Yeast exo-beta-glucanases can be used as efficient and readily detectable reporter genes in Saccharomyces cerevisiae.
Yeast exo-1,3-beta-glucanases are secretable proteins whose function is basically trophic and may also be involved in cell wall glucan hydrolytic processes. Since fluorescein di(beta-D-glucopyranoside) is a fluorogenic substrate detectable and quantifiable by flow cytometry, it was used for testing the ability of the EXG1 gene product of Saccharomyces cerevisiae and its homologous gene in Candida albicans to function as reporter genes. These open reading frames were coupled to different promoters in multicopy plasmids, and exoglucanase activity quantified at flow cytometry. Exoglucanases were found to be useful tools for the study of promoter regions in S. cerevisiae. This technique has the advantage over other reporter gene systems--such as beta-galactosidase fusions--that it does not require permeabilization of yeast cells and therefore it allows the recovery of viable cells--by sorting--after flow cytometry analysis.
Departamento de Microbiología II, Facultad de Farmacia, Universidad Complutense de Madrid, Spain.
V J
VJ
Cid
A M
AM
Alvarez
A I
AI
Santos
C
C
Nombela
M
M
Sanchez
D005434:Flow Cytometry; D005800:Genes, Fungal; D017930:Genes, Reporter; D043326:Glucan 1,3-beta-Glucosidase; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D001617:beta-Glucosidase / Q000032:analysis / Q000235:genetics*
D001617:beta-Glucosidase; D043326:Glucan 1,3-beta-Glucosidase