Reduction In RNA Synthesis Research Articles

Reduced RNA synthesis levels in isolated mouse liver nuclei following reaction with [(H2O)(NH3)5Ru(II)]2+

The goal of this study is to establish the effect of [(H2O)(NH3)5Ru(II)]2+ reaction of nuclei on their RNA transcription levels. This question is important because ammineruthenium compounds share chemical and biological properties with the chemotherapeutic agent cis-dichlorodiammineplatinum(II) or cisplatin. First we demonstrate that mouse liver nuclei are active in RNA transcription in vitro and characterize the optimum conditions for in vitro transcription. Synthetic rates in the presence of inhibitors actinomycin D and alpha-Amanitin and measurements of oligo(dT)-cellulose RNA binding levels suggest that all three RNA Polymerases are active in synthesis at about the following percentages-RNA Polymerase I(30%), II(50%) and III(20%). Mouse liver nuclei reacted with [(H2O)(NH3)5Ru(II)]2+ and then oxidized had (NH3)5 Ru(III)3+n-DNA adduct levels inversely related to total RNA synthetic rates. Oligo(dT) cellulose RNA binding levels did not vary with DNA adduct density. These data suggest that direct DNA lesions rather than [(NH3)5Ru(III)]3+ effects on other aspects of the transcription system are responsible for the diminished RNA synthesis levels. Ammineruthenium complexes remain desirable candidates for chemotherapeutic agents that may be safely administered in the unreactive ruthenium(III) state and be activated toward DNA binding by reduction in the hypoxic environment of many tumour cells.

Multiple mechanisms for the inhibition of rRNA synthesis during HL-60 leukemia cell differentiation.

Treatment of HL-60 human promyelocytic leukemia cells with inducers of granulocytic differentiation produces a depletion of cellular rRNA, with the anthracycline antibiotics aclacinomycin A (ACM) and marcellomycin (MCM) causing a more rapid loss than dimethylsulfoxide (DMSO). This action is associated with a large reduction in RNA synthesis, which precedes any decreases in protein or DNA synthesis, and is specific for rRNA relative to total polyadenylated RNA synthesis. A 70% reduction in rRNA synthesis occurs within 20 minutes of ACM treatment and by 30 hours of DMSO exposure. Relative to the amount of 28S and 18S rRNA in the cells, there is a nearly complete depletion of the amount of 45S rRNA and other large rRNA precursors in cells treated with ACM, MCM, and the intercalating agent actinomycin D. In contrast, DMSO treatment produces a more coordinated decrease in 18S and 28S rRNA and rRNA precursors. The anthracycline antibiotics inhibited the synthesis of 5' proximal and 3' distal regions of the pre-rRNA transcript, while actinomycin D had a relatively sparing effect on the transcription of the 5' external transcribed spacer region of the gene relative to depletion of 3' transcripts. These studies demonstrate that different inducers of HL-60 differentiation have varying sites of action on rRNA synthesis and/or processing, with depletion of cellular rRNA as a common consequence.

Inhibition of amino acid uptake and incorporation into proteins in Friend erythroleukemia cells by the anthracycline antitumor antibiotic aclacinomycin A

Treatment of Friend erythroleukemia cells with aclacinomycin A caused a concomitant inhibition of the uptake of 14C-α-aminoisobutyric acid (AIB) and of the incorporation of 3H-alanine into proteins. The decrease in amino acid uptake and incorporation into proteins was dose-dependent and reached a maximum of 60% within 3 hours at the concentration of aclacinomycin A, 200 mg/ml. A comparison of the effect of protein incorporation of 3H-alanine and cell proliferation by various anthracycline antitumor antibiotics in a concentration range of 50–200 mg/ml revealed that two other N-alkylated anthracyclines, pyrromycin and marcellomycin, are also potent inhibitors of the incorporation of amino acids into proteins. Inhibition of amino acid incorporation into proteins correlated well with the reduction of cell number at a later time. In contrast, adriamycin and daunomycin inhibited the incorporation of 3H-alanine into proteins only weakly, although these substances were highly active at inhibiting cell proliferation. Studies with an inhibitor of RNA synthesis, antinomycin D, suggest that the concomitant inhibition of amino acid uptake and incorporation into proteins observed with aclacinomycin A is not due to a reduced RNA synthesis. In addition, aclacinomycin A, up to a concentration of 10 μg/ml, did not inhibit protein synthesis in a cell-free translational system from rabbit reticulocytes. These results indicate that the reduction of amino acid incorporation into protein after treatment of Friend erythroleukemia cells with aclacinomycin A may be due to a reduced uptake of amino acids. Inhibition of the transport of 14C-AIB may be indicative for an interaction of aclacinomycin A with the plasma membrane.

Phosphorylation within a specific domain of the phosphoprotein of vesicular stomatitis virus regulates transcription in vitro

We have investigated the functional significance of phosphoserine residues that lie in the L protein-binding domain between amino acids 213 and 247 of the phosphoprotein (NS) of vesicular stomatitis virus. A series of mutant NS proteins were made by cell-free translation of mRNAs transcribed from the cloned gene. Site-directed substitution of alanine for both serine 236 and serine 242 essentially abolished RNA synthesis catalyzed by the NS-L complex. Substitution of either of these serines reduced RNA synthesis by 75%. Serine 218 played no major role in RNA synthesis. Phosphorylation of NS by the L protein was abrogated by substitution of either serine 236 or serine 242. These results indicate that phosphorylation of serines 236 and 242 in the NS protein regulates its binding with the L protein and the N-RNA template and is essential for activation of viral RNA synthesis.

Reduction in RNA synthesis following red cell-mediated microinjection of antibodies to RNA polymerase I

Antibody molecules directed against RNA polymerase I, the enzyme responsible for rRNA synthesis, were introduced into rat hepatoma cells by red cell-mediated microinjection. Access of the antibodies to the nucleolus, the site of rRNA synthesis, was facilitated by microinjecting mitotic cells. Using indirect immunofluorescence, anti-RNA polymerase I immunoglobulins, but not control immunoglobulins, were found localized in the nucleoli of microinjacted cells. To assess whether intracellular antibodies could alter RNA synthesis, cultures were labeled with [ 3H]uridine at various times after microinjection. Reduction in RNA synthesis, relative to cells microinjected with non-immune immunoglobulins, was observed within three hours. These results demonstrate that antibodies introduced into the cytoplasm of mitotic cells via red cell-mediated microinjection have free access to nuclear components and that they remain functional within the nuclei of living cells.

Autoradiographic detection of HPRT variants of human lymphocytes resistant to RNA synthesis inhibition

The feasibility of using RNA synthesis in freshly isolated, human peripheral blood lymphocytes to detect 6-thioguanine (TG)- and 8-azaguanine (AG)-resistant variants in an autoradiographic assay similar to that of Strauss and Albertini (1979) has been evaluated. In phytohemagglutinin (PHA)-stimulated cultures RNA synthesis and HPRT activity began well in advance of DNA synthesis and increases in parallel during the first 44 h of culture. Introduction of TG or AG with PHA at the beginning of culture completely inhibited DNA synthesis during the first 44 h and reduced RNA synthesis to low levels within 24 h. When TG or AG was added after cells had been in culture for 38 h, DNA synthesis was reduced quickly while RNA synthesis was inhibited more slowly. An autoradiographic assay is described in which freshly isolated lymphocytes are cultured with PHA for 24 h, with or without TG or AG, then labeled with [ 3H]uridine for 1 h. TG-resistant and AG-resistant variant frequencies for 2 normal individuals and a Lesch-Nyhan individual were determined with this assay. The variant frequencies for the normal individuals ranged from 0.46 to 10.6 × 10 −5 depending upon the selective conditions used. All the Lesch-Nyhan cells were resistant to 0.2 μM–2 mM AG; some were sensitive to 0.2 mM TG and most were sensitive to 2.0 mM TG.

Nuclear Chromatin Structure in Relation to Cell Differentiation and Cell Activation in the Cap and Quiescent Centre ofZea maysL.

Barlow, P. W. 1985. Nuclear chromatin structure in relation to cell differentiation and cell activation in the cap and quiescent centre of Zea mays L.—J. exp. Bot. 36: 1492-1503. Nuclear chromatin structure has been analysed by electron microscopy of thin sections of cells in four zones of the root cap—meristem, central, slime-secreting and outermost cells—and also in the quiescent centre of the root before and after decapping. The chromatin pattern has been related to the DNA and RNA synthetic activity of the nuclei. During cap cell maturation there was a progressive condensation of the chromatin and this was accompanied by some reduction of RNA synthesis. The degree of condensation was estimated from the area and number of pieces of electron dense chromatin which increased and decreased, respectively, during cap maturation. The volume fraction of condensed chromatin was also estimated but, in the cap, was not found to be a good indicator of nuclear activity. The outermost cells of the cap showed the greatest degree of chromatin condensation but were still active in RNA synthesis. Microdensitometry of their nuclear DNA contents gave an indication of loss of DNA in some of the nuclei. Decapping activated DNA and RNA synthesis in the quiescent centre and also stimulated a decondensation of chromatin: the number of condensed pieces of chromatin increased, and their size and volume fraction both decreased 4 h after decapping. The number of pores per unit length of nuclear envelope profile was also estimated. In the cap this number increased during cap maturation; in the activated quiescent centre the number remained constant except for a small rise 4 h after decapping. Key words—Zea mays, chromatin, root cap, quiescent centre. Correspondence to: University of Bristol Research Station, Long Ashton, Bristol BS18 9AF, U.K.

Gene identification in polytene chromosomes: some Balbiani ring 2 gene sequences are located in an interband-like region of Chironomus tentans

Injection of Chironomus tentans larvae with the nucleoside analogue DRB (5,6-dichloro-1-β-d-ribofuranosylbenzimidazole) led to a drastic reduction of RNA synthesis in the polytene salivary gland chromosomes, accompanied by a dissociation of RNA polymerase B (or II) from the chromosomal template. The DRB injection also led to a recondensation of the Balbiani ring 2 (BR2) puff and the formation of a well-defined banding pattern at the site of previous puffing. In the recondensed condition in situ hybridization of cloned BR2 DNA demonstrated that BR2 gene sequences are located in a region that light microscopically appears to be an interband between bands IV-3B9 and IV-3B10. No hybridization exists over bands IV-3B8 and IV-3B10. The in situ hybridization over interband IV-3B9/10 differs from the result of previous hybridization of BR2 RNA to polytene chromosomes from Malpighian tubules of C. tentans that showed that the BR2 gene is located in band IV-3B10 (Derksen et al. 1980). Due to the different hybridization probes used by the investigators both results are compatible with the location of the BR2 gene in a band-interband structure. These data seem to indicate: (1) that the repressed BR2 gene consists of two segments with different chromatin conformation (one with relatively extended interband chromatin and one with condensed band chromatin) and (2) that the transcriptional and regulatory unit of the BR2 gene may extend from the interband into the band or vice versa.

Heterocyst differentiation in Cylindrospermum licheniforme: studies on the role of transcription.

Heterocysts of the cyanobacterium Cylindrospermum licheniforme occur at the ends of the filaments. These cells, specialized for aerobic N2 fixation, synchronously differentiate after the fragmentation of filaments grown in a medium free of combined nitrogen. This study has examined the role of transcription during and after heterocyst differentiation. Autoradiography of intact filaments pre-labelled for 3 h with [3H]uracil revealed that RNA synthesis occurred at similar rates in vegetative cells, developing (pro)heterocysts and mature heterocysts. Since mature heterocysts no longer divide and contain a similar amount of DNA as vegetative cells, transcription must continue under conditions where replication no longer occurs. Rifampicin, when added to fragmented filaments at concentrations that reduced RNA synthesis by 95% in all cell types, inhibited proheterocyst formation but not the final morphological step of differentiation (pore plug deposition). However, chloramphenicol blocked all stages of heterocyst differentiation. Since the average mRNA half-life was 29 min, and the rifampicin-insensitive stage lasted 3-6 h, the later stages of differentiation may depend on long-lived mRNAs.

Control of RNA and protein synthesis by the concentration of Trp-tRNA Trp in Escherichia coli infected with bacteriophage MS2

The effect of varying the concentration of Trp-tRNATrp in Escherichia coli infected with bacteriophage MS2 has been studied by varying the amount of exogenously added tryptophan (Trp) to cells bearing a mutation which results in a tryptophanyl-tRNA synthetase with a higher Km value for Trp. The phenotype of the mutant has been confirmed by measuring the level of tRNATrp which can be aminoacylated in vivo, and the mutant has also been shown to have elevated tRNATrp levels compared to wild-type. The growth of MS2 decreases continuously with decreasing Trp concentration (and hence, decreasing Trp-tRNATrp concentration). This appears to be due to reduced gene expression, since at later times in infection the amount of MS2 coat protein synthesized likewise decreases continuously with decreasing Trp concentration. However, there is little decrease in the amount of coat protein or replicase synthesized during the first few minutes after the Trp concentration shift. A continuous increase in the average polysome size distribution is seen as the Trp concentration is decreased. MS2 RNA synthesis also decreases continuously with decreasing Trp concentration, and is shut off in the absence of Trp. This does not seem to be due to ppGpp as these cells are functionally relaxed under these conditions, nor does it seem to be due to degradation of pre-existing template. Addition of chloramphenicol abolishes the effect of Trp concentration on RNA synthesis. The data are consistent with a model in which ribosomes pause at Trp codons in the absence of Trp-tRNATrp, while other ribosomes queue behind and continue to load onto the message. The reduction of RNA synthesis would then be a consequence of coupling to translation.

Inhibition of RNA synthesis by oxolinic acid is unrelated to average DNA supercoiling.

Oxolinic acid reduced RNA synthesis rates whether chromosome supercoiling decreased, increased, or remained unchanged. Thus, inhibition of RNA synthesis by oxolinic acid appears to involve factors other than average DNA supercoiling level. Coumermycin A1 caused RNA synthesis rates to increase or decrease roughly in parallel with DNA supercoiling.

Does protein synthesis decline in lithium-treated sea urchin embryos because RNA synthesis is inhibited?

Vegetalization of sea urchin embryos by Li + is characterized by rates of protein synthesis which are normal during cleavage, and decline after hatching. This paper tests the hypothesis that Li + interferes with RNA synthesis during cleavage, resulting in the decline in protein synthesis at hatching when newly synthesized mRNA becomes critical for further normal development. Treatment with Li + does cause a decline in the incorporation of [ 3H]guanosine into RNA. However, this decline could be accounted for by reduced uptake of the labeled precursor with a concomitant reduction in precursor pool specific activity. Therefore, reduced protein synthesis after hatching in Li +-treated embryos cannot be accounted for by a comparable reduction in RNA synthesis.

Anomalies in the guanosine polyphosphate metabolism: Effect of the [formula omitted] mutation

The hisU1820 mutant (TA799) of Salmonella , typhimurium shows a substantial increase in the levels of ppGpp (MSI) and of pppGpp (MSII) during several types of metabolic shifts. Noticeable amounts of ppGp (MSIII) are also present post-carbon/energy source downshifts and temperature up-shifts. The increased levels of these guanosine polyphosphates were observed despite the absence of the expected reduction in RNA synthesis upon a nutritional downshift. We, therefore, suggest that the hisU mutation causes an increase in the accumulation of MSI and MSII; and that ppGpp alone is not sufficient to promote restriction of RNA synthesis during a nutritional transition.

Guanine nucleotide depletion and toxicity in mouse T lymphoma (S-49) cells.

Incubation of mouse T lymphoma (S-49) cells with the inosinate dehydrogenase inhibitor mycophenolic acid produced a depletion of both GTP and dGTP, and resulted in growth inhibition, partial reduction in RNA synthesis, and drastic inhibition of DNA synthesis. Similar results suggested to others that the depletion of dGTP is primarily responsible for toxicity. However, guanosine was as effective as deoxyguanosine at preventing mycophenolic acid toxicity although deoxyguanosine was more effective at elevating dGTP levels. Moreover, in hypoxanthine-guanine phosphoribosyltransferase-deficient mutants of S-49 (6MPR-3-3) deoxyguanosine was unable to prevent mycophenolic acid toxicity or to re-establish normal DNA synthesis, although it returned cellular dGTP but not GTP levels to normal. No other nucleotide levels changed in a way which could account for the toxicity. Incubation of cells with a combination of deoxyadenosine, deoxycytidine, and erythro-9-(2-hydroxy-3-nonyl)adenine produced a selective depletion of dGTP to levels similar to that produced by mycophenolic acid, but did not affect cell growth. Studies with cells synchronized by centrifugal elutriation show that the toxicity of mycophenolic acid is specific to the S-phase of the cell cycle. Addition of actinomycin D at a concentration that inhibited RNA synthesis increased the availability of GTP and re-established normal DNA synthesis in mycophenolic acid-treated S-49 cells. These results suggest that the depletion of GTP rather than that of dGTP produces toxic effects in S-49 cells and that GTP is required for DNA synthesis.

The stringent response to unacylated tRNA, energy- and temperature-downshift in Bacillus stearothermophilus

The response of the thermophile Bacillus stearothermophilus to inhibition of tRNA acylation, energy starvation and temperature downshift was characterized. We found that B. stearothermophilus, like other prokaryotic organisms, reacts with the so-called stringent response, which includes the accumulation of the unusual nucleotides guanosine 3',5' bis (diphosphate) [ppGpp] and guanosine 3'-diphosphate, 5'-tri-phosphate [pppGpp] and concomitantly the reduction of RNA synthesis and growth rate. The amount of (p)ppGpp formed depended on the cause of the stringent response: when tRNA acylation was inhibited (p)ppGpp synthesis was much higher than after energy starvation or temperature downshift whereas RNA synthesis was totally blocked in each case.

Inhibition of Moloney leukaemia virus production by N-methylisatin-beta-4':4-diethylthiosemicarbazone.

N-methylisatin-beta-4':4'-diethylthiosemicarbazone (M-IBDET) inhibited the production of Moloney leukaemia virus (MLV). Virus inhibition was related to drug concentrations and time of treatment. The effective antiviral drug concentrations ranged between 3.4 muM and 34 muM. Virus reverse transcriptase activity even at concentrations of 34 muM-M-IBDET was not inhibited. At virus inhibitory concentrations the drug reduced RNA synthesis only very slightly and did not affect protein synthesis at all, although growth and DNA synthesis of host cells were suppressed. The inhibition of cellular DNA synthesis was reversible. Comparison of M-IBDET with actinomycin D, cycloheximide and alpha-amanitin in terms of their inhibitory effect on the release of MLV into the culture medium showed that M-IBDET was comparable to the other antimetabolites. The inhibition of MLV production by M-IBDET was confirmed by various parameters of virus assay. It was concluded from the experimental evidence that M-IBDET specifically inhibits MLV-production.

Effects of d-glucosamine on the UTP pool and on nucleic acid synthesis in embryonal retina of chicken

d-glucosamine lowers in embryonal retinal of chicken the content of UTP and causes a reduction of RNA synthesis.

Relationship between ultrastructure and function of hepatocyte chromatin: A study with adrenalectomized rats after cortisol administration

Using either the routine staining procedure or the Feulgen-like Gautier's method the condensed pattern of hepatocyte chromatin in adrenalectomized rats appeared unchanged 1 hr after cortisol administration, whereas at 4 hr and especially at 8 hr it looked more dispersed, in parallel with an increased synthesis of RNA and nuclear nonhistone proteins (NHP). The quantity of [3H]cortisol bound to deoxyribonucleoproteins (DNP) was the same at 1 and 4 hr after hormone treatment and it was unaffected by amanitin, which induced a chromatin condensation. Eight hours after cortisol injection almost all the DNA structures, revealed by the Gautier technique, were in the unraveled form; concurrently, a large quantity of perichromatin fibrils was visualized with the Bernhard method for ribonucleoproteins (RNP). Amanitin poisoning of 8-hr stimulated rats induced, 1 hr after treatment, a light condensation of the DNA structures and a strong condensation after 2 hr; the perichromatin fibrils were lightly reduced 1 hr after toxin injection and had almost disappeared 2 hr thereafter. Amanitin reduced RNA synthesis to about 25% of control value both 1 and 2 hr after poisoning. No modification of NHP synthesis was observed after toxin treatment. Our results demonstrating a strict relationship only between the chromatin pattern and the quantity of perichromatin fibrils, morphological expression of heterogeneous nuclear (hn) RNA which has been synthesized (J. P. Bachellerie, E. Puvion, and J. P. Zalta,Eur. J. Biochem. 58, 327, 1975; S. Fakan, E. Puvion, and G. Spohr,Exp. Cell Res. 99, 155, 1976), suggest that the chromatin ultrastructural changes induced by cortisol are a consequence and not a cause of gene transcriptional activity.

Decreased 3H-uridine incorporation and increased 3H-adenosine incorporation by HeLa cells exposed to autologous culture fluid.

Actively growing HeLa monolayer cultures briefly exposed to the culture fluids (CF) from confluent HeLa cultures and labeled simultaneously or subsequently, incorporated less 3H-uridine (3H-UR) but more 3H-adenosine (3H-AR) than control cultures similarly exposed to fresh medium and labeled. Exposure to CF inhibited the uptake as well as the incorporation of 3H-UR by cultures. The inhibition of 3H-UR incorporation by CF-exposed cultures could be reduced by increasing the concentration of 3H-UR in the labeling medium. Both the inhibition of 3H-UR incorporation and the stimulation of 3H-AR incorporation were prevented by washing the CF-treated cultures with phosphate buffered saline before labeling. Similarly, both effects could be producted in HeLa cultures exposed to fresh medium containing 1 X 10(-5) M uridine instead of to CF. Therefore, the observed effects of CF on label incorporation were probably due to the presence of uridine or a related compound, and the inhibition of 3H-UR incorporation resulted from reduced uptake of 3H-UR rather than from reduced RNA synthesis by exposed cells. The active agent in the CF, formed only when cultures were incubated at physiological temperatures, was not a product of medium decay. It was a cellular product formed equally well by cultures incubated in medium containing dialysed or whole serum.

An Investigation on the Mechanism of Action of Propachlor

Studies were conducted to examine over time the effects of propachlor (2-chloro-N-isopropylacetanilide) on the growth of cucumber (Cucumus sativusL. ‘Straight Eight’) roots and associated biosynthetic reactions. Complete inhibition of root elongation occurred within 16 hr after exposure to propachlor. Inhibition of growth was not found to be a result of an effect on ATP formation or respiration. Protein biosynthesis was reduced several hours before the observed inhibition of growth therefore implicating it as the causal factor. Inhibition of protein synthesis occurred prior to an observed reduction in RNA synthesis suggesting that the primary effect of propachlor is on protein biosynthesis and that its effect on nucleic acid synthesis is secondary. It is concluded that the primary mechanism of action of propachlor is its effect on nascent protein biosynthesis.