Fig Chapter 12: Alternative approaches to mutational dissection

Types of mutational analysis 1. “Classical” “forward genetics” approach to understanding gene function: –Collect mutations. –Select those that affect the biological process of interest. –Study the mutant phenotype to discern the role of genes in the process –Clone the gene and carry out molecular analysis 2. “Post-genomics” “reverse genetics” approach: –Start with the cloned/sequences gene of unknown function –Create mutants of the gene –Study the mutant phenotype to discern the biological role of the gene

Selecting general mutagenic agents

Genetic screening versus selection Genetic screen: produce and sort through many non-mutant individuals to find the rare desired mutation Genetic selection: only the desired mutation survives

Fig. 16-4

Genetic screens can be carried out for a wide variety of biological functions (phenotypes): biochemical mutations morphological mutations lethal mutations conditional mutations (restrictive/permissive conditions) behavioral mutations secondary screens: modifier mutations gene expression mutations (using “reporters”)

Fig Forward selection criteria: testing for auxotrophy

Fig Forward selection criteria: testing for phototaxis

Fig Forward selection criteria: cell cycle progression Aspergillus nidulans

Fig Forward selection criteria: developmental morphology Danio rerio

Fig Screen strategy: survey haploids for mutant phenotypes

Genetic screen strategies Haploid screen Diploid screen for dominant mutations (“F1 screen”) Diploid screen for recessive mutations (“F2 screen”) Diploid screen for recessive mutations – specific locus screen “Special tricks” screens

Fig Enhancer trap screen to identify tissue-specific enhancers

Reverse genetics Knowing the sequence of a gene permits experiments to determine its function by directed mutation or phenocopy analysis Targeted gene knockout

Fig Knowing a gene sequence, it can become a target for knockout or replacement

Reverse genetics Knowing the sequence of a gene permits experiments to determine its function by directed mutation or phenocopy analysis Targeted gene knockout Site-directed mutagenesis

Fig Knowing a gene sequence, it can become a target of in vitro mutagenesis

Fig Knowing a gene sequence, it can become a target of in vitro mutagenesis

Reverse genetics Knowing the sequence of a gene permits experiments to determine its function by directed mutation or phenocopy analysis Targeted gene knockout Site-directed mutagenesis Produce phenocopies with antisense RNA

Fig Knowing a gene sequence, it can become a target for RNA-interference experiments dsRNA induces cellular complexes that degrade dsRNA

Fig Knowing a gene sequence, it can become a target for RNA-interference experiments Can induce RNA-specific degradation by deliberately introducing dsRNA into cells Look for phenotypes in RNAi-treated cells/organisms

Fig

Fig Understanding the functional basis of dominant mutations

Fig Understanding the functional basis of dominant mutations

Fig Understanding the functional basis of dominant mutations

Fig Understanding the functional basis of dominant mutations

Fig. 16-