Isolation of an episomal yeast gene and replication origin as chromatin

Abstract

A multicopy yeast plasmid containing the TRP1 gene (coding for N-5'-phosphoribosylanthranilate isomerase) and ARS1 (autonomously replicating sequence 1) has been purified as chromatin. Electrophoretic analysis of nucleic acid and proteins and electron microscopy show that the plasmid chromatin is largely free of contaminants. Electron-microscopic and linking-number analyses indicate that the plasmid chromatin contains seven nucleosomes, as predicted by the indirect end-label analyses of Thoma, Bergman, and Simpson [J. Mol. Biol. (1984) 177, 715-733]. Indirect end label mapping of micrococcal nuclease cuts demonstrates that nucleosome positions and nuclease-sensitive regions are not altered by the purification. The plasmid chromatin behaves homogeneously with respect to its elution from nuclei, template activity, and intrinsic buoyant density. Taken together, these observations suggest that different copies of the TRP1ARS1 plasmid do not differ from each other grossly in chromatin structure. We discuss the potential for understanding eukaryotic gene regulation offered by the ability to isolate unique genes as chromatin.