A """"""""protein linkage map"""""""" is a description of the multiple protein-protein interactions that exist within a cell. This map would consist of the protein complexes responsible for: processes such as transcription and replication, pathways that function in signaling and metabolism, and structures such as the cytoskeleton. Protein-protein interactions can be detected by a simple genetic assay, the two-hybrid system. This assay reconstitutes transcriptional activity by the interaction of a protein fused to a DNA-binding domain with a protein fused to a transcriptional activation domain, and has been successfully applied to a wide diversity of proteins from different organisms. In preliminary studies using the bacteriophage T7, we have demonstrated that use of the two-hybrid system can be extended to a genome-wide approach. This approach allows us to search all proteins encoded by the phage against each other to detect interacting pairs. We propose to expand this approach to generate a protein linkage map for the yeast Saccharomyces cerevisiae. This map should provide critical information concerning a multitude of yeast proteins identified by genome sequencing efforts. Furthermore, it should illuminate the organization and function of a eukaryotic cell. Specifically, we aim: (1) to generate suitable vectors, yeast reporter strains and yeast genomic libraries; (2) to screen one to two million random DNA-binding hybrids against a library of activation domain hybrids to identify protein-protein interactions; (3) to enter these interactions into a database constructed for this purpose; and (4) to use these data to derive and analyze linked sets of proteins, which should comprise various cellular pathways and structures.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM054415-04
Application #
2771050
Study Section
Genome Study Section (GNM)
Project Start
1995-09-01
Project End
2000-08-31
Budget Start
1998-09-01
Budget End
2000-08-31
Support Year
4
Fiscal Year
1998
Total Cost
Indirect Cost
Name
University of Washington
Department
Genetics
Type
Schools of Arts and Sciences
DUNS #
135646524
City
Seattle
State
WA
Country
United States
Zip Code
98195