title: In vitro site selection of a consensus binding site for the Drosophila melanogaster Tbx20 homolog midline.
abstract: We employed in vitro site selection to identify a consensus binding sequence for the Drosophila melanogaster Tbx20 T-box transcription factor homolog Midline. We purified a bacterially expressed T-box DNA binding domain of Midline, and used it in four rounds of precipitation and polymerase-chain-reaction based amplification. We cloned and sequenced 54 random oligonucleotides selected by Midline. Electromobility shift-assays confirmed that 27 of these could bind the Midline T-box. Sequence alignment of these 27 clones suggests that Midline binds as a monomer to a consensus sequence that contains an AGGTGT core. Thus, the Midline consensus binding site we define in this study is similar to that defined for vertebrate Tbx20, but differs from a previously reported Midline binding sequence derived through site selection.
authors.affiliation: Genes and Development Research Group, Alberta Children's Hospital Research Institute, Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta, Canada.
authors.forename: Nima
authors.initials: N
authors.lastname: Najand
authors.forename: Jae-Ryeon
authors.initials: JR
authors.lastname: Ryu
authors.forename: William J
authors.initials: WJ
authors.lastname: Brook
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D019521:Body Patterning; D029721:Drosophila Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004331:Drosophila melanogaster / Q000378:metabolism*; D018507:Gene Expression Regulation, Developmental; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D020825:T-Box Domain Proteins / Q000737:chemistry* / Q000378:metabolism
chemical_list: D029721:Drosophila Proteins; D009841:Oligonucleotides; D020825:T-Box Domain Proteins; C495571:mid protein, Drosophila