Isolation of peroxisome-defective CHO mutant cells using green fluorescent protein.
The authors constructed a recombinant green fluorescent protein (GFP) (PTS-GFP), which carries peroxisome targeting signal (PTS1 or PTS2) as an additional sequence, by polymerase chain reaction. The gene encoding for the recombinant GFP was constructed into an eukaryotic expression vector, and stable transformant of CHO cell expressing PTS-GFP was isolated, following the transfection of the plasmid encoding for the GFP. Each expressed PTS-GFP appeared to be localized in peroxisomes, because the GFP was observed in cellular structures, as was catalase. The observation proposed a visual screening procedure for isolating peroxisome-defective mutant. Following an enrichment of mutant cells by use of 9-(1'-pyrene)nonanol/ultraviolet irradiation (P9OH/UV) method, five peroxisome-defective mutants were isolated by pursuing the fluorescent signals from GFP. Two mutants (SK24 and SK32) were isolated from CHO cells expressing PTS1-GFP, and three mutants (PT13, PT32, and PT54) were isolated from cells expressing PTS2-GFP. Four mutants, except for PT13, showed cytosolic distributions of both PTS-GFP and catalase. On the other hand, mutant PT13 showed a cytosolic distribution on PTS2-GFP, but a peroxisomal distribution on catalase. Cell fusion analysis between SK24 mutant and other mutants indicated that PT54 mutant is in the same complementation group (CG) as SK24, but that SK32, PT13, and PT32 mutants are in different complementation group(s) from SK24.
Ito
M
M
Department of Biology, Saga Medical School, Saga 849-8501, Japan. itohm1@post.saga-med.ac.jp
Ito
R
R
Miura
S
S
Huang
Y
Y
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D009154:Mutation; D020675:Peroxisomes / Q000235:genetics*
D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins