Fused bacterial luciferase subunits catalyze light emission in eukaryotes and prokaryotes.
A monocistronic luxAB gene containing the luxA and luxB genes coding for bacterial luciferase has been generated by site-specific mutagenesis so that it is now possible to express luciferase under control of a single promoter in eukaryotes as well as in prokaryotes. The fused luciferase subunits (alpha and beta), linked by a decapeptide, were synthesized in yeast under the Gal-4 promoter, in Escherichia coli under the T7-phage promoter, and in vitro in a rabbit reticulocyte lysate after transcription and capping of the mRNA under the SP6 phage promoter. Replacement of the ATG codon initiating the luxB sequence in the fused luxAB gene with CAG prevented internal initiation and allowed purification of a highly active fused luciferase in the absence of the beta-luciferase subunit. Consequently luciferase activity can be directly attributed to the fused luciferase alone and does not require complementation with free beta subunit of luciferase. Light emission could be measured in yeast and bacterial cells without the need for cell lysis providing the basis for measuring gene expression directly in vivo. These results demonstrate the potential applicability of the fused bacterial luciferase genes as a reporter of gene expression both in prokaryotic and eukaryotic systems.
Boylan
M
M
Department of Biochemistry, McGill University, Montreal, PQ Canada.
Pelletier
J
J
Meighen
E A
EA
D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005796:Genes; D005798:Genes, Bacterial; D008027:Light; D008156:Luciferases / Q000235:genetics / Q000378:metabolism*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D010957:Plasmids; D012441:Saccharomyces cerevisiae / Q000235:genetics
D001426:Bacterial Proteins; D046911:Macromolecular Substances; D008156:Luciferases