DNA-protein binding in interphase chromosomes.
The metachromatic dye, azure B, was analyzed by microspectrophotometry when bound to DNA fibers and DNA in nuclei with condensed and dispersed chromatin. The interaction of DNA and protein was inferred from the amount of metachromasy (increased beta/alpha-peak) of azure B that resulted after specific removal of various protein fractions. Dye bound to DNA-histone fibers and frog liver nuclei fixed by freeze-methanol substitution shows orthochromatic, blue-green staining under specific staining conditions, while metachromasy (blue or purple color) results from staining DNA fibers without histone or tissue nuclei after protein removal. The dispersed chromatin of hepatocytes was compared to the condensed chromatin of erythrocytes to see whether there were differences in DNA-protein binding in "active" and "inactive" nuclei. Extraction of histones with 0.02 N HCl, acidified alcohol, perchloric acid, and trypsin digestion all resulted in increased dye binding. The amount of metachromasy varied, however; removal of "lysine-rich" histone (extractable with 0.02 N HCl) caused a blue color, and a purplish-red color (micro-peak absorption) resulted from prolonged trypsin digestion. In all cases, the condensed and the dispersed chromatin behaved in the same way, indicating the similarity of protein bound to DNA in condensed and dispersed chromatin. The results appear to indicate that "lysine-rich" histone is bound to adjacent anionic sites of a DNA molecule and that nonhistone protein is located between adjacent DNA molecules in both condensed and dispersed chromatin.
D000818:Animals; D001001:Anura; D002455:Cell Division; D002467:Cell Nucleus / Q000378:metabolism*; D004396:Coloring Agents; D004247:DNA / Q000378:metabolism*; D004912:Erythrocytes / Q000378:metabolism; D006657:Histones / Q000378:metabolism; D008099:Liver / Q000378:metabolism; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D013053:Spectrophotometry; D014357:Trypsin / Q000378:metabolism
D004396:Coloring Agents; D006657:Histones; D011506:Proteins; D004247:DNA; D014357:Trypsin