Transcription in Isolated Wheat Nuclei: II. CHARACTERIZATION OF RNA SYNTHESIZED IN VITRO

Abstract

Nuclei free of RNase activity were isolated from 3-hour-imbibed wheat (var. Yamhill) embryos by centrifugation through a discontinuous gradient of Percoll. The maximum rate of RNA synthesis observed in these nuclei was approximately 5 picomoles [(3)H]UTP per milligram DNA per minute. Two monovalent cation optima were found when measured in the presence of 15 millimolar MgCl(2) or 2 millimolar MnCl(2); 15 and 75 millimolar (NH(4))(2)SO(4). At the high monovalent cation optimum, the rate of RNA synthesis was linear for the first 10 to 15 minutes of incubation and still increasing after 3 hours. RNA synthesized in vitro (30-minute pulse followed by a 30-minute chase) showed distinct 18 and 26S RNA peaks comprising 13 and 17% of the total radioactivity, respectively. The over-all pattern of RNA synthesized in vitro was similar to the in vivo pattern. Approximately 40 to 50% of the RNA synthesized was inhibited by alpha-amanitin at 4 micrograms per milliliter. The newly synthesized 6 to 10S RNA appeared to be selectively inhibited by alpha-amanitin.