GENETIC-EVIDENCE SUPPORTING THE ROLE OF PEROXISOME ASSEMBLY FACTOR (PAF)-1 IN PEROXISOME BIOGENESIS - POLYMERASE CHAIN-REACTION DETECTION OF A MISSENSE MUTATION IN PAF-1 OF CHINESE-HAMSTER OVARY CELLS

The peroxisome/plasmalogen-deficient Chinese hamster ovary (CHO) mutan t cell line ZR-78.1 contains a mis-sense mutation in its cDNA-encoding peroxisome assembly factor-1 (PAF-1). Using a rapid polymerase chain reaction assay, we now demonstrate that the genome of ZR-78.1 contains only the mutant allele. When mutant ZR-78.1 is fused with wild-type k aryoplasts, occasional ''negative nuclear hybrids'' are observed that lack peroxisomes (Allen, L.-A. H., Morand, O. H., and Raetz, C. R. H. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 7012-7016). Despite the fac t that negative nuclear hybrids are tetraploid, they do not contain th e wild-type PAF-1 gene, suggesting that a chromosome fragment bearing the wild-type copy of PAF-1 was lost. Negative nuclear hybrids reconst ituted with wild-type cytoplasts do contain a wild-type PAF-1 gene, in dicating that the cytoplasts somehow reintroduced the wild-type PAF-1 allele without increasing ploidy. These findings support the role of P AF-1 and exclude the hypothesis of an additional cytoplasmic requireme nt for reinitiation of peroxisome biogenesis in peroxisome-deficient C HO cells. The plasmalogen deficiency and some other biochemical proper ties of ZR-78.1 are partially corrected in 5-azacytidine-treated subcl ones. However, such pseudorevertants do not contain peroxisomes, consi stent with the fact that there is no wild-type PAF-1 gene to reactivat e by demethylation.