The fidelity of the mitotic transmission of minichromosomes in S. cerevisiae is monitored by a novel visual assay that allows one to detect changes in plasmid copy number in individual mitotic divisions. This assay is used to investigate the mitotic transmission of a plasmid containing a putative yeast origin of replication (ARS1) and a centromere (CEN3). The rate of improper segregation for the minichromosome is 200-fold higher than observed for a normal chromosome. However, the replication of the minichromosome is stringently controlled; it overreplicates less than once per one thousand mitotic divisions. We also use this assay to isolate and characterize mutations in ARS1 and CEN3. The mutations in ARS1 define a new domain required for its optimal activity, and the mutations in CEN3 suggest that the integrity of element II is not essential for centromere function. Finally, the phenotypes of the mutations in ARS1 and CEN3 are consistent with their function in replication and segregation, respectively.
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)