Rapid selection against truncation mutants in yeast reverse two-hybrid screens

The yeast reverse two-hybrid system is a powerful technique for isolating m utations in a protein that abolish its interaction with a known partner. Se lection is based on abrogation of growth suppression imposed when wild-type interactions confer 5-fluoroorotic acid (5-FOA) sensitivity to yeast cells . A laborious component of this system is to eliminate those mutations that cause protein truncation. By fusing the green fluorescent protein (GFP) to the C-terminus of a protein of interest, dynein light chain (LC8), we were able to rapidly isolate mutations that did not result in protein truncatio n.