Effect of prednisolone on thymus lymphocytes: II. Studies on [formula omitted] binding and RNA polymerase activity of isolated nuclei

Abstract

The rate of actinomycin D (AMD) binding to DNA of isolated nuclei is very rapid. Nearly 90% of the total AMD bound binds during the first minute of incubation, irrespective of AMD concentration or temperature. The amount of AMD bound depends on the concentration of AMD in the medium. Two distinct phases of this concentration dependency were detected. During the initial phase (up to 15 μg/ml AMD concentration) the amount of AMD bound increases in a 1:1 proportion to the increase in AMD concentration; during the second phase (15–500 μg/ml), the increase in bound AMD is in a proportion of 1:8 to the increase in AMD concentration. This phenomenon is considered to reflect AMD binding to DNA in derepressed and repressed chromatin, respectively; AMD “titration” of derepressed sites is completed when 14–16 AMD molecules are bound per 10 3 base pairs of DNA. Increase in ionic strength “opens” new sites for AMD binding while divalent cations specifically decrease the number of sites binding AMD. Treatment of isolated thymocyte nuclei with prednisolone does not significantly change AMD binding; this is in contrast with liver cell nuclei, which bind 10–12% more AMD in the presence of prednisolone. The nuclei isolated from prednisolone-treated thymocytes bind 10–18% less AMD than control nuclei. In some experiments this difference was diminished when AMD binding was studied at higher ionic strength. The mechanism of this phenomenon as well as the possible reasons for its irreproducibility are discussed. No change in RNA-polymerase activity was detected when isolated nuclei were treated with prednisolone. However, when nuclei were isolated from prednisolone-treated cells, they exhibited a decreased activity in the presence of both Mg 2+ or Mn 2+ ions. An increase in ionic strength resulted in stimulated RNA-polymerase activity, with a higher stimulation being observed in nuclei from prednisolone-treated cells. As a result, the difference between control nuclei and those of prednisolone-treated cells diminished at higher ionic strength. The mechanism of the action of prednisolone in modulating thymus chromatin is discussed.