title: Limited bidirectional activity of two housekeeping gene promoters: human HPRT and PGK.
abstract: Typical of other housekeeping genes, the promoter for the human hypoxanthine phosphoribosyl transferase-encoding gene (HPRT) is G + C-rich, lacks a TATA box and has multiple transcription start points. To test the hypothesis that these features may result in relaxed control over the direction of transcription, we examined the effect of orientation on the ability of the HPRT promoter to control expression of the following reporter genes in transfected cells: luc (firefly luciferase), cat (bacterial chloramphenicol acetyltransferase) and neo (neomycin resistance). A 376-bp fragment containing the HPRT promoter efficiently expressed the luc gene irrespective of orientation, and the 5' ends of luciferase RNA produced in cells transfected with inverted promoter constructs mapped to within the HPRT promoter, indicating that the HPRT promoter has bidirectional activity. However, in the presence of two divergently-flanking reporter genes expression from the inverted HPRT promoter was only 10-20% compared to the noninverted orientation. Furthermore, the inverted HPRT promoter expressed cat less well than luc, and was unable to express neo sufficiently well to produce any colonies under appropriate selection conditions. Attempts to detect endogenous divergent HPRT transcripts were unsuccessful. The promoter of another housekeeping gene, encoding 3-phosphoglycerate kinase (PGK), expressed moderate levels of cat (40%) but not luc (less than 5%) in the inverted orientation. By comparison, two TATA-box containing promoters functioned extremely poorly when inverted. This study indicates that two plasmid-borne housekeeping promoters have at least a limited potential for bidirectional activity, but the functional significance of this is unclear if the corresponding endogenous housekeeping promoters express divergent transcripts at similarly low levels. The poor activity of the HPRT and PGK promoters in the inverted orientation suggests that there is a mechanism which influences the direction of transcription from these promoters.
authors.affiliation: Center for Molecular Genetics, UCSD, La Jolla 92093.
authors.forename: P
authors.initials: P
authors.lastname: Johnson
authors.forename: T
authors.initials: T
authors.lastname: Friedmann
mesh_terms: D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D004352:Drug Resistance, Microbial / Q000235:genetics; D004790:Enzyme Induction; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics*; D008156:Luciferases / Q000235:genetics; D009355:Neomycin; D010735:Phosphoglycerate Kinase / Q000235:genetics*; D011401:Promoter Regions, Genetic; D014158:Transcription, Genetic
chemical_list: D008156:Luciferases; D015500:Chloramphenicol O-Acetyltransferase; D007041:Hypoxanthine Phosphoribosyltransferase; D010735:Phosphoglycerate Kinase; D009355:Neomycin