RNA Synthesis in Isolated Nuclei of the Dinoflagellate Crypthecodinium cohnii

Abstract

Atypical eukaryotic RNA polymerase activity was demonstrated in nuclei of C. cohnii, a eukaryote devoid of histones. Nuclei were isolated from growing cultures of this dinoflagellate and assayed for endogenous RNA polymerase (EC 2.7.7.6) activity. There was a biphasic response to Mg2+ with optima at .apprx. 0.01 and 0.02 M MgCl2, but in contrast to other eukaryotic RNA polymerases, this enzyme activity was inhibited by low MnCl2 concentrations. In the presence of 0.01 M MgCl2 the optimum (NH4)2SO4 concentration was 0.025 M, a concentration at which the nuclei were lysed. Incorporation of [3H]UMP into RNA was inhibited by actinomycin D and dependent on the presence of undegraded DNA, and the reaction product was sensitive to ribonuclease and KOH digestion. Omission of one or more ribonucleoside triphosphates greatly reduced the incorporation. Only a slight enhancement of RNA polymerase activity resulted from the addition of various amounts of native and denatured calf thymus DNA. Spermine caused a marked inhibition while spermidine had little effect on RNA synthesis in the nuclei. Under the optimum conditions described in the present paper the nuclei incorporated .apprx. 3 pmol of [3H]UMP/.mu.g DNA at 25 C for 15 min, and .apprx. 80% of this activity was inhibited by the eukaryotic RNA polymerase II inhibitor, .alpha.-amanitin (20 .mu.g/ml). A unique situation therefore exists in C. cohnii nuclei, in which absence of histones (a prokaryotic trait) is combined with .alpha.-amanitin-sensitive RNA polymerase activity (a eukaryotic trait).