Stories arising…
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Transcript of Stories arising…
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Stories arising…
Joe Pearson5/10/2009
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CG13333
•5’Frag widely expressed, including in midline•Repeated AGGTRG identified by PhyloGibbs•Mutating AGGTRG eliminates all expression•Mutating ACGTG specifically reduces midline expression
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CG13333 5’ motifs
CAGGTAGCAGGTAGCAGGTAGCAGGTAGCAGGTAGCAGGTAGCAGGTAGCAGGTAGCAGGTAGCAGGTACCAGGTAGCAGGTAC
DmelDsimDsecDyakDereDanaDpseDperDwilDgriDmojDvir
TAGGTGGTAGGTGGTAGGTGGTAGGTGGTAGGTGGTAGGTGGTAGGTGGTAGGTGGTAGGTAG
CAGGTAGCAGGTAGCAGGTAGCAGGTAGCAGGTAGCAGGTAGCAGGTAGCAGGTAGCAGGTAGCAGGTAGCAGGTAACAGGTAG
CACGTCACGTCACGTCACGTCTCGTCACGTCACGTCACGT
GAGGTAGGAGGAAGGAGGAAG
•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?
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Next Steps: CG13333
• Mutate “Zelda” sites to block Zelda binding• CG13333 expression in vielfaltig/zelda
mutants
vflStaudt et al., 2006
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argos
GFP SIM
midline expression no midline expression untested
•Intron1-1: Sporadic PMG, some AMG•Intron1-3: AMG only•Phylogibbs identified SCGGAAGTS in I1-3, similar to ETS consensus CMGGAWRY
I1-1 I1-3
EGFR MAPK Pnt Argos
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100%
0%
CMGGAWRYSCGGAAGTS
ACGTG
subC
subC subB subDsubA
SubA SubDI1f3 subB
argos
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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?
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•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
Glec locus dissection
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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?
1 1 CAGCAGTTAAACTCATGCGCAGCGCGTGCTCCATTTCCATTGATCGGTTGCGTTGGgttc-GGATTCATTCATTTCACTATCATTCAATGGTTTCTGACC 2 1 CAGCAGTTAACCTTATGCGCAGCGCGTGCGACAGTTCCATTGATCGGTTGCGTTGGctcctGGGTTCATTCATTTCATTATCATTCAATGGTTTCTGACC 1 100 TTTTGAGCCGGGGTTACATGGTACGTGGGCCATAAACTAATTTCTGCCAGCCAGTCGGgaaaaaAAAACAGCTTGAGTTTTCCGCAGACAAGCTGCGTGC 2 101 TTTTGAGCCGGGGTTACATGGTACGTGGCCCATAAACTAATTTCTGCCAGCCAGTCGGcgg---AAAACAGCTTGAGTTTTTCGCAGACAAGATGCGTGC 1 200 GGCGGAAGTGACGGCGGAAAAGTAGCCCATCAATAGACCCAGACACGTACCCAGAAAACATCTaAAAGATACAAAAATACACGCCGACATGCATTCaaat 2 198 GGCGGAAGTGACGACGGAAAAGTAGCCCATCAGTAGACCCAGACACGTACCCAGAAAACATCTgAAAGATACAAATATACGCGACGACATGCATTCga-- 1 300 aatgccCAGTGAAAAATCAGCGCAGCTACGAGATATCAAAATATAAGCAAAGCTGAA 2 295 ------CAGTGAAAAATCAGCGCAGCTACGAGATATTAAAAAAAAAACAAAGCTGAA
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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?
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• VUM expression– Not 100%
• Missing motif?• Not complete
element?• No Sim:Tgo site
• No midline primordium expression– Shared enhancer
with ect3?
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•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