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

Human centromeres are characterized by distinct subsets of alpha-satellite DNA and by a number of centromeric proteins (CENPs) at least one of which, CENP-B, binds specifically to alpha-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 autoantibodies extend over the entire length of the linear alpha-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 alpha-satellite DNA and an oligomer specific for the CENP-B box sequence, the regular spacing of CENP-B binding motifs within arrays of alpha-satellite DNA was visualized directly. FISH with elongated chromatin structures released from interphase nuclei with the drug N-[4-(9-acridinylamino)-3-methoxyphenyl]methanesulfonamide shows that D7Z1 and D7Z2, two distinct alpha-satellite 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 alpha-satellite arrays on homologous chromosomes is highly asynchronous. The newly replicated alpha-satellite lacks the CENP component.