RNA synthesis in fixed cells by endogenous RNA polymerases

Abstract

A method for the detection of endogenous DNA-dependent RNA polymerase activity in eukaryotic cells is described. Incubation of ethanol and acetone-fixed cells with ribonucleoside triphosphates ATP, GTP, CTP and [ 3H]UTP results in the incorporation of radioactivity into the nuclei. The reaction product is not removed by trichloroacetic acid (TCA). However, labelling is blocked by actinomycin D and RNase indicating that the reaction is dependent on DNA and that RNA is synthesized. Evidence suggests that incorporation occurs as a result of the elongation of RNA chains which were initiated, in vivo. The amount of RNA synthesized is limited and remains complexed within the chromatin. The sites of label incorporation may be localized in the nucleolus and nucleoplasm of the cell by autoradiography. Grain counts give a relative measure of polymerase I and II activities respectively. The use of fixed cells circumvents problems which are associated with labelling of RNA in vivo. Incorporation is not dependent either on the transport of precursors into the cell or their uptake into intracellular pools. The method permits the transcriptional activities of individual cells to be detected and quantitated during differentiation.