DNA-SYNTHESIS IN PERMEABLE MOUSE ASCITES SARCOMA-CELLS

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  • 1 January 1977
    • journal article
    • research article
    • Vol. 37  (1) , 137-144
Abstract
DNA synthesis was studied in mouse ascites sarcoma cells using a permeable cell system. The sarcoma was induced by the Schmidt-Ruppin strain of Rous sarcoma virus. The cells were made permeable to nucleoside triphosphates by treatment with a hypotonic buffer containing 10 mM Tris-Cl, 4 mM MgCl2, 1 mM EDTA and 6 mM 2-mercaptoethanol (pH 8.0). DNA synthetic activity in the permeable cells was dependent on 4 deoxyribonucleoside triphosphates, ATP, Mg2+ and a proper ionic environment. The activity was stimulated about 50% by the addition of an appropriate concentration of CTP, GTP and UTP in an assay mixture containing ATP and 4 deoxyribonucleoside triphosphates. DNA synthesis was confined to the nucleus and was sensitive to N-ethylmaleimide and DNase. The activity assayed by the permeable cell system correlated closely with the DNA replicating activity assayed by [3H]deoxythymidine incorporation in intact cells. The close correlation between DNA synthesis in vitro and in vivo was further confirmed in cultured sarcoma cells synchronized with DNA synthesis. Analysis of the DNA synthesized in vitro by alkaline cesium sulfate density gradient centrifugation showed that over half the DNA synthesized in permeable cells was due to elongation of strands initiated in vivo. The permeable cell system appears to be a useful method for examining DNA replication of cells in suspensions.