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.     

Ribosomal RNA on the surfaces of nonleukemic mouse ascites tumor cells

RNAs on the cell surfaces of two nonleukemic and two leukemic strains of mouse ascites tumor cells were studied by fractionating the RNAs released from the cell surface by gentle pronase treatment after sucrose density gradient centrifugation, by indirect membrane immunofluorescence that used anti-RNA antibody and by cell electrophoresis. RNA was extracted from the cell supernatants of Ehrlich ascites tumor and sarcoma 180 cells (nonleukemic) that had been treated or not treated with pronase (1 microgram/ml, 37 degrees C, 20 min) followed by sucrose density gradient centrifugation. It was clearly demonstrated that the amounts of ribosomal RNA (18S and 28S) released after pronase treatment were approximately 80% greater than that of nonpronase-treated cells. Ehrlich ascites tumor cells that had been treated with actinomycin D (100 micrograms/kg body weight of mouse, 16 h) in vivo released an amount of ribosomal RNA after pronase treatment only 20% greater than the value for untreated cells. Actinomycin D treatment greatly reduced both the cell surface negative charge and the cell surface immunofluorescence when rabbit anti-RNA antibody was used. Under the same experimental conditions with actinomycin D, only ribosomal RNA synthesis showed preferential inhibition, not the syntheses of poly A-containing messenger RNA, 4S or other small-size RNAs. In contrast, L1210 and C1498 cells (leukemic) showed no change in the amounts of ribosomal RNA released after pronase treatment. L1210 cells also showed no change in the surface negative charge after being treated with actinomycin D.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of low dose actinomycin D treatment in vivo on the biosynthesis of ribosomal proteins in rat liver

The long-term effects (up to 12 h) of low dose in vivo actinomycin D treatment, which selectively inhibits rRNA synthesis, on the activity of rat liver for the synthesis of ribosomal proteins relative to that for the synthesis of total protein were investigated. The effects of actinomycin D treatment in vivo and in vitro on the template activity of poly(A)-containing mRNA of rat liver for ribosomal proteins were examined by using a wheat germ cell-free system. The following results were obtained. 1. The activity of rat liver for synthesizing total protein observed in vivo and in vitro was inhibited by actinomycin D treatment even at a small dose. 2. A double-labeling technique using [3H] and [14C]leucine in vivo showed that the rate of synthesis of the ribosomal protein fraction relative to that of total protein in actinomycin-treated rat liver (6 + 6 h) was 1.45 times higher than that in the control rat. 3. By using a wheat germ cell-free system, it was shown that the template activity of poly(A)-containing mRNA for the synthesis of total protein was increased slightly by actinomycin D treatment in vivo. Furthermore, the template activity for the ribosomal protein fraction relative to that for total protein was increased. This increase was observed in most of the ribosomal proteins separated on two-dimensional acrylamide gel electrophoresis, although the extents of increase were different among individual ribosomal proteins examined. On the other hand, the selective increase of the template activity for the ribosomal protein fraction was not observed when poly(A)-containing mRNA was incubated with actinomycin D in vitro, although the template activity for total protein was increased slightly.     

Translocation of nucleolar phosphoprotein B23 ( ) induced by selective inhibitors of ribosome synthesis

To elucidate the possible role of nucleolar phosphoprotein B23 in ribosome synthesis, drugs which inhibit the processing of ribosomal RNA were employed. After treatment with actinomycin D, toyocamycin or high doses of α-amanitin, a uniform nucleoplasmic fluorescence was observed. Low doses of α-amanitin and the protein synthesis inhibitor puromycin and cycloheximide had no effect on protein B23 translocation. By ELISA immunoassay, there was a 60% decrease in the amount of protein B23 in the nucleoli of the actinomycin D-treated cells as compared with the control nucleoli. Conversely, the amount of protein B23 in the nucleoplasm (excluding nucleoli) was 3-fold higher in the actinomycin D-treated cells. Preribosomal ribunucleoprotein particles (pre-rRNPs) were extracted from isolated nucleoli of Novikoff hepatoma ascites cells and fractionated on sucrose density gradients. Protein B23 was found co-localized with the pre-rRNPs as determined by ELISA assays which agrees with previous studies. The proteins in these 80 S and 55 S pre-ribosomal ribonucleoprotein particles were fractionated by 10% gel electrophoresis. Immunoblots showed protein B23 was present in both pre-rRNPs.     

Effect of actinomycin D on cell proliferation induced by phytohemagglutinin in human lymphocytes]

Actinomycin D at a dose inhibiting the synthesis of ribosomal RNA (0.15 MUG/ML) BLOCKS THE TRANSITION OF CELLS INTO ACTIVE PROLIFERATION DURING THE FIRST 6 HOURS AFTER PHYTOHAEMAGGLUTININ TREATMENT. The effect of actinomycin D becomes weaker as the cells proceed through the prereplicative period. 1 mug/ml actinomycin D blocks the synthesis of all RNSs. It has been found that actinomycin D at this concentration blocks the transition of cells into the proliferation during the entire prereplicative period.     

Alterations of protein synthesis in arbovirus-infected L cells

Lust, George (Fort Detrick, Frederick, Md.). Alterations of protein synthesis in arbovirus-infected L cells. J. Bacteriol. 91:1612-1617. 1966.-Cellular protein synthesis and ribonucleic acid (RNA) synthesis in mouse L cells were markedly depressed 1 hr after infection with Venezuelan equine encephalomyelitis virus. Host RNA and protein synthesis were inhibited more rapidly by the virus infection than by actinomycin D. In cells infected 4 hr, a cytoplasmic RNA polymerase was demonstrated which was absent in uninfected cells. At this time, deoxyribonucleic acid-directed RNA synthesis catalyzed by the nuclear RNA polymerase was inhibited in vitro in enzyme preparations from nuclei of virus-infected cells. For optimal activity, the cytoplasmic RNA polymerase required the four nucleoside triphosphates, Mg(++), and RNA. The enzyme was insensitive to actinomycin D and deoxyribonuclease, indicating that it catalyzed RNA-directed RNA synthesis. Attempts to purify the induced polymerase further were unsuccessful. Fresh preparations had to be used because the enzymatic activity was unstable.     

Translocation of nucleolar phosphoprotein B23 (37 kDa/pI 5.1) induced by selective inhibitors of ribosome synthesis

To elucidate the possible role of nucleolar phosphoprotein B23 in ribosome synthesis, drugs which inhibit the processing of ribosomal RNA were employed. After treatment with actinomycin D, toyocamycin or high doses of alpha-amanitin, a uniform nucleoplasmic fluorescence was observed. Low doses of alpha-amanitin and the protein synthesis inhibitor puromycin and cycloheximide had no effect on protein B23 translocation. By ELISA immunoassay, there was a 60% decrease in the amount of protein B23 in the nucleoli of the actinomycin D-treated cells as compared with the control nucleoli. Conversely, the amount of protein B23 in the nucleoplasm (excluding nucleoli) was 3-fold higher in the actinomycin D-treated cells. Preribosomal ribonucleoprotein particles (pre-rRNPs) were extracted from isolated nucleoli of Novikoff hepatoma ascites cells and fractionated on sucrose density gradients. Protein B23 was found co-localized with the pre-rRNPs as determined by ELISA assays which agrees with previous studies. The proteins in these 80 S and 55 S pre-ribosomal ribonucleoprotein particles were fractionated by 10% gel electrophoresis. Immunoblots showed protein B23 was present in both pre-rRNPs.     

Inhibition of host protein synthesis by vaccinia virus: fate of cell mRNA and synthesis of small poly (A)-rich polyribonucleotides in the presence of actinomycin D.

Purified vaccinia virus rapidly inhibited HeLa cell protein synthesis in the presence of actinomycin D. Under these conditions host polyribosomes were extensively degraded but the mRNA was stable as indicated by a greater than 90% recovery of prelabeled polyadenylylated RNA. Although actinomycin D prevented the synthesis of host mRNA and poly(A) in uninfected cells, incorporation of adenosine into poly(A) was inhibited by less than 50% in infected cells. Further analysis indicated that there was little or no normal size viral mRNA but that a unique class of small poly(A)-rich RNA was made in the presence of actinomycin D. From measurements of the RNase resistance and base composition of the RNA, approximately 40% of the nucleotide sequence was estimated to be poly(A). The poly(A)-rich RNA was found associated with small polyribosomes and monoribosomes that were inactive in protein synthesis. It was suggested that the poly(A) segment of the RNA is formed by the poly(A) polymerase previously found in vaccinia virus cores and that the inactive RNA, by competing with host mRNA, may contribute to the virus-mediated inhibition of host protein synthesis observed in the presence of actinomycin D.     

REPOPULATION OF THE POSTMITOTIC NUCLEOLUS BY PREFORMED RNA

This study is concerned with the fate of the nucleolar contents, particularly nucleolar RNA, during mitosis Mitotic cells harvested from monolayer cultures of Chinese hamster embryonal cells, KB6 (human) cells, or L929 (mouse) cells were allowed to proceed into interphase in the presence or absence (control) of 0.04–0 08 µg/ml of actinomycin D, a concentration which preferentially inhibits nucleolar (ribosomal) RNA synthesis 3 hr after mitosis, control cells had large, irregularly shaped nucleoli which stained intensely for RNA with azure B and for protein with fast green. In cells which had returned to interphase in the presence of actinomycin D, nucleoli were segregated into two components easily resolvable in the light microscope, and one of these components stained intensely for RNA with azure B. Both nucleolar components stained for protein with fast green In parallel experiments, cultures were incubated with 0.04–0 08 µg/ml actinomycin D for 3 hr before harvesting of mitotic cells, then mitotic cells were washed and allowed to return to interphase in the absence of actinomycin D. 3 hr after mitosis, nuclei of such cells were devoid of large RNA-containing structures, though small, refractile nucleolus-like bodies were observed by phase-contrast microscopy or in material stained for total protein. These experiments indicate that nucleolar RNA made several hours before mitosis persists in the mitotic cell and repopulates nucleoli when they reform after mitosis     

Dependence of the nature of the action of metabolic inhibitors on ribosomal RNA synthesis in Ehrlich ascites carcinoma cells on cell integrity

Ribosomal RNA (rRNA) synthesis in the intact Ehrlich ascite carcinoma cells is selectively inhibited by papaverin (ED50 = 0.01 mM), 2,4-dinitrophenol (DPN; ED50 = 5 microM), and actinomycin D (ED50 = 0.1 microgram/ml). The inhibition of rRNA synthesis is not connected with a direct action of these agents on the rRNA synthesis apparatus, and they had no effect on isolated cell nuclei. The rRNA synthesis in cells permeabilized with triton X-100 (0.05%) becomes insensible to the action of papaverine and DPN, but is inhibited by actinomycin D in low doses. In cells permeabilized with digitonin (0.01%) the rRNA synthesis shows no sensibility to the action of low doses of actinomycin D. The results suggest that the action of these agents on the rRNA synthesis may depend on cell integrity and on the permeabilization method employed.     

Translocation of nucleolar phosphoprotein B23 (37kDapI 5.1) induced by selective inhibitors of ribosome synthesis

To elucidate the possible role of nucleolar phosphoprotein B23 in ribosome synthesis, drugs which inhibit the processing of ribosomal RNA were employed. After treatment with actinomycin D, toyocamycin or high doses of α-amanitin, a uniform nucleoplasmic fluorescence was observed. Low doses of α-amanitin and the protein synthesis inhibitor puromycin and cycloheximide had no effect on protein B23 translocation. By ELISA immunoassay, there was a 60% decrease in the amount of protein B23 in the nucleoli of the actinomycin D-treated cells as compared with the control nucleoli. Conversely, the amount of protein B23 in the nucleoplasm (excluding nucleoli) was 3-fold higher in the actinomycin D-treated cells. Preribosomal ribunucleoprotein particles (pre-rRNPs) were extracted from isolated nucleoli of Novikoff hepatoma ascites cells and fractionated on sucrose density gradients. Protein B23 was found co-localized with the pre-rRNPs as determined by ELISA assays which agrees with previous studies. The proteins in these 80 S and 55 S pre-ribosomal ribonucleoprotein particles were fractionated by 10% gel electrophoresis. Immunoblots showed protein B23 was present in both pre-rRNPs.     

Cytoplasmic microinjection of immunoglobulin Gs recognizing RNA helices inhibits human cell growth

We report here that nucleolar and cytoplasmic RNA in mammalian cells is recognized specifically by both experimentally induced monoclonal IgG unique for left-handed Z-RNA and by autoimmune mouse monoclonal IgG specific for ribosomal RNA. Nucleolar Z-RNA synthesis, like nucleolar ribosomal RNA synthesis, is inhibited by actinomycin D treatment and dimethylsulfoxide-induced differentiation. Immune anti-Z-RNA IgGs microinjected into living nuclei bind nucleolar RNA, and these complexes appear to be removed from the nucleus within minutes. Cytoplasmically microinjected monoclonal or polyclonal anti-Z-RNA IgGs specifically bind cytoplasmic RNA and inhibit cell multiplication. Microinjection of antibodies directed against double-stranded RNAs. Elevated ionic conditions, which in energy-minimized models can cause the walls of the groove in Z-RNA (but not Z-DNA) to approach each other and close, also prevent antibody binding to specific synthetic or cellular Z-RNA determinants. Our antibodies binding unique Z-RNA structures probably recognize antigens determined by the exposed 2'-OH ribose sugar-phosphate groups.     

Cell division and nucleolar ribonucleic acid synthesis in synchronous cultures

Summary Nucleolar RNA synthesis is inhibited and cell division delayed in synchronous cultures of mouse fibroblasts (strain L-929) treated with actinomycin D (0.04 μg per ml). The gradual loss of actinomycin D from the cells during a 2-hr period following incubation is accompanied by an increase in the rate of nucleolar RNA synthesis to the control level. Following this the rate of protein synthesis is decreased by 25% for approximately 9 hr. The length of time that nucleolar RNA and protein synthesis are inhibited accounts for the delay in mitosis 1 1/2 cell cycles later. These data support the contention that certain proteins produced during one interphase are prerequisite for division in a subsequent cycle.