Metabolism of [5-3H]uridine in mouse and specificity for labeling of liver ribonucleic acid

Yngner, Torsten; Petersen, Inga; Lewan, Lillemor

doi:10.1016/0020-711x(77)90011-8

Cited by 18 publications

(6 citation statements)

References 30 publications

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“…The combined fractions from the Partisil-SAX column could be freeze-dried and rechromatographed without desalting, because they contained low concentrations of phosphate. We also collected fractions con [17][18][19][20] and attributable to high uridine catabolism in the liver [17,21,22]. As reported by Moyer et al [20] and Dahnke [23], [3H]cytidine was much better incorporated into soluble nucleotides and RNA.…”

Section: Resultsmentioning

confidence: 99%

Pyrimidine metabolism and sugar nucleotide synthesis in rat liver

Rijcken

Hooghwinkel

Ferwerda

1990

Biochemical Journal

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With radioactive precursors, the labelling kinetics of the soluble pyrimidine nucleotides and of RNA were measured in rat liver to determine the contribution of the metabolic flows through synthesis de novo and the salvage pathway. To separate and quantify all pyrimidine nucleotides, an h.p.l.c. technique was developed using anion-exchange chromatography and reversed-phase chromatography. The concentrations of cytidine nucleotides were in the range of 30-45 nmol/g wet weight, and the concentrations of the uridine phosphates and of the UDP-sugars were approx. 6 and 20 times higher respectively. After a single injection of [14C]orotic acid and of [3H]cytidine, the specific radioactivities were determined as a function of time. The 1'C/3H ratio was calculated and gave a good indication of the involvement of the different flows. It could be concluded that UTP derived from synthesis de novo and from the salvage pathway is not completely mixed before being utilized. The flow of the salvage pathway is relatively more directed to RNA synthesis in the nucleus and that of synthesis de novo to cytoplasmic processes. For CTP it could also be concluded that the flow of the salvage pathway was relatively more directed to RNA synthesis in the nucleus. Because of the nuclear localization of the enzyme CMP-NeuAc (N-acetylneuraminate) synthase, special attention was paid to CMP-NeuAc. However, a conclusion about a location about the synthesis of CMP-NeuAc could not unequivocally be drawn, because of the small differences in 14C/3H ratio and the different values for the CDP-lipids.

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Section: Resultsmentioning

confidence: 99%

Pyrimidine metabolism and sugar nucleotide synthesis in rat liver

Rijcken

Hooghwinkel

Ferwerda

1990

Biochemical Journal

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“…It has been shown by biochemical method that RNA in various tissues may be derived from labeled uridine (22)(23)(24)(25)(26)(27)(28) and labeled orotic acid (22,23,29,30) . In the small intestine of the rat, however, Raisonnier et al (31) reported incorporation of ['°C]uridine, but of only traces of [ t 'C]orotic acid, which implies that orotic acid was not a suitable precursor for studies of RNA in rat small intestine .…”

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confidence: 99%

Radioautographic visualization of differences in the pattern of [3H]uridine and [3H]orotic acid incorporation into the RNA of migrating columnar cells in the rat small intestine.

Uddin¹,

Altmann²,

Leblond³

1984

The Journal of Cell Biology

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The epithelium of rat small intestine was radioautographed to examine whether RNA is synthesized by the salvage pathway as shown after [3H]uridine injection or by the de novo pathway as shown after [3H]orotic acid injection. The two modes of RNA synthesis were thus investigated during the migration of columnar cells from crypt base to villus top, and the rate of synthesis was assessed by counting silver grains over the nucleolus and nucleoplasm at six levels along the duodenal epithelium--that is, in the base, mid, and top regions of the crypts and in the base, mid, and top regions of the villi. Concomitant biochemical analyses established that, after injection of either [5- 3H]uridine or [5-3H]orotic acid: (a) buffered glutaraldehyde fixative was as effective as perchloric acid or trichloracetic acid in insolubilizing the nucleic acids of rat small intestine; (b) a major fraction of the nucleic acid label was in RNA, that is, 91% after [3H]uridine and 72% after [3H]orotic acid, with the rest in DNA; and (c) a substantial fraction of the RNA label was in poly A+ RNA (presumed to be messenger RNA). In radioautographs of duodenum prepared after [3H] uridine injection, the count of silver grains was high over nucleolus and nucleoplasm in crypt base cells and gradually decreased at the upper levels up to the villus base. In the rest of the villus, the grain count over the nucleolus was negligible, while over the nucleoplasm it was low but significant. After [3H]-orotic acid injection, the number of silver grains over the nucleolus was negligible at all levels, whereas over the nucleoplasm the number was low in crypt cells, but high in villus cells with a peak in mid villus. The interpretation is that, except for a small amount of label incorporated into DNA from either precursor by crypt cells, the bulk of the label is incorporated into RNA as follows. In the crypts, cells make almost exclusive use of uridine, that is, of the salvage pathway, for the synthesis of ribosomal RNA in the nucleolus and of messenger and transfer RNA in the nucleoplasm. However, when cells pass from crypt to villus, they mainly utilize orotic acid--i.e., the de novo pathway--for the synthesis of messenger and transfer RNA within the nucleoplasm.

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“…Radioactively labelled orotate and uridine have been used interchangeably to label uridine nucleotides and to study RNA synthesis in vivo and in vitro in animal cells or tissues. The degree of incorporation of these precursors into RNA varies widely for different tissues in the same animal (Witschi, 1972;Lewan et al, 1975;Yngner et al, 1977). The predominant incorporation of one over the other has largely determined the selection of the precursor to be used in studying a particular tissue or cell type.…”

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confidence: 99%

“…The labelling characteristics and turnover rates of different classes of RNA have been extensively studied in the kidney (Hill, 1975;Melvin et al, 1976;Ouellette & Malt, 1976). The kidney efficiently utilizes both exogenous uridine and orotate for RNA synthesis (Lewan et al, 1975;Yngner et al, 1977), but there are marked differences in the relative uptakes of these precursors in both the medullary and cortical regions (Toback et al, 1974a,b;Liberti & Kline, 1974). These differences in uptake between cortex and medulla would seem to preclude the useful study of pyrimidine metabolism in the whole organ.…”

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confidence: 99%

Incorporation of exogenous precursors into uridine and ribonucleic acid. Nucleotide compartmentation in the renal cortex in vivo

Cortés

Levin

Dumler

et al. 1979

Biochemical Journal

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The possibility of compartmentation of UTP in vivo was investigated in the renal cortex of unanaesthetized rats. In addition, liver and spleen were studied in order to compare tissues with different utilization of precursors for pyrimidine nucleotide synthesis. After continuous 2h infusions of [(3)H]uridine or [(3)H]orotate, their incorporation into UTP, UDP-sugars and RNA was quantified. Rates of RNA synthesis were calculated by dividing the incorporation of precursor into RNA by the average specific radioactivity of the UTP pool. Although similar RNA-synthesis rates might have been expected with the two precursors, higher rates were found with uridine than with orotate. The relative incorporation into UDP-sugars of these precursors was also different. Similar results were obtained in the liver. In the spleen, equal amounts of both precursors were incorporated into UTP, but [(3)H]orotate incorporation did not lead to labelling of RNA. To evaluate the heterogeneity of cells with respect to the metabolism of pyrimidines, precursor incorporation was studied in isolated glomeruli and by radioautography. Incorporation into glomeruli was qualitatively similar to but quantitatively different from results in the renal cortex. Although there is obvious tissue heterogeneity, compartmentation of UTP pools is the most credible explanation for the results obtained with the renal cortex and liver. Consequently RNA and UDP-sugars may originate from two different UTP pools. Tissue heterogeneity is the likely explanation for the results obtained in the spleen. Studies of synthesis of pyrimidine and RNA, particularly in relation to growth and regeneration, must take into consideration the precursor used, the apparent existence of UTP compartmentation and the degree of cellular heterogeneity.

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Metabolism of [5-3H]uridine in mouse and specificity for labeling of liver ribonucleic acid

Cited by 18 publications

References 30 publications

Pyrimidine metabolism and sugar nucleotide synthesis in rat liver

Pyrimidine metabolism and sugar nucleotide synthesis in rat liver

Radioautographic visualization of differences in the pattern of [3H]uridine and [3H]orotic acid incorporation into the RNA of migrating columnar cells in the rat small intestine.

Incorporation of exogenous precursors into uridine and ribonucleic acid. Nucleotide compartmentation in the renal cortex in vivo

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