Characterisation of DNA from condensed and dispersed human chromatin |
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Authors: | J.R. Gosden A.R. Mitchell |
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Affiliation: | Medical Research Council, Clinical and Population Cytogenetics Unit, Western General Hospital, Edinburgh EH42XU, Scotland |
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Abstract: | Condensed and dispersed chromatin fractions were isolated from human placental nuclei. The DNA of each fraction was purified and characterised by isopycnic centrifugation, thermal fractionation on hydroxylapatite (HAP) and sequence complexity studies. The DNAs had identical buoyant densities in neutral CsCl (1.698 g/cm3) and similar melting profiles on HAP. Analytical ultracentrifugation in Ag+-Cs2SO4, however, showed that satellite DNAs were present in the condensed fraction DNA (DNAC) but were not visible in the dispersed fraction DNA (DNAD). In addition, DNAC was found to be enriched in highly reiterated sequences (20% reassociated by C0t 10?3) which can be correlated with the presence of satellite DNAs, whereas DNAD contained only 3% of these fast reassociating sequences. In contrast DNAD contained 30% intermediate sequences (reassociating between C0t 10?3 and C0t 100) which represent only 10% of DNAC. The reassociated highly repeated sequences of DNAC showed the presence of two components in both CsCl density gradients and HAP thermal elution studies. This suggests that either there are sequence relationships resulting in partial mismatching between the different highly repeated DNA sequences in this fraction, or that highly repeated sequences are associated with less repetitious DNA. The results are discussed in terms of possible differences in genetic activity between the chromatin fractions. |
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