Isolation and characterization of novel peroxisome biogenesis-defective Chinese hamster ovary cell mutants using green fluorescent protein.
We developed an improved method for isolation of peroxisome biogenesis-defective somatic animal cell mutants, using a combination of green fluorescent protein (GFP) expression and the 9-(1'-pyrene)nonanol/ultraviolet (P9OH/UV) selection method. We used TKaG1 and TKaG2 cells, the wild-type Chinese hamster ovary (CHO) cells, CHO-K1, that had been stably transfected with cDNAs each encoding rat Pex2p as well as GFP tagged at the C-terminus with peroxisome targeting signal type 1 (PTS1) or N-terminally PTS2-tagged GFP. P9OH/UV-resistant cell colonies were examined for intracellular location of GFP on unfixed cells, by fluorescence microscopy. Seven each of the mutant cell clones isolated from TKaG1 and TKaG2 showed cytosolic GFP-PTS1 and PTS2-GFP, respectively, indicating the defect in peroxisome assembly. By transfection of PEX2, PEX5, PEX6, and PEX12 cDNAs and cell fusion analysis between the CHO cell mutants, five different complementation groups (CGs) were identified. Two mutant clones, ZPG207 and ZPG208, belonged to novel CGs. Further CG analysis using fibroblasts from patients with peroxisome biogenesis disorders, including rhizomelic chondrodysplasia punctata (RCDP), revealed that ZPG208 belonged to none of human CGs. ZPG207 was classified into the same CG as RCDP. Taken together, ZPG208 is in a newly identified, the 12th, CG in peroxisome-deficient CHO mutants reported to date and represents a novel mammalian CG.
Ghaedi
K
K
Faculty of Science, Kyushu University, Fukuoka, 812-8581, Japan.
Kawai
A
A
Okumoto
K
K
Tamura
S
S
Shimozawa
N
N
Suzuki
Y
Y
Kondo
N
N
Fujiki
Y
Y
D000074183:ATPases Associated with Diverse Cellular Activities; D000251:Adenosine Triphosphatases / Q000235:genetics; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D005816:Genetic Complementation Test; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics; D008565:Membrane Proteins / Q000235:genetics; D008830:Microbodies / Q000235:genetics* / Q000378:metabolism*; D009154:Mutation; D000074428:Peroxisomal Biogenesis Factor 2; D018901:Peroxisomal Disorders / Q000235:genetics*; D000074437:Peroxisomal Targeting Signal 2 Receptor; D000074435:Peroxisome-Targeting Signal 1 Receptor; D051381:Rats; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics
D008164:Luminescent Proteins; D008565:Membrane Proteins; C105251:PEX12 protein, human; C000614230:PEX2 protein, human; C000614264:PEX5 protein, human; D000074437:Peroxisomal Targeting Signal 2 Receptor; D000074435:Peroxisome-Targeting Signal 1 Receptor; C491336:Pex12 protein, rat; C000614232:Pex2 protein, rat; D018160:Receptors, Cytoplasmic and Nuclear; D000074428:Peroxisomal Biogenesis Factor 2; D049452:Green Fluorescent Proteins; D000251:Adenosine Triphosphatases; D000074183:ATPases Associated with Diverse Cellular Activities; C490759:PEX6 protein, human; C096998:Pex6 protein, rat