Cytochemical characterization of deoxyribonucleoprotein by UV-microspectrophotometry on heat denatured cell nuclei

Abstract

Denaturation of double-stranded DNA into a single-stranded state can be studied by heating fixed cells attached to quartz slides and then determining the increase in nuclear UV-absorption at 265 nm by microspectrophotometry at room temperature. In order to prevent renaturation as the slides are cooled, formaldehyde is added to the solution in which heat denaturation is performed. The influence of formaldehyde concentration, duration of heating and ionic strength on the stability of DNA to heat denaturation has been examined. The standard method involves heating of ethanol/acetone fixed cells to temperatures between 22°C and 100°C in 0.15 M NaCl, 0.015 M sodium citrate containing 4% formaldehyde for 20 min followed by cooling to room temperature and mounting in glycerol. Different cell types examined by the cytochemical method show markedly different heat denaturation curves. These differences appear to reflect differences in DNA-protein interaction in the deoxyribonucleoprotein complexes of nuclear chromatin. DNA in nuclei which are completely repressed and which have a tightly condensed chromatin, e.g. sperm heads and hen erythrocyte nucleim show greater stability to heat denaturation than does DNA in more active cell nuclei. The thermal stability of DNA in rat thymocyte nuclei, the most active cell nuclei examined, is as low as that of calf thymus DNA in solution. Hen erythrocyte nuclei show a two-step transition from the double-stranded to the single-stranded state. The first step occurs at a temperature which is slightly lower than that of isolated DNA, whereas the second step occurs at a higher temperature. Bull spermatozoa show a single-step transition and a T m value which is higher than that for bovine DNA. In all cell types examined, 85–100% of the DNA in the fixed cell nucleus appears to be in the double-stranded form.