Site-directed mutagenesis was performed using the Quikchange II site-directed mutagenesis kit (Stratagene) according to manufacturer's instructions. For large deletions (greater than 48 bp) a modification of the manufacturer's protocol was used with the primers containing ,30 bp of complementary sequence either side of the site of deletion, as described previously [28] . Mutagenesis to introduce insertions longer than 6 bp was performed in two steps [29] , by first subjecting mutagenesis reactions (containing either the sense or antisense primer) to three cycles of PCR, then mixing the reactions and performing a further 18 cycles according to manufacturer's instructions.