Reporter Gene Expression Upon Stable Transfection When Only a TATA Box or a TATA Box Plus Sp1 Sites Are Present 5′ to the Gene

摘要

ABSTRACTEpisomal plasmids for stable transfection of mammalian cell cultures were constructed that have a G418-resistance (neo) gene immediately downstream of a highly truncated promoter. These plasmids had a functional hygromycin-resistance gene (hyg) as a selectable marker. Surprisingly, in LTK−cells, but not HeLa cells, stably transfected with these BK virus-based plasmids having no promoter elements adjacent to theneogene, readthrough transcription, probably from about 1 kb upstream, gave almost as efficient expression of theneogene as of thehyggene with a full-length promoter immediately upstream. When the transfecting plasmids contained Epstein-Barr virus (EBV) DNA sequences for episomal maintenance and had multiple Sp1 sites and a TATA box as the only promoter elements 5′ to theneogene, only about 3–9 of HeLa transfectants were G418 resistant (G418R). In transfections with analogous plasmids lacking these promoter elements 5′ to theneogene, no G418Rcolonies were seen. The establishment of the G418Rphenotype probably required integration of plasmid DNA into favorable chromosomal sites and was aided by the presence of the TATA box plus Sp1 sites as a subminimal promoter. The absence of detectable G418-resistance in most of the HeLa transfectant clones obtained with EBV-type plasmids, even at a high plasmid copy number and even when a TATA box and six Sp1 sites were present immediately upstream of theneogene, indicates that these elements do not suffice for appreciable gene expressionin vivoand that this is a suitable model system for studying DNA rearrangements that can potentiate expression of the