Alessandra L. Starling, Kenneth J. Gollob, Elisabete J. Vicente, Gisele M. Andrade-Nóbrega, Mônica B. Rodriguez
J. Miguel Ortega
Published: March 31, 2003
Genet. Mol. Res. 2 (1) : 124-135
Cite this Article:
A.L. Starling, K.J. Gollob, E.J. Vicente, G.M. Andrade-Nóbrega, M.B. Rodriguez, M. Ortega (2003). Evaluation of alternative reporter genes for the yeast two-hybrid system. Genet. Mol. Res. 2(1): 124-135.
About the Authors
Alessandra L. Starling, Kenneth J. Gollob, Elisabete J. Vicente, Gisele M. Andrade-Nóbrega, Mônica B. Rodriguez
J. Miguel Ortega
Corresponding author
M.B.Rodriguez
E-mail: monicabar@icb.ufmg.br
ABSTRACT
The yeast two-hybrid system is a powerful tool for screening protein-protein interactions and has also been used for large-scale studies. We evaluated two protein-coding sequences as reporter genes for the yeast two-hybrid system, to determine if it was suitable as an alternative screening strategy. Aspergillus awamori glucoamylase activity results in clear haloes around colonies producing this enzyme after growth on starch plates and staining with iodine vapors. However, transcription activation by Gal4 on Gal-regulated promoters was insufficient for this type of phenotypic visualization. A modified gene of Aequoria victoria enhanced green fluorescent protein (EGFP) was tested to determine its suitability for interaction screenings with flow cytometry. When the EGFP reporter gene system was incorporated into the cells, Gal4 transcriptional activation produced sufficient fluorescence for detection with the flow cytometer, especially when there were strong interactions.
Key words: Yeast two-hybrid system, Reporter genes, Glucoamylase, Green fluorescent protein.