Assay of centromere function using a human artificial chromosome

In order to define a functional human centromere sequence, an artificial ch romosome was constructed as a reproducible DNA molecule. Mammalian telomere repeats and a selectable marker were introduced into yeast artificial chro mosomes (YACs) containing alphoid DNA from the centromere region of human c hromosome 21 in a recombination-deficient yeast host. When these modified Y ACs were introduced into cultured human cells, a YAC with the alphoid DNA f rom the alpha 21-I locus, containing CENP-B boxes at a high frequency and a regular repeat array, efficiently formed minichromosomes that were maintai ned stably in the absence of selection and bound CENP-A, CENP-B, CENP-C and CENP-E. The minichromosomes, 1-5 Mb in size and composed of multimers of t he introduced YAC DNA, aligned at metaphase plates and segregated to opposi te poles correctly in anaphase. Extensive cytological analyses strongly sug gested that the minichromosomes had not acquired host sequences and were fo rmed in all cases by a de novo mechanism. In contrast, minichromosomes were never produced with a modified YAC containing alphoid DNA from the alpha 2 1-II locus, which contains no CENP-B boxes and has a less regular sequence arrangement. We conclude that alpha 21-I alphoid DNA can induce de novo ass embly of active centromere/kinetochore structures on minichromosomes.