The genes for firefly luciferase and chloramphenicol acetyltransferase (CAT) were used as reporter genes to explore the activation of heterologous promoters by 8-Br-cAMP. Cells were transfected with a CAT gene/tyrosine hydroxylase promoter, which contains a cAMP response element. Extracts from cells treated with 8-Br-cAMP had 340% more enzyme activity than untreated cells. In contrast, treated cells transfected with a tyrosine hydroxylase/luciferase construct had 30% less activity than control cells. Simian virus and rous sarcoma virus promoters/luciferase constructs also had lower activities in cells treated with 8-Br-cAMP than untreated cells. The inhibition of luciferase enzyme activity by cAMP appears to be posttranscriptional since both luciferase and CAT RNA levels were similarly increased in cells treated with 8-Br-cAMP or 1-methyl-3-isobutylmethylxanthine. The lower level of luciferase activity was not due to simple allosteric inhibition. We conclude that constructs using the firefly luciferase as a reporter gene are unsuitable for studying the effects of cAMP on the regulation of promoters.
|Number of pages||3|
|State||Published - Jun 5 1989|
- Bromo cyclic AMP, 8-
- Methyl-3-isobutylmethylxanthine, 1-
- Reporter gene