Protein-fragment complementation assays (PCAs) can be used to study protein-protein interactions (PPIs) in any living cell, in vivo or in vitro, in any subcellular compartment or membranes. Here, we present a detailed protocol for performing and analyzing a high-throughput PCA screening to study PPIs in yeast, using dihydrofolate reductase (DHFR) as the reporter protein. The DHFR PCA is a simple survival-selection assay in which Saccharomyces cerevisiae DHFR (scDHFR) is inhibited by methotrexate, thus preventing nucleotide synthesis and causing arrest of cell division. Complementation of cells with a methotrexate-insensitive murine DHFR restores nucleotide synthesis, allowing cell proliferation. The methotrexate-resistant DHFR has two mutations (L22F and F31S) and is 10,000 times less sensitive to methotrexate than wild-type scDHFR, but retains full catalytic activity. The DHFR PCA is sensitive enough for PPIs to be detected for open reading frame (ORF)-PCA fragments expressed off of their endogenous promoters.
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| Evidence ID | Analyze ID | Gene/Complex | Systematic Name/Complex Accession | Qualifier | Gene Ontology Term ID | Gene Ontology Term | Aspect | Annotation Extension | Evidence | Method | Source | Assigned On | Reference |
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| Evidence ID | Analyze ID | Gene | Gene Systematic Name | Phenotype | Experiment Type | Experiment Type Category | Mutant Information | Strain Background | Chemical | Details | Reference |
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| Evidence ID | Analyze ID | Gene | Gene Systematic Name | Disease Ontology Term | Disease Ontology Term ID | Qualifier | Evidence | Method | Source | Assigned On | Reference |
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| Evidence ID | Analyze ID | Regulator | Regulator Systematic Name | Target | Target Systematic Name | Direction | Regulation of | Happens During | Regulator Type | Direction | Regulation Of | Happens During | Method | Evidence | Strain Background | Reference |
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| Site | Modification | Modifier | Source | Reference |
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| Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Allele | Assay | Annotation | Action | Phenotype | SGA score | P-value | Source | Reference | Note |
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Increase the total number of rows showing on this page by using the pull-down located below the table, or use the page scroll at the table's top right to browse through the table's pages; use the arrows to the right of a column header to sort by that column; filter the table using the "Filter" box at the top of the table; click on the small "i" buttons located within a cell for an annotation to view further details about experiment type and any other genes involved in the interaction.
| Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Assay | Annotation | Action | Modification | Source | Reference | Note |
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| Complement ID | Locus ID | Gene | Species | Gene ID | Strain background | Direction | Details | Source | Reference |
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| Evidence ID | Analyze ID | Dataset | Description | Keywords | Number of Conditions | Reference |
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