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Chapter 12: Alternative approaches to mutational dissection Fig. 16-1 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”) Forward selection criteria: testing for auxotrophy Fig. 16-6 Forward selection criteria: testing for phototaxis Fig. 16-7 Forward selection criteria: cell cycle progression Aspergillus nidulans Fig. 16-10 Forward selection criteria: developmental morphology Danio rerio Fig. 16-12 Screen strategy: survey haploids for mutant phenotypes Fig. 16-13 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 Enhancer trap screen to identify tissue-specific enhancers Fig. 16-14 Reverse genetics Knowing the sequence of a gene permits experiments to determine its function by directed mutation or phenocopy analysis • Targeted gene knockout Knowing a gene sequence, it can become a target for knockout or replacement Fig. 16-15 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 • Knowing a gene sequence, it can become a target of in vitro mutagenesis Fig. 16-16 Knowing a gene sequence, it can become a target of in vitro mutagenesis Fig. 16-16 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 Knowing a gene sequence, it can become a target for RNA-interference experiments dsRNA induces cellular complexes that degrade dsRNA Fig. 16-19 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. 16-18 Fig. 16-21 Understanding the functional basis of dominant mutations Fig. 16-22 Understanding the functional basis of dominant mutations Fig. 16-22 Understanding the functional basis of dominant mutations Fig. 16-22 Understanding the functional basis of dominant mutations Fig. 16-22 Fig. 16- Fig. 16-