ABSTRACT
The nuclei of the lymphocytes of rat spleen were isolated with the modified Kohji Ueno method and then were purified. Four kinds of nucleoside triphosphate (ATP, GTP, CTP, UTP) as well as 3H-UTP and certain inorganic ions were added in to study the transcriptional function, DNA-dependent RNA polymerase activity, of the nuclei in vitro. The influence of the substrate concentration, temperature, pH value, the concentration of certain inorganic ions(Mg, Mn, NH4 + ), actinomycin D and ?-amanitin on the polymerase activity was evaluated.It was found that Mn and (NH4)2SO4 in high concentration(0.2M) could markedly enhance the transcriptional activity while actinomycin D and a-amanitin inhibited it. A-amanitin in low concentration (1?g/ml) could inhibit 80% of the transcriptional activity. If proper ion concentration was given, relatively high activity of RNA polymerase could be shown. The transcriptional activity was in linear relation with certain range of the nucleus concentration. Substrate requires the existence of the four kinds of nucleoside triphosphate: this means that the transcriptional model reflects the activity of the DNA-dependent RNA polymerase and is suitable for the study of transcriptional mechanism.
ABSTRACT
The inhibitory effects of actinomycin D on chromatin (or DNA) template and the stimulating effects of synthetic template Poly [d(A-T)] on transcription were observed in the liver nucleic transcriptional system of the rat in vitor.By introducing proper concentrations of actinomycin D and Poly [d(A-T)] into each reactionary system,a method to assay free RNA polymerase activity in the nucleus was established.In addition,a comparison of the contents between chromatin-engaged and free polymerase in normal rat liver nuclei isolated with hypertonic sucrose (2.3 mol/L) was made.Eventually,the possible role free RNA Polymerases play in the eukaryotic transcriptional process was discussed.
