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6 clones of a Drosophila embryo cDNA library fused to the transcription activation domain of yeast GAL4 in the vector pGAD10 and isolated three independent partial clones of the d-axin cDNA. A larger partial d-axin cDNA clone (2.7 kb) was isolated by rescreening a λgt11 Drosophila embryo cDNA library with the smaller cDNAs, and the remaining 5′-end region was obtained by the 5′ RACE (rapid amplification of cDNA ends) technique (Clontech). Multiple independent polymerase; chain reaction (PCR) products were analyzed to exclude the possibility of PCR-induced errors. Sequence comparison analysis was performed with the blastp program [S. F. Altschul et al., Nucleic Acids Res. 25, 3389 (1997)].
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0344556823
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note
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35S-methionine with the coupled transcription-translation TNT system (Promega). CST and GST-fusion proteins (2 μg) immobilized to glutathione-Sepharose beads were mixed with in vitro-translated proteins in binding buffer [20 mM tris-HCl (pH 8.0), 140 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 10% glycerol, 1% Triton X-100, and 10 μg/ml each of aprotinin, leupeptin, and pepstatin) for 1 hour at 4°C. After washing five times with binding buffer, bound proteins were fractionated by SDS-polyacrylamide gel electrophoresis (PAGE) followed by autoradiography.
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20
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0344987756
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unpublished data
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F. Hamada et al., unpublished data.
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Hamada, F.1
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22
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0344125270
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note
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Using in situ hybridization to polytene chromosomes, we mapped the d-axin gene to 99D in the third chromosome. The genomic P1 clone DS00026 from the Berkeley Drosophila Genome Project contained the genomic region corresponding to the d-axin cDNA. The genomic organization of the d-axin locus and the intron-exon structure were determined by Southern (DNA) blot analysis and subsequent sequencing across the exon-intron boundaries.
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23
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p, Δ2-3 strain [H. M. Robertson et al., Genetics 118, 461 (1988)].
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Robertson, H.M.1
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0027160708
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The GAL4/UAS system was used for overexpression of d-axin [A. H. Brand and N. Perrimon, Development 118, 401 (1993)]. The entire d-axin open reading frames of various constructs were subcloned into the pUAST vector and introduced into the w germ line by the standard P-element transformation method These UAS-d-axin lines were crossed with several GAL4 drivers [ Y. Shiga, M. Tanaka-Matakatsu, S. Hayashi, Dev. Growth Differ. 38, 99 (1996); E. J. Rulifson, C. A. Micchelle, J. D. Axelrod, N. Perrimon, S. S. Blair, Nature 384, 72 (1996)].
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The GAL4/UAS system was used for overexpression of d-axin [A. H. Brand and N. Perrimon, Development 118, 401 (1993)]. The entire d-axin open reading frames of various constructs were subcloned into the pUAST vector and introduced into the w germ line by the standard P-element transformation method These UAS-d-axin lines were crossed with several GAL4 drivers [ Y. Shiga, M. Tanaka-Matakatsu, S. Hayashi, Dev. Growth Differ. 38, 99 (1996); E. J. Rulifson, C. A. Micchelle, J. D. Axelrod, N. Perrimon, S. S. Blair, Nature 384, 72 (1996)].
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The GAL4/UAS system was used for overexpression of d-axin [A. H. Brand and N. Perrimon, Development 118, 401 (1993)]. The entire d-axin open reading frames of various constructs were subcloned into the pUAST vector and introduced into the w germ line by the standard P-element transformation method These UAS-d-axin lines were crossed with several GAL4 drivers [ Y. Shiga, M. Tanaka-Matakatsu, S. Hayashi, Dev. Growth Differ. 38, 99 (1996); E. J. Rulifson, C. A. Micchelle, J. D. Axelrod, N. Perrimon, S. S. Blair, Nature 384, 72 (1996)].
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Germ-line clones of d-axin were generated by use of the FLP-DFS technique [T. B. Chou and N. Perrimon, Genetics 144, 1673 (1996)]. All embryos derived from homozygous d-axin germ-line clones die during embryonic development. The TM3 gfp chromosome was used to determine the zygotic genotype of embryos. Cuticles were prepared and mounted in Hoyer's medium [J. van der Meer, Drosophila Inf. Serv. 52, 160 (1977)], viewed, and photographed under dark field.
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Germ-line clones of d-axin were generated by use of the FLP-DFS technique [T. B. Chou and N. Perrimon, Genetics 144, 1673 (1996)]. All embryos derived from homozygous d-axin germ-line clones die during embryonic development. The TM3 gfp chromosome was used to determine the zygotic genotype of embryos. Cuticles were prepared and mounted in Hoyer's medium [J. van der Meer, Drosophila Inf. Serv. 52, 160 (1977)], viewed, and photographed under dark field.
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+ for imaginal discs [J. Jiang and G. Struhl, Nature 391, 493 (1998)]. Mutant phenotypes were induced in the mid-second instar larval stage by heat shock at 37°C for 30 min. For the induction of the hip70-myc-gfp gene, wandering larvae were heat shocked at 37°C for 60 min and then processed for immunostaining after a 60-min recovery period at 25°C. The mutant clones can be recognized by the y marker in adults and by the absence of GFP expression in the imaginal discs, respectively.
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+ for imaginal discs [J. Jiang and G. Struhl, Nature 391, 493 (1998)]. Mutant phenotypes were induced in the mid-second instar larval stage by heat shock at 37°C for 30 min. For the induction of the hip70-myc-gfp gene, wandering larvae were heat shocked at 37°C for 60 min and then processed for immunostaining after a 60-min recovery period at 25°C. The mutant clones can be recognized by the y marker in adults and by the absence of GFP expression in the imaginal discs, respectively.
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Wing imaginal discs from third instar larvae were dissected, fixed, and stained with mouse antibodies to Arm (anti-Arm, N2-7A1) [M. Peifer, D. Sweeton, M. Casey, E. Wieschaus, Development 120, 369 (1994)] or anti-DII [G. Vachon et al., Cell 71, 437 (1992)]. These antibodies were detected with rhodamine-conjugated goat anti-mouse immunoglobulin G (Jackson Immunochemicals). Confocal fluorescent images were obtained with a Zeiss LSM510 microscope.
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Wing imaginal discs from third instar larvae were dissected, fixed, and stained with mouse antibodies to Arm (anti-Arm, N2-7A1) [M. Peifer, D. Sweeton, M. Casey, E. Wieschaus, Development 120, 369 (1994)] or anti-DII [G. Vachon et al., Cell 71, 437 (1992)]. These antibodies were detected with rhodamine-conjugated goat anti-mouse immunoglobulin G (Jackson Immunochemicals). Confocal fluorescent images were obtained with a Zeiss LSM510 microscope.
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Vachon, G.1
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0344987755
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note
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We thank J. Jiang, G. Struhl, the Bloomington Stock Center, and the European Drosophila P-element-induced Lethal Stock Center (Szeged Center) for fly stocks; S. Yanagawa, S. Cohen, and E. Wieschaus for plasmids and antibodies; the Berkeley Drosophila Genome Project and E. Nitasaka for genomic P1 clones; A. Ogai, Y. Katoh, N. Kakinuma, T. Shikina, T. Wada, A. Furui, and M. Ichikawa for technical assistance; and S. Higashijima, E. Shishido, T. Tabata, and K. W. Cho for helpful discussions. Supported by grants from the Ministry of Education, Science, and Culture of Japan and the National Institute for Basic Biology Cooperative Research Program.
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