PCR strategy and identification of carboxylase coding substitutions. (A) The entire carboxylase gene was sequenced using PCR fragments generated with the primers that are indicated in Table 2. The double-headed arrows indicate exonic (E1-E15) and other fragments (whose nucleotide positions at the 5′ and 3′ ends are shown below the lines) whose names are the same as those used in Table 2. (B-D) Electropherograms corresponding to the 3 new mutations identified in this work are shown: (B) Asp31Asn in exon 2, (C) Trp157Arg in exon 4, and (D) Thr591Lys in exon 13. The positions of the mutations are indicated on top of the electropherograms using the mRNA numbering for the carboxylase (GenBank accession M81592). Wild-type and mutant nucleotides are indicated on top of the corresponding peaks, and wild-type (black) and mutant (red) nucleotide and amino acid sequences are shown below the electropherogram.