Effect of Actinomycin D on Virus-induced Ribonucleic Acid Polymerase Formation in Foot-and-Mouth Disease Virus-Infected Baby Hamster Kidney Cells

Actinomycin D, at a concentration that inhibits cellular ribonucleic acid (RNA) synthesis, inhibited the production of foot-and-mouth disease virus-induced RNA polymerase in baby hamster kidney cells. Inhibition was proportional to exposure time and reached 85% when actinomycin D was added 90 min before infection. Polymerase production was inhibited to the same extent in growth and minimal media, and the kinetics of its appearance were slightly different than in untreated cells. Enzyme preparations from actinomycin-treated cells having one-third to one-tenth the activity of untreated samples gave products with RNA profiles similar to those of controls. The 37S viral peak, 20S ribonuclease-resistant peak, and 26 to 28S peaks were present in all cases. Actinomycin D did not consistently inhibit virus production in either medium. Insulin did not prevent the actinomycin induced inhibition of polymerase and virus production from occurring.     

Inhibition of two-dimensional growth and suppression of ribonucleic acid and protein synthesis in the gametophytes of the fern, Asplenium nidus, by chloramphenicol, puromycin and actinomycin D

Chloramphenicol and puromycin at appropriate concentrations inhibited the induction of two-dimensional growth in the gametophytes of the fern Asplenium nidus without drastically inhibiting germination and continued filamentous growth. Similar responses to actinomycin D were reported earlier. Radioautographic techniques were employed to study the pattern of ribonucleic acid and protein synthesis in gametophytes which were treated with chloramphenicol, puromycin and actinomycin D. Uptake of H³-uridine into ribonucleic acid was strongly inhibited by all three antibiotics. Chloramphenicol and puromycin were not as effective as actinomycin D in inhibiting H³-leucine incorporation. The results are discussed in relation to the quality of light and antibiotics on two-dimensional growth in the gametophytes.     

Macromolecular synthesis, differentiation and cell division in Tetrahymena pyriformis, mating type I variety 1

In Tetrahymena pyriformis, mating type I, variety 1, cycloheximide rapidly and completely inhibited incorporation of ¹⁴C-L-leucine into protein. Actinomycin D (25 μg per ml) inhibited incorporation of ¹⁴C-uracil into cold-TCA-insoluble material, after a 5–10 minute lag. Frequently a subsequent decline in the amount of radioactivity was observed. Protein synthesis continued in actinomycintreated cultures for a variable time after cessation of RNA synthesis. Oral development was affected by cycloheximide virtually immediately, and by actinomycin D after a 10–15 minute lag. Cells affected by either drug before the onset of oral membranelle formation were permanently arrested in the stomatogenic field phase. Cells affected in the early and middle stages of membranelle formation completed development of membranelles, but did not invariably complete cell division. Cycloheximide, when added at the beginning of membranelle formation, brought about arrest or resorption of membranelles after they were completed. Actinomycin did not elicit resorption, but sometimes brought about blockage during cell division. Cells affected by either drug after membranelles were fully formed (and cell division was just beginning) completed oral development, nuclear divisions, and cell division. These results suggest that concurrent RNA and protein synthesis are essential for the initiation but not for the completion of membranelle differentiation. The results also suggest that a specific messenger RNA(s) with a very short half-life is required for the synthesis of proteins involved in the initiation of membranelle differentiation.     

Ribonucleic Acid Synthesis During the Differentiation of Sporangia in the Water Mold Achlya

The role of ribonucleic acid (RNA) synthesis in the development of sporangia in the saprolegniaceous mold Achlya was studied. Methods were developed for growing and treating large populations of mycelia so that the hyphal tips would differentiate into sporangia with considerable synchrony. Under the starvation conditions imposed for the differentiation of sporangia, net RNA, deoxyribonucleic acid (DNA), and protein synthesis ceased. However, incorporation of radioactive precursors into RNA continued at a high rate throughout the period of differentiation, showing that the enzymatic mechanism for RNA synthesis was still in an active state. Actinomycin D inhibited the differentiation of sporangia and the incorporation of labeled precursors into RNA. The level of actinomycin used did not inhibit the normal outgrowth and branching of the mycelia that occurred during differentiation. Thus, DNA-dependent RNA synthesis was required for the differentiation of sporangia. Sucrose gradient analysis of newly synthesized RNA showed that only the ribosomal and soluble fractions of RNA were labeled during vegetative growth. During the differentiation of sporangia, ribosomal and soluble RNA fractions were also labeled, and, in addition, a heterodisperse fraction of labeled RNA which was heavier than ribosomal RNA appeared; this fraction was not evident in the newly synthesized RNA from vegetative mycelia.     

Effect of Actinomycin D on the Transfer of Ribonucleic Acid from Nucleus to Cytoplasm in Lactobacillus acidophilus

After starvation for deoxyribosides, the deoxyribonucleic acid (DNA) of Lactobacillus acidophilus is restricted to a localized region of the cell. (3)H-uracil is first incorporated into such a restricted region but subsequently is found throughout the cell. This spread occurs despite the absence of protein synthesis and a major reduction in the rate of ribonucleic acid (RNA) synthesis. However, blocking RNA synthesis with actinomycin D restricts incorporation to a localized region of the cell. It is concluded that uracil is first incorporated into RNA in the bacterial nucleus from which it subsequently spreads through the cell. Actinomycin D could prevent this spread by preventing the completion of RNA molecules, which therefore do not dissociate from the DNA template.     

Dependence of Wound-induced Respiration in Potato Slices on the Time-restricted Actinomycin-sensitive Biosynthesis of Phospholipid

Actinomycin D prevents the full development in a 24-hour period of both wound respiration and cyanide resistance only when given in the first 10 to 12 hours following the cutting of potato tuber (Solanum tuberosum var. Russet) slices. The capacity for choline incorporation into phosphatidylcholine increases with slice aging and is inhibited by actinomycin D in the same time-restricted way. The time-restricted effectiveness of actinomycin D applies to the cutting-elicited enhanced synthesis of three critical enzymes of phosphatidylcholine synthesis, namely phosphorylcholine-glyceride transferase, phosphorylcholine-cytidyl transferase, and phosphatidylphosphatase. By contrast, actinomycim D given at any time is without effect on the measurable levels after 24 hours of a selection of glycolytic and mitochondrial respiratory enzymes. Neither succinic dehydrogenase nor cytochrome oxidase activity increases with time in aging potato slices in the presence or absence of chloramphenicol. The foregoing observations emphasize the central role of phospholipid, and ultimately membrane biosynthesis, in the development of wound-induced respiration.     

Differential inhibitory effects of actinomycin D among strains of poliovirus

Schaffer, Frederick L. (University of California, Berkeley), and Marjorie Gordon. Differential inhibitory effects of actinomycin D among strains of poliovirus. J. Bacteriol. 91:2309-2316. 1966.-Actinomycin D exerted a differential effect on the ability of strains of poliovirus to replicate in HeLa cells. LSc-2ab was studied as an example of a strain markedly inhibited by actinomycin; MEF(1) served as a control strain with minimal inhibition. The effect was noted at an actinomycin concentration of 0.1 mug/ml, but 2.5 mug/ml was used for most studies. Variability in the effect was attributed, in part, to physiological factors. Actinomycin was effective when present during the first 2 hr of LSc infection, but had little effect if present at later times. It did not block adsorption or initiation of ecilpse. It did block synthesis of ribonucleic acid in LSc-infected cells. Several possible modes of action are discussed, the most attractive being that actinomycin blocks synthesis of some cell component, the concentration of which is more critical for replication of some poliovirus strains than others.     

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.     

Requirement of RNA for the Auxin-induced Elongation of Oat Coleoptile

Using etiolated oat coleoptile segments the following results were obtained. Actinomycin D pretreatment for one hour produced about 50 per cent inhibition of RNA synthesis (labeled uracil incorporation), but the elongation caused by IAA was not inhibited in the following 5 hours at least. Actinomycin D pretreatment for three hours produced about 75 per cent inhibition of RNA synthesis and almost complete inhibition of subsequent IAA-induced elongation, which is accompanied by the inhibition of IAA-induced increase in cell wall extensibility. The inhibiting effect of actinomycin D seemed to be reduced when IAA was given within a certain period.     

Cytokinin-Mediated Translational Control of Protein Synthesis in Cultured Cells of Glycine max

Polyribosome formation was stimulated by cytokinin treatmentof cultured cells of Glycine max cv. Funk Delicious. When suspensioncultures were given 0·5 µM zeatin after 24 h inculture in medium lacking a cytokinin, a nearly 2-fold increasein the polyribosome/monoribosome ratio occurred over the subsequent3 h. The effect of actinomycin D and of 5-fluorouridine on RNAsynthesis and on the polyribosome/monoribosome ratios of thesecells was examined. Actinomycin D at 5 and 20 µg/ml^–1inhibitedtotal RNA synthesis by 39 and 60%, respectively, as measuredby [³H]uridine incorporation into acid-precipitable material.The degree of inhibition of precursor incorporation into polyribosomalRNA was similar. At 0·1 mM, 5-fluorouridine inhibited[³H]uridine incorporation by 76%, and [³H]guanosine incorporationby 66% into polyribosomal RNA after 3 h of treatment. Fractionationof the polyribosomal RNA by oligo(dT)-cellulose chromatographydemonstrated that low concentrations of both actinomycin D (5µg ml^–1) and 5-fluorouridine (0·1 mM) inhibitedthe synthesis of ribosomal RNA to a greater extent than thepoly(A)-containing fraction of the messenger RNA. Synthesisof the poly(A)-containing RNA was inhibited by 24% with 5µgml^–1 actinomycin D and by 30% with 0·1 mM 5-fluorouridine.At the above concentrations, these two inhibitors reduced thepolyribosome/monoribosome ratio of the cytokinin-deprived cellsover a 3 h period, but they did not prevent cytokinin-inducedpolyribosome formation. These results provide further evidencethat cytokinin regulates polyribosome levels through an effecton protein synthesis at the translational level     

Differential effects of actinomycin d and cordycepin in lettuce seed germination and RNA synthesis

Intact lettuce seed germination was inhibited by cordycepin but not by actinomycin D; however, when seeds were clipped at the cotyledonary end, actinomycin D partially inhibited germination. Uptake studies with intact seeds using ³H-actinomycin D showed that it was unable to reach the embryo prior to radical protrusion. ³H-Cordycepin uptake studies using intact seeds showed that cordycepin was able to reach the embryo during the first 3 hours of incubation and at subsequent times. The pericarp and endosperm offered resistance to penetration of cordycepin into the embryo. In contrast to actinomycin D, cordycepin markedly inhibited ³H-uridine incorporation into RNA of intact seeds during the first 10 and 12 hours of incubation. About 60% of ³H-adenosine incorporation into poly A-RNA was inhibited by cordycepin during 12 hours of incubation, whereas actinomycin D had little effect. RNA synthesis appears to be essential for seed germination.     

Effects of medium composition and metabolic inhibitors on glycosaminoglycan synthesis in chick embryo cartilage and its stimulation by serum and triiodothyronine

Incorporation of inorganic sulfate into glycosaminoglycans of chick embryo sternum is stimulated by serum and triiodothyronine. Variations in the amino acid content of the medium, and in particular in the concentration of glutamine, changed the incorporation in control and stimulated sterna to the same degree. Omission of Na⁺ from the medium greatly reduced incorporation in both control and stimulated sterna; incorporation, and its stimulation by triiodothyronine, was restored by raising the concentration of Na⁺. Ouabain and valinomycin inhibited incorporation more than 90%, and triiodothyronine did not stimulate under these conditions. Puromycin and cycloheximide also inhibited incorporation almost completely, and abolished the stimulation by triiodothyronine and serum. Addition of p-nitrophenyl-β-xyloside, in the presence of puromycin or cycloheximide, restored sulfation to a level of 5–10% of the control value; however, this level of incorporation was not increased by addition of serum or triiodothyronine.Actinomycin D, colchicine and vinblastine inhibited incorporation by 40% or less at the highest concentrations tested; however, these three agents completely abolished the ability of triiodothyronine to stimulate incorporation. Lumicolchicine and cytochalasin B decreased incorporation in controls slightly, but did not affect the stimulation by serum or triiodothyronine.The results indicate that thyroid hormones stimulate glycosaminoglycan synthesis only under conditions which support efficient synthesis in control incubations, and suggest that microtubule formation may be essential to the mode of action of thyroid hormones in this system.     

Ribonucleic acid and protein synthesis in Rhizophlyctis rosea zoospores

LéJohn, Herbert B. (Purdue University, Lafayette, Ind.), and James S. Lovett. Ribonucleic acid and protein synthesis in Rhizophlyctis rosea zoospores. J. Bacteriol. 91:709-717. 1966.-The uniflagellate zoospores of Rhizophlyctis rosea display active motility and a high endogenous respiratory metabolism, but neither growth nor net ribonucleic acid (RNA) or protein synthesis can be measured by ordinary procedures. Nevertheless, synthesis can be detected with isotopic precursors. Uracil-C(14) is incorporated slowly into both the soluble and ribosomal RNA. Analysis of zoospore extracts (on diethylaminoethyl cellulose columns or sucrose gradients) after various periods of labeling suggested that most of the uracil incorporation represents slow synthesis of ribosomal precursor RNA and, ultimately, ribosomes. Actinomycin D caused an 80% inhibition of uracil incorporation. The most rapidly labeled RNA was susceptible to extensive degradation in cells treated with actinomycin, but the percentage of stable RNA increased with the time of incorporation before addition of the antibiotic. Neither the effects of actinomycin nor the results of chase experiments have established unequivocally the existence of turnover or the presence of a short-lived "messenger" fraction in motile spores. Both leucine and methionine were slowly incorporated into a spectrum of cellular proteins. The methyl group of C(14)-methylmethionine also served as a methyl donor for the methylation of soluble RNA but not of ribosomal RNA. The observations that some of the newly synthesized RNA and protein occur in the intact 82S ribosomes and that actinomycin inhibits the low level of protein synthesis provide some indirect evidence for a very low rate of "messenger" synthesis and turnover in zoospores.     

Changes in the pattern of protein synthesis induced by 3-indolylacetic Acid

Experiments have been performed to investigate whether indoleacetic acid changes the balance between the rates of synthesis of different kinds of proteins. Sub-apical sections of etiolated peas were incubated with ¹⁴C- or ³H-labeled amino acid, and combined to give dual-labeled tissue. Cell fractions were prepared by differential centrifugation, and the dual-labeled protein of each fraction analyzed by gel-filtration. When 2 × 10⁻⁵ m indoleacetic acid was included with ¹⁴C-labeled amino acid, but not with the ³H-labeled amino acid, pronounced changes occurred in the pattern of incorporation of the ¹⁴C label into protein. These changes were greatest in the proteins of the particulate fraction which included nuclear material. Although the pattern of incorporation of lysine was shown to be different from that of leucine, the changes induced by indoleacetic acid were quantitatively similar whichever amino acid was used as a precursor. Dual-labeled protein was further fractionated using column chromatography on DEAE-cellulose. The results suggested that the effect of indoleacetic acid may not be completely general, and that the pattern of synthesis of many proteins may be unaltered by indoleacetic acid. When tissue was preincubated with 10 μg/ml actinomycin D for 30 minutes, incorporation of amino acid into protein was reduced but not abolished. Actinomycin D did, however, prevent the changes in the pattern of protein synthesis which were induced by indoleacetic acid.     

Deoxyribonucleic Acid Degradation in Bacillus subtilis During Exposure to Actinomycin D

At high concentrations (10 mug/ml), actinomycin D inhibited deoxyribonucleic acid (DNA) synthesis in Bacillus subtilis. Inhibition occurred quickly (in less than 1 min) and was complete. In strain 23 thy his, inhibition of DNA synthesis by actinomycin D was followed by partial degradation of one of the two daughter strands to acid-soluble products. Degradation began at the replication point and proceeded over a distance equal to about 12% of a chromosome in length. Actinomycin D played some essential part in degradation, since exposure of the cells to other treatments or agents which inhibit growth did not lead to the above result.     

ACTINOMYCIN D-INDUCED CHANGES IN GROWTH AND RIBONUCLEIC ACID METABOLISM IN THE GAMETOPHYTES OF BRACKEN FERN

Actinomycin D affected the morphological type of growth in the gametophytes of Pteridium aquilinum and the distribution of RNA and protein in their particulate fractions. Increasing concentrations of the drug progressively inhibited two-dimensional growth at the end of a period during which controls had formed typical two-dimensional plants. RNA was lost maximally from the nuclei-rich and ribosome-rich fractions of plants growing in a concentration of actinomycin D which inhibited two-dimensional growth. The magnitude of changes in protein content of the plants was less striking. Presence of actinomycin D in the medium also suppressed incorporation of uridine-H³ into cytoplasmic fractions of gametophytes. The possibility that two-dimensional growth in the gametophytes is under control of a newly synthesized messenger RNA, which is sensitive to actinomycin D, is discussed.     

The participation of the embryonic genome during early cleavage in the rabbit

Actinomycin D and the mushroom toxin α-amanitin similarly inhibit ribonucleic acid synthesis in the rabbit zygote. Actinomycin D also causes an immediate arrest of cleavage, whereas α-amanitin allows limited further development. The decreasing rate of amino acid incorporation caused by continuous exposure of cleaving rabbit embryos to α-amanitin suggests that a relatively homogeneous embryonic RNA is involved in the support of early protein synthesis and is turning over with a half-life of approximately 24 hr, or three cell generation times.     

Phospholipid Synthesis in Sindbis Virus-Infected Cells

We investigated the metabolic requirements for the decrease in phospholipid synthesis previously observed by Pfefferkorn and Hunter in primary cultures of chick embryo fibroblasts infected with Sindbis virus. The incorporation of (32)PO(4) into all classes of phospholipids was found to decline at the same rate and to the same extent; thus, incorporation of (14)C-choline into acid-precipitable form provided a convenient measure of phospholipid synthesis that was used in subsequent experiments. Experiments with temperature-sensitive mutants suggested that some viral ribonucleic acid (RNA) synthesis was essential for the inhibition of choline incorporation, but that functional viral structural proteins were not required. The reduction in phospholipid synthesis was probably a secondary effect of infection resulting from viral inhibition of the cellular RNA and protein synthesis. All three inhibitory effects required about the same amount of viral RNA synthesis; the inhibition of host RNA and protein synthesis began sooner than the decline in phospholipid synthesis; and both actinomycin D and cycloheximide inhibited (14)C-choline incorporation in uninfected cells. In contrast, incorporation of (14)C-choline into BHK-21 cells was not decreased by 10 hr of exposure to actinomycin D and declined only slowly after cycloheximide treatment. Growth of Sindbis virus in BHK cells did not cause the marked stimulation of phospholipid synthesis seen in picornavirus infections of other mammalian cells; however, inhibition was seen only late in infection.