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DETERMINATION OF RATES OF DNA SYNTHESIS IN CULTURED MAMMALIAN CELL POPULATIONS

In cultures of a murine mastocytoma, endogenous synthesis of thymidine phosphates, as determined by the incorporation of [(3)H]deoxyuridine into DNA, was reduced within 15 min to less than 3% of control values by the addition of amethopterin (10 µM) in combination with hypoxanthine and glycine. If [...

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Autores principales: Siegers, M. P., Schaer, J. C., Hirsiger, H., Schindler, R.
Formato: Texto
Lenguaje:English
Publicado: The Rockefeller University Press 1974
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2109404/
https://www.ncbi.nlm.nih.gov/pubmed/4214821
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author Siegers, M. P.
Schaer, J. C.
Hirsiger, H.
Schindler, R.
author_facet Siegers, M. P.
Schaer, J. C.
Hirsiger, H.
Schindler, R.
author_sort Siegers, M. P.
collection PubMed
description In cultures of a murine mastocytoma, endogenous synthesis of thymidine phosphates, as determined by the incorporation of [(3)H]deoxyuridine into DNA, was reduced within 15 min to less than 3% of control values by the addition of amethopterin (10 µM) in combination with hypoxanthine and glycine. If [(3)H]thymidine and unlabeled thymidine were added simultaneously with amethopterin, the increase with time of radioactivity in cellular DNA was linear at least between 30 and 90 min, while radioactivity in the acid-soluble nucleotide fraction remained constant during this time interval, indicating that intracellular thymidine nucleotides had the same specific activity as exogenously supplied [(3)H]thymidine. This permitted calculation of the amount of thymidine incorporated per hour into DNA of 10(6) cells. In conjunction with the base composition of mouse DNA, these results were used to calculate rates of DNA synthesis. Cell proliferation rate, cell cycle time, and the duration of the S period were not affected to any appreciable extent by the addition of amethopterin and thymidine. Rates of DNA synthesis, as derived from thymidine incorporation rates, were in good agreement with those derived from the measured mean DNA content of exponentially multiplying cells and rates of cell proliferation.
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spelling pubmed-21094042008-05-01 DETERMINATION OF RATES OF DNA SYNTHESIS IN CULTURED MAMMALIAN CELL POPULATIONS Siegers, M. P. Schaer, J. C. Hirsiger, H. Schindler, R. J Cell Biol Article In cultures of a murine mastocytoma, endogenous synthesis of thymidine phosphates, as determined by the incorporation of [(3)H]deoxyuridine into DNA, was reduced within 15 min to less than 3% of control values by the addition of amethopterin (10 µM) in combination with hypoxanthine and glycine. If [(3)H]thymidine and unlabeled thymidine were added simultaneously with amethopterin, the increase with time of radioactivity in cellular DNA was linear at least between 30 and 90 min, while radioactivity in the acid-soluble nucleotide fraction remained constant during this time interval, indicating that intracellular thymidine nucleotides had the same specific activity as exogenously supplied [(3)H]thymidine. This permitted calculation of the amount of thymidine incorporated per hour into DNA of 10(6) cells. In conjunction with the base composition of mouse DNA, these results were used to calculate rates of DNA synthesis. Cell proliferation rate, cell cycle time, and the duration of the S period were not affected to any appreciable extent by the addition of amethopterin and thymidine. Rates of DNA synthesis, as derived from thymidine incorporation rates, were in good agreement with those derived from the measured mean DNA content of exponentially multiplying cells and rates of cell proliferation. The Rockefeller University Press 1974-08-01 /pmc/articles/PMC2109404/ /pubmed/4214821 Text en Copyright © 1974 by The Rockefeller University Press This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/4.0/).
spellingShingle Article
Siegers, M. P.
Schaer, J. C.
Hirsiger, H.
Schindler, R.
DETERMINATION OF RATES OF DNA SYNTHESIS IN CULTURED MAMMALIAN CELL POPULATIONS
title DETERMINATION OF RATES OF DNA SYNTHESIS IN CULTURED MAMMALIAN CELL POPULATIONS
title_full DETERMINATION OF RATES OF DNA SYNTHESIS IN CULTURED MAMMALIAN CELL POPULATIONS
title_fullStr DETERMINATION OF RATES OF DNA SYNTHESIS IN CULTURED MAMMALIAN CELL POPULATIONS
title_full_unstemmed DETERMINATION OF RATES OF DNA SYNTHESIS IN CULTURED MAMMALIAN CELL POPULATIONS
title_short DETERMINATION OF RATES OF DNA SYNTHESIS IN CULTURED MAMMALIAN CELL POPULATIONS
title_sort determination of rates of dna synthesis in cultured mammalian cell populations
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2109404/
https://www.ncbi.nlm.nih.gov/pubmed/4214821
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