TY - JOUR
T1 - Isolation and characterization of novel peroxisome biogenesis-defective chinese hamster ovary cell mutants using green fluorescent-protein
AU - Ghaedi, Kamran
AU - Kawai, Atsushi
AU - Okumoto, Kanji
AU - Tamura, Shigehiko
AU - Shimozawa, Nobuyuki
AU - Suzuki, Yasuyuki
AU - Kondo, Naomi
AU - Fujiki, Yukio
N1 - Funding Information:
We thank T. Sakaguchi and N. Matsumoto for technical assistance, the members of the Fujiki laboratory for helpful comments, and R. Tanaka for secretarial work. We also thank T. Tsukamoto for pTZCMVPTS2-dhfr plasmid. This work was supported in part by CREST grant (to Y.F.) from the Japan Science and Technology Corporation and Grants-in-Aid for Scientific Research (07408016, 08249232, 08557011, 09258215, 09044094, and 10129224 to Y.F.) from The Ministry of Education, Science, Sports and Culture.
PY - 1999/5/1
Y1 - 1999/5/1
N2 - 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)o 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 PTS2GFP, 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.
AB - 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)o 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 PTS2GFP, 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.
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U2 - 10.1006/excr.1999.4413
DO - 10.1006/excr.1999.4413
M3 - Article
C2 - 10222140
AN - SCOPUS:0033134180
SN - 0014-4827
VL - 248
SP - 489
EP - 497
JO - Experimental Cell Research
JF - Experimental Cell Research
IS - 2
ER -