Toward a protein–protein interaction map of the budding yeast: A comprehensive system to examine two-hybrid interactions in all possible combinations between the yeast proteins
| Autor: | Satoru Kuhara, Ritsuko Ozawa, Kiyoshi Yamamoto, Tomoko Chiba, Takashi Ito, Yoshiyuki Sakaki, Shigeru Muta, Kosuke Tashiro, Mayumi Nishizawa |
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| Rok vydání: | 2000 |
| Předmět: |
Genetics
Multidisciplinary Mechanism (biology) Yeast Proteins Genes Fungal DNA Recombinant Cellular functions A protein Saccharomyces cerevisiae Computational biology Biological Sciences Biology Polymerase Chain Reaction Budding yeast Fungal Proteins Vesicular transport protein Open Reading Frames Genes Reporter Two-Hybrid System Techniques Feasibility Studies DNA Fungal Protein Interaction Map |
| Zdroj: | Proceedings of the National Academy of Sciences. 97:1143-1147 |
| ISSN: | 1091-6490 0027-8424 |
| Popis: | Protein–protein interactions play pivotal roles in various aspects of the structural and functional organization of the cell, and their complete description is indispensable to thorough understanding of the cell. As an approach toward this goal, here we report a comprehensive system to examine two-hybrid interactions in all of the possible combinations between proteins of Saccharomyces cerevisiae . We cloned all of the yeast ORFs individually as a DNA-binding domain fusion (“bait”) in a MAT a strain and as an activation domain fusion (“prey”) in a MAT α strain, and subsequently divided them into pools, each containing 96 clones. These bait and prey clone pools were systematically mated with each other, and the transformants were subjected to strict selection for the activation of three reporter genes followed by sequence tagging. Our initial examination of ≈4 × 10 6 different combinations, constituting ≈10% of the total to be tested, has revealed 183 independent two-hybrid interactions, more than half of which are entirely novel. Notably, the obtained binary data allow us to extract more complex interaction networks, including the one that may explain a currently unsolved mechanism for the connection between distinct steps of vesicular transport. The approach described here thus will provide many leads for integration of various cellular functions and serve as a major driving force in the completion of the protein–protein interaction map. |
| Databáze: | OpenAIRE |
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