Molecular and genetic characterization of an ornithine decarboxylase-deficient Chinese hamster cell line.

Abstract

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.