Targeted gene repair directed by chimeric RNA/DNA oligonucleotides has proven successful in eukaryotic cells including animal and plant models. In many cases, however, there has been a disparity in the levels of gene correction or frequency. While the delivery of these chimera into the nucleus and the long-term stability or purity of these molecules may contribute to this variability, understanding the molecular regulation of conversion is the key to improving or stabilizing frequency. To this end, we have identified genes that control targeted repair, using the genetically tractable organism, Saccharomyces cerevisae and a bank of yeast mutants. Results from experiments in cell-free extracts focused our attention on RAD52, RAD1 and RAD59 as central regulatory factors. RAD1 and RAD59 appear to be required for high levels of conversion whereas RAD52 appears to act, surprisingly, in a suppressive fashion. Results from the in vitro experiments were translated into targeting experiments in vivo. Here, mutations in a fusion construct, containing a marker gene, were converted to wild type, evidenced by the expression of green fluorescence in converted cells. Because the repaired fusion gene contains a corrected neomycin sequence, cells were subsequently placed under G418 selection and conversion confirmed at the genetic level. Taken together, these results establish, for the first time, genes that participate in the regulation of targeted gene repair and provide a novel system for evaluating true frequencies of correction. Importantly, this system enables visualization of corrected (green) and uncorrected (clear) cells enabling measurements of conversion in real time.
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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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