The effect of actinomycin D on RNA synthesis and nucleolar ultrastructure in the liver of rats treated with fluorouracil

Summary Rats were given cytidine-³H and 10 min later 50 mg fluorouracil. They were killed after 25 hours. Actinomycin D was given at various times before sacrifice. The collapse of the nucleolus and the segregation of its components, seen in rats sacrificed one hour after administration of actinomycin D only, was prevented by prior treatment with fluorouracil. In rats treated with fluorouracil and given actinomycin 12 or 20 hours prior to death, there was a more or less pronounced collapse of the nucleolus but no typical segregation of its components. Radioautographs of livers from untreated rats or rats given actinomycin only at the times mentioned, and killed 25 hours after administration of cytidine-³H, were labelled mainly over the cytoplasm. Radioautographs from rats, treated with fluorouracil only, or fluorouracil plus actinomycin, showed labelling over the nucleoli, but depressed labelling over the cytoplasm. Biochemical analysis of RNA labelling showed high ribosomal peaks in untreated rats and rats treated with actinomycin only. Rats treated with fluorouracil, or fluorouracil plus actinomycin showed no labelling of the 29S and 18S ribosomal peaks. The results indicate that fluorouracil blocks or delays the formation of ribosomal RNA and that the inhibition, at least in part, takes place in the nucleolus.This work was supported by grants from the Swedish Medical Research Council (Project K68-12X-623-04), the Swedish Cancer Society (Project 6831), the Medical Faculty of Uppsala and the Swedish Society for Medical Research.     

The effects of fluorouracil and actinomycin,single and combined,on the nucleolar ultrastructure of various tissues of the rat

Summary 5-fluorouracil and actinomycin D were given, single and combined, to rats. The nucleolar ultrastructure was examined in liver, pancreas, spleen, kidney cortex, adrenal cortex, in epithelial and smooth muscle cells of the small intestine and in the Purkinje cells of the cerebellum. 5-fluorouracil, given alone, provoked changes in all tissues examined. Actinomycin D, given alone, gave changes in all tissues examined, except the cerebellum. Treatment with fluorouracil prior to actinomycin delayed and modified the effect of the latter drug in all tissues examined.This work was supported by grants from the Swedish Medical Research Council (Project K68-12X-623-05) and the Swedish Cancer Society (Project 6831).The results were briefly presented at the XIIth International Congress of Cell Biology, Bruxelles 1968.     

RELATIONSHIP BETWEEN RNA SYNTHESIS, CELL DIVISION, AND MORPHOLOGY OF MAMMALIAN CELLS : I. Puromycin Aminonucleoside As An Inhibitor of RNA Synthesis and Division in HeLa Cells

Logarithmically growing HeLa cell monolayers were treated with a range of concentrations of puromycin aminonucleoside (AMS). The effects of AMS were studied by the following means: microscope examination of treated cells; enumeration of the cell number using an electronic particle counter; analyses for DNA, RNA, and protein content; incorporation of P³² and H³-thymidine into nucleic acids; and fractionation of nucleic acids by column chromatography. Taking the rate of incorporation of the isotopic precursor as a measure of nucleic acid synthesis, it was found that concentrations of the inhibitor which had a rapid effect on the rate of cell division inhibited the synthesis of all types of nucleic acids and of protein, but depressed ribosomal RNA synthesis most markedly. Lower concentrations of AMS selectively inhibited ribosomal RNA and, to a lesser extent, transfer RNA synthesis. Partial inhibition of ribosomal RNA synthesis with low doses had no effect on the rate of cell division within the period studied (3 generation times). The cell content of RNA returned to normal when the inhibitor was removed.     

Ribosomal RNA synthesis in mitochondria of Neurospora crassa

Ribosomal RNA synthesis in Neurospora crassa mitochondria has been investigated by continuous labeling with [5-³H]uracil and pulse-chase experiments. A short-lived 32 S mitochondrial RNA was detected, along with two other short-lived components; one slightly larger than large subunit ribosomal RNA, and the other slightly larger than small subunit ribosomal RNA. The experiments give support to the possibility that 32 S RNA is the precursor of large and small subunit ribosomal RNA's. Both mature ribosomal RNA's compete with 32 S RNA in hybridization to mitochondrial DNA. Quantitative results from such hybridization-competition experiments along with measurements of electrophoretic mobility have been used to construct a molecular size model for synthesis of mitochondrial ribosomal RNA's. The large molecular weight precursor (32 S) of both ribosomal RNA's appears to be 2.4 × 10⁶ daltons in size. Maturation to large subunit RNA (1.28 × 10⁶ daltons) is assumed to involve an intermediate ~1.6 × 10⁶ daltons in size, while cleavage to form small subunit RNA (0.72 × 10⁶ daltons) presumably involves a 0.9 × 10⁶ dalton intermediate. In the maturation process ~22% of the precursor molecule is lost. As is the case for ribosomal RNA's, the mitochondrial precursor RNA has a strikingly low G + C content.     

Cytological,radioautographic and ultrastructural studies on the effect of 5-fluorouracil on rat liver

Summary Young rats were given two doses of 50 mg 5-fluorouracil with a 22-hour interval. One hour after the second dose they were given either cytidine-³H or leucine-³H and killed three hours later. Radioautographs of the livers revealed a decrease in RNA labelling, whereas the protein labelling was essentially uninfluenced. The liver cells exhibited an increase in nucleolar volume. Electron microscopy revealed changes in the ultrastructure of the liver cell nucleoli, while other parts of the cells were essentially unaltered.The investigation was supported by a research grant (project No Y 515) from the Swedish Medical Research Council and Riksföreningen mot Cancer.     

Cell cycle analysis in asynchronous cultures using the BUdR-Hoechst technique.

During synchronized germination of spores of Dictyostelium discoideum, protein synthesis begins almost concomitantly with syntheses of messenger-like RNA (mlRNA) and 4–5S RNA (presumably tRNA) in the swollen spore stage and the initiation of ribosomal RNA (rRNA) synthesis is somewhat delayed. DNA synthesis occurs in the early stages of the amoeba emergence phase. Cycloheximide (200 μg/ml) blocked spore germination as well as total protein synthesis, whereas actinomycin D (60 μg/ml) did not affect either. This concentration of actinomycin D selectively inhibited formation of rRNA but did not influence the synthesis of mlRNA. Examinations of RNA labeled with [¹⁴C]uracil during germination indicated that polysomes initially detectable in the course of the germination process contain ¹⁴C-labeled mlRNA. It was concluded that at least some of mRNA synthesized during germination of D. discoideum spores is involved in protein synthesis required for the germination.     

Effect of the "RNA control" locus in Escherichia coli on RNA bacteriophage R23 replication.

The effect of the rel gene of Escherichia coli on the RNA synthesis induced by phage R23 was studied. This RNA phage has the property of inhibiting ribosomal RNA formation and completely dominating the RNA synthesis of the host. Phage-specific RNA formation was found to be dependent on the allelic state of the rel gene. Determinations of RNA synthesis were made by both cumulative and short-term incorporations of uracil and adenine. Variations in labeling of nucleotide pools were compensated for by determining specific activities of ATP and UTP and using these values to obtain true, relative rates of RNA synthesis.     

Selective inhibition of precursor incorporation into ribosomal RNA in gamma-irradiated Tetrahymena pyriformis.

Sublethal doses of gamma radiation are known to inhibit total RNA synthesis in the ciliate protozoan Tetrahymena. To determine if the synthesis of a particular class of RNA is preferentially inhibited, pulse-labeled RNA was isolated from normal exponentially growing cells, irradiated cells, and cells in which total RNA synthesis had recovered to the pre-irradiation level. The RNAs were analyzed by SDS-polyacrylamide gel electrophoresis and oligo(dT)-cellulose column chromatography. Inhibition of RNA synthesis primarily involves ribosomal RNA. However, radiation does not cause a delay in the processing of precursor rRNA or a preferential loss of either of the mature rRNAs. Following irradiation, poly(A)-containing RNA [poly(A+)RNA] is synthesized at a rate up to three times greater than the control rate. The elevated poly(A+)RNA synthesis occurs during the period of depressed rRNA synthesis and even after rRNA synthesis has recovered to its pre-irradiation rate. While the sizes of the total cellular ribonucleoside triphosphate pools are depressed in the irradiated cells, these pools probably do not represent the actual compartments containing the precursors for RNA synthesis, and the observed changes cannot explain the modifications in macromolecular synthesis in irradiated Tetrahymena.     

The Synthesis of Ribosomes in E. coli: IV. The Synthesis of Ribosomal Protein and the Assembly of Ribosomes

The incorporation of C¹⁴ leucine into the protein moiety of ribosomes has been studied as a sequel to the studies of ribosomal RNA synthesis. In contrast to the latter studies, labeled leucine is incorporated directly into 50S and 30S ribosomes without measurable delay by precursor stages. There is, however, evidence of some transfer of radioactivity from the 43S group of particles to the 50S. The inhibition of protein synthesis by chloramphenicol results in the accumulation of material similar to the eosome—the primary precursor in ribosome synthesis. There is also evidence for the synthesis of some neosome. The results of the studies of ribosomal RNA and protein synthesis are combined into a model of ribosome synthesis. Finally, consideration is made of the significance of these studies of ribosome synthesis for general problems of protein synthesis and information transfer.     

The Synthesis of Ribosomes in E. coli: I. The Incorporation of C14-Uracil into the Metabolic Pool and RNA

C¹⁴-uracil is rapidly incorporated by E. coli at low concentrations. Approximately half the radioactivity passes directly into RNA with very little delay. The remaining half enters a large metabolic pool and later is incorporated into RNA. The total rate of uptake (growing cells) is not greater than the requirement for uracil and cytosine for RNA synthesis. The size of the metabolic pool is not influenced measurably by the external uracil concentration. No evidence is found for the existence of a fraction of RNA which is rapidly synthesized and degraded.     

Cytidine nucleotide biosynthesis and the level of cytochrome P-450 in rat liver microsomes after administration of colchicine.

Colchicine displays a biphasic effect on the biosynthesis of cytidine nucleotides in rat liver; an initial depression is followed by activation of synthesis. Depending on the duration of exposure to colchicine, the changes of the values of the ratio of specific radioactivity of cytosine to uracil in the acid-soluble pool and 3′-CMP to 3′-UMP of cytoplasmic ribosomal RNA are inversely proportional to changes of the levels of cytochrome P-450 in liver microsomes. The utilization of [2-¹⁴C]orotic acid for the biosynthesis of DNA cytosine as affected by the exposure to colchicine reflects the changes in the specific radioactivity of the cytidine components of the acid-soluble pool. However, the maximal radioactivity in DNA thymine is reached under these conditions only at longer time intervals.     

Effect of salt and water stress on RNA synthesis in the excised embryo axis of germinating mung bean (Phaseolus aureus ROXB).

The rate of synthesis of RNA was studied under salt and water stresses created by the addition of four different salts and polyethylene glycol-6000 (PEG-6000) in excised embryo axis of mung bean using^l4C-uracil. Iso-osmotio concentrations of salts and PEG resulted in differential effect on RNA synthesis. PEG inhibited, whereas salt stimulated incorporation of labelled uracil into RNA. Ionic differences on RNA synthesis were very clear as synthesis of RNA was more affected with sulphate than chloride and with sodium than potassium ions.     

Effect of 5-fluorouracil posttreatment on sensitivity to ultraviolet radiation and course of messenger RNA synthesis inEscherichia coli cells

The effect of 5-FU posttreatment and uracil starvation on sensitivity to ultraviolet radiation and on the course of messenger RNA synthesis was studied in the strainsEscherichia coli 15 T-U-his-,Escherichia coli BU- andEscherichia coli Br. 5-FU posttreatment in liquid medium increased the number of surviving cells, while posttreatment in uracil medium either did not affect resistance or lowered it. A correlation was sought between increased resistance and messenger RNA synthesis (or stabilization). Messenger RNA synthesis was studied by means of pulse-labelling with radioactive precursor and the amount of m-RNA was determined by measuring total RNA proteosynthetic activity. It was found that the amount of RNA with messenger activity increased in cells treated with 5-FU, but that it decreased during cultivation in uracil medium. It is assumed that the 5-FU-induced increase in resistance is based on the preferential promotion of repair processes under conditions of a reduced turnover and the consequent stabilization of the m-RNA molecules. The possible mechanism of this promotion is discussed.     

Synthesis of Protein, Ribonucleic Acid, and Ribosomes by Individual Bacterial Cells in Balanced Growth

Relative rates of protein synthesis in individual cells were determined by allowing random populations to incorporate tritiated leucine for very short periods (pulses) and then examining autoradiographs of these cells to assess the amount of incorporation (grains per cell) as a function of cell size. Relative rates of ribonucleic acid (RNA) synthesis were determined in the same way by using tritiated uracil. Unless the uracil pulse was very short (less than 1/20 generation), the RNA labeled during the pulse was predominantly ribosomal. The rate of protein synthesis in individual cells is directly proportional to cell size. The rate of RNA synthesis also increases linearly with size in larger cells, but there appears to be a slight delay in RNA synthesis immediately after cell division. Total cellular content of protein, RNA, and ribosomes is directly proportional to cell size. Thus, we conclude that, in individual cells during the cell cycle (i) the average rate of protein synthesis per ribosome is constant and (ii) the increase in macromolecular mass of the cell is exponential with age.     

RIBOSOME SYNTHESIS IN TETRAHYMENA PYRIFORMIS

The cellular site of synthesis of ribosomal RNA in Tetrahymena pyriformis was studied by analyzing the purified nuclear and cytoplasmic RNA from cells pulse labeled with uridine-³H. The results of studies using zonal centrifugation in sucrose density gradients show that the ribosomal RNA is synthesized in the nucleus as a large precursor molecule sedimenting at 35S. The 35S molecule undergoes rapid transformation through two main nuclear intermediates, sedimenting at about 30S and 26S. The smaller ribosomal RNA (17S) appears first in the cytoplasm and it seems to be absent from the nucleus. The apparent delay in the appearance of the larger ribosomal RNA (26S) in the cytoplasm is due to the presence of a larger pool of its precursors in the nucleus as indicated by pulse-chase experiments. The newly synthesized ribosomal RNA's appear in the cytoplasm as discrete 60S and 45S ribonucleoprotein particles, before their incorporation into the polysomes.     

Biosynthesis of cytidine nucleotides in rat liver after administration of D-galactosamine

Following the administration of D-galactosamine the utilization of [2-¹⁴C]orotic acid for the synthesis of the cytidine components of the acidsoluble extract and liver RNA cytosine is markedly decreased. The depression of the specific activity of the cytidine components takes place after application of low doses of the drug which do not interfere with the specific activity of the uridine components of the acid-soluble extract or of liver RNA uracil. Simultaneously the administration of [U-¹⁴C]cytidine paralleled by its enhanced liver uptake. The total amount of uridine as well as cytidine components of the acid-soluble extract following the administration of D-galactosamine increases; however, the molar ratio of both pyrimidines does not change. The alterations of the cytidine metabolism after the administration of the drug are accompanied by the increased level of microsomal cytochrome P-450.