Characterization of human heterochromatin by in situ hybridization with satellite DNA clones

Biotinylated DNA from two satellite-related, repetitive DNA clones, pHuR 98 and pHuR 195 (specific for chromosomes 9 and 16, respectively), and from a Y-specific clone, pY-3.4A, were hybridized to human metaphase chromosomes using fluoresceinated avidin to detect binding. The chromosomes were simultaneously counterstained with distamycin-DAPI to identify the AT-rich heterochromatin of chromosomes 1, 9, 15, 16, and the Y chromosome. With this method, clear results were obtained under both normal and low stringency conditions, allowing hybridization between molecules sharing 80-85% and 60-65% identity, respectively. Thus, additional sites related to the probes could be identified. A close relationship was shown between the heterochromatin of chromosomes 1 and 16, both hybridizing with clone pHuR 195 under low stringency. Hybridization with clone pHuR 98 was highly specific for chromosome 9, even under low stringency. A relationship between chromosomes 9, 15, and the Y chromosome, however, was shown by hybridization with clone pY-3.4A. The chromosomal distribution of the three repetitive DNA clones used in this study, and data from the literature, are in accordance with the distribution of the heterochromatin types characterized by staining with different fluorescent dyes and dye combinations. Furthermore, our sequence data for clones pHuR 98 and pHuR 195 may explain the fluorescent properties on which the cytogenetic classification of the heterochromatin is based.