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Titolo:
Multiple cloning sites from mammalian expression vectors interfere with gene promoter studies in vitro
Autore:
Beliveau, A; Leclerc, S; Rouleau, M; Guerin, SL;
Indirizzi:
UnivPQaval, Med Res Ctr, Ctr Hosp Univ Quebec, Mol Endocrinol Lab, St Foy,Univ Laval St Foy PQ Canada G1K 7P4 v Quebec, Mol Endocrinol Lab, St Foy,
Titolo Testata:
EUROPEAN JOURNAL OF BIOCHEMISTRY
fascicolo: 2, volume: 261, anno: 1999,
pagine: 585 - 590
SICI:
0014-2956(199904)261:2<585:MCSFME>2.0.ZU;2-0
Fonte:
ISI
Lingua:
ENG
Soggetto:
NUCLEAR FACTOR-I; GROWTH-HORMONE; DNA; SEQUENCES; BINDING; PROTEIN; ELEMENT; COMPLEX; FAMILY; REGION;
Keywords:
transient transfection; multiple cloning site; promoter; misinterpretation;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
23
Recensione:
Indirizzi per estratti:
Indirizzo: Guerin, SL Ctr Hosp Univ Quebec, CHUL, Res Ctr, Mol Endocrinol Lab, 2705 Laurier Blvd, Ctr Hosp Univ Quebec 2705 Laurier Blvd St Foy PQ Canada G1V 4G2
Citazione:
A. Beliveau et al., "Multiple cloning sites from mammalian expression vectors interfere with gene promoter studies in vitro", EUR J BIOCH, 261(2), 1999, pp. 585-590

Abstract

When performing transcriptional analyses, reporter gene-expression vectorsare used to insert promoter fragments through the selected use of a multiple cloning site (MCS) located upstream of the reporter gene. The MCS from pBluescript(TM) has frequently been transferred into reporter plasmids (usually bearing the chloramphenical acetyltransferase reporter gene) and used to subclone various promoter fragments from diverse genes. Analyses in electrophoretic mobility shift assay using this MCS as labeled probe revealed that it specifically binds multiple nuclear proteins from a whole array of widely used cell types. Moreover, the presence of the MCS sequence dramatically altered promoter activity in a totally unpredictable fashion that depends on the distance between the MCS and the basal promoter start site of the gene, leading to severe misinterpretation of the transfection data. Finally, we provide evidence that the BamHI/SmaI/PstI restriction site combinationis likely one of the major binding site for nuclear proteins on the pBluescript(TM) MCS, therefore suggesting that this particular combination of restriction sites should be avoided in the MCS from plasmids that are to be used in promoter studies.

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Documento generato il 27/10/20 alle ore 04:50:16