2. Genetic interaction and interpretation of genetic interactions - Biosynthetic pathway/ genes acting in different steps. -Order genes in a genetic pathway - studies on yeast mating -pheromone response - Epistasis analysis using null mutations- The GAP story - Epistasis analysis using gf mutations - The Ras suppressors -Enhancer and synergistic effect between two alleles - -The Ras pathway. -Understanding at molecular level/biochemical level. -Limitation of genetics

4. Pheromone response in yeast Adopted from Hartwell et al. Genetics

5. Genetics study of mating response 1. How do you want to start it? 2. What is the assay (phenotype) for the screen? Makay and Manning 1978, Hartwell 1980 Isolated mutations in 12 genes that cause the sterile phenotype. They named them ste1-12. All with the non-response phenotype.

6. Let us go back to 1970s to re-live the genetics of yeast mating response Normal plates with yeast lawn+ a factor, no growth spread + EMS Ste1 Ste2 Ste3 Ste4 ste5 Makay and Manning 1978 Hartwell, 1980, get to ste 12 by isolating Ts alleles.

7. Now what would you do? If we assume all these genes act in the same pathway. Assuming ste1-12 mutations are all loss-of-function mutations, can we use them to determine the order of gene actions? A: yes. B: No. - If you made a double mutant containing ste1 and ste2, what would you see? What would you learn?

8. A test Signal Gene A signal response Gene A has a loss-of-function mutation If Gene A is a positive factor, the mutation should enhance the signal response A: Yes. B: No Signal Gene A signal response Gene A has a gain-of-function (hyperactive) mutation If Gene A is a negative factor, the mutation should enhance the signal response A: Yes. B: No

9. when two genes act in the same pathway, mutants with opposite phenotypes mean one gene act as a positive regulator, the other is the negative regulator.

10. Genetic epistasis Epistatic: one effect masks the other Epistasis is used to learn about the order of gene action - indirect - need to learn biochemistry to understand the molecular action - important to verify and biochemical assumption

11. Epistasis can only be done with two different mutant phenotypes Genes in the same pathway: Mutations with opposite mutant phenotypes Blinder et al. 1989 Cell: Mating constitutive = haploid lethal (please go read the paper) Use cleaver sector synthetic lethal screen isolated many haploid lethal mutations that are unable to mate. Several mutations define the Ga gene. always want to mate Unable to mate ?

13. About trimeric G protein  receptor RGS GTP GDP Pi GTP GDP  or , who interacts with the downstream target?     ? ? 

14. Who activates the target,  or  mating response G  (-): hyper response, always want to mate GG G  (-): No mating response mating response GG G  (-) & G  (-): No mating response mating response GG A: GG mating response GG GG B: C: not sure

15. Getting tricky G  (-): constitutive mating G  non-mating (ste mutants) G  (-) + G  (-): no mating.  Target GG A:  Target GG B: C: Could be either

16. Epistasis with lf mutations  Target GG Receptor lf  Target GG Receptor lf  is required for the target activation

17. About trimeric G protein  receptor RGS GTP GDP Pi GTP GDP       Target GG A:

18. epistasis analysis using loss-of-function mutations: genetic control of programmed cell death Gene AGene B ced-9(+) ced-9(-) ced-9(+) ced-9(-) ced-4(+) ced-4(-) Phenotype Normal programmed cell death Extra cell deaths Cells that normally die survive Conclusion: ced-4(-) phenotype is epistatic to that of ced-9(-) Results ced-9ced-4 cell death On Off On Off No death Extra death No death Death signal Linear model ced-9 Factor X Death signal On Off On Off On Off ced-4 cell death No death Extra death No death Parallel model Molecular Actions CED-4 No death signal CED-4 is inactive Death signal CED-9 Activates CED-3 caspase for the killing CED-9 A: linear B: parallel

19. A second example of epistasis analysis using loss-of-function mutations: dauer larva formation Gene AGene B age-1(+) age-1(-) daf-18(+) daf-18(-) daf-18(+) daf-18(-) Phenotype When starved, worms become dauer larvae Defective in dauer formation Constitutive dauer formation Defective in dauer formation Conclusion: daf-18(-) phenotype is epistatic to that of age-1 Results age-1daf-18 Dauer formation On Off On Off No dauer Constitutive dauer No dauer Linear Model signal Factor X signal On Off On Off On Off On Parallel Model age-1 daf-18 Dauer formation AGE-1 PI3Kinase DAF-18 PTEN PIP 3 Binds and activates AKT kinases to prevent dauer formation signal + P - P PIP 2 Molecular actions A: linear B: parallel

20. What if you only have positive factors? - in case of mating response, you only have ste genes, whose lf mutants are non-maters. You can use gain-of-function mutations (hypermorph). - gf mutations often generate opposite mutant phenotypes as that of lf mutations in the same gene.

21. When a hyperactive (gf) mutant phenotype is the winner Conclusion: the let-60(gf) phenotype is epistatic to that of sem-5(lf) Gene AGene B sem-5(+) sem-5(lf) let-60 (+) let-60 (gf) let-60 (+) let-60 (gf) Phenotype in vulval induction 3 of the 6 precursor cells are induced Extra cells induced (Multivulva) Less than 3 cells induced (Vulvaless) Extra cells induced (Multivulva) Results Sem-5 (GRB2) let-60 (Ras) Vulval induction On Off Off (lf) On Off On (gf) Induction No induction No induction induction Model and explanation signal + - + +/- EGFRSOS In a given precursor cell signalpathway function No parallel model

22. When a hyperactive (gf) mutant phenotype is the loser Conclusion: the lin-45 (lf) phenotype is epistatic to that of let-60 (gf) Gene AGene B lin-45 (+) lin-45 (lf) let-60 (+) let-60 (gf) let-60 (+) let-60 (gf) Phenotype in vulval induction 3 of the 6 precursor cells are induced Extra cells induced (Multivulva) Less than 3 cells induced (Vulvaless) Results lin-45 (Raf) let-60 (Ras) Vulval induction On Off Off (lf) On Off (lf) On Off On On (gf) Induction No induction No induction Induction No induction Model A signal + - + - + In a given precursor cell lin-45/Raf Gene X Vulval induction On Off (lf) On Off (lf) On Off On On (gf) Induction No induction No induction Induction No induction signal +-+-++-+-+ let-60/Ras On Off On Off Model B Biochemistry: Ras directly binds to raf for its activation

23. Why do we do epistasis analysis? - provide a critical guide for biochemical analysis -Add significance to relationship based on biochemical functions