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In Vitro Labeling of RNA in Isolated Nucleoli of the Walker Tumor and Liver^*

( Department of Pharmacology, Baylor University College of Medicine, Houston, Texas)

Isolated tumor and liver nucleoli were found to incorporate ATP-8-C¹⁴, UTP-2-C¹⁴, GTP-8-C¹⁴, and CTP-2-C¹⁴ into RNA. Optimal activity required the presence of all four nucleotide triphosphates and Mg⁺⁺. At low concentrations, Mn⁺⁺ stimulated the labeling, but high concentrations of Mn⁺⁺ inhibited the reaction. Mercaptoethanol was also required for the optimal activity. The pH optimum was 8.2 for both tumor and liver nucleoli. Maximum incorporation of isotope into RNA was found at 15–30 minutes after the incubation was begun. The reactions were DNA-dependent, as shown by sensitivity to actinomycin D. Evidence for internucleotide incorporation of ATP-P³² was obtained by analysis of P³² distribution into nucleotide monophosphates after alkaline hydrolysis of the RNA. The nearest neighbor frequency of the AMP moiety of labeled nucleolar RNA was determined with the aid of ATP-P³² and was found to differ in the liver and the Walker tumor.

^* Supported in part by grants from the Jane Coffin Childs Fund, the U.S. Public Health Service, the American Cancer Society, and the National Science Foundation.

Received 5/ 4/64.