The yeast (Saccharomyces cerevisiae) RAD54-GFP DNA repair reporter assay (GreenScreen assay, GSA) can be used for early genotoxicity screening in drug discovery. During the initial validation of this preregulatory assay, a subset of known genotoxic compounds that did not give reproducibly clear positive GSA results was identified. Cell permeability, inherent drug resistance mechanisms, metabolic activation and compound solubility were identified as possible barriers to the detection of specific compounds. In this study three types of modification to the existing assay protocol were explored in order to address these possibilities: (i) modification of the reporter host strain by deletion of genes involved in cell wall integrity or with products functioning as efflux pumps (PDR5, ERG6, SNQ2, YOR1); (ii) expression in the host yeast of human phase I metabolic activation genes and (iii) variation in the test solvent system for compounds with poor aqueous solubility. The modifications described and the assay results presented show how the assay may be tailored to suit specific classes of test compound in a more analytical mode. Improvements in assay sensitivity were seen in the detection of some genotoxins using yeast cell wall mutants and those expressing human cytochrome P450 genes.
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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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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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