|
rdf:type |
|
|
lifeskim:mentions |
|
|
pubmed:issue |
23
|
|
pubmed:dateCreated |
1991-2-1
|
|
pubmed:abstractText |
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.
|
|
pubmed:commentsCorrections |
http://linkedlifedata.com/resource/pubmed/commentcorrection/2263463-13584361,
http://linkedlifedata.com/resource/pubmed/commentcorrection/2263463-1715207,
http://linkedlifedata.com/resource/pubmed/commentcorrection/2263463-2188968,
http://linkedlifedata.com/resource/pubmed/commentcorrection/2263463-2197989,
http://linkedlifedata.com/resource/pubmed/commentcorrection/2263463-2204245,
http://linkedlifedata.com/resource/pubmed/commentcorrection/2263463-22164,
http://linkedlifedata.com/resource/pubmed/commentcorrection/2263463-2510298,
http://linkedlifedata.com/resource/pubmed/commentcorrection/2263463-2567734,
http://linkedlifedata.com/resource/pubmed/commentcorrection/2263463-2643115,
http://linkedlifedata.com/resource/pubmed/commentcorrection/2263463-2649403,
http://linkedlifedata.com/resource/pubmed/commentcorrection/2263463-2673007,
http://linkedlifedata.com/resource/pubmed/commentcorrection/2263463-2843296,
http://linkedlifedata.com/resource/pubmed/commentcorrection/2263463-2991927,
http://linkedlifedata.com/resource/pubmed/commentcorrection/2263463-3023048,
http://linkedlifedata.com/resource/pubmed/commentcorrection/2263463-3072472,
http://linkedlifedata.com/resource/pubmed/commentcorrection/2263463-320092,
http://linkedlifedata.com/resource/pubmed/commentcorrection/2263463-3290687,
http://linkedlifedata.com/resource/pubmed/commentcorrection/2263463-3290688,
http://linkedlifedata.com/resource/pubmed/commentcorrection/2263463-3321057,
http://linkedlifedata.com/resource/pubmed/commentcorrection/2263463-337691,
http://linkedlifedata.com/resource/pubmed/commentcorrection/2263463-3779841,
http://linkedlifedata.com/resource/pubmed/commentcorrection/2263463-3925456,
http://linkedlifedata.com/resource/pubmed/commentcorrection/2263463-4075392,
http://linkedlifedata.com/resource/pubmed/commentcorrection/2263463-498279,
http://linkedlifedata.com/resource/pubmed/commentcorrection/2263463-6537904,
http://linkedlifedata.com/resource/pubmed/commentcorrection/2263463-6791577,
http://linkedlifedata.com/resource/pubmed/commentcorrection/2263463-6895517,
http://linkedlifedata.com/resource/pubmed/commentcorrection/2263463-6933441,
http://linkedlifedata.com/resource/pubmed/commentcorrection/2263463-6991473,
http://linkedlifedata.com/resource/pubmed/commentcorrection/2263463-7326750
|
|
pubmed:language |
eng
|
|
pubmed:journal |
|
|
pubmed:citationSubset |
IM
|
|
pubmed:chemical |
|
|
pubmed:status |
MEDLINE
|
|
pubmed:month |
Dec
|
|
pubmed:issn |
0305-1048
|
|
pubmed:author |
|
|
pubmed:issnType |
Print
|
|
pubmed:day |
11
|
|
pubmed:volume |
18
|
|
pubmed:owner |
NLM
|
|
pubmed:authorsComplete |
Y
|
|
pubmed:pagination |
7033-9
|
|
pubmed:dateRevised |
2009-11-18
|
|
pubmed:meshHeading |
pubmed-meshheading:2263463-Amanitins,
pubmed-meshheading:2263463-Cell Nucleus,
pubmed-meshheading:2263463-Genetic Techniques,
pubmed-meshheading:2263463-Glucan Endo-1,3-beta-D-Glucosidase,
pubmed-meshheading:2263463-Multienzyme Complexes,
pubmed-meshheading:2263463-Peptide Hydrolases,
pubmed-meshheading:2263463-RNA Polymerase II,
pubmed-meshheading:2263463-Saccharomyces cerevisiae,
pubmed-meshheading:2263463-Spheroplasts,
pubmed-meshheading:2263463-TATA Box,
pubmed-meshheading:2263463-Temperature,
pubmed-meshheading:2263463-Time Factors,
pubmed-meshheading:2263463-Transcription, Genetic
|
|
pubmed:year |
1990
|
|
pubmed:articleTitle |
Preparation and characterization of yeast nuclear extracts for efficient RNA polymerase B (II)-dependent transcription in vitro.
|
|
pubmed:affiliation |
Centre d'Etudes Nucléaires de Saclay, Service de Biochimie, Gif-sur-Yvette, France.
|
|
pubmed:publicationType |
Journal Article,
Research Support, Non-U.S. Gov't
|
