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© 1994 Oxford University Press

RESEARCH-ARTICLE

Structural analysis of {alpha}-satellite DNA and centromere proteins using extended chromatin and chromosomes

Thomas Haaf* and David C. Ward

Department of Genetics, Yale University School of Medicine 333 Cedar Street, PO Box 3333, New Haven, CT 06510, USA

* To whom correspondence should be addressed

Received February 14, 1994; Accepted March 12, 1994

Human centromeres are characterized by distinct subsets of a-satellite DNA and by a number of centromeric proteins (CENPs) at least one of which, CENP-B, binds specifically to a-satellite DNA sequences. When the centromeres of metaphase chromosomes are mechanically stretched to five to 20 times their normal length, CENPs specifically recognized by CREST autoantlbodles extend over the entire length of the linear a-satellite array. For higher resolution analysis we spread Interphase chromatin across a slide resulting in highly extended chromatin fibers. By fluorescence in situ hybridization (FISH) with human a-satellite DNA and an oligomer specific for the CENP-B box sequence, the regular spacing of CENP-B binding motifs within arrays of a-satellite DNA was visualized directly. FISH with elongated chromatin structures released from interphase nuclei with the drug W-[4-<9-acridinylamlno)-3-methoxyphenyl]methanesulfonamide shows that D7Z1 and D7Z2, two distinct a-satelllte arrays on chromosome 7, are not interspersed with each other but are separated by as little as several hundred kilobases, consistent with previous long-range mapping data. The D7Z2 array, which does not bind detectable amounts of CENPs, can be assigned to the short arm side of the D7Z1 array using artificially stretched chromosomes. In interphase nuclei unreplicated segments give a singlet hybridization signal, whereas fully replicated loci appear as doublets. Although D7Z1 Is replicated prior to D7Z2 In the majority of cells, the replication timing of one array relative to the other is variable. The replication of a-satellite arrays on homologous chromosomes is highly asynchronous. The newly replicated a-satellite lacks the CENP component.


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