Engineering Ribonucleoside Triphosphate Specificity in a Thymidylyltransferase†

Biochemistry ◽  
2008 ◽  
Vol 47 (33) ◽  
pp. 8719-8725 ◽  
Author(s):  
David L. Jakeman ◽  
Jessica L. Young ◽  
Malcolm P. Huestis ◽  
Pauline Peltier ◽  
Richard Daniellou ◽  
...  
Keyword(s):  
Ribonucleoside Triphosphate

scholarly journals Initiation and elongation of polyribonucleotide chains on rat ventral-prostate chromatin transcribed by homologous ribonucleic acid polymerase B.

1977 ◽  
Vol 166 (2) ◽  
pp. 189-198 ◽  
Author(s):  
P Thomas ◽  
P Davies ◽  
K Griffiths
Keyword(s):  
Rna Polymerase ◽  
Rna Synthesis ◽  
Prostate Gland ◽  
Ventral Prostate ◽  
Control Of Gene Expression ◽  
Rat Ventral Prostate ◽  
Associated Proteins ◽  
Ribonucleoside Triphosphate ◽  
Androgenic Steroids ◽  
Transcriptional Process

The characteristics of initiation of RNA synthesis and the elongation of RNA chains on rat ventral-prostate chromatin by RNA polymerase B were investigated by two methods. 1. Initiation was carried out under low-salt conditions with three ribonucleoside triphosphates, and elongation was begun in the absence of reinitiation by the addition of the fourth ribonucleoside triphosphate and increasing the salt concentration. 2. Stable initiation complexes were formed by preincubation of enzyme with template at 37 degrees C, elongation was started by the addition of all four ribonucleoside triphosphates and reinitiation or spurious RNA synthesis was prevented by rifamycin AF/013. The latter method gave more reliable results. The dependence of those parameters on the androgenic status of the animal was studied. During the first 24h after castration, elongation was mainly affected, whereas after 72h a smaller number of initiation sites for RNA polymerase B on chromatin was evident. Considerable diurnal variations in the various parameters were observed. Changes in the relative concentrations of the chromatin-associated proteins were also observed after castration. In the rat ventral-prostate gland androgenic steroids may not only influence one stage of the transcriptional process, but may affect many factors involved in the control of gene expression.

Abstract 1440: Endothelial And Cardiomyocyte Toxicity Of Endogenously Produced Nucleoside: 4-pyridone-3-carboxamide-1-β-d-riboside

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Ewa M Slominska ◽  
Czeslawa Orlewska ◽  
Cesare M Terracciano ◽  
Ada H Yuen ◽  
Urszula Siedlecka ◽  
...  
Keyword(s):  
Renal Failure ◽  
Endothelial Cells ◽  
Chronic Renal Failure ◽  
Cell Line ◽  
Cell Function ◽  
Cell Types ◽  
Human Erythrocytes ◽  
Rat Hearts ◽  
Prolongation Of Action Potential ◽  
Ribonucleoside Triphosphate

Our recent studies identified hitherto unknown, naturally occurring nucleotide: 4-pyridone-3-carboxamide-1-β-D-ribonucleoside triphosphate (4PYTP). This nucleotide was found to accumulate in the erythrocytes of patients with chronic renal failure, a condition that precipitates heart failure and atherosclerosis. We found also increase in plasma concentration of nucleoside precursor of 4PYTP: 4-pyridone-3-carboxamide-1-β-D-ribonucleoside (4PYR) in patients with renal failure. This study was aimed to evaluate metabolism and potential toxic effect of this nucleoside in cardiomyocytes and endothelial cells. We used human erythrocytes isolated from peripheral blood, human endothelial cell line HMEC-1 cell line and cardiomyocytes isolated from rat hearts. Cells were incubated with chemically synthesized 4PYR. Metabolic assessments were performed with HPLC or liquid chromatography/mass spectrometry. We demonstrated formation of 4PYTP in human erythrocytes during incubation with 4PYR. We noted however, preferential accumulation of monophosphate of 4PYR (4PYMP) over 4PYTP. Concentration of 4PYMP increased in the erythrocytes from below 5 μM to 76.9±7.1, 254.7±13.9 and 674.3±34.3 μM after 6h incubation with 0.1, 0.3 and 1 mM 4PYR. 4PYMP progressively accumulated in the cultured endothelial cells during incubation with 4PYR up to 72 h. Cardiomyocytes were also shown to accumulate 4PYMP. In all cell types formation of 4PYR nucleotides was accompanied by decrease in cellular ATP concentration. Furthermore, treatment with 4PYR and formation of its nucleotides in endothelial cells reduced NO synthase pathway while in cardiac myocytes resulted in prolongation of action potential. We conclude that endogenously formed 4PYR is effectively metabolized to nucleotide derivatives in the erythrocytes, cardiomyocytes and endothelium. Depletion of ATP in these cells accompanying 4PYR metabolism and deleterious effects on cell function suggests that accumulation of 4PYR and its enhanced metabolism to nucleotides in endothelium and cardiomyocyted may contribute to cardiovascular pathology observed in chronic renal failure.

scholarly journals Ribonucleotides incorporated by the yeast mitochondrial DNA polymerase are not repaired

2017 ◽  
Vol 114 (47) ◽  
pp. 12466-12471 ◽  
Author(s):  
Paulina H. Wanrooij ◽  
Martin K. M. Engqvist ◽  
Josefin M. E. Forslund ◽  
Clara Navarrete ◽  
Anna Karin Nilsson ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Nuclear Dna ◽  
Excision Repair ◽  
Genome Replication ◽  
Dntp Pool ◽  
Frequent Exchange ◽  
Repair Mechanisms ◽  
Ribonucleoside Triphosphate ◽  
Deoxyribonucleoside Triphosphate ◽  
Mitochondrial Dna Polymerase

Incorporation of ribonucleotides into DNA during genome replication is a significant source of genomic instability. The frequency of ribonucleotides in DNA is determined by deoxyribonucleoside triphosphate/ribonucleoside triphosphate (dNTP/rNTP) ratios, by the ability of DNA polymerases to discriminate against ribonucleotides, and by the capacity of repair mechanisms to remove incorporated ribonucleotides. To simultaneously compare how the nuclear and mitochondrial genomes incorporate and remove ribonucleotides, we challenged these processes by changing the balance of cellular dNTPs. Using a collection of yeast strains with altered dNTP pools, we discovered an inverse relationship between the concentration of individual dNTPs and the amount of the corresponding ribonucleotides incorporated in mitochondrial DNA, while in nuclear DNA the ribonucleotide pattern was only altered in the absence of ribonucleotide excision repair. Our analysis uncovers major differences in ribonucleotide repair between the two genomes and provides concrete evidence that yeast mitochondria lack mechanisms for removal of ribonucleotides incorporated by the mtDNA polymerase. Furthermore, as cytosolic dNTP pool imbalances were transmitted equally well into the nucleus and the mitochondria, our results support a view of the cytosolic and mitochondrial dNTP pools in frequent exchange.

scholarly journals The relationship between the activity of DNA-dependent RNA polymerase I and the rate of synthesis of rRNA in hepatoma cells in culture

1981 ◽  
Vol 194 (1) ◽  
pp. 43-51 ◽  
Author(s):  
E A Thompson ◽  
R H Keith ◽  
A H Cavanaugh ◽  
K M Wood
Keyword(s):  
Steady State ◽  
Rna Polymerase ◽  
Cell Lines ◽  
Rna Polymerase I ◽  
28S Rrna ◽  
Pol I ◽  
Cell Enzyme ◽  
Ribonucleoside Triphosphate ◽  
Polymerase I ◽  
The Relationship

Cell culture lines were established from the transplantable mouse hepatomas H6 and H129. Both cell lines had a doubling time about 30 h when maintained in medium containing 5% foetal bovine serum. H6 cells contained about 3-4 times more DNA-dependent RNA polymerase I (Pol I; ribonucleoside triphosphate–RNA nucleotidyltransferase, EC 2.7.7.6) than did H129 cells. Moreover, the H6-cell enzyme was more heat-labile than that from H129 cells. Steady-state contents of 28S rRNA were measured in both cell lines. Exponentially growing cultures of H6 cells contained about 6.5pg of 28S rRNA/cell, and similar cultures of H129 cells contained about 5.8pg/cell. Stationary cultures of both cell lines contained about 2pg of 28S rRNA/cell. By two different techniques, the half-time for turnover of 28S rRNA was estimated to be 16-17h for both H6 and H129 cells. Knowing the turnover rate and the steady-state concentration, one may calculate that both H6 and H129 cells synthesize 28S rRNA at a rate of about 0.25 pg/h per cell. The amount of template-bound Pol I activity was similar in nuclei isolated from H6 and H129 cell cultures. These data indicate that, although H6 cells contained 3-4 times more Pol I than did H129 cells, both cell lines synthesized rRNA at about the same rate.

The cobamide-dependent ribonucleoside triphosphate reductase of lactobacilli

1965 ◽  
Vol 20 (4) ◽  
pp. 439-445 ◽  
Author(s):  
R.L. Blakley ◽  
R.K. Ghambeer ◽  
P.F. Nixon ◽  
E. Vitols
Keyword(s):  
Ribonucleoside Triphosphate

scholarly journals Reverse transcriptional mutagenesis induced by ribonucleoside triphosphate analogue, PTP and its availability for anti-HIV-1 therapy

2001 ◽  
Vol 1 (1) ◽  
pp. 255-256
Author(s):  
K. Moriyama ◽  
T. Okada ◽  
D. Loakes ◽  
N. Miyano-Kurosaki ◽  
H. Takaku ◽  
...  
Keyword(s):  
Transcriptional Mutagenesis ◽  
Ribonucleoside Triphosphate ◽  
Anti Hiv ◽  
Hiv 1
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