Isolation of chromosomal origins of replication in yeast

Abstract

Origins of replication have been identified in the DNA of viruses, mitochondria, bacterial plasmids and the bacterial chromosome. However, origins of replication of eukaryote chromosomes have remained elusive because of the large size and sequence complexity of chromosomes and in particular for want of a suitable assay for their detection. Recent development of techniques for genetic transformation of yeast by autonomously replicating cytoplasmic plasmids now makes it possible to search for eukaryote origins in a manner analogous to that used for bacteria. Here we describe the construction and properties of a plasmid which contains no effective eukaryote replication origin and whose efficiency of replication in yeast is greatly enhanced by insertion of certain fragments of yeast chromosomal DNA. We believe these to contain replication origins, since yeast transformants are shown to contain copies of the transforming plasmids.