New mutations and phenotypes associated with glutamate and aspartate transport in Chinese hamster ovary (CHO-K1) cells
1Department of Neurobiology and Anatomy, School of Medicine, University of Utah, Salt Lake City 84132, USA.
Abstract:
Two new Chinese hamster ovary cell (CHO-K1) mutants lacking amino acid transport System X-AG activity were isolated by [3H]aspartate suicide selection. These null mutants, Dd-B6 and Dd-B7, were analyzed by somatic cell hybridization, along with previously described partial-function mutants, Ed-A1 and Ed-B8. With respect to System X-AG activity, all four mutations fell into a single complementation group. By quantitative assay, the mutations in Ed-A1 and Ed-B8 behaved as simple recessives in fusions with wild type cells, while those in Dd-B6 and Dd-B7 were codominant. We have discovered that Ed-A1 and Ed-B8 are highly permeable to small neutral molecules. This high permeability phenotype was dominant to wild-type. Northern, Southern, and Western analyses indicated that System X-AG in CHO is not closely related to any of the three well characterized glutamate transporters represented by GLT-1, EAACI or GLAST.
Insights
Researchers identified new Chinese hamster ovary cell mutants affecting amino acid transport System X-AG. These mutants reveal insights into the genetic basis and characteristics of this crucial transport system.
Area of Science:
- Cell Biology
- Molecular Genetics
- Biochemistry
Background:
- Amino acid transport is vital for cellular function.
- System X-AG plays a role in amino acid uptake in Chinese hamster ovary (CHO-K1) cells.
- Understanding transport mechanisms requires genetic and molecular characterization.
Purpose of the Study:
- To isolate and characterize new mutants of amino acid transport System X-AG in CHO-K1 cells.
- To determine the genetic complementation and dominance relationships of these mutants.
- To investigate the molecular relationship of System X-AG to known glutamate transporters.
Main Methods:
- Isolation of mutants using [3H]aspartate suicide selection.
- Somatic cell hybridization for complementation analysis.
- Quantitative assays for transport activity and permeability.
- Molecular analyses including Northern, Southern, and Western blotting.
Main Results:
- Two new null mutants (Dd-B6, Dd-B7) and two partial-function mutants (Ed-A1, Ed-B8) were identified.
- All four mutations belong to a single complementation group for System X-AG activity.
- Mutations in Ed-A1 and Ed-B8 are recessive, while Dd-B6 and Dd-B7 are codominant.
- Ed-A1 and Ed-B8 exhibit a dominant high permeability to small neutral molecules.
- System X-AG is not closely related to GLT-1, EAACI, or GLAST glutamate transporters.
Conclusions:
- The identified mutants provide valuable tools for studying System X-AG.
- The genetic analysis reveals complex inheritance patterns for amino acid transport.
- System X-AG represents a distinct transporter family, not closely related to known glutamate transporters.
