Application of the beta-glucuronidase gene fusion system to Saccharomyces cerevisiae.
Bacterial beta-glucuronidase (GUS) has been described as a useful reporter enzyme for gene fusion studies in bacteria and plants. Here we report the expression of GUS in yeast to illustrate further applications of this enzyme as a quantitative tool for measuring gene activity, as a colour selection marker and as a versatile system for protein targeting studies. There is no intrinsic GUS activity in any yeast strain tested. GUS was expressed in transgenic yeast on a multiple-copy vector under the control of the alcohol dehydrogenase 1 (ADH1) promoter. The enzyme is stable in yeast and its activity may be monitored by very sensitive colorimetric or fluorometric methods in extracts, or by the histochemical reagent 5-bromo-4-chloro-3-indolylglucuronide (X-Gluc) on plates. To test the efficacy of GUS as a reporter for targeting proteins into different subcellular compartments in vivo, we fused the presequence of the mitochondrial tryptophanyl-tRNA-synthetase gene (MSW) to the amino terminus of GUS. The activity of the fusion protein is not substantially impaired and it is imported efficiently into yeast mitochondria.
Molecular Genetics Department, Institute of Plant Science Research, Trumpington, UK.
U K
UK
Schmitz
D M
DM
Lonsdale
R A
RA
Jefferson
D015153:Blotting, Western; D003001:Cloning, Molecular; D005822:Genetic Vectors; D005966:Glucuronidase / Q000096:biosynthesis / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D014170:Transformation, Genetic
D011993:Recombinant Fusion Proteins; D005966:Glucuronidase