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.
Schmitz
U K
UK
Molecular Genetics Department, Institute of Plant Science Research, Trumpington, UK.
Lonsdale
D M
DM
Jefferson
R A
RA
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