CENP-C is an evolutionarily conserved centromeric protein. We have used the chicken DT40 cell line to test the idea that CENP-C is sufficient as well as necessary for the formation of a functional centromere. We have compared the effects of disrupting the localization of CENP-C with those of inducibly overexpressing the protein. Removing CENP-C from the centromere causes disassembly of the centromere protein complex and blocks cells at the metaphase-anaphase junction. Overexpressed CENP-C is associated with an increase in errors of chromosome segregation and inhibits the completion of mitosis. However, the excess CENP-C does not disrupt the native centromeres detectably and does not associate with another conserved centromere protein, ZW10. The distribution of the excess CENP-C changes during the cell cycle. In metaphase, the excess CENP-C coats the chromosome arms. At the metaphase-anaphase transition, the excess CENP-C clusters, and during interphase it is present in large bodies which form around pre-existing centromeres which are also clustered. These results indicate that CENP-C is necessary but not sufficient for the formation of a functional centromere and suggest that the structure of CENP-C may be regulated during the cell cycle.

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We have introduced a 6.5 Mb human mini-chromosome with a complex centromere structure into DT40 cells and have used sequence targeting and telomere-directed chromosome breakage to dissect the sequence requirements for centromere function. These experiments proved that a vertebrate centromere with two blocks of functional alphoid DNA separated by 2.5 Mb can exist as a stable structure in some but not all vertebrate cells. Further experiments indicated that recovery of chromosomes with less than approximately 100 kb of alphoid DNA is very inefficient, suggesting that a functional centromere requires a minimum of approximately 100 kb of alphoid DNA. Mini-chromosomes with minimal centromeres segregate accurately in some but not all vertebrate cells and should be useful for the detection of sequence-specific factors required for vertebrate centromere maintenance.

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