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Genetic interaction and interpretation of genetic interactions

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

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Genetic interaction and interpretation of genetic interactions

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  1. 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

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

  3. Genetics study of mating response How do you want to start it? 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.

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

  5. 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?

  6. A test signal response Gene A Signal signal response Gene A Signal 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 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

  7. 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.

  8. 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

  9. ? Unable to mate Epistasis can only be done with two different mutant phenotypes Genes in the same pathway: Mutations with opposite mutant phenotypes always want to mate 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.

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

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

  12. bg Target Ga A: bg Target B: Ga Getting tricky Ga (-): constitutive mating Gb (-): non-mating (ste mutants) Ga (-) + Gb (-): no mating. C: Could be either

  13. bg Target Receptor bg Target Ga Receptor Ga lf lf lf lf lf lf lf lf Epistasis with lf mutations gis required for the target activation

  14. bg Target Ga A: About trimeric G protein GTP GTP GDP GDP receptor a a    RGS  Pi

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

  16. Dauer formation signal age-1 daf-18 Linear Model On Off Off Off On Off No dauer Constitutive dauer No dauer age-1 signal Dauer formation Factor X Parallel Model daf-18 On Off On Off On Off On Off Off AGE-1 PI3Kinase signal Binds and activates AKT kinases to prevent dauer formation + P Molecular actions PIP2 PIP3 - P DAF-18 PTEN Gene A Gene B Phenotype A second example of epistasis analysis using loss-of-function mutations: dauer larva formation Results age-1(+) age-1(+) age-1(-) age-1(-) daf-18(+) daf-18(-) daf-18(+) daf-18(-) 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 A: linear B: parallel

  17. 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.

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

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

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

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