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DNA Synthesis in Membrane-denuded Nuclei and Nuclear Fractions from Host Liver and Morris Hepatomas¹

Department of Anatomy and Cell Biology, University of Pittsburgh School Of Medicine, Pittsburgh, Pennsylvania 15261 [M. L. C., M. S., P. O.], and Howard University Medical Center, Washington, D. C. 20001 [H. P. M.]

Incorporation of [³H]TTP into membrane-denuded nuclei and fractions of these nuclei from host liver and Morris hepatomas has been compared. Treatment of sucrose nuclei with Triton X-100 removed 95% of the phospholipids and 15 to 20% of the protein. These membrane-denuded nuclei remained physically stable.

The Triton X-100-extracted nuclei incorporated label into their DNA in a nuclear-incorporating system similar to sucrose nuclei with their membranes intact. Triton X-100-treated nuclei from hepatoma 7777 incorporated six times more label and those from hepatoma 7800 incorporated three times more label than Triton X-100-treated host liver nuclei. Nuclei from the three sources incorporated more label when exogenous DNA was added to the incubation system, but the difference in incorporation between the hepatoma nuclei and the host liver nuclei disappeared.

When Triton X-100-treated nuclei, prepared from a tumor-bearing animal given injections of [³H]thymidine for 10 min were fractionated on sucrose gradients after disruption by high Mg²⁺ concentration, the fractions from hepatoma 7777 nuclei contained six times as much label as the host liver nuclear fractions. Nuclear fractions prepared from unlabeled hepatomas or host livers had DNA polymerase activity. The activity, however, is the same in fractions prepared from hepatoma 7777 or host liver nuclei.

It is suggested that the nuclear membrane does not play an important role in nuclear DNA synthesis. It is further suggested that the increased incorporation found with hepatoma nuclei is dependent on a physical or chemical arrangement of components within the nucleus and not solely on different enzyme levels.

¹ Supported by NIH Grant RO1 CM 1637.

Received 4/18/75. Accepted 6/ 9/75.

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