DEV01329 Supplementary Material

Files in this Data Supplement:

• Table S1 (PDF)
• Supplemental Figure 1 -

  Fig. S1. The identification of LSox5 isoforms expressed in chick embryos. (A) Amino acid sequence of chicken LSox5 that shares 95.7% identity with its human orthologue (EMBL/GenBank Accession Number AB081589). Only mismatch residues of the corresponding human protein are shown below. The string of amino acids involved in alternative splicing (positions 56-90) is underlined. A region that is absent in mammalian LSox5 sequences (positions 448-456) is boxed, this region being noticeably enriched in potential phosphorylation sites. The amino acids corresponding to the HMG domain are shaded in grey (positions 564-641). The sequences corresponding to the oligonucleotides used for the PCR amplification in B are underlined with arrows. The arrow under M-417 indicates the initial residue of SSox5 (see text). (B) RT-PCR amplification of mRNA from chick embryos. Two bands were obtained during the exponential phase of amplification when mRNA from E2 embryos was subjected to RT-PCR with the flanking primers also shown in A and the same result was obtained at the different embryonic stages tested (not shown). The larger band contains the additional sequence underlined in A (LSox5-II). (C) Detection of the LSox5 isoforms in western blots of whole chick embryo extracts using a rabbit antiserum raised against a recombinant fusion protein. A doublet was observed that most probably corresponds to the LSox5-I and LSox5-II proteins.

• Supplemental Figure 2 -

  Fig. S2. Differential splicing of the SRD (serine-rich domain) in Sox5 gene across species. The boundary between exon XI (capital letters) and the upstream intron (lowercase) in the Sox5 gene was found in the genome of the chicken (Gg), human (Hs), mouse (Mm), rat (Rn), zebrafish (Dr) and fugu (Fr) available in the Ensembl Genome Browser (http://www.ensembl.org). The amino acid sequences are shown in the lower row, with the conceptual translation of the human intronic region in lowercase. In the chicken, two different splice acceptor sites (shaded) provoke the alternative inclusion of the SRD (boxed sequence) in the LSox5 polypeptide. Whereas the first site is lost in the mouse and rat (red nucleotides) preventing the splicing of the SRD, the loss of the second site in zebrafish and fugu makes inclusion of the SRD constitutive. In humans, both splice acceptor sites are present, but a nucleotide change generates a stop codon (also in red) that invalidates the translation of this region.