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Stories arising…

Stories arising…. Joe Pearson 5/10/2009. CG13333. 5’Frag widely expressed, including in midline Repeated AGGTRG identified by PhyloGibbs Mutating AGGTRG eliminates all expression Mutating ACGTG specifically reduces midline expression. CG13333 5’ motifs. Dmel Dsim Dsec Dyak Dere Dana

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Stories arising…

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  1. Stories arising… Joe Pearson 5/10/2009

  2. CG13333 • 5’Frag widely expressed, including in midline • Repeated AGGTRG identified by PhyloGibbs • Mutating AGGTRG eliminates all expression • Mutating ACGTG specifically reduces midline expression

  3. CG13333 5’ motifs Dmel Dsim Dsec Dyak Dere Dana Dpse Dper Dwil Dgri Dmoj Dvir CAGGTAG CAGGTAG CAGGTAG CAGGTAG CAGGTAG CAGGTAG CAGGTAG CAGGTAG CAGGTAG CAGGTAC CAGGTAG CAGGTAC TAGGTGG TAGGTGG TAGGTGG TAGGTGG TAGGTGG TAGGTGG TAGGTGG TAGGTGG TAGGTAG CAGGTAG CAGGTAG CAGGTAG CAGGTAG CAGGTAG CAGGTAG CAGGTAG CAGGTAG CAGGTAG CAGGTAG CAGGTAA CAGGTAG CACGT CACGT CACGT CACGT CTCGT CACGT CACGT CACGT GAGGTAG GAGGAAG GAGGAAG • PhyloGibbs identified AGGTRG as a repeated, conserved motif • Perfectly conserved instances extend to CAGGTAG • Zelda binds CAGGTAG to promote widespread pre-blastoderm expression (Liang et al., 2008) • Is Zelda acting as ubiquitous activator in late embryo?

  4. Next Steps: CG13333 • Mutate “Zelda” sites to block Zelda binding • CG13333 expression in vielfaltig/zelda mutants vfl Staudt et al., 2006

  5. argos midline expression no midline expression untested I1-1 I1-3 • Intron1-1: Sporadic PMG, some AMG • Intron1-3: AMG only • Phylogibbs identified SCGGAAGTS in I1-3, similar to ETS consensus CMGGAWRY GFP SIM Argos EGFR MAPK Pnt

  6. argos 100% SCGGAAGTS CMGGAWRY ACGTG 0% subA subB subC subD SubA subB subC SubD I1f3

  7. Next Steps: argos • Mutate ETS-like sites • Mutate Sim site • Test argos role in AMG development? • Cell autonomous inhibition of EGFR signaling? • Inhibition of EGFR signaling in other AMG? • Inhibition of EGFR in other cells?

  8. Glec locus dissection • 5’-2 vs. 5’2sA • Separable enhancers • 5’-1 vs. 5’2sA • Derepression in MP1 • 5’-3 vs. 5’3sA • Derepression in anterior midline • 5’2 vs. 5’3sA • Repeated 300bp fragment with 2 Sim:Tgo sites

  9. repeated glec upstream sequence • Evolutionary history • Promoter-proximal (5’-3sA) instance ancestral • Highly conserved through D. pseudoobscura • Degenerate match through D. virilis • Unclear when duplication occurred • Repeated Anterior midline motif? • Two Sim:Tgo sites, both conserved • Why repeated at same gene?

  10. Next Steps: glec • Test remaining 5’-2 and 3’-1 subfragments • Mutate Sim:Tgo, CREB binding sites in 5’-3 • Test repeated motif for midline expression function • Test 5’-2 and 5’-3 in pBPGw-UCP (GAL4 reporter) • GAL4 amplification? • hsp70 vs. UCP?

  11. VUM expression • Not 100% • Missing motif? • Not complete element? • No Sim:Tgo site • No midline primordium expression • Shared enhancer with ect3?

  12. 600 bp fragment drives expression in all midline primordium (mesectoderm?) • 3 Sim:Tgo sites • 2 Su(H) sites • 2 Twist sites (also bound in vivo) • Caveat: overexpression of sim does not expand sema-1b expression

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