A fragment containing the origin of replication from Xenopus laevis mitochondrial DNA can promote the autonomous replication of recombinant DNA plasmids in Saccharomyces cerevisiae (V. A. Zakian, 1981, Proc. Nat. Acad. Sci. USA 78, 3128-3132). Plasmids containing Xenopus mitochondrial DNA (pXEY26 or pXY32) are maintained in transformed cells by selecting for expression of the URA3 gene, which is carried on the same plasmid but is derived from yeast chromosomal DNA. We have investigated the maintenance and replication of these plasmids in a number of ways. We report that respiratory-deficient strains of yeast can maintain plasmids containing Xenopus mitochondrial DNA with essentially the same facility as respiratory competent strains. In addition, the replication of pXEY26 is similar to that of yeast chromosomal DNA: its replication requires active CDC28, CDC4, and CDC7 gene products and does not occur in cells arrested in the G1 phase of the cell cycle by incubation with the natural yeast pheromone α factor. This pattern of replication differs from that of yeast mitochondrial DNA which replicates in cdc28, cdc4, or cdc7 cells at restrictive temperatures and in the presence of α factor. These data indicate that a recombinant DNA plasmid need not contain a yeast origin of replication in order for its replication to be under cell cycle control. We also report that pXEY26, like many other recombinant DNA plasmids in yeast, is found in only a fraction of the cells in a culture growing under selection for the plasmid (5-55%, depending on the strain). However, in all strains, pXEY26 is a high copy number plasmid: copy number ranged from about 40 to about 200 copies per cell in those cells which contain plasmid DNA. Therefore, the transmission of pXEY26 is highly nonrandom. Since the rate of replication of pXEY26 at permissive conditions in four strains was indistinguishable from that of 2-μm DNA, the endogenous yeast plasmid, its nonrandom pattern of segregation is unlikely to be due to a defect in its replication.
All Science Journal Classification (ASJC) codes
- Molecular Biology