Isolation of nucleoli from Tetrahymena pyriformis. |
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Authors: | T Higashinakagawa M Sezaki S Kondo |
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Affiliation: | Laboratory of Developmental Biology, Mitsubishi-Kasei Institute of Life Sciences, 11 Minamiooya, Machida, Tokyo 194, Japan |
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Abstract: | A procedure is described to isolate nucleoli from Tetrahymena pyriformis which contain extrachromosomal ribosomal DNA. Macronuclei isolated by the Nonidet procedure were sonicated at a reduced magnesium concentration, and the sonicate was fractionated by isopycnic centrifugation in a metrizamide density gradient. The heaviest band, designated Band IIb, contains exclusively ribosomal DNA, thus constituting the nucleolar fraction. The purity of the nucleolar fraction on a DNA basis, which is defined as the percentage of ribosomal DNA and determined by equilibrium centrifugation in a CsCl density gradient, was around 70%. Electron microscopic examination revealed that the isolated nucleoli retained fairly well the ultrastructure of the in situ nucleoli. Some of the biochemical properties of the isolated nucleoli are also presented. |
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