Expression and in vivo determination of firefly luciferase as gene reporter in Saccharomyces cerevisiae.
The LUC gene coding for Photinus pyralis firefly luciferase was cloned in different yeast episomal plasmids in order to assess its possibilities as an in vivo reporter gene. Activity of the enzyme in transformed cells in vivo was measured by following light emission and assay conditions optimized in intact cells, with regard to oxygen concentration, temperature, cell concentration in assay mixtures and external ATP concentration. Among the factors tested, light emission was drastically influenced by the external pH in the assay (which resulted in a ten-fold amplification signal) and by substrate permeability. The growth phase of the cells was also important for the level of activity detected. Cloning of firefly luciferase gene under the control of different yeast-regulated promoters (ADH1, GAL1-10) enabled us to measure their strength which correlated well with previously described data. We conclude that firefly luciferase is an adequate gene reporter for the in vivo sensitive determination of gene expression and promoter strength in yeast.
Vieites
J M
JM
Departamento de Microbiología II, Facultad de Farmacia, Universidad Complutense, Madrid, Spain.
Navarro-García
F
F
Pérez-Díaz
R
R
Pla
J
J
Nombela
C
C
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001517:Coleoptera / Q000201:enzymology*; D003902:Detergents / Q000494:pharmacology; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D008156:Luciferases / Q000235:genetics*; D011401:Promoter Regions, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development
D003902:Detergents; D000255:Adenosine Triphosphate; D008156:Luciferases