We improved the integration replacement/disruption method (Shortle, D., Novic, P., and Botstein, D. Proc. Natl. Acad. Sci. USA 81: 4889-4893, 1984) for isolating mutants in any of essential genes of the yeast Saccharomyces cerevisiae by integrating mutagenized DNA into the wild type gene of interest. We adopted this method to isolate temperature-sensitive mutants of the MPC1 gene encoding the YLL031C ORF. To facilitate integration of the mutagenic plasmid at a site near the 5' end of the ORF, a BamHI site was created at 300 bp downstream of the 5' end of the truncated ORF to be mutagenized. The MPC1 gene was disrupted in the wild type haploid strain by integrating a 5'-truncated derivative of the gene with mutations induced by in vitro mutagenesis. Transformants thus obtained were subjected for diagnosis of conditional lethality by replica-plating onto an appropriate selection medium to detect mutants. A primary mutant isolated by this method reverted in a high frequency due to a tandem repeat created by mutagenic integration. We deviced a method to obtain a stable temperature-sensitive strain by disrupting the tandem duplication. Two stable temperature-sensitive mutants thus obtained were found to be remedial either with 1 M sorbitol or with 0.1 M Mg2+ and to be sensitive to local anestheticum, tetracaine, at 25 degrees C.
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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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