Isolation and Characterization of Anew Peroxisomedeficient Cho Mutant Cell Belonging Tocomplementation Group 12

We searched for novel chinese hamster ovary (cho) cell mutants defective inperoxisome biogenesis by an improved method using peroxisome targetingsequence (pts) of pex3p (amino acid residues 1–40)-fused enhanced greenfluorescent protein (egfp). from mutagenized tkaeg3(1–40) cells, the wildtypecho-k1 stably expressing rat pex2p and of rat pex3p(1–40)-egfp,numerous cell colonies resistant to the 9-(10-pyrene) nonanol/ultraviolet treatmentwere grown. these colonies were examined for intracellular location of pex3p(1–40)-egfp. by this method, we have isolated one cho cell mutant, zpeg403,which was found to belong to complementation group g (cg-g). expression ofthe human peroxin, pex3p cdna encoding a 373-amino-acid peroxisomalmembrane protein morphologically and biochemically restored peroxisomebiogenesis, including peroxisomal membrane assembly, in zpeg403 cells.mutation and genomic dna pcr analyses showed that, the dysfunction of pex3pin zpeg403, was due to one base (a) substitution in place of (g) in the first baseof splicing site at the boundary of exon 6 and intron 6 of pex3 gene, giving riseto remaining of all of intron 6, thereby inducing 81 bp insertion between positions523–524 of pex3 orf, resulting in deletion of 200 amino acid residues from thec-terminus of pex3p and a frame shift inducing both 18-amino-acid substitutionand an early termination codon.