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Chinese hamster cells deficient in ornithine decarboxylase activity: reversion by gene amplification and by
Abstract:
A group of Chinese hamster ovary (CHO) cell mutants deficient in ornithine decarboxylase (ODC) activity are described and compared to the prototype mutant reported previously (21). Although all mutants belong to the same complementation group, they can be divided into two classes: those with some residual enzyme activity and those with no activity. All mutants are putrescine auxotrophs, but they differ in their ability to utilize the enzyme's substrate, ornithine, a property which correlates with the amount of residual enzyme activity. The mutants also differ in their frequency of reversion to prototrophy. The leaky mutants revert at a high rate by overproducing a partially defective enzyme by a gene amplification mechanism similar to that leading to the ornithine analog-resistant mutants which have elevated enzyme levels. Spontaneous reversion in the null mutants is rare. However, one null mutant, which was induced with ethyl methane sulfonate and which makes ODC mRNA but no active enzyme, is nevertheless revertible with 5-azacytidine. We conclude that CHO cells are at least diploid at the ODC locus, but that only one allele is active. Further studies suggest the possibility that ethyl methane sulfonate is not just a classical mutagen but may also induce gene inactivations that are revertible by 5-azacytidine.
Insights
Researchers studied Chinese hamster ovary (CHO) cell mutants lacking ornithine decarboxylase (ODC). They found these mutants vary in enzyme activity and reversion rates, suggesting diploidy at the ODC locus with only one active allele.
Area of Science:
- Biochemistry
- Cell Biology
- Genetics
Background:
- Ornithine decarboxylase (ODC) is a key enzyme in polyamine synthesis.
- Mutants deficient in ODC activity are essential tools for studying gene regulation and function.
- Previous studies established a prototype ODC-deficient Chinese hamster ovary (CHO) cell mutant.
Purpose of the Study:
- To characterize newly generated CHO cell mutants with deficiencies in ornithine decarboxylase (ODC) activity.
- To compare these mutants to a previously reported prototype.
- To investigate the genetic basis of ODC deficiency and reversion mechanisms in CHO cells.
Main Methods:
- Isolation and characterization of CHO cell mutants lacking ODC activity.
- Complementation analysis to group mutants.
- Assessment of residual enzyme activity and substrate utilization.
- Analysis of reversion frequencies to prototrophy.
- Investigation of gene amplification and potential epigenetic modifications.
Main Results:
- Mutants were classified into two groups: those with residual ODC activity and those with no activity.
- All mutants were auxotrophic for putrescine, but varied in their ability to utilize ornithine.
- Leaky mutants showed high reversion rates via gene amplification of a partially defective ODC enzyme.
- Null mutants exhibited rare spontaneous reversion, but one was revertible with 5-azacytidine.
- Evidence suggests CHO cells are at least diploid at the ODC locus, with monoallelic expression.
Conclusions:
- CHO cell mutants deficient in ODC activity provide insights into gene regulation and diploidy.
- Gene amplification is a mechanism for reversion in leaky ODC-deficient mutants.
- Ethyl methanesulfonate may induce reversible gene inactivations, potentially through epigenetic modifications.
