 30S RNA but inhibited the production of the 16S form. Several findings made it unlikely that these results were due to breakdown of parental RNA and reincorporation of (32)P into progeny structures. The results suggested that the mechanism of interferon action involves inhibition of protein synthesis by parental viral RNA, since a specific viral RNA polymerase had previously been demonstrated to be necessary for production of 16S RNA. No protein synthesis appears necessary for formation of 30S RNA from parental virus RNA.     

Effect of incubation conditions, inhibitors, 2-mercaptoethanol and testosterone on RNA synthesis in ram spermatozoa

In vitro studies on RNA synthesis using washed ram spermatozoa were carried out by measuring the incorporation of (3)H-uridine into RNA. Penicillin-G (100 mug/ml medium) was added to prevent contamination by microorganisms. Spermatozoa were quickly separated from seminal plasma by washing twice in Tris-HCl buffer (at pH 7.2) and centrifuged at 1,000 g for 5 min. Washed spermatozoa were then diluted to 1 10 , 1 20 or 1 40 (v/v) by the same buffer system (containing 400 mg% glucose) and were incubated in air at 37 degrees C for 1, 2 and 4 h. Results indicated that the rate of RNA synthesis was maximal at 1 40 semenbuffer dilution (5-8 x 10(7) spermatozoa/ml) and increased linearly up to 4 h of incubation. The rate of RNA synthesis at 1 40 dilution also increased linearly as the dose of exogenous glucose substrate was increased up to 400 mg%. Denaturation of the ram spermatozoa by 1% HgCl(2) caused almost complete inhibition of RNA synthesis that amounted to 97% of the control samples. Incubation of spermatozoa with 50, 100 or 200 mug/ml chloramphenicol also inhibited uridine incorporation by 86 to 94%, while equivalent doses of cycloheximide did not. On the other hand, the incorporation of (3)H-uridine into the RNA of ram spermatozoa was significantly enhanced by graded doses of 2-mercaptoethanol (0.2, 0.4 and 0.8 muM) and of testosterone (15 and 30 mug/ml). The results of this study indicate RNA synthesis, mainly of mitochondrial origin, by mature ram sperm. The data also suggest a role for intracellular cyclic adenosine monophosphate in the regulation of sperm RNA synthesis.     

Developmental and regional variations in ribonucleic acid synthesis on cerebral chromatin

1. Chromatin was prepared from purified nuclei isolated from liver and cerebral regions of the rat. 2. The capacity of these preparations to promote RNA synthesis in the presence of bacterial RNA polymerase was determined. 3. The rate of RNA synthesis on chromatin was normally 12-21% of the rate observed with native DNA, but was markedly stimulated on addition of 200mm-ammonium sulphate. 4. At physiological concentrations (80mug./ml.), the brain-specific S-100 protein inhibited RNA synthesis on DNA and chromatin. 5. Cerebral chromatin from foetal and newborn animals was more active in RNA synthesis than were the analogous preparations from liver. 6. Cerebellar chromatin maintained a high rate of RNA synthesis during brain maturation. In contrast, RNA synthesis on chromatin from other brain regions and liver declined with age of the rat. 7. RNA synthesized on chromatin stimulated amino acid incorporation in an Escherichia coli ribosomal system and hybridized with homologous DNA. 8. RNA synthesized on chromatin from adult cortex or hindbrain hybridized with DNA to a greater extent than that synthesized on cerebellar chromatin. 9. The proportion of RNA formed on cerebral-cortical chromatin that hybridized with DNA increased with age of the rat. 10. The results indicate that the total amount and the types of RNA synthesized on cerebral chromatin vary regionally and during development.     

Synthesis of Ribonucleic Acid in Cells Infected with LSc Poliovirus at Elevated Temperatures

The synthesis of viral ribonucleic acid (RNA) was detected within 2 hr after infection with LSc poliovirus at 35 C. This RNA eluted as a single peak with 0.9 m NaCl on methylated albumin celite columns, was sensitive to ribonuclease, precipitated in the presence of 2 m LiCl, and had an S(20) value at 34 +/- 2 in linear sucrose gradients. When cells were infected at 39 to 40 C, there was also early synthesis of RNA. However, 2 hr after infection this synthesis was drastically inhibited. The absence of net RNA synthesis at 39 to 40 C during the late stages of infection was not caused by rapid degradation of newly formed RNA, since the RNA produced between 1 and 2 hr at 39 to 40 C was still present 3.5 hr after infection. There was a 3 log(10) inhibition in the production of infectious virus when p-fluorophenylalanine was present in the medium at a concentration of 25 mug/ml. This concentration of analogue had little effect upon the production of viral polymerase and viral RNA. Virus grown in the presence of analogue at a concentration of 10 mug/ml exhibited increased heat sensitivity compared to control virus. However, viral polymerase exhibited no change in sensitivity to heat or manganese when cells were exposed to 25 mug of p-fluorophenylalanine per ml during infection. p-Fluorophenylalanine had a relatively selective effect on viral capsid protein but did not reverse the inhibition of synthesis of viral RNA at 39 to 40 C.     

Effect of Rifamycins and Related Antibiotics on the Deoxyribonucleic Acid-Dependent Ribonucleic Acid Polymerase of Vaccinia Virus Particles

A number of compounds related to rifampin which act as expected in the Escherichia coli system have been tested for their ability to inhibit the vaccinia particle deoxyribonucleic acid-dependent ribonucleic acid (RNA) polymerase in vitro. Some compounds are inactive even at concentrations of 500 mug/ml, others are able to produce partial inhibition, and others strongly inhibit the enzyme activity at 150 mug/ml or less. The inhibition, where present, operates immediately but appears to be at least partially reversible. At least one compound which is without effect against bacterial RNA polymerase is a potent inhibitor of the viral RNA polymerase. As the enzyme activity of rifampin-resistant mutants of vaccinia virus is inhibited to the same extent as that of the wild type, the observed in vitro effect on vaccinia virus RNA polymerase is not identical with the in vivo effect specifically directed against a vaccinia-specified protein.     

Mechanism of Action of the Fungicide Thiabendazole, 2-(4′-Thiazolyl) Benzimidazole

Thiabendazole, 2-(4'-thiazolyl) benzimidazole (TBZ) inhibited the growth of Penicillium atrovenetum at 8 to 10 mug/ml. Oxygen consumption with exogenous glucose was inhibited at 20 mug/ml, but endogenous respiration required more than 100 mug/ml. TBZ inhibited completely the following systems of isolated heart or fungus mitochondria: reduced nicotinamide adenine dinucleotide oxidase, succinic oxidase, reduced nicotinamide adenine dinucleotide-cytochrome c reductase, and succinic-cytochrome c reductase at concentrations of 10, 167, 10, and 0.5 mug/ml, respectively. Cytochrome c oxidase was not inhibited. Antimycin A and sodium azide caused the usual inhibition patterns for both fungus and heart terminal electron transport systems. In the presence of antimycin, the fungicide inhibited completely succinate-dichloro-phenolindophenol reductase and succinate-2, 2-di-p-nitrophenyl-(3, 3-dimethoxy-4, 4-biphenylene-5, 5-diphenylditetrazolium)-reductase at 2 and 4 mug of TBZ per ml, respectively. Coenzyme Q reductase required 15 mug/ml. TBZ reduced the uptake by P. atrovenetum of glucose and amino acids and decreased the synthesis of various cell components. At 120 mug/ml, the incorporation of labeled carbon from amino acids-U-(14)C was decreased: lipid, 73%; nucleic acids, 80%; protein, 80%; and a residual fraction, 89%. TBZ did not inhibit peptide synthesis in a cell-free protein-synthesizing system from Rhizoctonia solani. Probably the primary site of inhibition is the terminal electron transport system and other effects are secondary.     

Synthesis of ribonucleic acid by isolated rat liver mitochondria

Rat liver mitochondria isolated in sucrose-N-tris(hydroxymethyl)methyl-2-aminoethane-sulphonic acid (TES) incorporated [(3)H]UTP into RNA for 1h. Incorporation was inhibited 50% by 1mug of actinomycin D/ml, 1mug of acriflavine/ml and 0.5mug of ethidium bromide/ml but was insensitive to rifampicin, rifamycin SV, streptovarcin and deoxyribonuclease. After the first 10min of incubation, the synthesis was insensitive to ribonuclease. RNA synthesis by mitochondria isolated in sucrose-EDTA was insensitive to actinomycin D and sensitive to ribonuclease during the first 10min of the incubation but thereafter the sensitivities were the same as for mitochondria isolated in sucrose-TES. In a hypo-osmotic medium the relative extent of incorporation of the four ribonucleoside triphosphates into RNA was CTP>UTP=ATP>GTP. In an iso-osmotic medium the incorporation of CTP and GTP decreased. All four nucleotides were incorporated into RNA in a DNA-dependent process, as indicated by the inhibition by actinomycin D. In addition, CTP and ATP were incorporated into the CCA end of mitochondrial tRNA. ATP was also incorporated into an unidentified acid-insoluble compound, which hydrolysed in alkali to a product that was not ATP, ADP or 5'- or 2(3')-AMP. Atractyloside inhibited the incorporation of ATP into RNA with 50% inhibition at 2-3nmol/mg of protein. The [(3)H]UTP-labelled RNA had peaks of 16S and 13S characteristic of mitochondrial rRNA. In addition a peak at 20-21S was observed as well as heterogeneous RNA sedimenting throughout the gradient. The synthesis of all these species was inhibited by actinomycin D, indicating that rat liver mitochondrial DNA codes for mitochondrial rRNA as well as other as yet unidentified species.     

Physiological and biochemical effects of the mycotoxin patulin on Chang liver cell cultures.

Patulin exhibits both cytotoxic and cytopathic effects on cultured Chang liver cells. The LD50 found was 1.85 mug per ml of patulin. Effects on growth were observed with as little as 0.1 mug per ml of patulin; a 50% reduction in growth was observed at 0.38 mug per ml of patulin. Using a challenge dose of 2.5 mug per ml of patulin, the cytotoxic effect was reversible after an exposure of 10 min, but was not reversible after 20 min. Protein synthesis was depressed after 60 min and RNA synthesis after 20 min of contact with patulin. Neither protein nor RNA synthesis was completely inhibited after 260 min.     

Effect of cerulenin on the growth and differentiation of Dictyostelium discoideum.

The growth of Dictyostelium discoideum Ax-2 was inhibited completely by cerulenin at a concentration of 5 mug/ml. This inhibition of growth was found to be due to the inhibition of fatty acid synthesis. Acetate incorporation into a long-chain fatty acid was inhibited completely by cerulenin, and the growth inhibition could be reversed by inclusion of certain saturated fatty acids in the medium. Unsaturated fatty acids and sterols failed to reverse the inhibitory effect. The fatty acid and sterol compositions of cerulenin-treated cells were determined to establish whether the drug could be used to manipulate the organism's lipid composition. Only relatively small manipulations were obtained under the conditions employed in this study. Cerulenin inhibited differentiation but only at high concentrations (150 mug/ml). This inhibition could be reversed by palmitic acid, suggesting that the prime cause of the inhibition was an inhibition of fatty acid synthesis. Thus, it appears that continued fatty acid synthesis is required for the cellular process of differentiation in D. discoideum.     

DNA-directed RNA polymerase from HeLa cells. Isolation, characterization and cell-cycle distribution of three enzymes.

DNA-directed RNA polymerase was solubilized from total HeLa cells. Three distinct classes of the enzyme could be clearly differentiated by their sensitivity toward alpha-amanitin. While form A is completely resistant to high concentrations (133 mug/ml) of this toxin, enzyme B is highly sensitive and is completely inhibited by concentrations of 0.1 mug/ml. In contrast, RNA polymerase C shows an intermediate behaviour (50% inhibition at 30% mug/ml). Separation of the three individual enzymes was achieved by chromatography on DEAE-cellulose (to separate enzyme B from A and C) and DEAE-Sephadex (to separate polymerase A from C). All three RNA polymerases were subsequently purified by phosphocellulose chromatography followed by sedimentation through glycerol gradients. Analysis of the purified enzymes by gel electrophoresis under denaturating conditions showed that the A enzyme consists of five subunits with molecular weights of 185, 128, 65, 41 and 32 X 10(3). In contrast, polymerase B is composed of seven subunits in variable stoichiometry with molecular weights of 215, 175, 145, 123, 68, 43 and 31 X 10(3) respectively. The subunit structure of enzyme C is not entirely clear at present and remains to be established. In addition, RNA polymerase activities were solubilized from mitotic and middle-S phase cells in comparison to controls. With respect to amounts and/or activities of all three RNA polymerases A,B and C no significant differences were detectable between logarithmically growing, mitotic and middle-S phase cells.     

Transcription of specific genes in isolated nuclei from HeLa cells in vitro.

Isolated HeLa cell nuclei were employed to catalyze the synthesis of RNA in vitro. In the presence of low concentrations of alpha-amanitin (1 mug/ml), used to suppress the formation heterogeneous nRNA, these nuclei synthesize RNA very efficiently for extended periods of time (at least 60 min) at an elongation rate of about seven nucleotides per second. The product, analyzed on sucrose density gradients and polyacrylamide gels was found to exist of two predominant size classes. Synthesis of the 45-S ribosomal precursor was completely resistant even to high concentrations of alpha-amanitin (150 mug/ml) and hence was catalyzed by enzyme A (or I). A limited degree of processing of the 45-S precursor occurred in vitro. In addition, a second RNA class of low molecular weight (4-8 S) was synthesized by HeLa cell nuclei in the presence of 1 mug/ml alpha-amanitin in vitro. Analysis on 8% polyacrylamide gels resolved the RNA into four distinct components. Their synthesis was resistant to low (1 mug/ml) but clearly sensitive to high (150 mug/ml) concentrations of alpha-amanitin. Consequently the synthesis of all these small-molecular-weight RNA species is catalyzed by RNA polymerase C (or III). For the assessment of the initiation frequency of the individual classes of RNA, a new technique was developed independent of labelling the 5' end of the RNA molecule with the gamma-phosphate of the initiating nucleotide. It employs the double labelling of an RNA molecule with two different isotopes added sequentially at different stages of completion of the chain. From the incorporation ratio of the two isotopes into a particular class of RNA, conclusions can be drawn concerning their initiation frequency. The results obtained have shown a high reinitiation frequency for the small-molecular-weight RNA species at all stages of the incubation reaction. In contrast, reinitiation of the 45-S precursor RNA occurs only to a limited extent in isolated HeLa cell nuclei in vitro.     

Use of Miracil D to Suppress Bacterial Ribonucleic Acid and Protein Synthesis During Bacteriophage MS2 Infection

Under certain culture conditions, Miracil (35 mug/ml) halts the growth of uninfected Escherichia coli. Cellular ribonucleic acid (RNA) synthesis is almost completely suppressed, whereas deoxyribonucleic acid and protein synthesis are inhibited to a lesser extent. When the drug is added to host bacteria prior to infection with bacteriophage MS2, the phage adsorb to the cells, but penetration of the viral RNA is inhibited. Penetration may be achieved without further viral development by infection in the presence of chloramphenicol. If the bacteria are infected with MS2 in the presence of chloramphenicol, subsequently washed to remove the chloramphenicol, and then treated with Miracil at any time between 0 and 20 min postinfection, a second viral function is inhibited and the yield of progeny phage is reduced. Addition of the drug after 20 min postinfection does not inhibit the infection process. When Miracil is present from early times in infection, only a limited synthesis of both double- and single-stranded virus-specific RNA is observed. The viral RNA species thus produced do not appear to differ from those made in the absence of the drug. A comparison of the activities of the viral RNA synthetase produced during the course of infection in the presence and in the absence of Miracil suggests that a possible cause of the inhibition is the synthesis of an unstable enzyme in the presence of the drug.