Toxic effects of excess cloned centromeres

Abstract

Plasmids carrying a Saccharomyces cerevisiae centromere have a copy number of one or two, whereas other yeast plasmids have high copy numbers. The number of CEN plasmids per yeast cell was made artificially high by transforming cells simultaneously with several different CEN plasmids carrying different, independently selectable markers. Some host cells carried five different CEN plasmids and an average total of 13 extra copies of CEN3. Several effects were noted. The copy number of each plasmid was unexpectedly high. The plasmids were mutually unstable. Cultures contained many dead cells. The viable host cells grew more slowly than control cells, even in nonselective medium. There was a pause in the cell cycle at or just before mitosis. We conclude that an excess of centromeres is toxic and that the copy number of centromere plasmids is low partly because of selection against cells carrying multiple centromere plasmids. The toxicity may be caused by competition between the centromeres for some factor present in limiting quantities, e.g., centromere-binding proteins, microtubules, or space on the spindle pole body.