Yeast two-hybrid constructs
All cDNA and amino acid numbering is based on the human PAX6 cDNA and protein reference sequences available from the Human PAX6 Allelic Variant Database web site [27]. Standard PCR and subcloning techniques were used to make three PAX6 cDNA constructs in the pDBLeu expression vector (ProQuest Two-Hybrid System, Invitrogen), which generates a protein fused to the yeast GAL4 DNA binding domain. PAX6PST contains the whole PST domain (amino acids 278–422 of the full-length PAX6 protein). Primers were ST001 (forward) 5'-AAA AGT TCG ACT GCC AGC AAC ACA CCT AGT C-3' and ST005 (R) 5'-TTT TGC GGC TTT TTA CTG TAA TCT TGG CCA GTA TTG-3'. PAX6CTP contains the newly defined C-terminal peptide alone (391–422). Primers were ST004 (F) 5'-AAA AGT CGA CTA CCA CTT CAA CAG GAC TCA TT-3' and ST005 (R). PAX6PST-CT contains the PST domain minus the C-terminal peptide (278–390). This was made by cutting PAX6PST with NdeI and NotI to drop out the C-terminal peptide, and inserting a synthetic linker between the two restriction sites. All fragments generated by PCR or with linkers were sequenced to check that no errors had been introduced.
A PAX6 PST domain construct containing the mutation 1627A>G (Q422R) was generated in the same way as the PAX6PST construct, but using the reverse PCR primer ST006 5'-TTT TGC GGC CGC TTT TTA CCG TAA TCT TGG CCA GTA TTG AG-3', which contains the mutant nucleotide substitution (underlined).
cDNA sequences containing the mutations 1615del10 [28] and 1629insT (X423L) [12] were generated by PCR from reverse transcribed RNA (a gift from Dr K Williamson and Prof V van Heyningen). Primers were ST015 (F) 5'-CCC ACA TAT GCA GAC ACA C-3' and ST031 (R) 5'-TTG CGG CCG CAT CCA TCC AGT CTA CAT TGT TC-3'. The PAX6PST construct was cut with NdeI and NotI to release the normal C-terminal peptide, and the mutant sequence was inserted.