Comparison of the thermal denaturation behaviour of DNA-solutions and metaphase chromosome preparations in suspension

Hyperchromicity measurements are well established to analyse the thermal de naturation behaviour of pure DNA sequences in solution. Here, we show that under appropriate experimental conditions this technique can also be applie d to study thermally controlled conformation changes of higher order DNA-pr otein complexes as for instance metaphase chromosome preparations in suspen sion. A computer controlled sensitive, upright double beam photometer with a heatable cuvette was constructed. Measurements of the temperature depende nt extinction of both, solutions and particle suspensions are possible, sin ce sedimentation effects of particles can be neglected due to the vertical optical axis in the probe cuvette. Thermal denaturation of metaphase chromo some preparations of human and Chinese hamster cells was investigated and c ompared to melting profiles of DNA solutions for two excitation wavelengths , 256 and 313 nm. The influence of neutral and low pH was considered. The r esults indicate that metaphase chromosome preparations show a thermal denat uration behaviour different from pure DNA. Whereas DNA solutions showed one pH dependent melting peak at 256 nm only, the peak pattern of metaphase ch romosome preparations showed a large variability both at 256 and 313 nm. At neutral pH, in two temperature regions (40-55 degrees C and 75-82 degrees C) peaks were found indicating chromosome typical conformation changes inde pendently from the mammalian cell species (Chinese hamster, human). In cont rast to pure DNA, no typical reduction in the temperatures of peak maxima w ith decreasing pH was found for metaphase chromosome preparations of both c ell types.