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Mutagenesis and genetic analysis with Chinese hamster auxotrophic cell markers
Abstract:
Chinese hamster ovary cells were treated with various physical and chemical mutagens and subject to the bromodeoxyuridine plus visible light procedure for isolation of auxotrophic mutants. more than 200 auxotrophs have been isolated which require exogenous supplement of various nutrilites for growth, such as glycine, adenine, thymidine, inositol, etc. Fifty-five of these have been characterized by complementation tests and were shown to constitute 15 different loci. All these auxotrophs are highly stable and exhibit all-or-none growth response to the respective nutrilites. Enzyme deficiencies have been identified in several of these classes. When these auxotrophs are hybridized with human cells and grown in selective medium, the hybrids lose human chromosomes at a rapid rate and the analysis for synteny of the human genes can be successfully carried out. Moverover, in hybrids formed between an adenine-requiring auxotroph and human cells, a human esterase activator gene has been identified which appears to regulate the expression of esterase gene activities in the Chinese hamster genome. Studies of this kind may lead to the understanding of gene regulation in mammalian cells.
Insights
Researchers isolated over 200 auxotrophic mutants from Chinese hamster ovary cells. These mutants aid in understanding gene regulation and mapping human genes using cell hybridization techniques.
Area of Science:
- Cell Biology
- Genetics
- Molecular Biology
Background:
- Auxotrophic mutants are crucial for genetic analysis and understanding metabolic pathways.
- Chinese hamster ovary (CHO) cells are a widely used model system in biological research.
Purpose of the Study:
- To isolate and characterize auxotrophic mutants from CHO cells.
- To utilize these mutants for gene mapping and studying gene regulation in mammalian cells.
Main Methods:
- Treatment of CHO cells with mutagens.
- Isolation of auxotrophic mutants using the bromodeoxyuridine plus visible light procedure.
- Complementation tests to identify different genetic loci.
- Hybridization of auxotrophs with human cells for gene mapping.
Main Results:
- Over 200 auxotrophic mutants were isolated, requiring various nutrients.
- Fifty-five mutants were characterized, defining 15 distinct genetic loci.
- Enzyme deficiencies were identified in several mutant classes.
- Cell hybridization enabled rapid loss of human chromosomes and synteny analysis.
- A human esterase activator gene was identified in adenine-requiring auxotroph-human cell hybrids.
Conclusions:
- The isolated auxotrophs are stable and valuable tools for genetic studies.
- This methodology facilitates human gene mapping and understanding of gene regulation in mammalian systems.
- The identified esterase activator gene provides insights into mammalian gene expression control.