Multiple cloning sites from mammalian expression vectors interfere with gene promoter studies in vitro

When performing transcriptional analyses, reporter gene-expression vectors are used to insert promoter fragments through the selected use of a multipl e cloning site (MCS) located upstream of the reporter gene. The MCS from pB luescript(TM) has frequently been transferred into reporter plasmids (usual ly bearing the chloramphenical acetyltransferase reporter gene) and used to subclone various promoter fragments from diverse genes. Analyses in electr ophoretic mobility shift assay using this MCS as labeled probe revealed tha t it specifically binds multiple nuclear proteins from a whole array of wid ely used cell types. Moreover, the presence of the MCS sequence dramaticall y altered promoter activity in a totally unpredictable fashion that depends on the distance between the MCS and the basal promoter start site of the g ene, leading to severe misinterpretation of the transfection data. Finally, we provide evidence that the BamHI/SmaI/PstI restriction site combination is likely one of the major binding site for nuclear proteins on the pBluesc ript(TM) MCS, therefore suggesting that this particular combination of rest riction sites should be avoided in the MCS from plasmids that are to be use d in promoter studies.