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Titolo:
Isolation and characterization of polyadenylation complexes assembled in vitro
Autore:
Veraldi, KL; Edwalds-Gilbert, G; MacDonald, CC; Wallace, AM; Milcarek, C;
Indirizzi:
Univ Pittsburgh, Sch Med, Dept Mol Genet & Biochem, Pittsburgh, PA 15261 USA Univ Pittsburgh Pittsburgh PA USA 15261 Biochem, Pittsburgh, PA 15261 USA Texas Tech Univ, Hlth Sci Ctr, Dept Biochem & Cell Biol, Lubbock, TX 79430USA Texas Tech Univ Lubbock TX USA 79430 em & Cell Biol, Lubbock, TX 79430USA
Titolo Testata:
RNA-A PUBLICATION OF THE RNA SOCIETY
fascicolo: 5, volume: 6, anno: 2000,
pagine: 768 - 777
SICI:
1355-8382(200005)6:5<768:IACOPC>2.0.ZU;2-L
Fonte:
ISI
Lingua:
ENG
Soggetto:
PRE-MESSENGER-RNA; CLEAVAGE STIMULATION FACTOR; FACTOR I-M; SPECIFICITY FACTOR; POLY(A) POLYMERASE; 160-KD SUBUNIT; CROSS-LINKING; PROTEIN; 3'-END; SITE;
Keywords:
pre-mRNA 3 '-end processing; plasma cells;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
42
Recensione:
Indirizzi per estratti:
Indirizzo: Milcarek, C Univ Pittsburgh, Sch Med, Dept Mol Genet & Biochem, W127 Biomed Sci Tower,Pittsburgh, PA 15261 USA Univ Pittsburgh W127 Biomed Sci Tower Pittsburgh PA USA 15261
Citazione:
K.L. Veraldi et al., "Isolation and characterization of polyadenylation complexes assembled in vitro", RNA, 6(5), 2000, pp. 768-777

Abstract

We developed a two-step purification of mammalian polyadenylation complexes assembled in vitro. Biotinylated pre-mRNAs containing viral or immunoglobulin poly(A) sites were incubated with nuclear extracts prepared from mousemyeloma cells under conditions permissive for in vitro cleavage and polyadenylation and the mixture was fractionated by gel filtration; complexes containing biotinylated pre-mRNA and bound proteins were affinity purified on avidin-agarose resin. Western analysis of known components of the polyadenylation complex demonstrated copurification of polyadenylation factors with poly(A) site-containing RNA but not with control RNA substrates containing either no polyadenylation signals or a point mutation of the AAUAAA polyadenylation signal. Polyadenylation complexes that were assembled on exogenousRNA eluted from the Sephacryl column in fractions consistent with their size range extending from 2 to 4 x 10(6) M-r. Complexes endogenous to the extract were of approximately the same apparent size, but more heterogeneous in distribution. This method can be used to study polyadenylation/ cleavage complexes that may form upon a number of different RNA sequences, an important step towards defining which factors might differentially associate withspecific RNAs.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 30/11/20 alle ore 07:18:29