Oduce a motif for a different protein than that being studied. This possibility may explain the discrepancy between the Liu et al. motif and all other T-box transcription factors including the motif identified for Mid in the present study.for particular binding sites arises from the spacing and orientation of the two half-sites as well as the nucleotides 25033180 flanking the core AGGTGT of each half-site [8]. We employed a site selection technique and identified DRRGTGWBRARGCG as the DNA binding motif for the Drosophila melanogaster Mid protein (Figure 3). The CG found at positions 14 and 15 in this motif appear to be specifically selected by MidTbx but are not essential for binding in an EMSA (Figure 3C). The motif identified in Figure 3 resembles that of most other T-box transcription factors and in particular is very close to the motif identified for the vertebrate homologue of Mid, Tbx20 [6]. It does not, however, resemble the motif previously identified for Mid (Figure 1B) [18]. Furthermore, we find that MidTbx is unable to shift the sequence identified by Liu et al. in an EMSA (Figure 1C). Based on our results and Indolactam V analysis we propose that Mid binds DNA targets as a monomer. Five lines of evidence support this hypothesis: 1) Most oligonucleotides had a single site and when two half-sites were found (4/27 oligonucleotides) they were oriented and spaced randomly with respect to one another; 2) MidTbx 
is able to bind oligonucleotides 14636-12-5 site containing only a single binding site; 3) EMSAs using oligonucleotides containing two potential binding sites only display a single band that runs at approximately the same mobility as MidTbx bound to a half-site; 4) The residues required for dimerization of Xbra and the nonstabilizing monomer-monomer contacts of Tbx3 are not conserved in Mid; 5) in vivo binding sites responsive to Mid are halfsites [19]. The possibility that region 2 in our motif is a variant half-site bound by a second MidTbx monomer cannot be excluded and therefore a crystal structure of MidTbx bound to this motif would be necessary to definitively conclude the nature of the MidTbx-DNA complex.Materials and Methods Expression of Mid T-box DomainDrosophila melanogaster Midline T-box domain (residues 171?93), containing the T-box domain, were PCR amplified from clone RE27439 using 59 GGGGCCGGATCCCATATGGCACCConclusionsT-box transcription factors have been shown to bind variations of the 24 bp palindromic Brachyury DNA binding motif called the T-site. It has been suggested that the specificity of T-box proteinsIdentification of a Drosophila Tbx20 Binding SiteCAAAATTGTCGGCTCCTGCAAT and 59 GGGGCCCTCGAGCATCGGATCGCGATCGAAGTCGGTGAGGCG primers. The PCR product was digested with Nde I and Xho I and ligated to a pET-21a vector digested with the same enzymes, resulting in a C-terminal 6xHis-tagged Mid T-box domain. 25 ml of Lauri-Bertani medium was inoculated with an overnight culture of Rosetta-gami cells (Novagen) transformed with the MidTbx in pET-21a, grown to an OD of 0.6 and induced with 0.5 mM IPTG. After 3 hours the cells were harvested, resuspended and lysed in 500 ml of buffer containing 20 mM HEPES pH 7.9, 100 mM KCl, 0.2 mM EDTA, 0.2 mM EGTA, 10 glycerol, 0.5 mM DTT, 10 mM imidazole and Complete EDTA-free protease inhibitor (Roche). The lysate was added to 300 ml of Ni-NTA magnetic agarose beads (Qiagen) with the original buffer removed and rocked on ice for 1 hour. The beads were washed 3 times and eluted in the same buffer as above except th.Oduce a motif for a different protein than that being studied. This possibility may explain the discrepancy between the Liu et al. motif and all other T-box transcription factors including the motif identified for Mid in the present study.for particular binding sites arises from the spacing and orientation of the two half-sites as well as the nucleotides 25033180 flanking the core AGGTGT of each half-site [8]. We employed a site selection technique and identified DRRGTGWBRARGCG as the DNA binding motif for the Drosophila melanogaster Mid protein (Figure 3). The CG found at positions 14 and 15 in this motif appear to be specifically selected by MidTbx but are not essential for binding in an EMSA (Figure 3C). The motif identified in Figure 3 resembles that of most other T-box transcription factors and in particular is very close to the motif identified for the vertebrate homologue of Mid, Tbx20 [6]. It does not, however, resemble the motif previously identified for Mid (Figure 1B) [18]. Furthermore, we find that MidTbx is unable to shift the sequence identified by Liu et al. in an EMSA (Figure 1C). Based on our results and analysis we propose that Mid binds DNA targets as a monomer. Five lines of evidence support this hypothesis: 1) Most oligonucleotides had a single site and when two half-sites were found (4/27 oligonucleotides) they were oriented and spaced randomly with respect to one another; 2) MidTbx is able to bind oligonucleotides containing only a single binding site; 3) EMSAs using oligonucleotides containing two potential binding sites only display a single band that runs at approximately the same mobility as MidTbx bound to a half-site; 4) The residues required for dimerization of Xbra and the nonstabilizing monomer-monomer contacts of Tbx3 are not conserved in Mid; 5) in vivo binding sites responsive to Mid are halfsites [19]. The possibility that region 2 in our motif is a variant half-site bound by a second MidTbx monomer cannot be excluded and therefore a crystal structure of MidTbx bound to this motif would be necessary to definitively conclude the nature of the MidTbx-DNA complex.Materials and Methods Expression of Mid T-box DomainDrosophila melanogaster Midline T-box domain (residues 171?93), containing the T-box domain, were PCR amplified from clone RE27439 using 59 GGGGCCGGATCCCATATGGCACCConclusionsT-box transcription factors have been shown to bind variations of the 24 bp palindromic Brachyury DNA binding motif called the T-site. It has been suggested that the specificity of T-box proteinsIdentification of a Drosophila Tbx20 
Binding SiteCAAAATTGTCGGCTCCTGCAAT and 59 GGGGCCCTCGAGCATCGGATCGCGATCGAAGTCGGTGAGGCG primers. The PCR product was digested with Nde I and Xho I and ligated to a pET-21a vector digested with the same enzymes, resulting in a C-terminal 6xHis-tagged Mid T-box domain. 25 ml of Lauri-Bertani medium was inoculated with an overnight culture of Rosetta-gami cells (Novagen) transformed with the MidTbx in pET-21a, grown to an OD of 0.6 and induced with 0.5 mM IPTG. After 3 hours the cells were harvested, resuspended and lysed in 500 ml of buffer containing 20 mM HEPES pH 7.9, 100 mM KCl, 0.2 mM EDTA, 0.2 mM EGTA, 10 glycerol, 0.5 mM DTT, 10 mM imidazole and Complete EDTA-free protease inhibitor (Roche). The lysate was added to 300 ml of Ni-NTA magnetic agarose beads (Qiagen) with the original buffer removed and rocked on ice for 1 hour. The beads were washed 3 times and eluted in the same buffer as above except th.