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Direct selection of Chinese hamster ovary strains deficient in CTP synthetase activity
1Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Potters Bar, Herts., U.K.
Abstract:
Mutant CHO cell strains dependent upon cytidine for growth and survival were isolated by a direct selection protocol. The mutants obtained are deficient in CTP synthetase activity (less than 2% residual activity) and have low reversion frequencies (less than 10(-7)). Cytidine deprivation of these stains leads to rapid depletion of intracellular CTP pools, but not dCTP pools, and a surprisingly rapid loss of cell viability. These properties should make the cytidine auxotrophs useful for a number of biochemical and genetic studies.
Insights
Researchers developed mutant Chinese hamster ovary (CHO) cell strains requiring cytidine for survival. These cells, deficient in CTP synthetase, offer valuable tools for biochemical and genetic studies.
Area of Science:
- Biochemistry
- Cell Biology
- Genetics
Background:
- Cytidine is essential for cell growth and survival.
- CTP synthetase is a key enzyme in pyrimidine nucleotide biosynthesis.
- Understanding nucleotide metabolism is crucial for cell biology.
Purpose of the Study:
- To isolate and characterize mutant Chinese hamster ovary (CHO) cell strains with specific deficiencies in cytidine metabolism.
- To investigate the impact of CTP synthetase deficiency on cellular nucleotide pools and viability.
- To establish novel tools for biochemical and genetic research.
Main Methods:
- Direct selection protocol for isolating auxotrophic mutants.
- Enzyme activity assays to measure CTP synthetase levels.
- Analysis of intracellular nucleotide pools (CTP and dCTP) under cytidine deprivation.
- Cell viability assays.
Main Results:
- Successfully isolated CHO cell mutants dependent on cytidine for growth.
- Mutants exhibited significantly reduced CTP synthetase activity (<2% residual).
- Cytidine deprivation led to rapid depletion of intracellular CTP pools and a sharp decline in cell viability.
- Low reversion frequencies (<10(-7)) were observed, indicating genetic stability.
Conclusions:
- The isolated cytidine auxotrophs are valuable models for studying pyrimidine biosynthesis.
- These mutants provide a robust system for investigating the essential role of CTP in cellular processes.
- The characterized properties make these strains useful for biochemical and genetic research applications.