New tools for protein linkage mapping and general two-hybrid screening

The two-hybrid system has proved to be a facile method for detecting and an alyzing protein-protein interactions. An expanded application of this syste m, protein linkage mapping, provides a means of identifying interactions on a global scale and should prove a powerful tool in analyzing whole genomes as their sequences become available. To overcome some of the inherent diff iculties in such a large-scale approach, we have constructed a set of new s trains and vectors that will allow for more efficient screening. The strain s contain a GALI-URA3 reporter for positive and negative selection, as well as a UAS(G)-lacZ reporter. The strains are of opposite mating types, permi tting libraries present in one strain to be easily screened against a secon d library in the companion strain. We also constructed a family of CEN-base d vectors for expression of both Gal4 DNA-binding and activation domain fus ions. These plasmids include a hemagglutinin epitope tag and different poly linkers to increase the ease of subcloning. CEN-based vectors are maintaine d at 1-2 copies per cell, limiting the number of individual cells containin g multiple plasmids that can confuse further analyses, and ensuring that fu sions are not expressed at toxic levels. Using these vectors, both homo- an d heterodimeric interactions resulted in up to 10-fold higher reporter gene transcription than obtained with 2 mu-based plasmids, despite significantl y lower protein levels. In addition to protein linkage mapping, these reage nts should be generally useful in standard two-hybrid applications. Copyrig ht (C) 1999 John Wiley & Sons, Ltd.