Mutational Analysis: Classical vs. Post-genomics Approach

1. Chapter 12: Alternative approaches to mutational dissection Fig. 16-1

2. 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

3. Selecting general mutagenic agents

4. 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

5. Fig. 16-4

6. 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”)

7. Forward selection criteria: testing for auxotrophy Fig. 16-6

8. Forward selection criteria: testing for phototaxis Fig. 16-7

9. Forward selection criteria: cell cycle progression Aspergillus nidulans Fig. 16-10

10. Forward selection criteria: developmental morphology Danio rerio Fig. 16-12

11. Screen strategy: survey haploids for mutant phenotypes Fig. 16-13

12. 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

13. Enhancer trap screen to identify tissue-specific enhancers Fig. 16-14

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

15. Knowing a gene sequence, it can become a target for knockout or replacement Fig. 16-15

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

17. Knowing a gene sequence, it can become a target of in vitro mutagenesis Fig. 16-16

18. Knowing a gene sequence, it can become a target of in vitro mutagenesis Fig. 16-16

19. 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

20. Knowing a gene sequence, it can become a target for RNA-interference experiments dsRNA induces cellular complexes that degrade dsRNA Fig. 16-19

21. 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

22. Fig. 16-21

23. Understanding the functional basis of dominant mutations Fig. 16-22

24. Understanding the functional basis of dominant mutations Fig. 16-22

25. Understanding the functional basis of dominant mutations Fig. 16-22

26. Understanding the functional basis of dominant mutations Fig. 16-22

27. Fig. 16-

28. Fig. 16-