Analyzing a Biological Process
Genetically in a Tractable
Model Organism
Forward Genetics
vs.
Reverse Genetics
Forward Genetics
Start with a phenotype
Try to get (eventually) to the molecule
Reverse Genetics
Start with a molecule (gene, RNA,
protein)
Try to get (eventually) to a mutant,
hence a phenotype, to evaluate
function in vivo
Forward Genetics
Screens
Brute force
With enrichment
Selections
Primary screens (or selections)
vs.
Secondary Screens (or selections)
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.)
Secondary screens (or selections)
? = How many types?
After screening (or selecting), how
to get to the molecules?
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!
Cell Polarization in Budding Yeast
What? Why? When? How? Where?
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
Normal pattern of cell-surface
growth and cytokinesis
Actin system
Cytoplasmic microtubules )
Nuclear migration and
spindle orientation
Two projects:
1. Further characterize CDC24
2. Identify and characterize additional
mutants with defects in bud formation
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!)
cdc24-4
CDC42 suppressed
ts
cdc24
(and recall the similar phenotypes of cdc24ts and
cdc42ts mutants)
ts
cdc24
MSB1 suppressed
ts
AND cdc42 mutations
Cdc42: Rhofamily
small GTPase
Cdc24p:
Activator
Cdc42p
(GEF) of
rsr1
Axial-specific marker
[Bud3p, Bud4p, Axl2p]
General site-selection signaling module
[Rsr1p, Bud2p, Bud5p]
Polarity establishment and maintenance functions
[Cdc24p, Cdc42p, etc.]
Axial-specific marker
Bipolar-specific markers
[Bud3p, Bud4p, Axl2p]
[Bud8p, Bud9p]
General site-selection signaling module
[Rsr1p, Bud2p, Bud5p]
Polarity establishment and maintenance functions
[Cdc24p, Cdc42p, etc.]
msb1∆ mutants were viable and indeed grew well
msb1∆ mutants were viable and indeed grew well
So screened for mutations that were inviable in
combination with msb1∆
(a “synthetic-lethal screen”)
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,
(Rho1p, Rho3p, Rho4p,
, Rdi1p, ....)
, Bni1p, Msb3p, ....)
Cytoskeletal system
( Septin array
Normal pattern of cell-surface
growth and cytokinesis
Actin system
Cytoplasmic microtubules )
Nuclear migration and
spindle orientation
Two-hybrid screening
with CDC42 baits
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,
,
,
, Bem4p, Msb1p,
(Rho1p, Rho3p, Rho4p,
, Rdi1p, ....)
, Bni1p, Msb3p, ....)
Cytoskeletal system
( Septin array
Normal pattern of cell-surface
growth and cytokinesis
Actin system
Cytoplasmic microtubules )
Nuclear migration and
spindle orientation