Isolation of a condensed, intracellular form of the 2-micrometer DNA plasmid of Saccharomyces cerevisiae

Abstract

We have initiated an investigation of the proteins bound in vivo to the 2-micrometer DNA plasmid found in the yeast Saccharomyces cerevisiae by examining its intracellular form. To isolate 2-micrometer DNA without disturbing proteins bound to the plasmid, an extract was prepared from a strain lacking mitochondrial DNA and the nuclear chromatin was removed from the extract by centrifugation. When the DNA in this extract was sedimented through a sucrose gradient containing 0.15 M NaCl, plasmid DNA had a sedimentation coefficient of approximately 70. This S value is greater than the S value of 25 for naked, superhelical 2-micrometer DNA. Cosedimenting with the DNA were proteins of the same size as the histone proteins associated with yeast nuclear chromatin. Digestion of the plasmid DNA with micrococcal nuclease and electrophoresis of the resulting DNA fragments revealed that segments of discrete sizes are protected from degradation. Examination of the plasmid DNA protein complex by electron microscopy showed nucleosome structures. We conclude that 2-micron DNA exists as a condensed chromosome body within the cell.