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PMID: 2263463 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Preparation and characterization of yeast nuclear extracts for efficient RNA polymerase B (II)-dependent transcription in vitro.

Nucleic acids research ·Vol. 18 ·No. 23 ·1990-12-11 ·Pages 7033-9

Verdier JM, Stalder R, Roberge M, Amati B, Sentenac A, Gasser SM

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.

MeSH Terms
Amanitins/pharmacology Cell Nucleus/metabolism Genetic Techniques Glucan Endo-1,3-beta-D-Glucosidase/isolation & purification,metabolism Multienzyme Complexes/isolation & purification,metabolism Peptide Hydrolases/isolation & purification,metabolism RNA Polymerase II/metabolism Saccharomyces cerevisiae/enzymology,genetics Spheroplasts TATA Box Temperature Time Factors Transcription, Genetic
Chemicals
Amanitins Multienzyme Complexes lyticase RNA Polymerase II Glucan Endo-1,3-beta-D-Glucosidase Peptide Hydrolases
Authors & Affiliations
6 authors, click to expand affiliations / ORCID
Verdier J M
Centre d'Etudes Nucléaires de Saclay, Service de Biochimie, Gif-sur-Yvette, France.
Stalder R
Roberge M
Amati B
Sentenac A
Gasser S M
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Article Info
Journal
Nucleic acids research
Abbr.
Nucleic Acids Res
ISSN
0305-1048
DOI
10.1093/nar/18.23.7033
Published
1990-12-11
Pages
7033-9
Language
English
Region
England
NLM ID
0411011
PMCID
PMC332766
Subset
IM
External Links
PubMed source DOI: 10.1093/nar/18.23.7033 PMC Full Text View journal details
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