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 WT mutant gene Z WT mutant PCR amplification from wild type and mutant EMS mutagenize seed

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)

w+w+ w-w- FRT FLP FLP/FRT Targeted Mitotic Recombination Recombination Step

w+w+ w-w- FRT w + / w - red eyes w + / w - red eyes FLP/FRT Targeted Mitotic Recombination Segregation Option I: Outsides vs. Insides

w+w+ w-w- FRT w + / w + red eyes w - / w - white eyes FLP/FRT Targeted Mitotic Recombination Segregation Option II: Tops vs. Bottoms

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