Asymmetric distribution of exogenous thymidine among pyrimidine isostichs suggests compartmentalization of replicative DNA synthesis during regeneration in rat liver

Kizer, Donald E.; Ringer, David P.; Howell, Boyd A.

doi:10.1016/0167-4781(83)90088-x

Cited by 3 publications

(2 citation statements)

References 41 publications

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“…The incorporation for precursors was essentially linear through 22 h, slowed at 22-30 h, and leveled by 40-48 h. All culture periods were terminated by decanting the culture medium and immediately adding 25 ml of a solution containing 0.15 M NaC1, 0.1 M EDTA, 20 mg/ml of SDS, and nuclei from 40 g of rat liver. This mixture was heated 10 min at 60°C, and DNA was isolated as described [13]. DNA concentrations were based on a colorimetric assay following the method of Burton [14].…”

Section: Dna Labeling and Isolationmentioning

confidence: 99%

Assessment of salvage pathways utilized for incorporation of exogenous pyrimidine nucleosides into DNA of guinea pig lymphocytes stimulated by Con A

et al. 1987

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The organization of specific pyrimidine pathways to channel various nucleoside precursors into DNA is poorly understood. We show that concanavalin A-stimulated guinea pig lymphocytes incorporate [3H]dThd, pH]dCyd, [3H]dUrd, [3H]Cyd and [aH]Urd into DNA-thymines and DNA-cytosines in a highly conserved distribution pattern. DNA-thymines were labeled only by dThd and dUrd, while DNA-cytosines were labeled only by dCyd, Cyd and Urd. The kinetics for the incorporation of the [3H]nucleosides were essentially identical, indicating equivalent abilities to measure DNA synthesis. Pyrazofurin inhibition of the pyrimidine de novo synthetic pathway inhibited cell proliferation and the levels of [3H]nucleoside incorporation by approx. 50%, but did not alter restricted distribution of the [3H]nucleosides among DNA-thymines and DNAcytosines. These findings indicate the absence of Cyd and dCMP deaminase salvage pathways and suggest either subcellular compartmentalization or differential regulation of ribonucleoside diphosphoreductase which permits reduction of CDP but not UDP.

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Section: Dna Labeling and Isolationmentioning

confidence: 99%

Assessment of salvage pathways utilized for incorporation of exogenous pyrimidine nucleosides into DNA of guinea pig lymphocytes stimulated by Con A

et al. 1987

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“…Isolation of DNA and Pyrimidine Tracts. Tissues were homogenized, nuclear pellets were isolated by centrifugation, and DNA was isolated as previously described (Kizer et al, 1983). DNA was depurinated with diphenylamine in formic acid, and the liberated pyrimidine tracts were separated according to the number of contiguous pyrimidine moieties per 0006-2960/85/0424-7498S01.50/0 © 1985 American Chemical Society tract by elution from DEAE-cellulose columns (Cerny et al, 1969;Spencer et al, 1969;Lieberman et al, 1971).…”

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confidence: 99%

Distribution patterns for 5-methylcytosine among apurinic DNAs from several sources

Ringer¹,

Howell²,

Beck³

et al. 1985

Biochemistry

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Purified DNA from the liver of rats, mice, rabbits, and guinea pigs, from guinea pig lymph nodes, from hyperplastic nodules induced in rat liver by feeding with 2-(acetylamino)fluorene, and from Escherichia coli cells was made apurinic by reaction with diphenylamine. After chromatographic separation of pyrimidine tracts (isostichs or isoplyths) according to the number of contiguous pyrimidines, semilog plots of tract frequency vs. the number of contiguous pyrimidines were linear, plots for DNA from several sources differed from one another, and all deviated significantly from randomness. Similar semilog plots for coding sequences among 60 mammalian genomes or 28 rat tissue genomes were intermediate among slopes for isolated DNA. Individual isostichs were hydrolyzed, and their constituent pyrimidine bases were analyzed by high-pressure liquid chromatography. Among isostichs from isolated DNAs, the distribution of Thy and Cyt contents differed markedly from the distribution of 5-methylcytosine (5-Me-Cyt); e.g., although isostich 1 contained 45-49% of 5-Me-Cyt, amounts of Thy or Cyt did not exceed 25%. Semilog plots of normalized values for tract frequency or the content of 5-Me-Cyt vs. isostich number were essentially superimposable; thus, among the first five pyrimidine tracts of a particular tissue or E. coli DNA, the number of tracts per 5-Me-Cyt moiety was essentially constant. The data showed that 5-Me-Cyt and/or dCyd-dGuo dinucleotides have a distribution throughout DNA structure that superimposes the distribution of pyrimidine tract frequency and suggests that regulatory 5-Me-Cyt moieties are principally located at 3' termini of pyrimidine tracts.

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Alteration in de novo pyrimidine biosynthesis during uridine reversal of pyrazofurin‐inhibited dna synthesis

Ringer

Howell

Etheredge

1991

J. Biochem. Toxicol.

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Pyrazofurin, a pyrimidine nucleoside analogue with antineoplastic activity, inhibits cell proliferation and DNA synthesis in cells by inhibiting uridine 5'-phosphate (UMP) synthase. It has been previously shown in concanavalin A (con A)-stimulated guinea pig lymphocytes (23) that pyrazofurin-inhibited DNA synthesis could be selectively reversed by exogenous uridine (Urd). In this report, we have examined possible mechanisms for the Urd reversal with experiments that determine the ability of exogenous Urd to (a) interfere with either the intracellular transport of pyrazofurin, or the conversion of pyrazofurin to its intracellularly active form, pyrazofurin-5'-phosphate; (b) reverse the pyrazofurin block of [14C]orotic acid incorporation into DNA; and (c) alter the pattern of exogenous [3H]Urd incorporation into DNA-thymine (DNA-Thy) and DNA-cytosine (DNA-Cyt) during pyrazofurin inhibition of pyrimidine de novo biosynthesis. The results of these experiments showed that Urd reversal does not occur through altered pyrazofurin transport or intracellular conversion to pyrazofurin-5'-phosphate, nor does it alter the distribution of [3H]Urd in DNA-Thy and DNA-Cyt. Instead, these findings indicate that the primary mechanism for exogenous Urd reversal of pyrazofurin inhibition of DNA synthesis involves the reversal of pyrazofurin inhibition of UMP synthase, thus restoring orotic acid incorporation into lymphocyte DNA through the pyrimidine de novo pathway.

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Asymmetric distribution of exogenous thymidine among pyrimidine isostichs suggests compartmentalization of replicative DNA synthesis during regeneration in rat liver

Cited by 3 publications

References 41 publications

Assessment of salvage pathways utilized for incorporation of exogenous pyrimidine nucleosides into DNA of guinea pig lymphocytes stimulated by Con A

Assessment of salvage pathways utilized for incorporation of exogenous pyrimidine nucleosides into DNA of guinea pig lymphocytes stimulated by Con A

Distribution patterns for 5-methylcytosine among apurinic DNAs from several sources

Alteration in de novo pyrimidine biosynthesis during uridine reversal of pyrazofurin‐inhibited dna synthesis

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