Characterization of a yeast replication origin ( ars2) and construction of stable minichromosomes containing cloned yeast centromere DNA ( CEN3)

A yeast DNA sequence ( ars2), capable of supporting autonomous replication of plasmids in yeast, has been characterized. The ars2 replicator occurs about 7 kb from the ARG4 gene on yeast chromosome VIII. Plasmids containing ars2 and the ARG4 gene transform yeast arg4 mutants to ARG4 + with high frequency (about 10 3 transformants/μg DNA) and replicate autonomously in the transformed cells. The ars2 plasmids are mitotically unstable and are readily lost from yeast cultures when grown under nonselective conditions. The addition of a DNA segment containing a functional yeast centromere ( CEN3) and an ars2 plasmid effectively stabilizes the plasmid against both mitotic and meiotic loss. The ars2-CEN3 minichromosomes replicate autonomously in controlled copy number while segregating in a typical Mendelian pattern (2+:2−) during meiosis. The requirement for a ceparate replicator sequence for stable mitotic and meiotic maintenance of centromere-containing minichromosomes is equally satisfied by the presence of either ars1 or ars2. The centromere controls plasmid copy number to a low value (usually one) regardless of the type of replicator used.