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M267 Larry Zipursky Lecture 3 Dissection of Receptor Tyrosine Kinase Pathways in Drosophila

M267 Larry Zipursky Lecture 3 Dissection of Receptor Tyrosine Kinase Pathways in Drosophila. Isolation of Sos Gain-of-Function Allele. EMS. E4. LY3. E1. sev. or. sev. or. sev. E4. LY3. E1. (. ). sev. sev. or. sev. X. (. ). E4. LY3. E1. sev. or. sev. or. sev.

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M267 Larry Zipursky Lecture 3 Dissection of Receptor Tyrosine Kinase Pathways in Drosophila

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  1. M267 Larry Zipursky Lecture 3 Dissection of Receptor Tyrosine Kinase Pathways in Drosophila

  2. Isolation of Sos Gain-of-Function Allele EMS E4 LY3 E1 sev or sev or sev E4 LY3 E1 ( ) sev sev or sev X ( ) E4 LY3 E1 sev or sev or sev ( ) Screen for restoration of R7: 1/30,000 progeny tested reverted to the phenotype --> Son of sevenless (SosJC2). Rogge RD (1991) Cell. 64, 39.

  3. Is SosJC2 Required in R7? X-ray larvae to induce mitotic recombination JC2 E4 Sos , P[w] w, sev ; + E4 Sos w, sev Identify white patches and score boundary ommatidia. Rogge RD (1991) Cell. 64, 39.

  4. Is SosJC2 Required in R7? Table 2. Analysis of Mosaic Ommatidia The table lists data from 69 mosaic ommatidia, each of which contained an R7 cell. These ommatidia were from the patches of 12 different eyes.

  5. Dissecting the Sev Signal Transduction Pathway Sensitization of the R7 Pathway ~ sevB4= a temperature sensitive allele of sev introduced as a transgene sevd2= a null allele F1 progeny screened = 30,000 E(sev) mutations = 20 Simon, M. A. et al. (1992) Cell. 1991 Nov 15;67(4):701-16.

  6. Screening for Modifiers of sevB4 Phenotype Simon, M. A. et al. (1992) Cell 67, 701-16.

  7. Interactions with Ellipse to Further Characterize Enhancers of sevenless Simon, M. A. et al. (1992) Cell 67, 701-16.

  8. Receptor Tyrosine Kinase (Sev or EGF) drk (E(sev)2B (a.k.a Grb-2) Sos (E(sev)2A) ras (E(sev)3C) Biochemistry: Drk binds to receptor, Sos binds to Drk, and Ras binds to Sos Simon, M.A. et al. (1993) Cell. 73, 169

  9. Sev-RasV12 very rough Screen for dosage-sensitive suppressors e.g., sev-RasV12 WT or weak rough * Isolation of Components Acting Downstream from Ras Rough refers to a disorganized array of ommatidia Fortini, M. et al. (1992) Nature 355, 559

  10. Ras Raf MAP Kinase Kinase MAP Kinase Results of sev-RasV12Screen Biochemistry:MAPKK activates MAPK, Raf activates MAPKK Ras binds to but does not activate Raf Therrien, M. et al. (1995). Cell. 83, 879. Karim, F.D. et al. (1996) Genetics 143, 315

  11. Genetic Approach to Identify Genes Between Ras and Raf Identify S(Ras) which do not suppress sev-RafD: SR3-1, SR2-2 Identify mutations in genes regulating post-translational modification of ras: Ras is prenylated; prenylation is required for activity Therrien, M. et al. (1995). Cell. 83, 879.

  12. Phenotype rough rough wild type + SR3-1 + + + SR2-2 + rough Rasval12 Farnesylation Myristylation Identification of Genes between ras and raf Therrien, M. et al. (1995). Cell. 83, 879.

  13. Molecular Identities of SR2-2 and SR3-1 SR2-2 = encodes the Drosophila b-subunit of Type 1 geranylgeranyl transferase SR3-1 = kinase suppressor of ras (ksr) Therrien, M. et al. (1995). Cell. 83, 879.

  14. The Canonical RTK Pathway RTK drk raf Sos ras KSR MAPK MAPKK Nuclear Factors

  15. KSR as a Signaling Scaffold Claperon and Therrien, 2007

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