The effect of cycloheximide upon polyribosome stability in two yeast mutants defective respectively in the initiation of polypeptide chains and in messenger RNA synthesis

Summary The effect of cycloheximide upon protein synthesis, RNA metabolism, and polyribosome stability was investigated in the parent and in two temperature-sensitive mutant yeast strains defective respectively in the initiation of polypeptide chains and in messenger RNA synthesis. Cycloheximide at high concentrations (100 g/ml) severely inhibits but does not completely stop protein synthesis (Fig. 1); the incorporation of ¹⁴C-amino acids into polyribosome-associated nascent polypeptide chains continues at a slow but measurable rate (Figs. 2 and 3). Polyribosome structures are stable in the parent strain at 36° whether or not cycloheximide is present (Fig. 5). However, in Mutant ts^- 136, a mutant defective in messenger as well as in stable RNA production, polyribosomes decay at the restrictive temperature (36° C) at the same rate whether or not cycloheximide is present (Fig. 5). Thus the maintenance of polyribosome structures is dependent upon the continued synthesis of messenger RNA even under conditions of extremely slow polypeptide chain elongation. In mutant ts^- 187, a mutant defective in the initiation of polypeptide chains, all of the polyribosomes decay to monoribosomes within 2 minutes after a shift to the restrictive temperature; cycloheximide completely prevents this decay demonstrating that this mutant is capable of continued messenger RNA synthesis at 36° C. Consistent with these observations is the fact that a newly synthesized heterogeneously sedimenting RNA fraction continues to enter polyribosomes in the presence of cycloheximide whereas the entrance of newly synthesized ribosomal RNA is severely inhibited (Figs. 7, 8, 9). The decay or lack of decay of polyribosomes at the restrictive temperature is, therefore, a rapid and discriminating test for the analysis of mutants defective in macromolecule synthesis. Mutants which exhibit a decay of polyribosomes in the presence of cycloheximide are likely to be defective directly or indirectly in the synthesis of messenger RNA whereas mutants in which decay is prevented or slowed by cycloheximide are likely to be defective in some factor required for the association of ribosomes and messenger RNA.     

The effect of thioacetamide on the maturation of high-molecular-weight ribonucleic acid in tumour cells

1. Although thioacetamide treatment of Krebs II ascites-tumour cells did not markedly affect the rate of RNA synthesis in vivo, it caused the formation of an unusual single-stranded RNA component sedimenting at approx. 26s. 2. The maturation process leading to the formation of methylated RNA was examined by following the kinetics of incorporation into RNA of radioactivity from [G-(3)H]uridine and l-[Me-(14)C]methionine. In treated and untreated tumour cells extensive methylation was observed, not only of the ribosomal RNA species, but also of their precursors, especially the precursor species sedimenting at 35s. 3. Evidence is also presented to suggest that methylation of low-molecular-weight RNA species occurs both in the nucleus and in the cytoplasm of these tumour cells. 4. Thioacetamide did not appear to have an effect on RNA methylation in vivo, and in thioacetamide-treated cells the 26s RNA accumulated within the nucleus, where it was methylated. 5. It is postulated that the 26s RNA is most likely to arise as a result of a fault in the scission process that gives rise to the ribosomal RNA components from their high-molecular-weight precursors.     

Effect of cycloheximide on protein and ribonucleic acid synthesis in cultured human lymphocytes

1. Phytohaemagglutinin stimulates the transformation into blast cells of human lymphocytes incubated in vitro. This transformation is accompanied by an increase in the incorporation of [(14)C]leucine into protein and [(3)H]uridine into RNA. 2. The incorporation of [(14)C]leucine by cultures grown in the presence or absence of phytohaemagglutinin is inhibited to the same extent by cycloheximide, a known inhibitor of protein synthesis. 3. Lymphocytes grown without phytohaemagglutin synthesize mainly non-ribosomal RNA. [(3)H]Uridine incorporation by these cells was increased by cycloheximide. 4. Lymphocytes incubated with phytohaemagglutinin begin to synthesize substantial quantities of ribosomal RNA. Under these conditions [(3)H]uridine incorporation was partially inhibited by cycloheximide. This inhibition is shown to be largely a result of inhibition of the synthesis of ribosomal RNA.     

Effect of 5-fluorouracil on the synthesis and stability of ribosomal RNA in Saccharomyces carlsbergensis

A study has been made of the effects of 5-fluorouracil on the synthesis and stability of ribosomal RNA in yeast. The analog causes ribosomal precursor RNA to accumulate. Mature ribosomal RNA species synthesized in the presence of 5-fluorouracil are unstable and are degraded. Pulse chase experiments showed that the 26 S ribosomal RNA is more rapidly degraded than the 17 S component, explaining our observations that in long term experiments apparently less 26 S than 17 S ribosomal RNA is formed. Possible reasons for the instability of ribosomal RNA containing 5-fluorouracil are discussed.     

Methionine-Dependent Synthesis of Ribosomal Ribonucleic Acid During Sporulation and Vegetative Growth of Saccharomyces cerevisiae

Methionine limitation during growth and sporulation of a methionine-requiring diploid of Saccharomyces cerevisiae causes two significant changes in the normal synthesis of ribonucleic acid (RNA). First, whereas 18S ribosomal RNA is produced, there is no significant accumulation of either 26S ribosomal RNA or 5.8S RNA. The effect of methionine on the accumulation of these RNA species occurs after the formation of a common 35S precursor molecule which is still observed in the absence of methionine. During sporulation, diploid strains of S. cerevisiae produce a stable, virtually unmethylated 20S RNA which has previously been shown to be largely homologous to methylated 18S ribosomal RNA. The appearance of this species is not affected by the presence or absence of methionine from sporulation medium. However, when exponentially growing vegetative cells are starved for methionine, unmethylated 20S RNA is found. The 20S RNA, which had previously been observed only in cells undergoing sporulation, accumulates at the same time as a methylated 18S RNA. These effects on ribosomal RNA synthesis are specific for methionine limitation, and are not observed if protein synthesis is inhibited by cycloheximide or if cells are starved for a carbon source or for another amino acid. The phenomena are not marker specific as analogous results have been obtained for both a methionine-requiring diploid homozygous for met13 and a diploid homozygous for met2. The results demonstrate that methylation of ribosomal RNA or other methionine-dependent events plays a critical role in the recognition and processing of ribosomal precursor RNA to the final mature species.     

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.     

Noncoordinate control of RNA synthesis in eucaryotic cells

Inhibition of protein synthesis in confluent monolayers of chick fibroblasts stimulates selectively the synthesis of 4S RNA, resulting in a net accumulation of 4S RNA in the inhibited cells. Under these conditions, inhibition of ribosomal RNA synthesis and processing occurs, as does a decrease in soluble uridine phosphate concentrations; increased pools of certain amino acids are also apparent. Recovery of cells from inhibition is accompanied by a rapidly increasing rate of protein synthesis that lasts for several hours. The small molecular weight RNA synthesized during inhibition of protein synthesis appears properly methylated, and in the presence of cycloheximide and actinomycin D shows a precursor-product conversion. Radiolabeled RNA synthesized during inhibition of protein synthesis is stable following the recovery of cells from inhibition. Stimulation of uridine incorporation into 4S RNA during arrest of protein synthesis is also demonstrated in high-density cultures of L- and Hep-2 cells, suggesting that this non-coordinate stimulation of 4S RNA may be a general property of eucaryotic cells.     

THE EFFECT OF PUROMYCIN ON INTRANUCLEAR STEPS IN RIBOSOME BIOSYNTHESIS

Inhibition of protein synthesis by puromycin (100 γ/ml) is known to inhibit the synthesis of ribosomes. However, ribosomal precursor RNA (45S) continues to be synthesized, methylated, and processed. Cell fractionation studies revealed that, although the initial processing (45S → 32S + 16S) occurs in the presence of puromycin, the 16S moiety is immediately degraded. No species of ribosomal RNA can be found to have emerged from the nucleolus. The RNA formed in the presence of puromycin is normal as judged by its ability to enter new ribosomal particles after puromycin is removed. This sequence of events is not a result of inhibition of protein synthesis, for cycloheximide, another inhibitor of protein synthesis, either alone or in combination with puromycin allows the completion of new ribosomes.     

Ribonucleic acid synthesis during the early action of thyroid hormones

1. The effect on RNA synthesis in rat liver of thyroidectomy and the administration of thyroid hormone, especially during its physiological latent period, was studied by determining: (a) the activity of DNA-dependent RNA polymerase in isolated nuclei; (b) the rate of synthesis of nuclear and cytoplasmic RNA in vivo; (c) polyribosomal sedimentation profiles; (d) the response of microsomes and ribonucleoprotein particles to polyuridylic acid; (e) the effect of inhibitors of RNA and protein synthesis on the biological activity of hormones. 2. The DNA-dependent RNA-polymerase activity of isolated rat-liver nuclei was lowered by thyroidectomy and stimulated by the administration of tri-iodo-l-thyronine or l-thyroxine (2-25mug./100g. body wt.) to both normal and thyroidectomized rats. In thyroidectomized rats, the activity of the Mg(2+)-activated RNA-polymerase reaction (for which the product is mainly ribosomal type of RNA) was stimulated at 10-12hr. after a single injection of tri-iodothyronine, reaching a peak value of 60-90% stimulation at 45hr. after hormone administration. The Mn(2+)/ammonium sulphate-activated RNA-polymerase reaction (for which the RNA product is more DNA-like) was not affected for 24hr. after hormone administration but stimulated by 30-40% at 45hr. The response of both RNA-polymerase reactions to the hormone in vivo paralleled the physiological response but the enzyme was not stimulated by the addition in vitro of the hormone to isolated nuclei. 3. Within 3-4hr. after tri-iodothyronine administration to thyroidectomized rats, the specific activity of rapidly labelled nuclear RNA, after a 10min. pulse of [6-(14)C]orotic acid, was 30-40% greater than the control values, the stimulation reaching 100 and 200% at 11 and 16hr. respectively after hormone administration. Longer exposures to [6-(14)C]orotic acid and [(32)P]phosphate showed that the hormone accelerated the synthesis of mitochondrial, microsomal (or ribosomal) and soluble RNA. The greater part of the labelled nuclear RNA was of the ribosomal type. The hormone-induced increases in the incorporation of radioactive precursors into RNA were not preceded, but followed, by enhanced uptake of the precursor. There was no change, per g. of liver, of DNA, nuclear RNA or soluble RNA, but there was a 40-60% increase in the amount of ribosomal RNA between 35 and 45hr. after a single injection of tri-iodothyronine to thyroidectomized rats. 4. Coinciding with the increase in ribosomal RNA after hormone administration was an increase in the average size and amount of polyribosomes. The newly formed ribonucleoprotein particles, or messenger RNA attached to them, or both, were more firmly bound to microsomal membranes after hormone treatment. 5. Polyuridylic acid caused a bigger stimulation of incorporation of [(14)C]phenyl-alanine by ribonucleoprotein particles, but not by microsomes, from thyroidectomized rats as compared with preparations from normal animals. The response of ribonucleoprotein particles to polyuridylic acid was lowered after tri-iodothyronine treatment of thyroidectomized rats. 6. Actinomycin D, 5-fluorouracil, puromycin and cycloheximide caused a 70-100% inhibition of the stimulatory effect of l-thyroxine and tri-iodo-l-thyronine on basal metabolic rate and growth rate in both normal and thyroidectomized animals. Administration of actinomycin D also abolished the stimulation of RNA polymerase by tri-iodothyronine. 7. It is concluded that regulation of nuclear and ribosomal RNA synthesis is an essential step leading to the biological action of thyroid hormones and that the formation of new ribosomes is an important aspect of the control of cytoplasmic protein synthesis by these hormones.     

Lomofungin as an inhibitor of nucleic acid synthesis in Saccharomyces cerevisiae

1. The antibiotic lomofungin was found to be a potent inhibitor of both DNA and RNA synthesis in Saccharomyces cerevisiae. Under selected growth conditions inhibition of DNA synthesis by the drug preceded inhibition of RNA synthesis. 2. Although in general lomofungin inhibited synthesis of ribosomal RNA and polydisperse RNA more effectively than that of low-molecular-weight RNA, under certain conditions the drug inhibited almost completely synthesis of both 4S and 5S RNA. 3. Inhibition of both RNA and DNA synthesis may be explained if RNA synthesis is required for DNA synthesis in yeast. Alternatively, lomofungin, in addition to interacting with DNA-dependent RNA polymerase, might interfere with a component(s) of the DNA-synthetic apparatus. The drug may thus prove to be of considerable value in studies of DNA synthesis in eukaryotes.     

Synthesis of acid phosphatase during dehydration of the yeast Saccharomyces cerevisiae

Summary During the dehydration of exponentially growing yeast cells for 24 h at 37° C, a 2–3 fold increase in the activity of acid phosphatase was observed. This increase is inhibited by cycloheximide and 2-deoxy-D-glucose and therefore is indicative of de novo synthesis. The presence of exogenous orthophosphate during drying does not affect the specific activity of this enzyme, thus indicating the constitutive character of the newly formed acid phosphatase.Freeze-etching showed some rearrangement of the plasmalemma structure of yeast cells during dehydration.     

Nucleolar 4s ribonucleic acid in dipteran salivary glands in the presence of inhibitor

1. Salivary glands of insect larvae accumulate newly made transfer RNA in the nucleolus when maintained in the presence of nucleoside antagonists that inhibit RNA synthesis preferentially at the chromosome. 2. The nucleus contains precursor transfer RNA, which, on the basis of the general evidence, may originate in the chromosome and then be methylated in the nucleolus. 3. The maturation of precursor ribosomal RNA is blocked in the nucleolus during inhibition. 4. The transport of nuclear RNA to cytoplasm is also blocked. 5. It is suggested that, if the transfer RNA accumulated in the nucleolus does indeed originate in the chromosome, the accumulation may result from the blockage of an obligatory transient association of the RNA with the nucleolus.     

The effect of ethionine on ribonucleic acid synthesis in rat liver.

1. By 1h after administration of ethionine to the female rat the appearance of newly synthesized 18SrRNA in the cytoplasm is completely inhibited. This is not caused by inhibition of RNA synthesis, for the synthesis of the large ribosomal precursor RNA (45S) and of tRNA continues. Cleavage of 45S RNA to 32S RNA also occurs, but there was no evidence for the accumulation of mature or immature rRNA in the nucleus. 2. The effect of ethionine on the maturation of rRNA was not mimicked by an inhibitor of protein synthesis (cycloheximide) or an inhibitor of polyamine synthesis [methylglyoxal bis(guanylhydrazone)]. 3. Unlike the ethionine-induced inhibition of protein synthesis, this effect was not prevented by concurrent administration of inosine. A similar effect could be induced in HeLa cells by incubation for 1h in a medium lacking methionine. The ATP concentration in these cells was normal. From these two observations it was concluded that the effect of etionine on rRNA maturation is not caused by an ethionine-induced lack of ATP. It is suggested that ethionine, by lowering the hepatic concentration of S-adenosylmethionine, prevents methylation of the ribosomal precursor. The methylation is essential for the correct maturation of the molecule; without methylation complete degradation occurs.     

Characterization of the ribonucleic acid synthesized by isolated rat-liver nuclei

1. Isolated rat-liver nuclei incorporated [¹⁴C]UMP into RNA when incubated in the presence of Mg²⁺ and all four ribonucleoside triphosphates. The addition of bentonite to the system diminished the breakdown of the newly synthesized RNA. 2. AMP and CMP were incorporated in the absence of the other added triphosphates, and in the presence of deoxyribonuclease. 3. RNA synthesized in the presence of Mg²⁺ contained a high proportion of CMP and GMP, and sedimented in the regions of ribosomal RNA and of heavier molecules. About 1% of this RNA hybridized with homologous DNA, and hybrid formation was more effectively inhibited by nuclear RNA than by ribosomal RNA. 4. RNA synthesized in the presence of Mn²⁺ plus ammonium sulphate had a composition intermediate between that of ribosomal RNA and of DNA, and about 4% of this RNA formed hybrids with DNA. 5. Less than 2% of the newly synthesized RNA was capable of forming ribonuclease- and deoxyribonuclease-resistant complexes. 6. It was concluded that the newly synthesized RNA arose as a result of an asymmetric process and included both ribosomal and DNA-like species.     

Macromolecule synthesis in yeast spheroplasts

Conditions have been established for the preparation of spheroplasts of Saccharomyces cerevisiae which are able to increase their net content of protein, ribonucleic acid (RNA), and deoxyribonucleic acid (DNA), several-fold upon incubation in a medium stabilized with 1 m sorbitol. The rate of RNA and protein synthesis in the spheroplasts is nearly the same as that occurring in whole cells incubated under the same conditions; DNA synthesis occurs at about half the whole cell rate. The spheroplasts synthesize transfer RNA and ribosomal RNA. The newly synthesized ribosomal RNA is incorporated into ribosomes and polysomes. The polysomes are the site of protein synthesis in these spheroplasts. Greater than 90% of the total RNA can be solubilized by treatment of the spheroplasts with sodium dodecyl sulfate or sodium deoxycholate. These spheroplast preparations appear to be a useful subject for the study of RNA metabolism in yeast.     

Effect of Purine and Pyrimidine Analogues on Growth and RNA Metabolism in the Soybean Hypocotyl-the Selective Action of 5-Fluorouracil

The effects of several base analogues and cycloheximide on RNA synthesis, protein synthesis, and cell elongation were studied in excised soybean hypocotyl. None of the pyrimidine analogues tested affected growth or protein synthesis; only 5-fluorouracil appreciably inhibited RNA synthesis. 8-Azaguanine and 6-methylpurine markedly inhibited RNA and protein synthesis and cell elongation. Cycloheximide effectively inhibited both cell elongation and protein synthesis.The results show that 5-fluorouracil selectively inhibited ribosomal and soluble RNA synthesis without affecting the synthesis of D-RNA. These results indicate that the requirement for RNA synthesis to support continued protein synthesis and cell elongation is restricted to the synthesis of D-RNA.5-Fluorouracil was incorporated into all classes of RNA in a form believed to be 5-fluorouridylic acid.Cycloheximide markedly inhibited the accumulation of ribosomal RNA, but the results indicate that CH did not inhibit, per se, the synthesis of ribosomal RNA. The accumulation of newly synthesized D-RNA was only slightly affected by cycloheximide. These results show that the inhibition of cell elongation by cycloheximide correlates with the inhibition of protein synthesis, but not with the effect on RNA metabolism.