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Pleiotropic mutants of Chinese hamster cells with altered cytidine 5'-triphosphate synthetase
Abstract:
Following chemical mutagenesis and multiple-step indirect selection, four clones of Chinese hamster V79 cells were isolated which exhibited auxotrophy for thymidine, deoxycytidine, or deoxyuridine but not for cytidine or uridine. All were resistant to uridine, 3-deazauridine, 5-fluorouridine, thymidine, and cytosine arabinoside at concentrations that were toxic to wild-type V79 cells. The cytidine 5'-triphosphate (CTP) and deoxycytidine 5'-triphosphate (dCTP) pools in the mutants were expanded, but the uridine 5'-triphosphate (UTP) pool either decreased or remained unchanged relative to the wild-type level. Furthermore, since the parental cells appear to be deficient in dCMP deaminase activity and CTP (or one of its metabolites) has been shown to inhibit uridine 5'-diphosphate (UDP) reduction, an elevated CTP level should lead to the observed thymidine auxotrophy. It also explains the joint resistance of mutant clones to thymidine and cytosine arabinoside. The change in the ratio of intracellular dCTP to thymidine 5'-triphosphate (dTTP) may be responsible for the elevation in the rates of spontaneous mutations in these mutants.
Insights
Mutant V79 cell clones resistant to nucleoside analogs were identified. These cells exhibit altered nucleotide pools and increased mutation rates, offering insights into DNA synthesis and repair mechanisms.
Area of Science:
- Cell Biology
- Genetics
- Biochemistry
Background:
- Chinese hamster V79 cells are a standard model for mutagenesis studies.
- Understanding nucleotide metabolism is crucial for cancer research and drug development.
Purpose of the Study:
- To isolate and characterize V79 cell mutants with altered nucleoside metabolism.
- To investigate the biochemical basis of resistance to nucleoside analogs.
- To explore the relationship between nucleotide pool alterations and spontaneous mutation rates.
Main Methods:
- Chemical mutagenesis of V79 cells.
- Multi-step indirect selection for auxotrophic mutants.
- Analysis of nucleotide triphosphate pools (CTP, dCTP, UTP, dTTP).
- Assessment of resistance to various nucleoside analogs.
- Measurement of spontaneous mutation rates.
Main Results:
- Four V79 cell clones auxotrophic for thymidine, deoxycytidine, or deoxyuridine were isolated.
- Mutants displayed resistance to uridine, 3-deazauridine, 5-fluorouridine, thymidine, and cytosine arabinoside.
- Expanded cytidine 5'-triphosphate (CTP) and deoxycytidine 5'-triphosphate (dCTP) pools were observed.
- Uridine 5'-triphosphate (UTP) pools were decreased or unchanged.
- Elevated CTP levels correlated with thymidine auxotrophy and resistance to thymidine and cytosine arabinoside.
- Altered dCTP to thymidine 5'-triphosphate (dTTP) ratios may increase spontaneous mutation rates.
Conclusions:
- Mutations affecting nucleotide metabolism can confer resistance to nucleoside analogs.
- Altered nucleotide pools in V79 cells provide a model for studying drug resistance.
- These mutants may serve as valuable tools for investigating DNA synthesis fidelity and mutagenesis.