Preparation and characterization of yeast nuclear extracts for efficient RNA polymerase B (II)-dependent transcription in vitro.
AUTOR(ES)
Verdier, J M
RESUMO
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.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=332766Documentos Relacionados
- Accurate transcription of cloned Neurospora RNA polymerase II-dependent genes in vitro by homologous soluble extracts.
- Accurate transcription of cloned Neurospora RNA polymerase II-dependent genes in vitro by homologous soluble extracts
- RNA polymerase II-dependent transcription in trypanosomes is associated with a SNAP complex-like transcription factor
- Activation of yeast RNA polymerase II transcription by a thymidine-rich upstream element in vitro.
- RNA polymerase II ternary transcription complexes generated in vitro.