AluI in situ digestion of human alphoid and classical satellite DNA regions: High-resolution digital image analysis of FISH signals from condensed and extended chromatin

Human lymphocyte chromatin either extended or condensed in interphase nuclei and chromosomes was in situ digested by the restriction endonuclease AluI and then hybridized with alphoid probes specific for chromosome 1 (D1Z5 locus), for chromosome X (DXZ1 locus), and with a classical satellite DNA probe specific for chromosome 9 (D9Z1 locus). Fluorescent hybridization signals were quantified by digital analysis of high-resolution images obtained by a Photo-CD system in an attempt to analyze the differential DNA removal produced by AluI in specific repetitive DNA sequences with known restriction site frequency and distribution. The analysis of area and average pixel grey count of hybridization signals suggests that the greater the degree of chromatin stretching, the higher the accessibility of the probe and/or reporter molecules to the target. Nevertheless, this greater hybridization efficiency does not result in a higher fluorescence intensity due to dispersion of individual signals. Specific repetitive DNA at D9Z1 locus (classical) remained impervious to digestion, while that at DXZ1 (alphoid) was extensively removed, according to the frequency and distribution of restriction sites. Nevertheless, though the restriction sites were at least as frequent as at the DXZ1 locus, DNA at the D1Z5 locus (alphoid) was only partially removed. This indicates that chromatin organization within the C-band partially prevents extraction of alphoid sequences, supporting the hypothesis that alphoid DNA sequences are differentially organized among chromosomes. Overall, the same results were obtained from condensed and extended chromatin, suggesting that higher-order chromatin organization does not influence the in situ DNA cleavage and removal by AluI.