Centromeric DNA cloned from functional kinetochore fragments in mitotic cells with unreplicated genomes - PubMed (original) (raw)

Comparative Study

. 1993 Jun:105 ( Pt 2):359-67.

doi: 10.1242/jcs.105.2.359.

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Comparative Study

Centromeric DNA cloned from functional kinetochore fragments in mitotic cells with unreplicated genomes

I I Ouspenski et al. J Cell Sci. 1993 Jun.

Abstract

Treatment of cells arrested in the cell cycle at the G1/S-phase boundary with 5 mM caffeine induces premature mitosis, resulting in chromosomal 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 centromere, we have developed a procedure for isolation of a centromere-enriched fraction of mitotic chromatin. Using this method, we have isolated and cloned DNA from the centromere-enriched material of Chinese hamster 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 is devoid of any extensive repetitive structure. This sequence is present in a single copy on chromosome 1 and is species-specific. Distinctive features of the clone include the presence of several A+T-rich regions 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 more compact packaging of centromeric chromatin in interphase nuclei and mitotic chromosomes.

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