Synthesis of simian virus 40 DNA in isolated nuclei

Nuclei isolated from African green monkey kidney cells infected with simian virus 40 at different times after infection maintain in vitro the same temporal sequence of host and viral DNA synthesis as that seen in intact cells. The viral DNA synthesized by the nuclei of cells previously infected for 32-35 hr was characterized by centrifugation through neutral and alkaline sucrose gradients, and by isopycnic banding in a propidium iodide-cesium chloride gradient. DNA synthesis in this system is maintained for only 4-5 min. Neutral sucrose gradient analysis showed that most of the radioactivity is associated with the replicative intermediate of simian virus 40 DNA and the rest sediments at 5-7 S. Alkaline gradient analysis showed that 50-60% of the radioactivity sediments as 3-7S fragments, and the rest between 7 and 16 S. Pulsechase experiments showed that in this system 3-7S fragments do not mature into long chains. A model is presented to explain the failure of these fragments to join into long chains in this in vitro nuclear system.