PREPARATION AND CHARACTERIZATION OF YEAST NUCLEAR EXTRACTS FOR EFFICIENT RNA POLYMERASE-B (II)-DEPENDENT TRANSCRIPTION INVITRO

We present a reproducible method for the preparation of nuclear extracts from the yeast Saccharomyces cerevisiae that support efficient RNA polymerase B (II)-dependent transcription. Extracts from both a crude nuclear fraction and Percoll-purified nuclei are highly active for site-specific initiation and transcription of a G-free cassette under the Adenovirus major late promoter. At optimal extract concentrations transcription is at least 5 times more efficient with the yeast extracts than with HeLa whole cell extracts. We show that the transcriptional activity is sensitive to alpha-amanitin and to depletion of factor(s) recognizing the TATA-box of the promoter. The in vitro reaction showed maximal activity after 45 min, was very sensitive to Cl-, but was not affected by high concentration s of potassium. We find that the efficiency of in vitro transcription in nuclear extracts is reproducibly high when spheroplasting is performed with a partially purified-beta 1,3-glucanase (lyticase). Therefore a simplified method to isolate the lyticase from the supernatant of Oerskovia xanthineolytica is also presented.