(A) A brief description of mutants made in this paper.
Rat PAH coding sequence was cloned in to an expression vector pET26b(+)
(Novagen), which contains a T7 promoter sequence (T7). The broken bold
line represents part of the resulting plasmid pET26-rPAH, corresponding
to the 5′ coding sequence. The numbers under the line indicate the
position of nucleotides; the initiation codon starts with number 1.
(Bottom) The first 40 amino acids from rat (RAT) and
human PAH (HUMAN). Shaded areas represent identical sequences in rat
and human PAH. The primers (arrows) and restriction sites used for
mutagenesis are shown at relative positions. (Middle)
Amino acids at site 29 of rat PAH and their corresponding enzyme names.
wt, wild type. (B) Primer usage for the first round of
PCR as described in Materials and Methods.
a, primers in combination with primer o265; b,
primers in combination with primer N-rev; c, the template
was human PAH sequence.