Heterogeneity and maintenance of centromere plasmid copy number in Saccharomyces cerevisiae

We developed a novel approach to quantitate the heterogeneity of centromere number in yeast, and the cellular capacity for excess centromeres. Small circular plasmids were constructed to contain the CUP1 metallothionein gene. ARS1 (autonomously replicating sequence) and a conditionally functional centromere (GAL1-GAL10 promoter controlled centromere). The CUP1 gene provided a gene dosage marker, and therefore a genetic determinant of plasmid copy number. Growth of cells on glucose is permissive for centromere function, while growth on galactose renders the centromere nonfunctional and the plasmids are segregated in an asymmetric fashion. We identified "lines" of cells containing increased numbers of plasmids after transformation. Cell lines containing as many as five to ten active centromeres are stably maintained in the absence of genetic selection. Thus haploid yeast cells can tolerate a 50% increase in their centromere number without affecting progression through the cell cycle. This system provides the opportunity to address issues of specific cellular controls on centromere copy number.