Cytofluorometric studies on conformation of nucleic acids in situ. II. Denaturation of deoxyribonucleic acid.

Abstract

Thermal denaturation of deoxyribonucleic acid (DNA) in situ in individual unbroken cells is studied by a cytofluorometric method. This method allows us to investigate DNA denaturation in the presence of divalent cations at concentrations reported to be necessary to maintain native structure of nuclear chromatin. Under these conditions the pattern of DNA denaturation is very different than when studied in the presence of ethylenediaminetetraacetate or citrate. The results suggest that with divalent cations present, the histone basic charges are more uniformly distributed along whole nuclear DNA. Various cell types exhibit great differences in sensitivity to DNA denaturation when assayed in the presence of 1 mM MgCl2. Human lymphocytes, monocytes and certain kinds of human leukemic cells show differences large enough to be used as a parameter for their recognition in mixed samples. Possible applications of the method in basic research on chromatin conformation and as a tool for cell recognition in diagnostic cytology or in the classification of human leukemia are proposed.