In Vitro Site Selection of a Consensus Binding Site for the Drosophila Melanogaster Tbx20 Homolog Midline

PLoS One. 2012;7(10):e48176. doi: 10.1371/journal.pone.0048176. Epub 2012 Oct 25.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Motifs
  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Binding Sites
  • Body Patterning
  • Drosophila Proteins / chemistry*
  • Drosophila Proteins / genetics
  • Drosophila Proteins / metabolism
  • Drosophila melanogaster / metabolism*
  • Gene Expression Regulation, Developmental
  • In Vitro Techniques
  • Molecular Sequence Data
  • Oligonucleotides / genetics
  • Sequence Homology, Amino Acid
  • T-Box Domain Proteins / chemistry*
  • T-Box Domain Proteins / metabolism

Substances

  • Drosophila Proteins
  • Oligonucleotides
  • T-Box Domain Proteins
  • mid protein, Drosophila