Microfluorometric Investigations of Chromatin Structure - III. Estimation of Histones and DNA in Thymocyte and Hepatocyte Nuclei. Effects of Extraction at pH 3.0

Experiments were undertaken in which the histones of mouse thymocyte and hepatocyte nuclei were stained by a fluorescent version of the Alfert and Geschwind (1953) procedure and measured by microfluorometry. These measurements were compared with those obtained after treatment of the nuclei with a pH 3.0 phosphate-citrate buffer which is known to displace fraction H1 histones from the chromatin of HeLa cell nuclei. Estimates of DNA with the fluorochromes Hoechst 33258, mithramycin, and proflavine were obtained in the same nuclei, with and without extraction at pH 3.0. In addition, the effects of extracting the nuclei in a phosphate-citrate buffer at pH 2.5 on DNA measurements were also determined. The results of hydrolyzing the nuclei over a period of 30 min to 5 h in 5% TCA at 60° C and staining at pH 8.0 with brilliant sulfaflavine for the demonstration of histones revealed that the fluorescence of 2c hepatocyte nuclei was always much greater than that of thymocyte nuclei. In addition, hepatocyte nuclei responded more slowly than thymocyte nuclei to hydrolysis; and the ratios of 4c:2c, 8c:4c, and 8c:2c hepatocyte nuclei generally exceeded the expected 2:2:4 values. While the fluorescence of thymocyte nuclei extracted at pH 3.0 and stained by the histone procedure was slightly greater than that of control thymocyte nuclei at all times during the hydrolysis period except for the earliest stages, the fluorescence of pH 3.0-extracted hepatocyte nuclei was always less than that of control hepatocyte nuclei; and the 2, 4, and 8c ploidy classes were not resolved clearly. Extraction at pH 3.0 and 2.5 generally produced an increase in fluorescence of both thymocyte and hepatocyte nuclei stained by the various DNA methods; but the absolute amount of change could not be related directly to known dependencies of the dyes on AT or GC base pairs. The results of the fluorescent histone procedure and those obtained with the various DNA methods after extraction at pH 3.0 and 2.5 indicate that these methods function as probes of nucleoprotein organization, rather than as absolutely quantitative procedures. It is presently unclear how the measurements are related to specific differences in the organization of the chromatin and/or transcriptional activities of the two types of nuclei