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Quantitative Aspects of Isolation of Nucleoli of the Walker Carcinosarcoma and Liver of the Rat^*

Masami Muramatsu, Karel Smetana and Harris Busch

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

A method is described for isolation of nucleoli of the Walker tumor and liver in which nuclei were sonicated in a Model DF-101 Raytheon Sonic Oscillator in which the temperature was maintained at 2° C. The medium used for the initial homogenization of the tissue was 0.25 M sucrose containing 0.0033 M CaCl₂. This concentration of calcium ion was found to be optimal for the successful release of nucleoli with maximal destruction of nuclei. The volume was maintained at 20 ml., and the time required for destruction of more than 99.8 per cent of the nuclei was 25–40 seconds. The amperage of the output current was 1.0–1.1. Selective centrifugation of the nucleoli of the sonicate was accomplished by initial centrifugation at 2500 x g for 5 minutes followed by rehomogenization of the pellet in 0.88 M sucrose and sedimentation of the nucleoli at 800 x g (1000 x g for the liver) for 15 minutes. A repetition of the last step produced a preparation of nucleoli which was 95–99 per cent pure by direct particle count. For further purification, this preparation was treated either with DNase I or with 2 M NaCl. The content of the nucleic acids and protein in these preparations was determined. Tumor nucleoli purified with DNase contained an average of 1.8 pg. of RNA and 16.1 pg. of protein per nucleolus. Liver nucleoli contained one-sixth the amount of RNA and protein of nucleoli of the Walker tumor. These preparations contained very little DNA. RNA comprised about 10 per cent of the dry weight of nucleoli of both liver and Walker tumor.

^* These studies were supported in part by grants from the Jane Coffin Childs Fund, the U.S. Public Health Service and the American Cancer Society.

Postdoctoral Trainee in Pharmacology.

On leave from the National Academy of Science, Prague.

Received 8/ 6/62.

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