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Mutagenesis and Genetic Screens Genome-Wide Phenotypic Analysis: “Phenomics” TILLING • Method for finding mutations produced by chemical mutagens in specific genes • Chemical mutagenesis – Usually produces point mutations – Very high mutagenic efficiency – Generally gives more subtle phenotypes than insertions • e.g., hypomorphs, temperature sensitive mutants TILLING in Arabidopsis I • EMS used to mutagenize Arabidopsis • Grow individual mutagenized lines • Make primers flanking gene of interest • Amplify using PCR EMS mutagenize seed gene Z WT gene Z mutant PCR amplification from wild type and mutant WT mutant TILLING in Arabidopsis II • Denature DNA from pools of mutant lines • Allow to hybridize to wild-type DNA • Detect mismatches in hybridized DNA – Denaturing HPLC – Cel I enzyme cuts at mismatches • Sequence to identify site of mutation ATGCGGACTG TACGCCGGAC ATGCGG CTG TACGCC GAC Cel 1 MOSAIC ANALYSIS MOSAIC ANALYSIS - WHY? Purposes of Mosaic Analysis • Examine later and/or tissue-specific functions of a gene required for viability • Bypass lethality to examine later function • Determine where gene function is required • Find which tissue is the source of gene activity • Determine “autonomy” of gene function • Cell lineage analysis SEM of Drosophila Compound Eye Eye Differentiation in Drosophila RTK Signal Transduction Pathway MOSAIC ANALYSIS – HOW? General Strategies for Mosaic Analysis • Nuclear or Cellular Transplantation • Mitotic Chromosomal Loss • Mitotic Recombination • Interchromosomal • Intrachromosomal Mitotic Recombination • Must be induced (not normal) • DNA breaks (eg., X-rays) • Site-specific enzymes » FLP recombinase » Cre recombinase FLP/FRT-Mediated Mitotic Recombination (Interchromosomal) FLP/FRT Targeted Mitotic Recombination •Recombination Step w+ FRT FLP w- FRT FLP/FRT Targeted Mitotic Recombination •Segregation Option I: Outsides vs. Insides w+ / wred eyes w+ FRT •Segregation Option I: Outsides vs. Insides w- FRT w+ / wred eyes FLP/FRT Targeted Mitotic Recombination •Segregation Option II: Tops vs. Bottoms w+ / w+ red eyes w+ FRT •Segregation Option II: Tops vs. Bottoms w- FRT w- / wwhite eyes What do we need? • • • • Mutation of interest distal to the FRT FRT near the centromere (preferably) Source of FLP recombinase Cell autonomous marker of genotype Employing cell markers for mitotic recombination