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pubmed:abstractText |
The peroxisome/plasmalogen-deficient Chinese hamster ovary (CHO) mutant cell line ZR-78.1 contains a missense 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 karyoplasts, 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 fact that negative nuclear hybrids are tetraploid, they do not contain the wild-type PAF-1 gene, suggesting that a chromosome fragment bearing the wild-type copy of PAF-1 was lost. Negative nuclear hybrids reconstituted with wild-type cytoplasts do contain a wild-type PAF-1 gene, indicating that the cytoplasts somehow reintroduced the wild-type PAF-1 allele without increasing ploidy. These findings support the role of PAF-1 and exclude the hypothesis of an additional cytoplasmic requirement for reinitiation of peroxisome biogenesis in peroxisome-deficient CHO cells. The plasmalogen deficiency and some other biochemical properties of ZR-78.1 are partially corrected in 5-azacytidine-treated subclones. However, such pseudo-revertants do not contain peroxisomes, consistent with the fact that there is no wild-type PAF-1 gene to reactivate by demethylation.
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