Molecular and genetic characterization of an ornithine decarboxylase-deficient Chinese hamster cell line.
The ornithine decarboxylase (ODC)-deficient Chinese hamster ovary (CHO) cell line C55.7 has normal amounts of ODC mRNA with very low amounts of immunologically detectable ODC protein, suggesting a structural mutation; however, 5-azacytidine treatment leads to phenotypical reversion (Steglich, C., and Scheffler, I. E. (1985) Somat. Cell Mol. Genet. 11, 11-23). We have demonstrated by chemical cleavage a single base mismatch in DNA heteroduplexes composed of wild-type and mutant cDNA strands. DNA sequencing showed that the mutant phenotype results from an aspartate-glycine substitution at amino acid 381 of the protein. When 5-azacytidine-revertant cell lines were selected for resistance to alpha-difluoromethylornithine, the resulting amplified ODC gene was structurally indistinguishable from the wild type gene. These results suggested the existence of a single active ODC locus in CHO cells. Using the methylation-sensitive restriction endonucleases AvaI and HpaII, we found evidence for two differentially methylated alleles in wild type, ODC-deficient and alpha-difluoromethylornithine-resistant cells. One of the alleles appeared completely inactivated by hypermethylation but could be reactivated by demethylation in spontaneous or 5-azacytidine-induced revertants.
Department of Biology, University of California San Diego, La Jolla 92093.
R B
RB
Pilz
C
C
Steglich
I E
IE
Scheffler
D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000235:genetics; D005260:Female; D008745:Methylation; D008969:Molecular Sequence Data; D009154:Mutation; D009692:Nucleic Acid Heteroduplexes / Q000235:genetics / Q000302:isolation & purification; D015345:Oligonucleotide Probes; D009955:Ornithine Decarboxylase / Q000172:deficiency* / Q000235:genetics; D010053:Ovary; D016133:Polymerase Chain Reaction; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D015183:Restriction Mapping
D009692:Nucleic Acid Heteroduplexes; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D012313:RNA; D004247:DNA; D009955:Ornithine Decarboxylase