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Figure 2


Fig. 2. The tbx-35 gene encodes a T-box factor. (A) Gene structure of tbx-35 on LGII. The left end of the gene is preceded by the start codon of ZK177.1. The deleted regions in tm1789 and tm618 are shown above the gene. The predicted mRNA is shown below. The coding region is shaded, with the T-box emphasized. The locations of MED-1 binding sites are shown as grey circles for the RAGTATAC site defined by MED-1 footprinting of the end-1 and end-3 promoters (Broitman-Maduro et al., 2005), and white circles for three lower-affinity RGGTATAC sites based on in vitro competition assays (G.B.-M., K.L., W.H. and M.M., unpublished). No additional MED sites are found elsewhere in the gene. We were unable to amplify the 5' end of the transcript using primers to detect SL1 or SL2, suggesting that the tbx-35 mRNA is not trans-spliced (Conrad et al., 1993); hence, the bona fide 5' end of the transcript is not known. A polyadenylation sequence (AATAAA) is found 115 bp downstream of the predicted stop codon, but we have not confirmed the precise 3' end of the transcript. (B) Alignment of the conserved T-box region of TBX-35 with those of other Tbx genes. TBX-35 is 25-28% identical (35-39% similar) to vertebrate eomesodermin, mouse brachyury, Drosophila Dorsocross2 and its closest homolog in C. elegans, TBX-37. The TBX-37 T-box is more closely related to the other T-box regions shown (e.g. 37% identical and 50% similar to brachyury). This alignment was generated with AlignX (within Vector NTI 6) using the default settings. An asterisk indicates a conserved glutamic acid residue that was mutated in a heat shock fusion construct (see text). Xl, Xenopus laevis; Hs, Homo sapiens; Mm, Mus musculus; Dm, Drosophila melanogaster. Accession numbers: eomesodermin, P79944 (Xenopus) and NP005433 (human); brachyury, CAA35985; Dorsocross2, AAM11545. (C) MED-1 binds the tbx-35 promoter. A 190 bp fragment of the tbx-35 promoter containing the proximal MED site cluster was incubated with increasing concentrations of recombinant DNA-binding domain of MED-1 as described (Broitman-Maduro et al., 2005). Double-stranded competitor oligonucleotides: wild type, 5'-TCATTTGTATACTTTATCTACAATAT; mutant, 5'-TCATTTGTTATCATTATCTACAATAT-3'. Underlined nucleotides represent the wild-type and mutated core MED-1 binding sites, respectively.





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