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 analyzing protein-protein interactions. An expanded application of this system, 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 difficulties in such a large-scale approach, we have constructed a set of new strains and vectors that will allow for more efficient screening. The strains contain a GAL1-URA3 reporter for positive and negative selection, as well as a UAS(G)-lacZ reporter. The strains are of opposite mating types, permitting libraries present in one strain to be easily screened against a second library in the companion strain. We also constructed a family of CEN-based vectors for expression of both Gal4 DNA-binding and activation domain fusions. These plasmids include a hemagglutinin epitope tag and different polylinkers to increase the ease of subcloning. CEN-based vectors are maintained at 1-2 copies per cell, limiting the number of individual cells containing multiple plasmids that can confuse further analyses, and ensuring that fusions are not expressed at toxic levels. Using these vectors, both homo- and heterodimeric interactions resulted in up to 10-fold higher reporter gene transcription than obtained with 2micro;-based plasmids, despite significantly lower protein levels. In addition to protein linkage mapping, these reagents should be generally useful in standard two-hybrid applications.
Durfee
T
T
Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720, USA. durf@nature.berkeley.edu
Draper
O
O
Zupan
J
J
Conklin
D S
DS
Zambryski
P C
PC
D001483:Base Sequence; D015153:Blotting, Western; D002503:Centromere / Q000737:chemistry*; D002874:Chromosome Mapping / Q000379:methods*; D004271:DNA, Fungal / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000737:chemistry*; D008969:Molecular Sequence Data; D010957:Plasmids / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics; D017422:Sequence Analysis, DNA; D020798:Two-Hybrid System Techniques; D001616:beta-Galactosidase / Q000032:analysis
D004271:DNA, Fungal; D001616:beta-Galactosidase