Evaluation of alternative reporter genes for the yeast two-hybrid system.
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.
Starling
Alessandra L
AL
Departamento de Biologia Geral, Instituto de Ciências Biologicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brasil.
Ortega
J Miguel
JM
Gollob
Kenneth J
KJ
Vicente
Elisabete J
EJ
Andrade-Nóbrega
Gisele M
GM
Rodriguez
Mônica B
MB
D001483:Base Sequence; D003001:Cloning, Molecular; D005434:Flow Cytometry; D017930:Genes, Reporter; D005087:Glucan 1,4-alpha-Glucosidase / Q000032:analysis / Q000235:genetics; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D020798:Two-Hybrid System Techniques; D015003:Yeasts / Q000235:genetics*
D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D005087:Glucan 1,4-alpha-Glucosidase