SEPARATION OF NUCLEI REPRESENTING DIFFERENT PHASES OF THE GROWTH CYCLE FROM UNSYNCHRONIZED MAMMALIAN CELL CULTURES |
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| Authors: | O. Wesley McBride and Elbert A. Peterson |
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| Affiliation: | From the Department of Health, Education, and Welfare, Laboratory of Biochemistry, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20014 |
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| Abstract: | Nuclei have been isolated from unsynchronized cultures of Chinese hamster fibroblasts after varying intervals of growth following the incorporation of thymidine -3H for 20 min. These nuclei were fractionated by unit gravity sedimentation in a stabilizing density gradient of sucrose, and fractions were analyzed for the concentration of nuclei, DNA, and radioactivity. A more rapidly sedimenting population of nuclei in the G2 phase of the cell cycle was separated from a group of nuclei in the G1 phase, and nuclei in progressive stages of DNA synthesis (S phase) were distributed between these two regions. The fractionation of intact cells by sedimentation according to their position in the cell cycle was found to be less satisfactory than the corresponding separation of nuclei. This probably results from the continuous accumulation of mass within individual cells throughout the entire cell cycle, whereas most of the mass of a nucleus is replicated during a relatively narrow interval of the total cell cycle. |
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