Graduate Thesis Or Dissertation

 

DNA precursor compartmentation in mammalian cells : distribution and rates of equilibration between nucleus and cytoplasm Public Deposited

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https://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/kh04dt006

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  • A rapid nuclear isolation technique was adapted in order to examine the question of DNA precursor compartmentation in mammalian cells. By using this method a reproducible proportion of the cellular nucleotides remained associated with the isolated nuclei. Examination, at several different cell densities, of exponentially growing HeLa cells showed that the nuclei contained a constant but distinct proportion of each dNTP. The nuclear dATP and dTTP concentrations were equal at all densities examined even though the dTTP pool was 150% of the dATP whole-cell pool. The nuclear portion of the whole-cell pools was roughly equal to the volume occupied by the nucleus. The nuclear-cytoplasmic dNTP pool distribution did not change throughout the cell cycle of synchronized Chinese hamster ovary (CHO) cells, with the exception of dCTP. dATP, dTTP, and dGTP pools rose less than 2-fold between G1 and S phase. The whole-cell dCTP pool rose 10-fold while the nuclear pool rose only 2-fold. The rates at which either radiolabeled cytidine or deoxycytidine equilibrated with the nuclear and whole-cell dCTP pools of G1 and S phase CHO cells were compared. Nuclear and whole-cell dCTP pools equilibrated at the same rate, regardless of the nucleoside used or the phase of the cell cycle. This indicates the existence of a single cellular dCTP pool. Interestingly, the deoxycytidine-derived dCTP specific activity was thirteen times larger in G1 than in S phase. Experiments comparing the labeling kinetics of ³H-thymidine in G1, S phase, and exponentially growing cells revealed that the S phase dTTP pool equilibrated with exogenously added thymidine faster than the G1 phase pool. The rate of equilibration in exponentially growing cells appeared to be a combination of that seen in G1 and S phases. A linear rate of ³H-thymidine incorporation into DNA occurred at the same rate in S phase and exponentially growing cells.
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