STRUCTURAL-ANALYSIS OF ALPHA-SATELLITE DNA AND CENTROMERE PROTEINS USING EXTENDED CHROMATIN AND CHROMOSOMES

Human centromeres are characterized by distinct subsets of alpha-satel lite 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 stretc hed to five to 20 times their normal length, CENPs specifically recogn ized by CREST autoantibodies extend over the entire length of the line ar alpha-satellite array. For higher resolution analysis we spread int erphase chromatin across a slide resulting in highly extended chromati n fibers. By fluorescence in situ hybridization (FISH) with human alph a-satellite DNA and an oligomer specific for the CENP-B box sequence, the regular spacing of CENP-B binding motifs within arrays of alpha-sa tellite DNA was visualized directly. FISH with elongated chromatin str uctures released from interphase nuclei with the drug N-[4-(9-acridiny lamino)-3-methoxyphenyl] 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, consiste nt with previous long-range mapping data. The D7Z2 array, which does n ot bind detectable amounts of CENPs, can be assigned to the short arm side of the D7Z1 array using artificially stretched chromosomes. In in terphase nuclei unreplicated segments give a singlet hybridization sig nal, whereas fully replicated loci appear as doublets. Although D7Z1 i s replicated prior to D7Z2 in the majority of cells, the replication t iming of one array relative to the other is variable. The replication of alpha-satellite arrays on homologous chromosomes is highly asynchro nous. The newly replicated alpha-satellite lacks the CENP component.