PubMed Health⌕ Search
Home/Archive/PubMed

PubMed · 2555292

An improved nuclear extract preparation method.

K Zerivitz·G Akusjärvi
View original source ↗Export metadata ↓

Abstract

A rapid, efficient, and highly reproducible procedure for nuclear extract preparation is described. The method uses lysolecithin (lysophosphatidylcholine) to disrupt plasma membranes and requires no detergents or douncing. Soluble extracts prepared by this method are comparable to conventional nuclear extracts in all assays tested. Lysolecithin nuclear extracts are competent for RNA polymerase II and III transcription, DNA replication, pre-mRNA splicing, and sequence specific DNA-protein binding. Nuclear extracts can be prepared on a small scale (10(7) cells) as well as for preparative purposes by this method.

Explore related subjects

AdenoviridaeCell NucleusDNA ReplicationDNA, ViralDNA-Binding ProteinsDNA-Directed RNA PolymerasesHeLa CellsHumansLysophosphatidylcholinesPromoter Regions, GeneticRNA, MessengerSimian virus 40Subcellular FractionsTranscription, GeneticJournal ArticleResearch Support, Non-U.S. Gov't

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

K Zerivitz, G Akusjärvi. An improved nuclear extract preparation method.. https://doi.org/10.1016/0735-0651(89)90016-2

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related citations

PubMed2005

SUMO modification of the Ets-related transcription factor ERM inhibits its transcriptional activity.

A variety of transcription factors are post-translationally modified by SUMO, a 97-residue ubiquitin-like protein bound covalently to the targeted lysine. Here we describe SUMO modification of the Ets family member ERM at positions 89, 263, 293, and 350. To investigate how SUMO modification affects the function of ERM, Ets-responsive intercellular adhesion molecule 1 (ICAM-1) and E74 reporter plasmids were employed to demonstrate that SUMO modification causes inhibition of ERM-dependent transcription without affecting the subcellular localization, stability, or DNA-binding capacity of the protein. When the adenoviral protein Gam1 or the SUMO protease SENP1 was used to inhibit the SUMO modification pathway, ERM-dependent transcription was de-repressed. These results demonstrate that ERM is subject to SUMO modification and that this post-translational modification causes inhibition of transcription-enhancing activity.

Adenoviridae↗
PubMed1992

Different binding site requirements for binding and activation for the bipartite enhancer factor EF-1A.

The human transcription factor EF-1A binds to the purine-rich E1A core enhancer sequence in the adenovirus E1A and E4 and polyomavirus enhancer regions. The consensus binding site for EF-1A resembles that of members of the ets domain protein family. EF-1A activation of transcription requires a dimeric binding site. Analysis of binding sites containing point mutations revealed that EF-1A binding is determined by the core nucleotides of the binding site, while transcriptional activation is determined both by the core and some peripheral nucleotides that do not affect binding. We have purified EF-1A and analyzed its two constituent subunits, EF-1A alpha and EF-1A beta. EF-1A alpha (MW approximately 60kD) makes the primary DNA contacts. EF-1A beta (MW approximately 50 kD) forms a heteromultimeric complex with EF-1A alpha both in solution and on a dimeric binding site. Binding of both EF-1A subunits is necessary, but not sufficient, for transcriptional activation. We present immunochemical and functional evidence that EF-1A alpha is related to the murine ets-related protein GABP alpha and that EF-1A beta is related to the murine protein GABP beta.

Adenoviridae↗