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) 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.
Allen
L A
LA
Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin, Madison 53706.
Hope
L
L
Raetz
C R
CR
Thieringer
R
R
D000217:Acyltransferases / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001374:Azacitidine / Q000494:pharmacology; D001483:Base Sequence; D016466:CHO Cells; D002999:Clone Cells; D006224:Cricetinae; D017931:DNA Primers; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism*; D008830:Microbodies / Q000502:physiology* / Q000648:ultrastructure; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D000074428:Peroxisomal Biogenesis Factor 2; D010955:Plasmalogens / Q000378:metabolism; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods; D012333:RNA, Messenger / Q000096:biosynthesis
D017931:DNA Primers; D008565:Membrane Proteins; D009838:Oligodeoxyribonucleotides; D010955:Plasmalogens; D012333:RNA, Messenger; D000074428:Peroxisomal Biogenesis Factor 2; D000217:Acyltransferases; C020051:glycerone-phosphate O-acyltransferase; D001374:Azacitidine