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Analyzing a Biological Process Genetically in a Tractable Model Organism

Analyzing a Biological Process Genetically in a Tractable Model Organism. Forward Genetics vs. Reverse Genetics.

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Analyzing a Biological Process Genetically in a Tractable Model Organism

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  1. Analyzing a Biological Process Genetically in a Tractable Model Organism

  2. Forward Geneticsvs.Reverse Genetics

  3. Forward GeneticsStart with a phenotype Try to get (eventually) to the moleculeReverse GeneticsStart with a molecule (gene, RNA, protein) Try to get (eventually) to a mutant, hence a phenotype, to evaluate function in vivo

  4. Forward GeneticsScreens Brute force With enrichmentSelections

  5. Primary screens (or selections)vs.Secondary Screens (or selections)

  6. Primary screens (or selections)(You know only the phenotype that you are interested in.)vs.Secondary Screens (or selections)(You already have one mutant or gene.)

  7. Secondary screens (or selections)? = How many types?

  8. After screening (or selecting), how to get to the molecules?

  9. After screening (or selecting), how to get to the molecules?Answer = it depends on what you have in hand and on what organism you are working with!

  10. Cell Polarization in Budding Yeast What? Why? When? How? Where?

  11. Axial-specific marker Bipolar-specific markers (Bud3p, Bud4p, Axl1p, Axl2p, ....) (Bud8p, Bud9p, Bud7p, Rax1p, Rax2p....) General site-selection functions (Rsr1p, Bud2p, Bud5p, ....) Polarity-establishment and polarity-maintenance functions (Cdc24p, Cdc42p, Bem3p, Rga1p, Cla4p, Ste20p, Bem4p, Msb1p, Bem1p, Bni1p, Msb3p, ....) (Rho1p, Rho3p, Rho4p, Bem2p, Rdi1p, ....) Cytoskeletal system ( Septin array Actin system Cytoplasmic microtubules ) Normal pattern of cell-surface growth and cytokinesis Nuclear migration and spindle orientation

  12. Two projects: 1. Further characterize CDC24 2. Identify and characterize additional mutants with defects in bud formation

  13. Ts--lethal mutants defective in bud formation: 1. The first 24 had cdc24 mutations. 2. The 25th had a mutation in a new gene, CDC42. (Actually, it had three mutations!)

  14. cdc24-4

  15. CDC42 suppressed cdc24ts (and recall the similar phenotypes of cdc24ts and cdc42ts mutants)

  16. MSB1 suppressed cdc24ts AND cdc42ts mutations

  17. Cdc42: Rho-family       small GTPase Cdc24p: Activator       (GEF) of Cdc42p

  18. rsr1

  19. Axial-specific marker [Bud3p, Bud4p, Axl2p] General site-selection signaling module [Rsr1p, Bud2p, Bud5p] Polarity establishment and maintenance functions [Cdc24p, Cdc42p, etc.]

  20. Axial-specific marker [Bud3p, Bud4p, Axl2p] Bipolar-specific markers [Bud8p, Bud9p] General site-selection signaling module [Rsr1p, Bud2p, Bud5p] Polarity establishment and maintenance functions [Cdc24p, Cdc42p, etc.]

  21. msb1∆ mutants were viable and indeed grew well

  22. msb1∆ mutants were viable and indeed grew well So screened for mutations that were inviable in combinationwithmsb1∆ (a “synthetic-lethal screen”)

  23. Axial-specific marker Bipolar-specific markers (Bud3p, Bud4p, Axl1p, Axl2p, ....) (Bud8p, Bud9p, Bud7p, Rax1p, Rax2p....) General site-selection functions (Rsr1p, Bud2p, Bud5p, ....) Polarity-establishment and polarity-maintenance functions (Cdc24p, Cdc42p, Bem3p, Rga1p, Cla4p, Ste20p, Bem4p, Msb1p, Bem1p, Bni1p, Msb3p, ....) (Rho1p, Rho3p, Rho4p, Bem2p, Rdi1p, ....) Cytoskeletal system ( Septin array Actin system Cytoplasmic microtubules ) Normal pattern of cell-surface growth and cytokinesis Nuclear migration and spindle orientation

  24. Two-hybrid screening with CDC42 baits

  25. Axial-specific marker Bipolar-specific markers (Bud3p, Bud4p, Axl1p, Axl2p, ....) (Bud8p, Bud9p, Bud7p, Rax1p, Rax2p....) General site-selection functions (Rsr1p, Bud2p, Bud5p, ....) Polarity-establishment and polarity-maintenance functions (Cdc24p, Cdc42p, Bem3p, Rga1p, Cla4p, Ste20p, Bem4p, Msb1p, Bem1p, Bni1p, Msb3p, ....) (Rho1p, Rho3p, Rho4p, Bem2p, Rdi1p, ....) Cytoskeletal system ( Septin array Actin system Cytoplasmic microtubules ) Normal pattern of cell-surface growth and cytokinesis Nuclear migration and spindle orientation

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