OVEC, a versatile system to study transcription in mammalian cells and cell-free extracts

Abstract

We have developed a vector, OVEC (“oligonucleotide Vector”) to study DNA sequences involved in the regulation of transcription in mammalian cells. This vector is equally suitable for studying expression in vivo after transfection into cells, or for transcription studies in vitro with cell-free extracts. Putative cis-acting DNA segments from enhancers or promoters can be inserted at a position immediately upstream of the TATA box and coding sequence of the rabbit β-globin gene. A regulatory DNA segment can be tested by itself or in conjunction with an enhancer located either in an adjacent upstream position, or downstream of the β-globin gene. Sl nuclease mapping can be used to study transcription from circular and linear templates and run-off transcription in vitro is also feasible. Transcripts from a reference globine gene with a small delection around the transcription initiation site can be measured with the same Sl nuclease probe and thus serve as an internal standard. We demonstrate the usefulness of OVEC by inserting either short oligonucleotides comprising a metal responsive enhancer element, or the SV40 enhancer, directly upstream of the TATA box. Both constructs yield high levels of correctly initiated transcripts in a transient expression assay in HeLa cells. In a HeLa cell nuclear extract the SV40 enhancer stimulates transcription 40-fold.