One-step measurement of firefly luciferase activity in yeast.
Firefly luciferase is often used as a sensitive genetic reporter in various cell types. The pitfall in yeast, however, has been the need to break down the rigid cells in order to measure the enzyme activity. In this study we have removed the peroxisomal targeting codons from the Photinus pyralis luciferase gene (luc) and shown that in the yeast Saccharomyces cerevisiae this modified luciferase gives high levels of light emission that is easy to measure from intact living cells. Furthermore, cells with the modified luciferase grew essentially faster than those with the wild-type luciferase, indicating that peroxisomal targeting of a foreign enzyme puts some constraints to cellular viability. As a model system we used two different reporter constructs. In the first, expression of the luciferase gene is under control of CUP1-promoter, a well known yeast promoter that is inducible by copper ions. In the second, luciferase activity is dependent on activation of the human oestrogen receptor and its interaction with oestrogen-responsive elements incorporated in a yeast promoter. The luciferase activity measurement could be done on a 96-well plate by simple addition of the substrate, D-luciferin, at a moderately acidic pH of 5.0. The ease of use of the non-peroxisomal luciferase makes it an interesting alternative for reporter genes that are conventionally used in yeast, such as lacZ.
Leskinen
P
P
University of Turku, Department of Biotechnology, Turku, Finland. piiles@utu.fi
Virta
M
M
Karp
M
M
D003300:Copper / Q000378:metabolism; D004790:Enzyme Induction / Q000187:drug effects; D004967:Estrogens / Q000378:metabolism; D049454:Firefly Luciferin / Q000378:metabolism; D017930:Genes, Reporter / Q000235:genetics*; D008156:Luciferases / Q000235:genetics / Q000378:metabolism*; D008163:Luminescent Measurements; D020675:Peroxisomes / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics
D004967:Estrogens; D049454:Firefly Luciferin; D003300:Copper; D008156:Luciferases