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Highly purified micro nuclei and macro nuclei from tetrahymena thermophila isolated by percoll gradients



Highly purified micro nuclei and macro nuclei from tetrahymena thermophila isolated by percoll gradients



Journal of Protozoology 30(1): 21-30



A new procedure is described that utilizes Percoll gradients for purifying micronuclei (MIC) and macronuclei (MAC) from T. therophila. Separation of MIC from MAC during certrifugation in Percoll gradients occurs as a result of their difference in size rather than density. Three kinds of tests were used to evaluate the purity of the nuclei: visualization of the nuclei by light microscopy; examination of the nuclei by EM; and Southern blots of MIC and MAC DNA probed with the 5s rRNA genes or a fragment from the MAC extrachromosomal rDNA molecule. When examined under the light microscope, the isolated MIC and MAC have much lower nuclear cross contamination levels than previous methods have reported. MIC contaminated with < 1 MAC in 1000 MIC and MAC contaminated with < 1 MIC in 500 MAC can be routinely prepared. Quantitative analyses of electron micrographs gave higher estimates of cross contamination in the purified nuclei, which may, in part, be explained by the difficulty in identifying small MIC or MAC fragments. Southern blots of MIC and MAC DNA probed with 5s rDNA confirmed the level of MAC contamination in the MIC estimated by light microscopy during purification of the nuclei. The level of nucleolar contamination in the MIC was estimated at 10% by Southern blots of MIC and MAC DNA, derived from a heterokaryon with distinctive MIC and MAC Bam HI sites, using an rDNA probe.

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