RNA Synthesis Activity Research Articles

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.

Effects of insulin-like growth factors on chick embryo hepatocytes.

Biological effects of insulin-like growth factors (IGF) I and II on primary cultures of chick embryo liver cells have been investigated and compared 1) with the biological effect of insulin and 2) with competitive binding of the three hormones to their respective binding sites. IGF I and II stimulate the incorporation of D[U-14C]-glucose into liver cell glycogen in a time- and dose-dependent manner, but with a 5-10-fold lower potency than insulin. Both IGFs also lead to enhanced incorporation of 5-[3H]uridine and L[U-14C]valine into trichloroacetic acid (TCA) insoluble material and to activation of ornithine decarboxylase activity. Their potency in stimulating RNA synthesis and ornithine decarboxylase activity is comparable to that of insulin. Protein synthesis is maximally stimulated at 3 nM by all three hormones. In the competitive binding studies, IGF I and II are 10-fold less potent than insulin in competing for [125I]insulin binding, but 100-fold more potent than insulin in competing for [125I]IGF I or II binding. These studies show that IGF I and II stimulate the same metabolic indices as insulin in the chick embryo liver. By comparing these biological effects with competitive binding data it appears that IGFs act on glucose metabolism in the chick embryo liver via the insulin receptor, whereas stimulation of growth indices by IGFs and insulin appears to be mediated by their own specific receptors.

Calcium thresholds in the activation of DNA and RNA synthesis in cultured planarian cells: relationship with hormonal and DB cAMP effects

DNA and RNA syntheses were reduced to a basal level in dissociated planarian cells grown for 48 h in a Ca 2+-free medium. These syntheses could be triggered anew by raising the Ca 2+ concentration in the medium. Serotonin could be substituted for Ca 2+ in stimulating DNA synthesis by Ca 2+-depleted cells, while dopamine greatly enhanced RNA synthesis in these cells. When Ca 2+ concentration was raised in hormone-treated cultures, DNA synthesis was again slightly increased but RNA synthesis was depressed. Both hormonal effects were completely inhibited by the anticalmodulin drug trifluoperazine. As serotonin and dopamine are both known to stimulate the adenylate cyclase system, it was further investigated whether the hormonal effects were mediated by cAMP. Indeed, a DB cAMP concentration of 1 μM increased DNA labelling when applied for 8 h to Ca 2+-depleted cultures. However, when Ca 2+ was present, the 8-h treatment with 1 μM DB cAMP was inhibiting. A 4-h pulse with 1 μM DB cAMP just after Ca 2+ addition was a condition for a high stimulation of DNA labelling. The other DB cAMP concentrations used, 0.1 and 10 μM, reduced DNA labelling. In the absence of Ca 2+, RNA labelling was only slightly increased by 0.1 μM DB cAMP, but was highly stimulated by a 4-h treatment of 1 μM DB cAMP in the presence of Ca 2+. The noted effects with 1 or 0.1 μM DB cAMP on DNA or RNA labelling corresponded to true changes in synthesis rather than alterations of the specific activity of the nucleotide pool by DB cAMP. Besides, it was precluded that these effects were due to butyrate issued from DB cAMP degradation. It was further shown that DB cAMP at 1 μM increased Ca 2+ uptake in planarian cells, whereas the other concentration reduced it. This observation might explain the stimulating effect on nucleic acid synthesis of 1 μM DB cAMP applied at the appropriate moment. Based on these results it seems that, for triggering RNA synthesis, the threshold value of Ca 2+ was lower than for DNA synthesis. These Ca 2+ thresholds might be reached, in the absence of Ca 2+ in the medium, by treatments with DB cAMP or hormones at the appropriate doses and periods. This interpretation is in agreement with the succession of biochemical events described in regenerating planarians and suggests that these events might be causally related.

Dependence of Cyclic Nucleotide Production and Luteinizing Hormone Receptor Formation upon Ribonucleic Acid Synthesis in Cultured Rat Granulosa Cells

The role of newly synthesized RNA in the differentiation of granulosa cells isolated from diethylstilbestrol-treated immature rats was studied during culture with actinomycin D. Choleragen-induced LH receptor formation and cGMP production at 48 h were completely inhibited by actinomycin D (greater than or equal to 100 ng/ml) added as late as 20 h after the initiation of culture and were partially reduced by addition of the antibiotic from 30-48 h. In contrast, addition of actinomycin D to freshly prepared cells enhanced choleragen-stimulated cAMP accumulation during the 48-h culture period. This effect was caused by both elevation of adenylate cyclase activity at 3 and 6 h of culture and inhibition of choleragen-induced phosphodiesterase activity during culture. The increase in cAMP production by actinomycin D was confined to the first few hours of culture, since the antibiotic did not enhance cAMP levels when added after 3 h and significantly reduced cAMP accumulation when added from 20-48 h of culture. Actinomycin D inhibited choleragen-stimulated incorporation of [3H]uridine into RNA of freshly prepared cells by 65% and reduced both RNA synthesis and incorporation of [3H]leucine into protein at 20 and 48 h of culture by approximately 90%. In untreated cells, RNA and protein synthesis and phosphodiesterase activity increased to a larger extent from 20-48 h than after choleragen treatment, but did not lead to elevated cAMP levels or LH receptors. These results suggest that the cAMP-induced syntheses of RNA and protein that are specific for increases in cGMP production and LH receptor formation occur predominantly during the second day of granulosa cell culture. In contrast, cAMP production can be markedly altered by changes in RNA and protein syntheses during the first hours of culture.

REPAIR OF 8‐METHOXYPSORALEN INDUCED DNA INTERSTRAND CROSS‐LINKS IN Tetrahymena thermophila

Abstract— The protozoan Tetrahymena thermophila was treated with 8‐methoxypsoralen in combination with long wavelength ultraviolet irradiation and the DNA‐repair response was studied. Following the treatment a lag‐period in cell proliferation was observed, the duration of which was proportional to the amount of psoralen used, and both swelling and deformation of the cells were observed. The treatment with 3 μg 8‐methoxypsoralen/mℓ and a light dose of 8 kJ m‐2 resulted in a 10‐fold decrease in DNA and RNA synthesis activity, while the protein synthesis was only moderately affected. Using the same conditions the lag‐period was 30 h, and during this time the psoralen induced DNA interstrand cross‐links were removed. Alkaline elution experiments showed that the repair process involves DNA single strand scissions, whereas no double strand DNA scissions were detected.

The Effect of 20-Hydroxyecdysone on the Synthesis of Fibroin mRNA in the Cultured Posterior Silk Gland of Bombyx mori

The activity of total RNA synthesis including fibroin mRNA in the cultured posterior silk gland of Bombyx mori was dependent on the developmental stage of larva from which the silk gland was dissected out. The majority of the RNA synthesized in the posterior silk gland cultured for 24 hr was ribosomal RNA and transfer RNA at all investigated stages. The synthesis of fibroin mRNA primarily decrease by the application of the molting hormone, 20-hydroxyecdysone, whereas the hormone stimulated the synthesis of both ribosomal and transfer RNAs.

Cytokine modulation of chondrocyte proteinase release.

Nonenzymatic, trypsin sensitive cytokines derived from lectin stimulated normal human mononuclear cells have been shown to induce release of proteoglycan and collagen degrading proteinase activity from chondrocytes in cartilage organ and isolated suspension culture systems. Active chondrocyte protein and RNA synthesis were required to induce activity. Cytokines responsible were of both monocyte and T cell origin. Direct monokine catabolic induction and monokine/lectin-triggered lymphokine inducing activity could be demonstrated. Cyclooxygenase inhibitors and direct or indirect modulation of mononuclear cell or chondrocyte cAMP levels had no effect on factor synthesis or activity. Hydrocortisone abrogated the effect. Cytokines responsible were heat labile, unaffected by reduction/alkylation or neuraminidase exposure, and stable over a pH range of 3-10.

Developmental changes in gene expression during pollen differentiation and maturation inNicotiana tabacum L

Total and polysome-bound ribosomes and the uptake and incorporation of3H-uridine and14C-leucine were examined in dividing microspores and in pollen grains isolated from anthers of 6 different developmental stages. Direct evidence was obtained that the formation of cytoplasm of the vegetative cell following microspore division is related to a rapid activation of RNA and protein synthesis and of ribosomes in differentiating pollen. Total ribosomes associated with gametophytic programme rose about 10times and the process of differentiation was accompanied by a rapid increase in uptake capacity of pollen grains for both uridine and leucine. Pollen development after cytoplasm synthesis and starch deposition continued by pollen maturation, which was characterized by a decline in RNA synthesis, dissociation of polysomes and by a further rise of transport activity of pollen grain wall for exogenous substrates, indicating probable pollen adaptation for utilization of metabolites from the degenerating tapetal cytoplasm.

Effects of prostaglandins on the prolactin stimulation of lipid biosynthesis in mouse mammary gland explants

The effects of prostaglandins E 1 and E 2, indomethacin, arachidonic acid, and 8,11,14-eicosatrienoic acid on the rate of ( 14C)-acetate incorporation into lipids in mouse mammary gland explants were studied. Prostaglandins El and E 2, as well as their precursors 8,11,14-eicosatrienoic acid and arachidonic acid, inhibited the rate of ( 14C)-acetate incorporation into lipids, possibly through increased cAMP levels or through end product inhibition of acetyl CoA carboxylase and fatty acid synthetase by long chain fatty acids. Indomethacin at concentrations of 50 ug/ml and above significantly reduced the basal rate of 14C-acetate incorporation into lipids, but it did not abolish the prolactin response. Since the prostaglandins, at low concentrations, have prolactin-like actions on RNA synthesis and ornithine decarboxylase activity, and since indomethacin attenuates the actions of prolactin on RNA synthesis, casein synthesis, and ornithine decarboxylase activity, it seems apparent that all of the metabolic actions of prolactin in the mammary gland may not occur via the same primary mechanism.

Alterations in myocardial RNA synthesis and RNA polymerase activity during normal growth.

The effect of normal growth (hypertrophy) on myocardial nuclear activity was investigated using male Wistar rats at 21, 50, and 100 days of age. Cardiac mass increased sevenfold during this age range. The concentration of RNA (mg X g-1) was the highest at 21 days and decreased 48% by 50 days of age and 68% after 100 days of development. RNA synthesis, corrected for alterations in the specific activity of the cytoplasmic nucleotide pool, was the highest at 21 days of age. After 50 days of growth, uridine incorporation was decreased fivefold. With continual growth (100 days), RNA synthesis was still reduced compared with the 21-day animals. RNA polymerase activity in myocyte nuclei showed little change in activity from 21 to 100 days of age. However, in the nonmyocyte fraction, RNA polymerase decreased threefold after 50 days of development. Collectively, these data suggest that the large decrease in myocardial RNA synthesis cannot be accounted for by a change in nuclear RNA polymerase activity and that an alteration in chromatin template capacity may be involved during this form of cardiac growth.

Inhibition of RNA and protein synthesis in isolated cerebellar cells by in vitro and in vivo methyl mercury

Cerebellar perikarya isolated from neonatal rats were exposed to 0-20 microM methyl mercury to simultaneously compare the effect on RNA and protein synthesis. Although 50% inhibition was found at approximately 8 microM for both [3H]uridine and [3H]phenylalanine incorporation, lower concentrations of methyl mercury produced 10-15% greater inhibition of RNA than protein synthesis. In vivo methyl mercury experiments also indicated a greater sensitivity of RNA synthesis in isolated cerebellar perikarya. The observed inhibition of RNA synthesis was not caused by a defect in cellular [3H]uridine uptake or by increased degradation of RNA. Both of these activities were altered by less than 10% at concentrations of methyl mercury that produced greater than 60% inhibition of RNA synthesis. Experiments showing that the specific activity of cerebellar cell RNA synthesis peaks and remains high between 4 and 10 d of age, whereas the specific activity of protein synthesis declines rapidly emphasize the potential importance of transcriptional perturbation in neonatal rats.

The activation of RNA synthesis by somatic nuclei injected into amphibian oocytes

Previous work has shown that nuclei of cultured cells or erythrocytes are transcriptionally activated when injected into the germinal vesicle of Xenopus oocytes. We now find that the total amount of stable RNA synthesized by an oocyte with injected nuclei is about twice that of an uninjected oocyte (∼15 ng/day). At least half of the RNA transcribed by the injected nuclei is low-molecular-weight RNA of discrete sizes, and is transcribed by RNA polymerase III. Part of this is attributable to a new class of gene (referred to as OAX) which makes a tanscript about 180 nucleotides long, and which is activated by at least 50-fold in somatic nuclei injected into oocytes. OAX RNA is not a degradation or processing variant of ribosomal or 5 S RNA, and is not coded by mitochondrial DNA. It enters oocyte cytoplasm, but turns over with a half-life of less than 3 hr, as judged by the culture of enucleate oocyte cytoplasms for several days during which 4 and 5 S RNAs are stable. OAX RNA synthesis is not detected by labelling cultured cells or their nuclei. The results emphasize the selectivity with which components of an oocyte germinal vesicle activate genes in injected somatic nuclei.

Nucleic acid aggregation geometry and the possible evolutionary origin of ribosomes and the genetic code

It is proposed that the primitive translational complex responsible for the evolutionary origin of the genetic code was an ordered RNA aggregate, stabilized not only by base-pairing, but also by the multivalent ionic interactions known to be responsible for bundle aggregation and condensation of DNA. A triplet code is the logical consequence of the structural symmetry and information coding capacity and efficiency of the proposed class of aggregates. The model requires rod-like adaptor, or primitive transfer RNA, molecules, and predicts that they were about 42 nucleotides in length. There are plausible reasons for preferring 5′ → 3′ reading of the message, and for favoring a primitive GNC code. It is further proposed that the primitive coding aggregate was the starting point for the evolution of ribosomes, and that the vestigial skeleton of the early translational complex may be discernible in present ribosomes. Amino acid specificity in coding interactions could have been generated spontaneously in a “bootstrap” process, given formation of an RNA aggregate in which RNA adaptors act as catalytic cofactors in the synthesis of peptides having appreciable catalytic activity for RNA synthesis and amino acid activation.

Denervation modulated changes in mouse skeletal muscle RNA concentration.

We have examined normal and denervated mouse skeletal muscle for histological evidence of changes in RNA concentration using acridine orange/nucleic acid fluorescence. In denervated muscle, cytoplasmic RNA concentration increases dramatically. The earliest increase is in the subsarcolemmal region, 7 days after denervation. By 28 days after denervation, the increase in RNA is distributed throughout the cytoplasm of the atrophic cells, non-atrophic cells show little or no increase. Prior to the increase in cytoplasmic RNA concentration, many myocyte nuclei exhibit enlarged nucleoli, indicative of active ribosomal RNA synthesis. Such active nucleoli are present throughout the 42 day study period. We conclude that both new RNA synthesis, and preferential accumulation of cytoplasmic RNA are prominent events in denervation atrophy. There is an ordered progression from nucleolar synthesis to subsarcolemmal accumulation to diffuse accumulation in atrophying cells. These observations complement biochemical findings of others which indicate an increase in both RNA and protein synthesis after denervation.

Number of transcribing RNA polymerase molecules and polyribonucleotide elongation rates in regenerating rat liver. Effect of cycloheximide treatment

Increased labelling of UMP internal residues is more marked in form I — than in form II — promoted RNA synthesis by regenerating liver nuclei in vitro and only form I RNA polymerase seems to enhance labelling of uridine in the 3′-termini. Increase in the number of transcribing polymerase I molecules and of their elongation rate is responsible for the activation of RNA synthesis — which affects essentially pre-ribosomal RNA synthesis — in regenerating livers. Pre-treatment of the rats with cycloheximide, at a dose which suppresses protein synthesis but not RNA synthesis in normal rats, prevents both effects and therefore the increase in RNA synthesis in regenerating liver.

RNA synthesis and RNA polymerase activity during natural cardiac hypertrophy

RNA synthesis and RNA polymerase activity during natural cardiac hypertrophy

Effect of 3-isobutyl-1-methylxanthine on prolactin actions on RNA synthesis, casein synthesis, lipid synthesis and ornithine decarboxylase activity in mouse mammary gland explants.

The effect of 3-isobutyl-1-methylxanthine (IBMX), an inhibitor of cyclic nucleotide phosphodiesterase, was tested on several actions of prolactin in cultured mouse mammary tissues. At concentrations of 0.5 mM and above, IBMX abolished the actions of prolactin on RNA and casein synthesis. IBMX by itself, stimulated ornithine decarboxylase (ODC) activity in a dose-response fashion; but the IBMX at concentrations up to 1 mM had no effect on the magnitude of the prolactin-stimulated ODC activity. IBMX inhibited in a dose-response fashion the rate of [14C]-acetate incorporation into lipids; however, prolactin stimulated lipid biosynthesis in the presence of IBMX concentrations of up to 1 mM.

Synchronous Alterations of RNA Synthesis and DNA-dependent RNA Polymerase Activities during Development of <italic>Dictyostelium discoideum</italic>

Synchronous Alterations of RNA Synthesis and DNA-dependent RNA Polymerase Activities during Development of <italic>Dictyostelium discoideum</italic>

Gametocytogenesis of Plasmodium falciparum in vitro: an electron microscopic study.

Plasmodium falciparum was grown in vitro in blood taken from naturally infected Gambian patients, and the development of the cultured sexual parasites was studied by light and electron microscopy. The young (Stage II and III) female gametocytes undergo a single cryptomitotic nuclear division. This division immediately follows the S phase which Sinden & Smalley (1979) have demonstrated in the Stage I and II gametocytes of both sexes. The male gametocytes, by contrast, do not undergo mitosis during their maturation period in the erythrocyte and thus remain polyploid. Hence the cell cycles of the male and female gametocytes differ significantly. The ultrastructural basis of the characteristic changes in shape of the developing gametocyte are shown to be due to the assembly and subsequent loss of components of the subpellicular membranous and microtubular cytoskeleton. Stage I-III gametocytes synthesize numerous ribosomes and endoplasmic reticulum. This correlates with the active synthesis of RNA and protein in these young parasites (Sinden & Smalley, 1979). The marked reduction in macromolecular synthesis in the mature parasites is paralleled by a reduction in cytoplasmic ribosome density in the male gametocyte only. In the female, however, this reduction in activity is correlated with the appearance of a nucleolus. These changes suggest that different mechanisms are being used to control RNA synthesis in the two sexes of gametocyte.

RNA synthesis and poly(adenosine diphosphoribosyl) synthetase activity in developing mouse testis.

RNA polymerase I and II and poly(ADP-ribosyl) synthetase activities were determined in isolated nuclei prepared from mouse testes at 1, 3, and 8 weeks after birth. RNA polymerase II and poly(ADP-ribosyl) synthetase activities increased with progression of spermatogenesis and age while RNA polymerase I activity decreased. The observed inverse relationship of RNA polymerase I and II parallels the shift in species of RNA, produced during spermatogenesis. Poly(ADP-ribosyl) synthetase activity increased with age, reaching a peak at 8 weeks. These enzymatic activities in aged mouse testis nuclei were equivalent to that of 8-week-old mice. Germ cells from the adult testis were separated by unit gravity velocity sedimentation. The enriched fractions containing pachytene and round spermatids possessed RNA polymerase and poly(ADP-ribosyl) synthetase activities. The present results suggest that transcriptional events during spermatogenesis may be modulated by changes in the activities of variants of RNA polymerase.