Forward Genetics What is forward genetics? Genetic screens designed to find genes that affect a trait of interest. PHENOTYPE GENE Random mutagenesis Screen for interesting phenotypes Track down the gene(s) responsible Traits can include morphology, physiology, behavior Reverse genetics? Genetic screens designed to identify phenotypes association with disruption of a particular gene or DNA sequence of interest. GENE PHENOTYPE

Forward Genetics Retinotectal projections in zebrafish Wild type never mind belladonna astray Baier et al., Kalstrom et al., and Trowe et al., 1996, Development

Saturation mutagenesis Large scale genetic screens that aim to create a mutation in every gene required for a trait. How do you know when you get there? When new mutations represent a second or multiple hit in a previously identified gene. The saturation point defines the set of genes required INDIVIDUALLY for a particular trait. Genes whose function are redundantly provided by another loci will be missed.

Mutagens Radiation Chemicals Insertions + Strong mutations – deletions, inversions, translocations +/- May disrupt multiple genes - Can be difficult to clone + Full spectrum of mutations, usually small + Randomly distributed - Difficult to clone + Full spectrum of mutations + Mutation is tagged, and easy to clone + Often reversible - Nonrandom distribution

Genetic Screens Screen for dominant mutations (F1 screen) Screen for recessive mutations (F3 screen) * ¼ of families will produce m/m offspring!

Genetic Screens

? ? Classification of Mutants: Complementation If you have two mutants that have the same phenotype, how to test if they are mutations in the same or different genes? ? ? Complement: provide the function of Complementation group: a set of alleles that fail to complement one another Complementation testing reduces a large number of mutations to a smaller set of loci. Non-allelic non-complementation can occur if mutations affect genes in the same pathway Hartl and Jones, 1998

Classification of Mutants: Complementation X X Mutant phenotype Non-Complementation Mutations are in the same gene WT phenotype Complementation Mutations are in different genes

Classification of Mutants: The ‘Morphs’ Loss of Function alleles Amorph null allele, no WT activity. genetic definition m/m = m/Df Hypomorph partial l-o-f, reduced WT activity. Usually recessive, however can be dominant if it is haploinsufficient (eg mutations on the X in males) genetic definition m/m < m/Df

Classification of Mutants: The ‘Morphs’ Gain of function alleles Hypermorph extra WT function (usually dominant) Examples include constitutive promoters, constitutively active proteins genetic definition: m/m> m/+ > m/Df Neomorph novel function (usually dominant) Examples include chimeric proteins due to translocations genetic definition: additional alleles (+ or Df) don’t affect the phenotype. So m/+ = m/Df

Classification of Mutants: The ‘Morphs’ Gain of function alleles Antimorph dominant negative protein product antagonizes (poisons) the WT protein. Occurs with proteins that function as multimers. B-galactosidase functions as a tetramer Wild Type Mutant

Molecular Characterization of Mutants Chromosomal Mapping (recombination mapping) Molecular identification Positional cloning Rescue with candidate genes Identification of altered transcriptional profiles Cloning based for insertions