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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 Pheromone response in yeast QuickTime™ and a Photo - JPEG decompressor are needed to see this picture. Adopted from Hartwell et al. Genetics 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. Let us go back to 1970s to re-live the genetics of yeast mating response spread Normal plates with yeast lawn Ste1 Ste2 Ste3 Ste4 ste5 + EMS Makay and Manning 1978 Hartwell, 1980, get to ste 12 by isolating Ts alleles. + a factor, no growth 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? A test Signal Signal Gene A signal response Gene A signal response GeneA A has has aa gain-of-function loss-of-function (hyperactive) mutation Gene mutation GeneA A is is aa negative positive factor, the mutation should IfIf Gene enhance the the signal signal response response enhance A: Yes. Yes. A: B: No No B: 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. 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 Epistasis can only be done with two different mutant phenotypes Genes in the same pathway: Mutations with opposite mutant phenotypes Unable to mate ? 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. About trimeric G protein GDP GDP GTP receptor a GTP a RGS Pi ? ? a or , who interacts with the downstream target? Who activates the target, a or Ga (-): hyper response, always want to mate Ga mating response G (-): No mating response G mating response Ga(-) & G(-): No mating response A: G Ga mating response C: not sure B: Ga G mating response Getting tricky Ga (-): constitutive mating G (-): non-mating (ste mutants) Ga (-) + G (-): no mating. A: B: Ga Target Ga C: Target Could be either Epistasis with lf mutations Receptor Ga Target lf lf lf lf Receptor Ga lf lf lf lf is required for the target activation Target About trimeric G protein GDP GDP GTP receptor a a RGS Pi A: GTP Ga Target epistasis analysis using loss-of-function mutations: genetic control of programmed cell death Results Gene A ced-9(+) Gene B ced-4(+) Phenotype Normal programmed cell death ced-9(-) ced-4(+) Extra cell deaths ced-9(+) ced-4(-) Cells that normally die survive ced-9(-) ced-4(-) Cells that normally die survive Conclusion: ced-4(-) phenotype is epistatic to that of ced-9(-) Linear model Parallel model Death signal ced-9 On Off Off Death signal No death signal Death signal Off On Off cell death No death Extra death No death ced-9 On Off Off Molecular Actions ced-4 ced-4 Off On Off CED-9 CED-4 CED-9 Factor X cell death Off On Off No death Extra death No death CED-4 is inactive Activates CED-3 caspase for the killing A: linear B: parallel A second example of epistasis analysis using loss-of-function mutations: dauer larva formation Results Gene A age-1(+) Gene B daf-18(+) Phenotype When starved, worms become dauer larvae age-1(+) daf-18(-) Defective in dauer formation age-1(-) daf-18(+) Constitutive dauer formation age-1(-) daf-18(-) Defective in dauer formation Conclusion: daf-18(-) phenotype is epistatic to that of age-1 Linear Model signal age-1 Dauer formation daf-18 On Off Off Off On Off No dauer Constitutive dauer No dauer age-1 signal Factor X Parallel Model Dauer formation daf-18 On Off Off On Off On Off On Off AGE-1 PI3Kinase signal +P Molecular actions PIP3 PIP2 -P DAF-18 PTEN Binds and activates AKT kinases to prevent dauer formation A: linear B: parallel 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. When a hyperactive (gf) mutant phenotype is the winner signal Results pathway function Gene A sem-5(+) Gene B let-60 (+) Phenotype in vulval induction 3 of the 6 precursor cells are induced sem-5(+) let-60 (gf) Extra cells induced (Multivulva) sem-5(lf) let-60 (+) Less than 3 cells induced (Vulvaless) sem-5(lf) let-60 (gf) Extra cells induced (Multivulva) Model and explanation Conclusion: the let-60(gf) phenotype is epistatic to that of sem-5(lf) In a given precursor cell EGFR signal + + +/- Sem-5 (GRB2) SOS On Off Off (lf) Off (lf) let-60 (Ras) On Off Off On (gf) Vulval induction Induction No induction No induction induction No parallel model When a hyperactive (gf) mutant phenotype is the loser Results Gene A lin-45 (+) Gene B let-60 (+) Phenotype in vulval induction 3 of the 6 precursor cells are induced lin-45(+) let-60 (gf) Extra cells induced (Multivulva) lin-45 (lf) let-60 (+) Less than 3 cells induced (Vulvaless) lin-45 (lf) let-60 (gf) Less than 3 cells induced (Vulvaless) Conclusion: the lin-45 (lf) phenotype is epistatic to that of let-60 (gf) In a given precursor cell Model A signal + + + Model B let-60 (Ras) lin-45 (Raf) On Off Off (lf) On Off (lf) On Off On On (gf) On (gf) signal Vulval induction let-60/Ras Gene X Vulval induction On Off Off On Off Induction No induction No induction Induction No induction lin-45/Raf + + + On Off On On (gf) On (gf) On On Off (lf) On Off (lf) Induction No induction No induction Induction No induction Biochemistry: Ras directly binds to raf for its activation Why do we do epistasis analysis? - provide a critical guide for biochemical analysis -Add significance to relationship based on biochemical functions