Preparation and characterization of yeast nuclear extracts for efficient RNA polymerase B (II)-dependent transcription in vitro.
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.
Verdier
J M
JM
Centre d'Etudes Nucléaires de Saclay, Service de Biochimie, Gif-sur-Yvette, France.
Stalder
R
R
Roberge
M
M
Amati
B
B
Sentenac
A
A
Gasser
S M
SM
D000546:Amanitins / Q000494:pharmacology; D002467:Cell Nucleus / Q000378:metabolism; D005821:Genetic Techniques; D004693:Glucan Endo-1,3-beta-D-Glucosidase / Q000302:isolation & purification / Q000378:metabolism; D009097:Multienzyme Complexes / Q000302:isolation & purification / Q000378:metabolism; D010447:Peptide Hydrolases / Q000302:isolation & purification / Q000378:metabolism; D012319:RNA Polymerase II / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D013104:Spheroplasts; D016385:TATA Box; D013696:Temperature; D013997:Time Factors; D014158:Transcription, Genetic
D000546:Amanitins; D009097:Multienzyme Complexes; C025310:lyticase; D012319:RNA Polymerase II; D004693:Glucan Endo-1,3-beta-D-Glucosidase; D010447:Peptide Hydrolases