Preparation and characterization of yeast nuclear extracts for efficient RNA polymerase B (II)-dependent transcription<i>in vitro</i>
Nucleic Acids ResearchPublished 1 January 1990Open access
Jean‐Michel Verdier, Rolf Stalder, Michel Roberge, Bruno Amati, André Sentenac, Susan M. Gasser
Citations31
SJR quartileQ1
SJR score7.78
SNIP4.71
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TL;DR
It is found 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).
Abstract
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 concentrations 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.
Keywords
Biochemistry, Genetics and Molecular Biology
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