Abstract
Evaluation of the technology for analysis of incorporation of [ 3H]TTP into nuclei of Chinese hamster fibroblast and HeLa nuclei was performed. The synthesis of the DNA was dependent on all four deoxyribonucleotide triphosphates, Mg 2+ and ATP. Nuclei pelleted after 5% trichloroacetic acid or 3 : 1 (methanol-acetic acid) precipitation at 4 °C resulted in an aggregate which could not be adequately resuspended for further washing. This problem was circumvented using 50% acetic acid fixation at 4 °C. Using this procedure the pellet of nuclei could be washed, removing much of radioactivity with minimal loss of nuclei, indicating adventitious adsorption of radioactive DNA. This was confirmed by sedimentation of unlabeled nuclei through the reaction mixture containing radioactive chromatin. We conclude that the existing methodologies for studying DNA synthesis in isolated nuclei have been inadequate.
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