Removing auto-activators from yeast-two-hybrid assays by conditional negative selection

Area of Science:

• Molecular Biology
• Biochemistry
• Genomics

Background:

• Yeast two-hybrid (Y2H) is a key method for detecting protein-protein interactions (PPIs).
• Genome-wide PPI network analysis is advancing rapidly with next-generation sequencing.
• Auto-activators (AAs) are a major source of false-positive results in Y2H assays.

Purpose of the Study:

• To develop and validate a negative selection strategy to minimize false positives caused by auto-activators in Y2H screens.
• To improve the accuracy of large-scale PPI network construction.

Main Methods:

• Integration of the pGAL2-URA3 fragment into the yeast genome.
• Utilizing 5-Fluoroorotic acid (5-FOA) to inhibit the growth of yeast cells expressing auto-activators.
• Application of the method to a Marchantia polymorpha cDNA library.

Main Results:

• Successfully minimized auto-activators from a Marchantia polymorpha cDNA library.
• Demonstrated efficacy in both liquid and solid-grown cultures.
• Confirmed the selective inhibition of yeast cells expressing auto-activators.

Conclusions:

• The pGAL2-URA3 and 5-FOA negative selection method is effective in reducing Y2H false positives.
• This approach is a valuable tool for large-scale Y2H screening and PPI network analysis.
• The URA3 marker also facilitates uracil autotrophy selection.