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1. ~ 1000 cells, small, easy to use for genetics

Caenhorhabditis elegans. 1. ~ 1000 cells, small, easy to use for genetics. 2. Entire lineage and nerve system mapped. 3. 3 day life cycle, easy to use for genetics. 4. hermaphrodite. Lineage by the John Sulston. The story of the lineage work.

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1. ~ 1000 cells, small, easy to use for genetics

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  1. Caenhorhabditis elegans 1. ~ 1000 cells, small, easy to use for genetics 2. Entire lineage and nerve system mapped. 3. 3 day life cycle, easy to use for genetics. 4. hermaphrodite

  2. Lineage by the John Sulston The story of the lineage work

  3. 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.

  4. Using vulval development as a model system Vulva: laying eggs mating

  5. Reproductive system in C. elegans

  6. Three steps of vulval development 1, precursor cells dorsa l gonad P 1 P1 2 cell migration hox genes 2, vulval induction Cell signaling E E V V V E Cell fate Lineage 3, morphogenesis c ell division, fusion, migration, etc.

  7. AC WT 3° 3° 2° 1° 2° 3° X - AC 3° 3° 3° 3° 3° 3° Indicating: A: AC is required for vulval induction B: AC may send a signal to induce vulval cells C: Both. Judith Kimble: gonad

  8. Paul Sternberg: AC WT 3° 3° 2° 1° 2° 3° VPC ablation X X X X X X 1°/2° 1°/2° 1°/2° Indicating:

  9. pathway function signal E E E E E E Vulvaless 0% V V V V V V Multivulva 200% anchor cell inductive signal E E V V V E Wild type 100% induction

  10. signal pathway function - use genetics to get the major players in the pathway

  11. lin-1 lin-3 EGF Vulval induction let-23 EGFR Earlier direct screens for vulval induction mutations Mutagen Vulvaless wild type Multivulva Horvitz and Sulston 1980, Genetics Ferguson and Horvitz 1985, Genetics Ferguson et al. 1987. Nature Aroian et al. 1991. Nature Hill and Sternberg 1992. Nature

  12. 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) Exchange reaction GDP GTP RAS GDP RAS GTP target protein Inactive Active state Hydrolysis reaction Pi

  13. Vote: X Vulval induction A. lin-15 X B. Vulval induction lin-15 Supressors of lin-15 mutation EMS lin-15(-); ; X(-) Vulvaless lin-15(-) , Muv X = EGFR, RAS and others. Han and Sternberg 1990, Cell; Beitel et al. 1990, Nature; Aroian et al. 1990 Nature

  14. EGFR(lf) Vulvaless RAS (gf) Multivulva EGFR(lf) + RAS (gf) Multivulva Vote: Vulval induction A. RAS EGFR Vulval induction RAS B. EGFR Relationship between RAS and EGFR EGFR RAS Vulval induction lin-15

  15. Pathway in early 1992 RAS EGFR

  16. signal signal Strategy 1: moving up from Ras Exchange reaction GDP GTP RAS GDP RAS GTP target protein Inactive Active state Hydrolysis reaction Pi

  17. The student and PI were mammalian biochemists. What system will they likely chose to identify the “something”? A: Mammalian cells. B. Drosophila. C. Xenopus oocyte D. Yeast. First strike: discovery of GTPase activating protein (GAP) 1. Questions addressed RAS:GTP RAS:GDP GTPase of Ras Pi In vitro weak t 1/2 = 30 min In vivo strong t 1/2 < 1 min A: Something in cells can stimulate the GTPase activity B: Something in intro inhibits the GTPase activity

  18. Approach: Using Xenopus oocytes - ease to manipulate due to large size - good assay for biological activity, oocyte maturation.

  19. Does GTP or GDP bind to injected Ras? In vivo In vitro WT Ras mostly bind to GDP Oncogenic Ras mostly to GTP

  20. GTPase activity: speed in vivo T 1/2 = 2-3 min for wild type. >1000 min for Asp12

  21. WT vs. Oncogenes WT Gly 12 RAS:GDP RAS:GTP GTPase activity Pi Oncogenes Val 12 Asp 12 RAS:GDP RAS:GTP X GTPase activity Pi

  22. 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

  23. EGFR EGFR How is Ras regulated by the signal? Exchange reaction GDP GTP RAS GDP RAS GTP target protein Inactive Active state GAP Pi

  24. Cell 1990. It is all GAP Kaplan 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 Target Ras

  25. Doug Lowy: not so fast, GNEF is more important EGFR Exchange reaction GDP GTP RAS GDP RAS GTP target protein Inactive Active state GAP Pi EGFR

  26. Exchange Model 1. EGFR GNEF RAS GDP GTP RAS GDP RAS GTP Model 2. EGFR GAP RAS Inactive Active state GAP Pi Ras (His116) mutant cause GDP to GTP exchange 10 x faster but its sensitivity to GAP is the same. 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 fast Adding EGF would have a small effect. (A. Yes, B. No) Results: model 1 should be right. Zhang et al. 1991. Science

  27. The End

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