A method is presented for isolating DNA segments capable of autonomous replication from Saccharomyces cerevisiae chromosomal DNA based on the differential transforming ability of autonomously replicating plasmids and nonreplicating plasmids. DNA plasmids that are capable of self-replication in yeast transform yeast spheroplasts at about 1000-fold higher frequency than nonreplicating plasmids. We have cloned from total yeast DNA a number of DNA segments that permit the pBR322 plasmid carrying the yeast LEU2 gene to replicate autonomously. These plasmid clones are characterized by their ability to transform Leu- spheroplasts to Leu+ at a high frequency and their ability to replicate autonomously. Analysis of the insert DNAs carried in some of these self-replicating plasmids divides them into two categories: those that are unique in the yeast genome and those that are repetitive.
