Chromosome segregation requires a functional spindle apparatus, microtubules, chromosomal attachment sites, and a centromere specific DNA sequence. Disruptions of any of these organelles can lead to chromosomal malsegregation and aneuploidy. We are addressing two aspects of the function of centromeres within yeast cells: 1) the ability of cells to modify the number of centromeres; and 2) the ability of cells to deal with damage in the centromere. We developed a plasmid system which allows for the genetic detection of the number of centromere-containing plasmids within a cell. This is being done by including within a centromere plasmid the gene for copper resistance CUP1 and a gene for beta-galactosidase. Increases in plasmid number lead to increased resistance and more beta-galactosidase. We have observed that haploid cells can tolerate at least 8 additional centromeres and that this does not disturb growth or the process of meiosis. This system will enable an analysis of the relationship of the spindle apparatus organization to centromere function. We have shown that toleration of extra centromeres is greatly reduced in cells of higher ploidy (i.e., diploids, triploids, and tetraploids), indicating a limitation of components for segregation. It appears that the temporary presence of large numbers of centromeres can inhibit meiosis. Because of the systems we have available for detecting aneuploidy, it will be possible to determine consequences of altered centromere number on genome stability with a high degree of detection (<10-5). Cells containing a large number of centromere plasmids are being used to examine repair in the centromere DNA.
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