RNA Synthesis in Isolated Nuclei of the Dinoflagellate Crypthecodinium cohnii

SYNOPSIS Atypical eukaryotic RNA polymerase activity was demonstrated in nuclei of Crypthecodinium 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 Mg 2+ with optima at ˜ 0.01 and 0.02 M MgCl 2 , but in contrast to other eukaryotic RNA polymerases, this enzyme activity was inhibited by low MnCl 2 concentrations. In the presence of 0.01 M MgCl 2 the optimum (NH 4 ) 2 SO 4 concentration was 0.025 M, a concentration at which the nuclei were lysed. Incorporation of [ 3 H]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 ˜ 3 pmoles of [ 3 H]UMP/muml; DNA at 25 C for 15 min, and ˜ 80% of this activity was inhibited by the eukaryotic RNA polymerase II inhibitor, α‐amanitin (20 m̈/ml). A unique situation therefore exists in C. cohnii nuclei, in which absence of histones (a prokaryotic trait) is combined with α‐amanitin‐sensitive RNA polymerase activity (a eukaryotic trait).