Nucleosome organization of the yeast 2-micrometer DNA plasmid: a eukaryotic minichromosome

Abstract

The eukaryotic microorganism Saccharomyces cerevisiae contains 50-100 copies per cell of a circular plasmid called 2-micrometer DNA. The intracellular structure of these molecules, which represent about 4% of the total DNA, was examined by digestion of total cellular chromatin with micrococcal nuclease (nucleate 3'-oligonucleotidohydrolase, EC 3.1.31.1). Nuclease-resistant DNA fragments were fractionated by gel electrophoresis and 2-micrometer DNA sequences were detected by hybridization. The 2-micrometer and chromosomal DNA digestion patterns were very similar indicating that both types of DNA are condensed into nucleosomes. An analysis of these digestion patterns showed that the kinetics of digestion of 2-micrometer chromatin and total chromatin are similar and that both have the same nucleosome repeat length of about 165 base pairs. Native 2-micrometer plasmids were examined by zone sedimentation in sucrose gradients containing 0.15 M NaCl and were found to have a sedimentation constant of 75 S, about 3 times the sedimentation constant of protein-free 2-micrometer DNA. This sedimentation property is what would be expected for a 2-micrometer DNA minichromosome. We conclude that within the cell 2-micrometer DNA molecules are organized in a chromatin structure very similar to that of the yeast chromosomes.