The influence of formamide on thermal denaturation profiles of DNA and metaphase chromosomes in suspension

Systematic photometric studies are presented to analyze the thermal denatur ation behaviour with and without formamide of metaphase chromosome suspensi ons in comparison to DNA solutions. Temperature dependent hyperchromicity m easurements at 256 nm and 313 nm were performed using an appropriately desi gned computer-controlled photometer device. Due to an upright optical axis, this allowed absorbance measurements with negligible sedimentation effects not only for solutions of pure DNA, but also for particle suspensions of i solated metaphase chromosomes. This device has a temperature resolution of +/- 0.5 degreesC and an optical sensitivity of 10(-3) to 10(-4) optical den sity. For calf thymus DNA the reduction of the melting point with the incre ase of formamide in the solution was measured at pH 7.0 and pH 3.2. The goo d correlation of the theoretical approximation to experimental data indicat ed the suitability of the apparatus to quantitatively describe DNA conforma tion changes induced by thermal denaturation. For metaphase chromosome prep arations of Chinese hamster culture cells, absorbance changes were measured between 20 degreesC and 95 degreesC with a temperature gradient of 1 degre esC/min. These measurements were performed at pH 7.0 and at pH 3.2. The den aturation profiles (= first derivative of the absorbance curve) resulted in a highly variable peak pattern at 256 nm and 313 nm indicating complex con formation changes. A statistical evaluation of the temperature values of th e peak maxima resulted in temperature ranges typical for chromosomal confor mation changes during thermal treatment. Especially the range of highest te mperature values was independent from pH modifications. For pH 3.2 the infl uence of formamide on the denaturation behaviour of metaphase chromosome pr eparations was analyzed. In contrast to pure DNA solutions, a reduction of the "melting point" (i.e. the maximum temperature at which a conformation c hange takes place) was not found. However, the denaturation behaviour depen ded on the duration of formamide treatment before the measurement.