Mechanism of Apparent Transcription Inhibition by Methyl Mercury in Cerebellar Neurons

Abstract: We have investigated the mechanism of inhibition of RNA synthesis by methyl mercury (MeHg) in isolated neonatal rat cerebellar cells. Each of the three component steps involved in the incorporation of exogenous [³H]uridine into cellular RNA was examined separately in whole-cell and/or subcellular preparations. Nuclear RNA polymerase activity was measured in preparations containing both free nuclei and whole cells. Incorporation of [³H]UTP into nuclear RNA was found to be unimpaired at concentrations of MeHg that inhibited whole-cell incorporation of [³H]uridine by > 75%. Cellular uptake of [³H]uridine was assayed in cerebellar cells treated with KCN to deplete ATP levels and block subsequent phosphorylation reactions of transported uridine. Uptake activity under these conditions was unaffected by MeHg. Measurement of intracellular phosphorylation of [³H]uridine indicated that inhibition of this activity closely paralleled that of RNA synthesis. Quantitation of individual uridine nucleotides by polyethyleneimine-cellulose TLC revealed reduced levels of UTP and UDP whereas levels of UMP were elevated, suggesting that impairment of phosphorylation was not the result of cellular ATP depletion but, more likely, a direct effect on phosphouridine kinase enzymes. This mechanism of MeHg-induced inhibition of RNA synthesis was confirmed by assays of uridine phosphorylation using cell-free extracts in which exogenous ATP was supplied.     

Inhibition of nucleic acid synthesis in Ehrlich tumor cells by periodate-oxidized adenosine and adenylic acid

Periodate-oxidized adenosine and AMP were inhibitory to both RNA and DNA synthesis in Ehrlich tumor cells in culture. With periodate-oxidized adenosine, the inhibition of RNA synthesis paralleled the inhibition of DNA synthesis. Periodate-oxidized AMP, however, was more inhibitory to DNA synthesis than to RNA synthesis. With both compounds, there was a decrease in the conversion of [¹⁴C]cytidine nucleotides to [¹⁴C]deoxycytidine nucleotides in the acid-soluble pool. The borohy-dride-reduced trialcohol derivative of the periodate-oxidized adenosine compound was not inhibitory to DNA or RNA synthesis in the tumor cells. The incorporation of [³H]uridine into 28S and 18S ribosomal RNA was inhibited by both periodate-oxidized adenosine and AMP, but the incorporation of [³H]uridine in 45S, 5S, and 4S RNA was essentially unaffected by these compounds. Periodate-oxidized adenosine inhibited Ehrlich tumor cell growth in vivo.     

In vitro DNA and RNA synthesis by human platelets.

In vitro incorporation of [Me-3H] thymidine and [5-3H] uridine into human platelets was demonstrated. Thymidine incorporation was inhibited by three specific inhibitors of DNA synthesis: hydroxyurea, cytosine arabinoside and daunomycin. The effect was dose-dependent. Uridine uptake by platelets was found to be inhibited by specific inhibitors of RNA synthesis such as actinomycin D, rifampicin and vincristine, the effect of actinomycin D being dose dependent. The drug also led to a time-dependent inhibition of protein synthesis when preincubated with platelets. The platelet RNA profile on polyacrylamide gel was demonstrated to be similar to that of embryonic mouse erythroblast RNA. Synthesis of all three fractions, 28 S, 18 S and 4 S, was inhibited by actinomycin D. These findings show that human platelets are capable of DNA and RNA synthesis, and that these activities play a role in controlling protein synthesis in these cells. Detectable amounts of DNA have been found in whole human platelets, and in isolated mitochondria derived from these cells. Isolated platelet mitochondria incorporated [3H] thymidine and [3H] uridine into their macromolecules. These activities were inhibited by daunomycin and by both rifampicin and actinomycin D, respectively. These results support the assumption that DNA and RNA synthesis found in intact cell preparations takes place most probably in platelet mitochondria.     

Covalent binding of 1-nitro-9-(3-dimethyl-n-propylamino) acridine, a new antitumor drug, to DNA of Ehrlich ascites tumor cells in vivo.

After exposing a line of rat liver epithelial cells to a single dose of the carcinogen N-acetoxy-2-acetylaminofluorene (N-acetoxy-AAF), a dose-dependent decrease in [³H]uridine incorporation into total cellular RNA was found. Approx. 50% inhibition occurred with 0.5 μg/ml of the compound. The kinetics of the response, the effects of actinomycin D, and the fractionation of the newly synthesized RNA by polyacrylamide gel electrophoresis indicated preferential inhibition of the synthesis of 45S ribosomal RNA precursor and a relative sparing of the synthesis of heterogeneous nuclear RNA.     

Ribonucleic acid synthesis and loss of viability in pea seed

Summary RNA synthesis and protein synthesis in embryonic axis tissue of viable pea (Pisum arvense L. var. N.Z. maple) seed commences during the first hour of germination. Protein synthesis in axis tissue of non-viable pea seed is barely detectable during the first 24 h after the start of imbibition. Nonviable axis tissue incorporates significant levels of [³H]uridine into RNA during this period but the level of incorporation does not increase significantly over the first 24 h of imbibition. In axis tissue of non-viable seed during the first hour of imbibition most of the [³H]uridine was incorporated into low molecular weight material migrating in advance of the 4S and 5S RNA species in polyacrylamide gels but some radioactivity was incorporated into a discrete species of RNA having a molecular weight of 2.7×10⁶. After 24 h, non-viable axis tissue incorporates [³H]uridine into ribosomal RNA, the low molecular weight material migrating in advance of the 4S and 5S RNA peak in polyacrylamide gels and a heterogeneous RNA species of molecular weight ranging from 2.2×10⁶ to 2.7×10⁶. No 4S or 5S RNA synthesis is detectable after 24 h of imbibition in non-viable axis tissue. Axis tissue of viable pea seed synthesises rRNA, 4S and 5S RNA, the low molecular weight material migrating in advance of the 4S and 5S RNA peak in polyacrylamide gels and the rRNA precursor species at both periods of germination studied. Loss of viability in pea seed appears to be accompanied by the appearance of lesions in the processing of rRNA precursor species and a significant loss of RNA synthesising activity.Abbreviations rRNA ribosomal RNA - TCA trichloroacetic acid - SLS sodium lauryl sulphate - PPO 2,5 Diphenyloxazole - POPOP 1,4-Bis-2-(4-methyl-5-penyloxazolyl)-benzene     

RNA synthesis during spore germination in Bacillus subtilis.

Summary We have analyzed the RNA synthesized during spore germination in Bacillus subtilis. Early in germination there is little incorporation of [³H]uridine into RNA. A large increase in incorporation into RNA was found at 45–60 min into germination which was in part due to increases in the specific activity of the UTP pool. When corrected for specific activity changes, the instantaneous rate of RNA synthesis showed a seven to tenfold increase between 30 and 45 min of germination. Polyacrylamide gel electrophoresis studies showed that the RNA synthesized during germination appeared very similar to the RNA made during vegetative growth. DNA-RNA hybridization studies indicated that mRNA and rRNA were synthesized throughout germination. Their relative proportions remained constant and were very similar to the composition of RNA synthesized during vegetative growth.In partial fulfillment of the requirements for the doctoral degree by A.S. in the Department of Microbiology at the New York University School of Medicine     

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     

The Incorporation of Phenylalanine and Uridine in Tetrahymena: A Pressure Study1

SYNOPSIS. The action of pressure was studied on the incorporation of labelled phenylalanine and uridine, and on the synthesis of water-soluble proteins in heat-synchronized Tetrahymena pyriformis. Incorporation of [¹⁴C] phenylalanine and [³H] uridine into TCA insoluble fractions was markedly decreased in pressuretreated Tetrahymena. Moreover, the incorporation was dependent upon the age of the cells. Water-soluble protein synthesis was unaffected. These results appear consistent with the proposal that pressure-induced division-delays occur by inhibiting the accumulation of “division proteins” which are essential for cell division.     

RNA and protein synthetic patterns during germination of Polysphondylium pallidum microcysts

During microcyst germination in the cellular slime mould Polysphondylium pallidum, an immediate rapid increase in the rate of protein synthesis ([3H]leucine incorporation) is observed within 15 min after the initiation of germination. The data, corrected for amino acid pool changes, reveal that the rate of protein synthesis reaches its peak at 1 1/2 h, after which it decreases. A low level of RNA synthesis ([3H]uridine incorporation) is observed after 1 h and this rate increases markedly after 2 h. Analysis of the RNA species shows a low level of synthesis of all ribosomal RNA's which begins between 1 and 2 h and increases after 2 h. The synthesis of a heterogeneously distributed, poly(A)-containing fraction of RNA (presumptive mRNA) is initiated some time after 2 h and the synthesis of a small molecular weight species in the 4-5S region is observed after 3 h. Thus, it seems that Polysphondylium microcysts show sequentially initiated synthesis of RNA during germination.     

RNA and protein synthesis in sperm cells isolated from Zea mays L. pollen

Summary Sperm cells are thought to be quiescent in pollen and activated upon pollen germination. To test this hypothesis, protein, RNA and DNA synthesis were assessed in Zea mays sperm cells at different times after isolation from pollen. Protein synthesis changed with time; while some proteins were found to be constitutive in both 0 and 24 h cells, others were synthesized and some disappeared. Overall, the number of proteins detected at 24 h doubled compared with freshly isolated cells. Incorporation of [³H]leucine in 24 h cells was about 50 times that in freshly isolated cells, and that of [5, 6-³H]uridine, about 7 times. Very low incorporation of [6-³H]thymidine into the cells was detected; there was no difference between freshly isolated and 24 h cells. It is possible that the differences in synthetic activity between freshly isolated and 24-h-old cells might correspond to sperm cell activation during pollen tube growth. If so, these metabolic changes may play an important role in fertilization.Supported by funds from a Strategic Grant (D.D.C.) and an Operating Grant (D.J.G.) from the Natural Sciences and Engineering Research Council of Canada     

Protein and nucleic acid synthesis during imbibition of dormant and after-ripened Vaccaria pyramidata seeds.

The regulation of nucleic acid and protein synthesis in dormant, thermodormant, and after-ripened embryos of Vaccaria pyramidata (Caryophyllaceae) has been studied. Germination of after-ripened V. pyramidata seeds is prevented by inhibitors of protein, RNA, and DNA synthesis. The synthesis of both protein and RNA is activated at the beginning of imbibition, whereas [³H]thymidine incorporation does not start until the second period of the imbibition phase. [³H]Thymidine incorporation is greatly reduced in embryos treated with cycloheximide or 6-methylpurine. There is no correlation between the level of [³H]uracil and l-[¹⁴C]leucine incorporation into macromolecules and the physiological state of the seeds: tRNA, ribosomal RNA, and poly(A)-containing RNA (probably mRNA) as well as proteins are synthesized at the same rate in both dormant and thermodormant embryos as in after-ripened embryos. The protein patterns of dormant and after-ripened embryos are similar, as shown by electrophoresis and electrofocusing of double-labeled proteins. The level of DNA synthesis, measured as [³H]thymidine incorporation, may, on the other hand, indicate the physiological activity of the seeds: [³H]Thymidine is incorporated at a high rate in after-ripened embryos only and remains at a low level in dormant or thermodormant embryos. This correlation is, however, observed only in the axes. DNA synthesis in the cotyledons does not show any relation to the developmental stage of the seeds. These results are discussed in relation to the regulation of dormancy and after-ripening of seeds.     

Effect of catecholamines and serotonin on RNA-synthesizing activity in isolated nuclei and chromatin of the rat brain and liver

It has been shown that intraperitoneal injections of L-DOPA cause an increase in the matrix activity of chromatin and stimulate the incorporation of [3H]uridine into the nuclear fraction of rat brain cells by 35%. In vitro studies have shown that preincubation of brain chromatin with L-DOPA diminishes the inhibiting effect of actinomycin D on RNA synthesis. It has been found that the rate of RNA synthesis in vitro depends on concentrations of catecholamines (L-DOPA, dopamine, norepinephrine) and serotonin.     

In vitro studies of cerebral cortical RNA and nucleotide metabolism in hypothyroidism.

—Thyroid hormone deficiency induced during the neonatal period in the rat, resulted in an enhanced incorporation of [2-¹⁴C]uridine and [8-¹⁴C]adenosine in vitro into cerebral cortical RNA at 25 days of age. An examination of the acid-soluble pool constituents separated by polyethyleneiminecellulose TLC, revealed that all phosphorylated derivatives were more highly labelled compared to controls. These differences were not apparent at a lower incubation temperature (4°C). When the average specific activity of precursor pool ATP labelled from adenosine was utilized for the calculation of the rate of RNA synthesis, no change was observed in hypothyroidism. The results are compatible with a maturational-dependent increase in nucleoside transport and rate of phosphorylation in hypothyroidism which is reflected in the stimulated incorporation into cerebral RNA. The apparent normal rate of RNA synthesis coupled with a diminished cellular RNA concentration in thyroid hormone deficiency, suggests an increased RNA turnover. Experiments with actinomycin D revealed no apparent difference in the rate of decay of rapidly-labelled (nuclear) RNA. The possibility is discussed that the processing of nuclear RNA, the formation of stable ribosomal complexes and events at the translational level are subject to modification in developing hypothyroid rats.     

Cytochemical properties of interphase chromatin condensed as a result of treatment with caffeine

Treatment of living cultured cells with caffeine (10 mg/ml, 2 h, 37 °C) brings about marked chromatin condensation which results in the appearance of small, distinct chromatin clumps in the majority of interphase nuclei. The changes taking place in the chromatin properties under the action of caffeine are rather similar to those observed in mitotic condensation (an increase in acridine orange and berberine binding and a sharp decrease in [³H]actinomycin D binding in situ; inhibition of [³H]uridine incorporation in vivo) and are reversible from the point of view of the criteria studied (nuclear morphology, ligand binding, [³H]uridine incorporation, culture viability). It is concluded that caffeine treatment can be regarded as a promising approach to the study of events occurring in chromatin condensation.     

RNA synthesis during spermiogenesis in the sea urchin: a high resolution autoradiographic study of [3H]uridine incorporation

Summary

RNA synthesis has been studied during spermiogenesis of Paracentrotus lividus by high resolution autoradiography using [³H]uridine as a labeled precursor. Under the experimental conditions used [³H]uridine incorporation is detectable only at the early spermatid stage. Labeling is distributed mainly over the nucleus and it appears completely absent from the mitochondria. After RNase digestion radioactivity falls to a background level, demonstrating that RNA synthesis actually occurs in early spermatids of P. lividus. It follows that in P. lividus during spermiogenesis cell differentiation is not entirely dependent on stored premeiotic RNAs.     

Stable Messenger Ribonucleic Acid and Germination of Myxococcus xanthus Microcysts

We have examined germination, protein synthesis and ribonucleic acid (RNA) synthesis by microcysts of the fruiting myxobacterium Myxococcus xanthus. The morphological aspects of microcyst formation were completed at about 2 hr after induction had begun. In such microcysts, germination, RNA synthesis, and protein synthesis were inhibited by actinomycin D (Act D). At 6 hr after induction, germination and protein synthesis had become relatively resistant to Act D, whereas RNA synthesis was inhibited by about 95%. Experiments with (3)H-Act D indicated that the deoxyribonucleic acids of both young and old microcysts bind Act D equally. Resistance of germination to Act D was acquired 4 to 5 hr after induction of microcyst formation, and was due to an Act D-sensitive synthesis at that time. Vegetative cells and microcysts were pulsed with uridine-5-(3)H and chased for 60 min; the RNA was extracted and analyzed by means of sucrose density gradient centrifugation and gel electrophoresis. Both microcysts and vegetative cells were found to contain grossly the same types of RNA in the same proportions. RNA pulse-labeled in microcysts was more stable than that in vegetative cells. No particular portions of the microcyst pulse-labeled RNA were selectively stabilized. These data indicate that a stable messenger RNA required for synthesis of germination proteins was synthesized during microcyst formation. This may be the same as the RNA synthesized 4 to 5 hr after initiation of microcyst formation. We suggest that the existence of such stable messenger RNA in microcysts is consistent with the limited biosynthetic activities of such cells.     

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.     

Comparison of overall rates of RNA synthesis in implanting and delayed implanting mouse blastocysts in vitro

Implanting and delayed implanting mouse embryos were incubatedin vitro with [³H]uridine for 2–24 hr. The size and specific activity of the [³H]UTP pools were determined by means of a double isotope technique using copolymer synthesis with the [³H]UTP in the embryos, exogenous [¹⁴C]ATP, andE. coli RNA polymerase. Using the rate of incorporation of [³H]uridine into acid-insoluble material and the specific activity of the [³H]UTP pools, it was possible to calculate the overall rate of incorporation of uridine into RNA by the embryos. In implanting embryos it was constant for 24 hr. In contrast, the initial rate of uridine incorporation by the delayed implanting embryos was only 31% of that in implanting embryos (i.e., per cell); this increased steadily during the incubation period, reaching 81% of the rate in implanting embryos after 24 hr. This activation of RNA synthesis by delayed implanting embryosin vitro occurred in the absence of any uterine stimulatory factors. Further, it was shown that although 10% mouse serum would support trophoblastic outgrowthin vitro, it did not influence uptake, distribution of label into nucleotides, or rate of uridine incorporation into RNA in either implanting or delayed implanting embryos. Therefore, it is suggested that if depression and activation of metabolic activity in blastocysts are part of the mechanims of delayed implantation, and if trophoblast outgrowthin vitro is analogous to the process of implantationin vivo, then these two aspects of embryo activation are under different controls.     

An autoradiographic method of detectingA. laidlawii andM. hyorhinis in cell cultures

Summary The autoradiographic investigation of L cells and Chinese hamster cells for the presence of mycoplasmas (A. laidlawii andM. hyorhinis) using uridine/uracil (UdR/U) testing is a rapid and reliable method suitable for the serial checking of even a small number of cells. It depends on a reduced incorporation of [³H]uridine and an increased uptake of [³H]uracil into the RNA of mycoplasma-infected cells, shown in autoradiograms by the density of the grains and their distribution. Results obtained by the autoradiographic technique correspond approximately to specific activity values of RNA-infected cells after the incorporation of [³H]uridine and [³H]uracil.     

Brief report: Synthesis of ribonucleic acid in oocytes collected from squirrel monkeys and humans following chorionic gonadotropin administration

The time between administration of human chorionic gonadotropin (hCG) and follicle aspiration was analyzed for alterations in [³H]uridine incorporation [as an indicator of relative synthesis of ribonucleic acid (RNA)] of oocytes from squirrel monkeys and humans. There was a significant decline in both the uptake and incorporation of [³H]uridine in squirrel monkey oocytes by 36 hr following hCG administration to the animals, as compared with 16 hr after hCG. Similarly, RNA synthesis diminished in oocytes collected 35 hr after hCG in humans, as compared with 12 hr after hCG. This reduction in RNA synthesis of maturing oocytes is similar to that of other mammalian species. This provides evidence for an increased interval between hCG administration and follicle aspiration in order to recover mature oocytes for in vitro fertilization studies.