Gene structure of moa tbx5 and detection of forelmb-specific tbx5 transcripts in ostrich and kiwi. A. Comparison of moa tbx5 gene structure with those of chicken, ostrich, and kiwi. Moa tbx5 exons were identical in length to those of chicken and kiwi. Complete genome data for ostrich was not obtained. The coding region is shown in grey and is 521 amino acids long for all transcripts shown. The 183 amino acid Tbox region is shown in black and is identical between chicken, kiwi, ostrich, and moa. The four amino acids unique to moa Tbx5 at the NH2 terminus are shown by white vertical lines. The position of Nuclear Localisation Signals 1 and 2 (NLS1, NLS2) and the Nuclear Export Signal (NES) are shown [52, 53]. B. A primer (OsF1) designed to tbx5 exon1 sequences obtained from ostrich forelimb cDNA by 5’ RACE was used to try and amplify tbx5 transcripts from heart and forelimb cDNA. Primers to exon 2 and exon 3 amplified the correct sized product from both tissues while the exon1 primer amplified transcripts from forelimb only. OH - ostrich heart cDNA, OFl - ostrich forelimb cDNA, KH - kiwi heart cDNA, KT - kiwi tissue cDNA. C. 5’ RACE analysis of A-tailed ostrich heart and forelimb transcripts identified forelimb and heart specific exons. Cross-hatched boxes represent exons expressed in forelimb only. Diagonally lined boxes are exons expressed in heart only. Primer walking of the chicken genome was used to determine the exon boundaries for kiwi and ostrich heart and forelimb tbx5 cDNA. Several clones were sequenced and compared with sequences upstream of chicken tbx5. Two heart-specific exons were located and a single forelimb exon. Analysis of chicken sequences directly upstream of these exons detected no obvious promoter-like sequences.