Analyzing a Biological Process Genetically in a Tractable Model Organism

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Analyzing a Biological Process Genetically in a
Tractable Model Organism
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Forward Geneticsvs.Reverse Genetics
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Forward Genetics Start with a phenotype Try to
get (eventually) to the moleculeReverse
Genetics Start with a molecule (gene, RNA,
protein) Try to get (eventually) to a mutant,
hence a phenotype, to evaluate function in
vivo
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Forward Genetics Screens Brute force With
enrichment Selections
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Primary screens (or selections)
vs.Secondary Screens (or selections)
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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.)
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Secondary screens (or selections)? How many
types?
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After screening (or selecting), how to get to the
molecules?
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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!
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Cell Polarization in Budding Yeast
What? Why? When? How? Where?
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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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• Two projects
• 1. Further characterize CDC24
• 2. Identify and characterize additional
  mutants with defects in bud formation

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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!)
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cdc24-4
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CDC42 suppressed cdc24ts (and recall the similar
phenotypes of cdc24ts and cdc42ts mutants)
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MSB1 suppressed cdc24ts AND cdc42ts mutations
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Cdc42 Rho-family       small GTPase Cdc24p
Activator       (GEF) of Cdc42p
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rsr1?
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Axial-specific marker Bud3p, Bud4p, Axl2p

General site-selection signaling module Rsr1p,
Bud2p, Bud5p
Polarity establishment and maintenance
functions Cdc24p, Cdc42p, etc.
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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.
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msb1? mutants were viable and indeed grew well
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msb1? mutants were viable and indeed grew
well So screened for mutations that were
inviable in combination with msb1? (a
synthetic-lethal screen)
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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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Two-hybrid screening with CDC42 baits
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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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