CENTROMERIC DNA CLONED FROM FUNCTIONAL KINETOCHORE FRAGMENTS IN MITOTIC CELLS WITH UNREPLICATED GENOMES

Treatment of cells arrested in the cell cycle at the G1/S-phase bounda ry with 5 mM caffeine induces premature mitosis, resulting in chromoso mal fragmentation and detachment of centromere-kinetochore fragments, which are subsequently attached to the mitotic spindle and segregated in anaphase. Taking advantage of this in vivo separation of the centro mere, we have developed a procedure for isolation of a centromere-enri ched fraction of mitotic chromatin. Using this method, we have isolate d and cloned DNA from the centromere-enriched material of Chinese hams ter cells. One of the clones thus obtained was characterized in detail . It contains 6 kb of centromere-associated sequence that exhibits no recognizable homology with other mammalian centromeric sequences and i s devoid of any extensive repetitive structure. This sequence is prese nt in a single copy on chromosome 1 and is species-specific. Distincti ve features of the clone include the presence of several A+T-rich regi ons and clusters of multiple topoisomerase II consensus cleavage sites and other sequence motifs characteristic of nuclear matrix-associated regions. We hypothesize that these features might be related to the m ore compact packaging of centromeric chromatin in interphase nuclei an d mitotic chromosomes.