Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We evaluated the effects of natural purine and pyrimidine nucleosides on protection from or reversal of 3'-azido-3'-deoxythymidine (AZT) cytotoxicity in human bone marrow progenitor cells by using clonogenic assays. The selectivity of the "protection" or "rescue" agents was examined in evaluating the antiretroviral activity of AZT in combination with these modulating agents and of AZT alone. Following exposure of human granulocyte-macrophage progenitor cells for 2 h to 5 microM AZT (70% inhibitory concentration), increasing concentrations of potential rescue agents were added. Cells were cultured, and colony formation was assessed after 14 days. At concentrations of up to 50 microM no natural 2'-deoxynucleosides, including thymidine, were able to reverse the toxic effects of AZT. Dose-dependent reversal was observed with uridine and cytidine, and essentially complete reversal was achieved with 50 microM uridine. In the protection studies, 100 microM thymidine almost completely antagonized the inhibition of granulocyte-macrophage colony formation produced by 1 microM AZT (50% inhibitory concentration), and 50 microM uridine effected 60% protection against a toxic concentration of AZT (5 microM) (70% inhibitory concentration). The antiretroviral activity of AZT in human peripheral blood mononuclear cells, assessed by revere transcriptase assays, was substantially decreased in the presence of thymidine, whereas no impairment of suppression of viral replication was observed in the presence of uridine in combination with AZT at a molar ratio (uridine/AZT) as high as 10,000. This demonstration of the capacity of uridine to selectively rescue human bone marrow progenitor cells from the cytotoxicity of AZT suggests that use of uridine rescue regimen with AZT may have potential therapeutic benefit in the treatment of acquired immunodeficiency syndrome.
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PMID:Uridine reverses the toxicity of 3'-azido-3'-deoxythymidine in normal human granulocyte-macrophage progenitor cells in vitro without impairment of antiretroviral activity. 319 Feb 1

When salmon total DNA was transcribed in a HeLa cell extract, a discrete 6S RNA was found to be synthesized by RNA polymerase III. We isolated several phage clones containing the 6S RNA gene from a salmon genomic library and determined the sequences of two representative clones. The 5' part of the gene showed remarkable sequence homology with the lysine tRNA1 molecule. This homology extended to secondary structures, and the numbers of nucleotides in the stem and loop structures in the 6S RNA were the same as those in lysine tRNA1. Further, the pseudouridylic acid residues synthesized by HeLa pseudouridylate synthase(s) were determined to be at uridine-27 and uridine-55, which are the positions of these modified nucleosides in lysine tRNA1. These results strongly suggest that the lysine tRNA1 gene is a progenitor of the highly repetitive and transcribable sequences in the salmon genome.
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PMID:Gene for lysine tRNA1 may be a progenitor of the highly repetitive and transcribable sequences present in the salmon genome. 345 71

We have used the gene for tRNAMet1 as a hybridization probe to measure the production of tRNAMet1 in the Friend erythroleukemia cell. In this cell, the relative concentration of tRNAMet1 (i.e., the percentage of total steady-state tRNA representing tRNAMet1) is 1.60 +/- 0.18. To study the relative synthesis of tRNAMet1, cells were labeled in vivo with [3H]uridine for periods ranging from 4 to 24 h, and the tRNA was isolated. The fraction of newly-synthesized tRNA representing tRNAMet1 (1.72% +/- 0.11) does not change when different in vivo labeling times are used. This value is similar to the relative concentration of tRNAMet1 in the older steady-state tRNA (1.61% +/- 0.18). The similar relative synthesis values using different labeling times, plus evidence presented that the total tRNA population decays homogeneously (t 1/2 = 110 h) indicate that tRNAMet1 has a cytoplasmic stability similar to the general tRNA population, and that its concentration relative to the tRNA population is established within the nucleus or soon after exiting the nucleus. Measurements of the synthesis of tRNAMet1 in isolated nuclei, relative to the synthesis of total RNA polymerase III transcripts, showed that this relative synthesis (0.291% +/- 0.017) is only 17% of the relative concentration of tRNAMet1 in the cytoplasm, which may reflect the presence of sequences other than tRNA in total nuclear polymerase III transcripts.
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PMID:The measurement of the production of tRNAMet1 in the Friend erythroleukemia cell. 346 48

In order to extend to the immune system previous findings that there is an age-related loss of hybridizability of the genes for ribosomal RNA (rRNA) in several tissues of mice, dogs and humans, we have investigated the function of the genes for rRNA in human T lymphocytes. These cells were chosen because they show a substantial decline in function with age, greater than that of other components of the immune system. rRNA synthesis was determined by measuring tritiated-UTP incorporation into acid precipitable counts as a result of the action of RNA polymerase I in nuclei isolated from phytohemagglutinin (PHA) stimulated peripheral-blood lymphocytes from 24 young adult and old human donors. The number of PHA-responsive cells from each donor was determined by counting grains in autoradiographs after a pulse of tritiated-uridine had been administered to them. The aggregate PHA induced synthesis of rRNA in the cultures decreased as a function of the age of the donor. However, the number of PHA-responsive cells also dropped with age. When the data are normalized for the number of PHA-responsive cells in each culture, it appears that rRNA synthesis per PHA-responding cell does not significantly decline with age, even though there is a suggestion of a decrease after corrections are made. On the average, differences between individuals of the same age group were as great or greater than age-related differences.
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PMID:Age-related RNA polymerase I activity in isolated nuclei of PHA stimulated human lymphocytes. 348 68

Effects of cyclosporin A (CsA) on rRNA synthesis in vivo and in vitro were studied using lymphosarcoma P1798 in culture. Pulse labeling with [3H]uridine indicated that treatment of P1798 cells with 1 microgram/ml of CsA for 24-h reduced rRNA levels by 50-60%, whereas rRNA levels of cells rescued from CsA and grown for 24 h were similar to those of controls. Transcription experiments using nuclei from control, treated, and rescued cells indicated that the reduction in rRNA synthesis in treated cells was due to reversible inhibition of transcription of rDNA. Transcription studies in vitro indicated that S100 extracts from CsA-treated cells were unable to carry out faithful transcription of cloned mouse rDNA, even though RNA polymerase I levels of control and treated cell extracts were similar. Mixing experiments indicated that the inability of the CsA-treated cell extract to transcribe cloned rDNA in vitro was not due to the presence of inhibitor(s) or nuclease(s) in such extracts. Supplementation of CsA-treated cell extract with partially or highly purified preparations of a transcription initiation factor for RNA polymerase I, obtained from control cell extracts, conferred transcriptional ability on the CsA-treated cell extract. Extracts from cells treated with cyclosporin H, an inactive analogue of CsA, faithfully transcribed rDNA, indicating the specificity of CsA action. These data indicate that CsA-treated cells lack the ability to initiate rDNA transcription in vivo and in vitro, due to specific, reversible reduction in the amount or activity of transcription factor IC. Significance of these results in understanding the mechanisms of the lymphostatic activity of CsA is discussed.
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PMID:Cyclosporin A inhibits rDNA transcription in lymphosarcoma P1798 cells. 368 Feb 46

The self-excision of a 413-base intervening sequence of the 26S rRNA of Tetrahymena thermophila has been investigated using phosphorothioate-substituted RNA. Transcripts containing this intron were prepared by T7 RNA polymerase-catalyzed polymerisation using a M13 mICE10 vector in the presence of various nucleoside alpha-thiotriphosphate analogues. Wild-type transcripts incorporating phosphorothioates 5' to adenosine or uridine were inactive, whereas incorporation 5' to cytidine or guanosine allowed splicing. The first two substitutions place phosphorothioates inter alia at the 5' and 3' splice sites respectively. Mutagenesis at either site allowed phosphorothioate substitution 5' to guanosine at each splice site. This did not block splicing, suggesting that substitution at internal sites within the intron has more effect.
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PMID:Self-splicing of Tetrahymena rRNA can proceed with phosphorothioate substitution at the splice sites. 369 41

The purpose of this study was to examine the effects of actinomycin D on localization of acridine orange (AO) binding to DNA in rat astrocytoma C6 cells and to discuss briefly the significance of AO chromatin interaction products. Actinomycin D markedly inhibited 3H-uridine incorporation into RNA and the percentage of AO positive cells was reduced to approximately 40% of that of the untreated control cells, whereas no distinct decrease of 3H-thymidine incorporation was induced by actinomycin D. Electron microscopic radioautography combined with the AO ultracytochemistry revealed that silver grains indicating binding of 3H-actinomycin D are located mostly over the euchromatin portion near the segregated nucleolus and heterochromatin and that no or only a few AO chromatin complex was found in nuclei labeled heavily with 3H-actinomycin D. These results of the present study together with the results of the previous studies seem to indicate that AO might selectively bind to active or derepressed DNA template sites for DNA dependent RNA polymerase in the euchromatin portion of the cell nucleus.
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PMID:Effects of actinomycin D on localization of acridine orange chromatin interaction complex in rat astrocytoma C6 cells. 371 93

Micronuclei have been induced by colchicine in rat kangaroo (Potorous tridactylis) PtK1 cells. The synthesis of RNA was investigated both in isolated micronuclei by quantifying RNA polymerase activities at different ionic strengths with or without inhibitors, and in micronucleated cells by radioautography after [3H]uridine pulse labeling. In vitro transcription shows that isolated micronuclei are able to take up [3H]UTP. The rate curves of incorporation are close to those of isolated diploid nuclei, though the level of incorporation was relatively lower (65-70%) than control nuclei. This indicates that micronuclei react to the ionic environment and to inhibitors in the same manner as described for many species of isolated diploid nuclei. The labelling distributions plotted from radioautographs show that micronuclei were able to efficiently incorporate the hot precursor. Furthermore, for short pulses there is no homogeneity in the labelling density among the different micronuclei and there is no correlation between the labelling intensity and the size of micronuclei. After 60-min pulse time, there is an enhanced uptake of [3H]uridine and all the micronuclei exhibit considerable labelling, although less than control cells. Thus, the micronuclei exhibit some characteristic RNA transcriptional activity in situ as well as after isolation. This material should be a particular interesting model with which to study the physiological activity and the role of each individual interphasic chromosome.
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PMID:RNA polymerase activity in PtK1 micronuclei containing individual chromosomes. An in vitro and in situ study. 381 15

The growth of MCF-7 cells was arrested by 24 h of isoleucine deprivation. Following replenishment of the medium, the incorporation of uridine and thymidine into trichloroacetic acid-precipitable material began to increase slowly and gradually rose to the level of cycling cells. The addition of 5 X 10(-9) M estradiol to growth-arrested cells dramatically shortened the time of onset of macromolecular synthesis and increased the overall amount of precursor incorporation 2- to 4-fold over the level obtained by arrested control cells. The increase in uridine incorporation preceded the increase in thymidine incorporation by 6 h. Inhibition of protein synthesis with cycloheximide blocked the recovery of macromolecular synthesis in both control and estrogen-treated cells. Actinomycin D was ineffective in blocking the estrogen-stimulated recovery of macromolecular synthesis at concentrations known to inhibit pre-rRNA synthesis (10(-8) M). At higher concentrations, uridine and thymidine incorporation were inhibited in a dose-dependent manner. Inhibition of RNA polymerase II activity with alpha-amanitin similarly blocked both the recovery of the cells from isoleucine starvation and the potentiation of this by estradiol. Dihydrofolate reductase and thymidine kinase activities are both stimulated by estradiol in MCF-7 cells. In cycling cells, estrogen stimulates a 2-fold increase in their messenger RNAs (mRNAs) within 24 h. The level of dihydrofolate reductase mRNA is unaffected by isoleucine starvation, and estrogen caused no change in dihydrofolate reductase mRNA levels over a 24-h period following reversal of growth arrest. Similar results were observed for the 600-nucleotide pS2 mRNA that has been identified as an estrogen-induced RNA in MCF-7 cells. In contrast, thymidine kinase mRNA was found to be increased by estrogen at 24 h, but not at 12 h, following reversal of growth arrest. This increase correlates with increases in thymidine, but not uridine incorporation. These data indicate that the estrogen-stimulated increase in thymidine incorporation following release from growth arrest is dependent on new RNA synthesis. However, the hormone did not increase the levels of three estrogen-regulated mRNAs coordinately with the increases observed in uridine incorporation.
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PMID:Relationship between the expression of estrogen-regulated genes and estrogen-stimulated proliferation of MCF-7 mammary tumor cells. 398 99

The incorporation of 3H-uridine in different regions of polytene chromosomes in live cells of the Drosophila melanogaster salivary glands was compared with the incorporation of 3H-UTP in the same regions under the incubation of cytological preparations of these chromosomes with the E. coli RNA polymerase. The label distribution by regions was compared with the DNA content in them. Individual regions of chromosomes differ by 3H-uridine incorporation in live cells to a much greater extent than by 3H-UTP incorporation in vitro under the incubation with a non-homologous enzyme. RNA synthesis in an exogenous enzyme depends on the DNA content in different chromosome regions to a much greater extent than RNA synthesis in vivo. The correlation of label distribution after 3H-uridine incorporation in live cells and after RNA synthesis in vitro on the preparations by the bacterial RNA polymerase is, correspondingly, very low. This enzyme forms, however, RNA's on puffs 2-3 times more actively than on the same regions in non-puffing state but this difference is dozens of times greater in live cells. RNA synthesis in vitro is, thus, non-specific and does not correspond practically to the intensity of RNA synthesis on the same chromosome regions in live cells. At the same time, as in live cells, the E. coli enzyme synthesizes twice more RNA on the single X-chromosome of males (1X2A) than on each of X-chromosomes of diploid (2X2A) and triploid (3X3A) females or superfemales (3X2A), whereas in intersexes (2X3A) X-chromosomes display intermediate template activity. Thus, RNA synthesis by a heterologous enzyme in vitro does not differ by this index from the synthesis in live cells. It is suggested that differences in the template activity of X-chromosomes in vitro depending on the sex index (X : A) are due to different degree of DNP condensation in these chromosomes. In spite of differences in the degree of condensation, the male X-chromosome binds on the fixed preparation approximately the same amount of thymus histone F1 carrying fluorochrome as each of two female X-chromosomes. Hence, there is no sharp difference between the male and female X-chromosomes by the number and length of DNA regions accessible for interaction with exogenous proteins. On the basis of the data obtained, a hypothesis about two levels and, respectively, two mechanisms of control gene activity in animal chromosomes is considered. The first mechanism is, supposedly, based on decondensation of DNP appears to result in that the same proteins-regulators in the same amount activate corresponding genes in X-chromosome in males twice more strongly than in females.
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PMID:[A comparison of bacterial RNA-polymerase RNA synthesis on polytene Drosophila chromosomes with transcription in living cells]. 421 55


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