ISOLATION OF A CHINESE-HAMSTER FIBROBLAST VARIANT DEFECTIVE IN DIHYDROXYACETONEPHOSPHATE ACYLTRANSFERASE ACTIVITY AND PLASMALOGEN BIOSYNTHESIS - USE OF A NOVEL 2-STEP SELECTION PROTOCOL

We have developed a two-step selection protocol to generate a populati on of Chinese hamster ovary (CHO) cell variants that are plasmalogen-d eficient, but contain intact, functional peroxisomes (plasmalogen(-)/p eroxisome(+)). This involved sequential exposures of a mutagenized cel l population to photodynamic damage by using two different pyrene-labe lled sensors, 9-(1'-pyrene)nonanol and 12-(1'-pyrene)dodecanoic acid. By this procedure we generated several isolates, all except one of whi ch displayed a severe decrease in plasmalogen biosynthesis. Further ch aracterization of one of the plasmalogen-deficient isolates, NRe1-4, s howed that it contained intact, functional peroxisomes. Whole-cell hom ogenates from NRe1-4 displayed severely decreased dihydroxyacetone pho sphate acyltransferase, which catalyses the first step in plasmalogen biosynthesis. NRe1-4 and another, recently described, plasmalogen-defi cient cell line, NZel-1 [Nagan, Hajra, Das, Moser, Moser, Lazarow, Pur due and Zoeller (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 4475-4480] we re hypersensitive to singlet oxygen, supporting the notion of plasmalo gens as radical oxygen scavengers. Wild-type-like resistance could be conferred on NRe1-4 upon restoration of plasmalogen content by supplem entation with a bypass compound, sn-1-hexadecylglycerol. NRe1-4 and ot her plasmalogen(-)/peroxisome(+) strains will allow us to examine furt her the role of ether lipids in cellular functions without complicatio ns associated with peroxisome deficiency, and might serve as an animal cell model for certain forms of the human genetic disorder rhizomelic chondrodysplasia punctata.