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1. ~ 1000 cells, small, easy to use for genetics
2. Entire lineage and nerve system mapped.
Caenhorhabditis elegans
3. 3 day life cycle, easy to use for genetics.
4. hermaphrodite
Upper Panel: Nomarski photomicrograph of one gonad arm of an adult C. elegans hermaphrodite. The distal portion of the gonad arm is up; the proximal portion is down.
Lower Panel: Nomarski photomicrograph of an adult C. elegans male gonad. The distal portion of the testis is above; the proximal region is below.
Three steps of vulval development
cell migrationhox genes
1, precursor cells
3, morphogenesis cell division, fusion, migration, etc.
Cell fate E E V V V E
Lineage
2, vulval induction Cell signaling
gonadP1 P12dorsal
AC
3° 3° 2° 1° 2° 3°WT
X
3° 3° 3° 3° 3° 3°- AC
Indicating:
A: AC is required for vulval inductionB: AC may send a signal to induce vulval cellsC: Both.
Judith Kimble:
gonad
anchor cellinductive signal
E E V V V E
Wild type 100% induction
E E EEEE
Vulvaless 0%
Multivulva 200%
V V V V V V
signal pathway function
Earlier direct screens for vulval induction mutations
wild type
Mutagen
Vulvaless
Multivulva
Horvitz and Sulston 1980, GeneticsFerguson and Horvitz 1985, GeneticsFerguson et al. 1987. NatureAroian et al. 1991. NatureHill and Sternberg 1992. Nature
lin-3EGF
let-23EGFR
lin-1
Vulval induction
Identification of Ras
Mammal: identified through oncogenic mutations in 1980
Ras homolog in yeast was identified in 1985.
Ras function in development was first identified in 1990 (C. elegans)
RASGDP
RASGTP
Pi
GTPGDP
Exchange reaction
Hydrolysis reaction
Active stateInactive
target protein
lin-15(-), Muv lin-15(-);; X(-) Vulvaless
EMS
Supressors of lin-15 mutation
X = EGFR, RAS and others. Han and Sternberg 1990, Cell; Beitel et al. 1990, Nature; Aroian et al. 1990 Nature
lin-15 Vulval induction
Vote:
XA.
B. lin-15 Vulval inductionX
Relationship between RAS and EGFR
EGFR(lf) Vulvaless
lin-15 Vulval inductionEGFRRAS
MultivulvaRAS (gf)
EGFR(lf) + RAS (gf) Multivulva
Vulval induction
Vote:
RASA.
B. Vulval induction
EGFR
RASEGFR
RASGDP
RASGTP
Pi
GTPGDP
Exchange reaction
Hydrolysis reaction
target protein
Active stateInactive
Strategy 1: moving up from Ras
signal
signal
QuickTime™ and aGIF decompressor
are needed to see this picture.
QuickTime™ and aGIF decompressor
are needed to see this picture.
First strike: discovery of GTPase activating protein (GAP)
1. Questions addressed
GTPase of Ras
In vitro weak t 1/2 = 30 minIn vivo strong t 1/2 < 1 min
A: Something in cells can stimulate the GTPase activityB: Something in intro inhibits the GTPase activity
The student and PI were mammalian biochemists. What system will they likely chose to identify the “something”?
A: Mammalian cells. B. Drosophila. C. Xenopus oocyteD. Yeast.
RAS:GTPRAS:GDP
Pi
Approach: Using Xenopus oocytes- ease to manipulate due to large size- good assay for biological activity, oocyte maturation.
Does GTP or GDP bind to injected Ras?
WT Ras mostly bind to GDPOncogenic Ras mostly to GTP
In vivo In vitro
WT vs. Oncogenes
RAS:GTPRAS:GDP
GTPase activityPi
RAS:GTPRAS:GDP
GTPase activityPi
WTGly 12
OncogenesVal 12Asp 12
X
What causes the difference between in vitro and in vivo?
Add cytoplasmic factor to in vitro > 200 fold difference
Trahey and McCormick Science Oct 1987
RASGDP
RASGTP
Pi
GTPGDP
Exchange reaction
GAP
target protein
Active stateInactive
How is Ras regulated by the signal?
EGFR
EGFR
Cell 1990. It is all GAPKaplan DR, Morrison DK, Wong G, McCormick F, Williams LT. PDGF beta-receptor stimulates tyrosine phosphorylation of GAP and
association of GAP with a signaling complex. Cell. 1990 61:125-33.
Receptor GAP Ras Target
Doug Lowy: not so fast, GNEF is more important
RASGDP
RASGTP
Pi
GTPGDP
Exchange reaction
GAP
target protein
Active stateInactive
EGFR
EGFR
Ras (His116) mutant cause GDP to GTP exchange 10 x fasterbut its sensitivity to GAP is the same.
Model 1. EGFR GNEF RAS
Model 2. EGFR GAP RAS
If model 2 is right, GAP activity determines the GTPRas/GDPRas ratio add EGF, activity should dramatically increase. (A. Yes, B. No)
If model 1 is right, His 116 already already cause the exchange 10 X fastAdding EGF would have a small effect. (A. Yes, B. No)
Results: model 1 should be right.
RASGDP
RASGTP
Pi
GTPGDP
Exchange
GAPActive stateInactive
Zhang et al. 1991. Science
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