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 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.
Ouspenski
I I
II
Department of Cell Biology, Baylor College of Medicine, Houston, TX 77030.
Brinkley
B R
BR
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells / Q000737:chemistry; D002458:Cell Fractionation; D002503:Centromere / Q000737:chemistry*; D016384:Consensus Sequence; D006224:Cricetinae; D003412:Cricetulus / Q000235:genetics*; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification; D004261:DNA Replication; D016678:Genome; D008938:Mitosis; D008969:Molecular Sequence Data; D012091:Repetitive Sequences, Nucleic Acid; D012689:Sequence Homology, Nucleic Acid; D013045:Species Specificity
D004247:DNA