Synthesis of dinucleoside tetraphosphates in transfected cells by a firefly luciferase reporter gene.
The firefly luciferase gene is widely used as a reporter gene and its expression is generally considered to be non-toxic. In addition to its light-producing reaction, luciferase can synthesise dinucleoside polyphosphates, intracellular signalling molecules, in vitro. Here we show that COS-7 cells transfected with a luciferase expression vector accumulate up to 0.5 mM adenine-containing dinucleoside tetraphosphates (Ap4N) during the 24 h following luciferin addition. The optimal external concentration of luciferin was 0.4-0.6 mM. In agreement with its poor ability to synthesise adenine-containing dinucleoside triphosphates in vitro, the level of these compounds did not increase after transfection. Consequently, the results of experiments involving luciferase-mediated light production by live cells should now be viewed in the light of the possible effects of an increased intracellular Ap4N concentration on the properties of the system under investigation. This observation also points to a useful non-invasive procedure for the specific enhancement of intracellular Ap4N for studies directed at understanding the functions of these compounds.
Murphy
G A
GA
School of Biological Sciences, University of Liverpool, Biosciences Building, Liverpool L69 7ZB, UK.
McLennan
A G
AG
D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D001517:Coleoptera / Q000235:genetics / Q000378:metabolism; D015226:Dinucleoside Phosphates / Q000096:biosynthesis*; D017930:Genes, Reporter; D008156:Luciferases / Q000235:genetics* / Q000378:metabolism; D013997:Time Factors; D014162:Transfection
D015226:Dinucleoside Phosphates; D008156:Luciferases