Subfractionation of soluble macronuclear chromatin and enrichment of specific genes as chromatin from Euplotes eurystomus.

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RESUMO

Euplotes eurystomus is a hypotrichous ciliated protozoan possessing within one cytoplasm a transcriptionally-inactive micronucleus with chromosomal-size DNA and a transcriptionally active macronucleus with "gene-size" DNA fragments. The chromatin in the macronucleus can be isolated in a soluble form without prior treatment by nucleases. In this study, macronuclear, soluble chromatin was subfractionated using isokinetic sucrose density gradient ultracentrifugation in a buffer consisting of 50 mM NaCl, 1 mM Na2 EDTA, 1 mM TEA HCl, pH 7.0, 0.1 mM TLCK and 0.1 mM PMSF. Fractions were collected and analyzed by DNA and protein gel electrophoresis, dot blot hybridization with specific gene probes, and modified Miller chromatin spreads. Analysis of the chromatin spreads showed that the sizes of the chromatin fragments in the various fractions correlate with the DNA size of the fragments. When dot blots of the fractions were hybridized with 5S rRNA, tubulin and rRNA gene probes we obtained about a 6 to 14-fold enrichment of hybridizable sequences in individual fractions. There appear to be differences in the non-histones present on each fraction as well as some overall similarities in histone and non-histone proteins.

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