CYTOCHEMICAL CHARACTERIZATION OF DEOXYRIBONUCLEOPROTEIN IN INDIVIDUAL CELL NUCLEI - TECHNIQUES FOR OBTAINING HEAT DENATURATION CURVES WITH AID OF ACRIDINE ORANGE MICROFLUORIMETRY AND ULTRAVIOLET MICROSPECTROPHOTOMETRY

Simple methods for obtaining heat denaturation curves on the deoxyribonucleoprotein DNP of individual cell nuclei or intact cells are described. Cells or nuclei adhering to microscopic slides were heated in a salt solution (SSC) containing formaldehyde. After heating the slides were rapidly cooled. Both the formaldehyde and the rapid cooling served to prevent renaturation of denatured DNA. The heated preparations were analyzed for changes in single- and double-strandedness by UV microspectrophotometry and by microfluorimetry after acridine orange staining. These techniques were used to obtain melting profiles of DNP from non-stimulated and PHA stimulated lymphocytes, erythrocyte nuclei activated by cell fusion with HeLa cells, normal erythrocyte nuclei and isolated HeLa nuclei. The results obtained show that the activation of a large number of genes in both human lymphocytes and hen erythrocyte nuclei is paralleled by striking changes in the melting profiles of nuclear DNP. © 1969.