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A rapid selection for animal cell mutants with defective peroxisomes
O H Morand1, L A Allen, R A Zoeller
1Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin, Madison.
Biochimica Et Biophysica Acta
|May 16, 1990
Summary
Chinese hamster ovary cells incorporate a pyrene-labeled alcohol into lipids. Mutants resistant to UV light show impaired alcohol metabolism, aiding in peroxisome biogenesis research.
Area of Science:
- Cell Biology
- Biochemistry
- Genetics
Background:
- Chinese hamster ovary (CHO) cells incorporate 9-(1'-pyrene)nonanol (P9OH) into cellular lipids.
- P9OH-labeled cells undergo cell death upon exposure to long-wavelength ultraviolet (UV) light due to reactive oxygen species generation.
- CHO mutants with defects in plasmalogen biosynthesis and peroxisome assembly exhibit resistance to P9OH/UV treatment.
Purpose of the Study:
- To investigate the mechanism behind the resistance of CHO mutants to P9OH/UV treatment.
- To elucidate the role of peroxisome assembly and plasmalogen biosynthesis in cellular response to P9OH and UV light.
- To establish a novel method for isolating animal cell mutants defective in peroxisome biogenesis.
Main Methods:
- Comparative analysis of P9OH incorporation into lipids of wild-type and mutant CHO cells.
- Measurement of [U-14C]hexadecanol incorporation into phospholipids of mutant cells.
- Assessment of 9-(1'-pyrene)nonanoic acid incorporation in wild-type and mutant cells.
Main Results:
- Mutant cells showed significantly reduced incorporation of P9OH into phospholipids (7.5-fold in ethanolamine-linked phospholipids) and triglycerides (2.4- to 6-fold).
- Impaired incorporation of [U-14C]hexadecanol into phospholipids was observed in mutant cells, indicating a defect in fatty alcohol metabolism.
- Mutant cells incorporated the fatty acid analog, 9-(1'-pyrene)nonanoic acid, twice as rapidly as wild-type cells.
Conclusions:
- The resistance of CHO mutants to P9OH/UV treatment is attributed to a general defect in fatty alcohol metabolism, specifically reduced P9OH incorporation.
- The findings highlight the importance of peroxisome assembly and plasmalogen biosynthesis in cellular lipid metabolism and response to oxidative stress.
- Resistance to P9OH/UV treatment offers a facile new approach for selecting animal cell mutants with deficiencies in peroxisome biogenesis.