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The cut 0.7-kb cis-regulatory element is the 0.7 kb at the 3′ end of the cut gene wing margin enhancer (12), which drives reporter gene expression along the presumptive wing margin. The sal 328-bp enhancer is a subfragment of the sal 10.2-kb enhancer (13), that drives expression in a wing pouch-restricted, albeit altered, pattern. Reporter constructs were made by cloning polymerase chain reaction (PCR)-generated fragments of the cut gene wing margin or sal enhancers into the Hsp lacZ-CaSpeR plasmid [H. Nelson, A. Laughon, Roux's Arch. Dev. Biol. 202, 341 (1993)]. The GenBank accession numbers for the cut 0.7-kb and sal 328-bp cis-regulatory elements are AF369916 and AF369917, respectively.
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0342850959
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note
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Generation of the SD TEA domain, DNase I footprinting, and electrophoretic mobility-shift assays were performed as described (7). Probes for footprinting were generated by end-filling subfragments of cis-regulatory elements with radioactively labeled nucleotides. Probes for shifts were annealed oligonucleotides that were radioactively labeled by kinase or end-filling with Klenow. Sequences protected by the SD TEA domain were aligned and analyzed using the PileUp program of GCG [Wisconsin Package Version 9.1, Genetics Computer Group (GCG), Madison, WI], with adjustments by hand.
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0342850958
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note
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Sequences of the upper strand of oligonucleotides used as probes for shifts and as PCR primers to mutate SD-binding sites (positions noted in parentheses) in the various cis-regulatory elements are listed in the 5′ to 3′ orientation. Altered bases are shown in lowercase in the mutant version, and the corresponding bases are underlined in the native sequence. At least three independent transgenic lines were analyzed for each set of SD sites mutated. sal (126) wild type: 5′ TTAAAGATGCTTTCTGGAATCCCACGAATGTCCATTGGATGG 3′ sal (126) mutant: 5′ TTAAAGATGCTTTCTctAATCagACtAATGaggATTGGATGG 3′ cut (558) wild type: 5′ TTTGTCAATGTAATTCGAAAAATGTCGTCAG 3′ cut (558) mutant: 5′ TTTGTCAATcTAATTCtActAATtTCGTCAG 3′ vgQ 1 (87) wild type: 5′ GCGTTGACAACATTCCAAACTCG 3′ vgQ 1 (87) mutant: 5′ GCGTTGACAtgAgctCttACTCG 3′ vgQ 2 (359) wild type: 5′ ATACGGGATGCCATGCCGCGTGC 3′ vgQ 2 (359) mutant: 5′ ATACGGGATctCATGagGCGTGC 3′ vgQ 3 (450) wild type: 5′ GAGGCCGTGGAATTCCCATTAATG 3′ vgQ 3 (450) mutant: 5′ GAGGCCGTctAATTagCATTAATG 3′ vgQ 4 (488) wild type: 5′ CTGCCAAAGATATTTCCTCTGCAG 3′ vgQ 4 (488) mutant: 5′ CTGCCAAActTATTTCCTCTGCAG 3′
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SD binds to discrete domains of CBP in vitro but does not interact with MAD or SU(H) (C. E. Nelson, S. B. Carroll, unpublished observations).
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0342850924
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We thank D. Nellen for identifying and providing the sal 328-bp element and transgenic flies;. S. Blair, K. Irvine, R. Schuh, and J. Jack for providing fruit fly stocks; T. Stone and J. Posakony for SU(H) protein; A. Laughon for MAD protein and probes; D. Dorsett for cut sequence and DNA; V. Kassner, J. Selegue, and K. Vacarro for invaluable technical assistance; L. Olds for figure preparation; J. Carroll for manuscript preparation; M. Levine for helpful discussion; and A. Kopp, G. Panganiban, and T. Wittkopp for comments on the manuscript. J. Magee initiated the studies of SD site mutations in the vg Q enhancer, and J.-Y. Sgro and the Institute for Molecular Virology provided access to GCG. Supported by NIH postdoctoral fellowships to K.A.G. and M.E.K., and the Howard Hughes Medical Institute, of which S.B.C. is an Investigator
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We thank D. Nellen for identifying and providing the sal 328-bp element and transgenic flies;. S. Blair, K. Irvine, R. Schuh, and J. Jack for providing fruit fly stocks; T. Stone and J. Posakony for SU(H) protein; A. Laughon for MAD protein and probes; D. Dorsett for cut sequence and DNA; V. Kassner, J. Selegue, and K. Vacarro for invaluable technical assistance; L. Olds for figure preparation; J. Carroll for manuscript preparation; M. Levine for helpful discussion; and A. Kopp, G. Panganiban, and T. Wittkopp for comments on the manuscript. J. Magee initiated the studies of SD site mutations in the vg Q enhancer, and J.-Y. Sgro and the Institute for Molecular Virology provided access to GCG. Supported by NIH postdoctoral fellowships to K.A.G. and M.E.K., and the Howard Hughes Medical Institute, of which S.B.C. is an Investigator.
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