Presentation on theme: "Forward Genetics What is forward genetics?"— Presentation transcript:
1 Forward Genetics What is forward genetics? Genetic screens designed to find genes that affect a trait of interest.PHENOTYPE GENERandom mutagenesisScreen for interesting phenotypesTrack down the gene(s) responsibleTraits can include morphology, physiology, behaviorReverse genetics?Genetic screens designed to identify phenotypes association with disruption of a particular gene or DNA sequence of interest.GENE PHENOTYPE
2 Forward Genetics Retinotectal projections in zebrafish Wild type never mindbelladonnaastrayBaier et al., Kalstrom et al., and Trowe et al., 1996, Development
3 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.
4 Mutagens Radiation Chemicals Insertions + Strong mutations – deletions, inversions,translocations+/- May disrupt multiplegenesCan be difficult to clone+ Full spectrum ofmutations, usually small+ Randomly distributed- Difficult to clone+ Full spectrum of mutations+ Mutation is tagged, and easy to clone+ Often reversible- Nonrandom distribution
5 Genetic Screens Screen for dominant mutations (F1 screen) Screen for recessive mutations (F3 screen)* ¼ of families will produce m/m offspring!
7 ? ? 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 ofComplementation group: a set of alleles that fail to complement one anotherComplementation 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 pathwayHartl and Jones, 1998
8 Classification of Mutants: Complementation XXMutant phenotypeNon-ComplementationMutations are in the same geneWT phenotypeComplementationMutations are in different genes
9 Classification of Mutants: The ‘Morphs’ Loss of Function allelesAmorphnull allele, no WT activity.genetic definition m/m = m/DfHypomorphpartial 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
10 Classification of Mutants: The ‘Morphs’ Gain of function allelesHypermorphextra WT function (usually dominant)Examples include constitutive promoters, constitutively active proteinsgenetic definition: m/m> m/+ > m/DfNeomorphnovel function (usually dominant)Examples include chimeric proteins due to translocationsgenetic definition: additional alleles (+ or Df) don’t affect the phenotype. So m/+ = m/Df
11 Classification of Mutants: The ‘Morphs’ Gain of function allelesAntimorphdominant negativeprotein product antagonizes (poisons) the WT protein.Occurs with proteins that function as multimers.B-galactosidase functions as a tetramerWild TypeMutant
12 Molecular Characterization of Mutants Chromosomal Mapping (recombination mapping)Molecular identificationPositional cloningRescue with candidate genesIdentification of altered transcriptional profilesCloning based for insertions