An origin of replication and a centromere are both needed to establish a replicative plasmid in the yeast Yarrowia lipolytica.

Two DNA fragments displaying ARS activity on plasmids in the yeast Yarrowia lipolytica have previously been cloned and shown to harbor centromeric sequences (P. Fournier, A. Abbas, M. Chasles, B. Kudla, D. M. Ogrydziak, D. Yaver, J.-W. Xuan, A. Peito, A.-M. Ribet, C. Feynerol, F. He, and C. Gaillardin, Proc. Natl. Acad. Sci. USA 90:4912-4916, 1993; and P. Fournier, L. Guyaneux, M. Chasles, and C. Gaillardin, Yeast 7:25-36, 1991). We have used the integration properties of centromeric sequences to show that all Y. lipolytica ARS elements so far isolated are composed of both a replication origin and a centromere. The sequence and the distance between the origin and centromere do not seem to play a critical role, and many origins can function in association with one given centromere. A centromeric plasmid can therefore be used to clone putative chromosomal origins coming from several genomic locations, which confer the replicative property on the plasmid. The DNA sequences responsible for initiation in plasmids are short (several hundred base pairs) stretches which map close to or at replication initiation sites in the chromosome. Their chromosomal deletion abolishes initiation, but changing their chromosomal environment does not.