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 endonuc lease AluI and then hybridized with alphoid probes specific for chromo some 1 (D1Z5 locus), for chromosome X (DXZ1 locus), and with a classic al satellite DNA probe specific for chromosome 9 (D9Z1 locus). Fluores cent hybridization signals were quantified by digital analysis of high -resolution images obtained by a Photo-CD system in an attempt to anal yze the differential DNA removal produced by AluI in specific repetiti ve DNA sequences with known restriction site frequency and distributio n. 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 ind ividual signals. Specific repetitive DNA at D9Z1 locus (classical) rem ained impervious to digestion, while that at DXZ1 (alphoid) was extens ively removed, according to the frequency and distribution of restrict ion sites. Nevertheless, though the restriction sites were at least as frequent as at the DXZ1 locus, DNA at the D1Z5 locus (alphoid) was on ly partially removed. This indicates that chromatin organization withi n the C-band partially prevents extraction of alphoid sequences, suppo rting the hypothesis that alphoid DNA sequences are differentially org anized among chromosomes. Overall, the same results were obtained from condensed and extended chromatin, suggesting that higher-order chroma tin organization does not influence the in situ DNA cleavage and remov al by AluI.