Related Experiment Videos
Selection of a lysine-resistant CHO-K1 mutant with reduced amino acid transport through multiple systems
1Department of Anatomy, School of Medicine, University of Utah, Salt Lake City 84132.
Abstract:
High levels of L-lysine were used to select for resistant variants of Chinese hamster ovary (CHO-K1) cells. Surviving colonies were screened for altered lysine transport and two with reduced uptake were picked. Clone CH-Kr, derived from the more severely affected colony, was analyzed in detail. In starved cells the Vmax of lysine uptake in CH-Kr was half that of CHO while Km was unaltered. The intracellular pool of lysine, a substrate of cationic amino acid transport system y+, was significantly lower in CH-Kr. However, transport and pools of other amino acids, which are not substrates of y+, were also reduced in CH-Kr, as was the internal sodium concentration, while hexose import was increased. It appears that the mutation in CH-Kr is pleiotropic, affecting some general aspects of amino acid transport.
Insights
Researchers selected for L-lysine resistant Chinese hamster ovary (CHO-K1) cells, identifying a variant (CH-Kr) with reduced lysine uptake. This mutation appears to pleiotropically affect general amino acid transport in the cells.
Area of Science:
- Cell Biology
- Biochemistry
- Genetics
Background:
- Amino acid transporters are crucial for cellular function.
- Understanding nutrient transport mechanisms is vital for cell physiology and disease research.
- Chinese hamster ovary (CHO-K1) cells are widely used in biological research and biopharmaceutical production.
Purpose of the Study:
- To investigate the molecular mechanisms underlying L-lysine resistance in CHO-K1 cells.
- To characterize the transport properties of a selected resistant variant.
- To determine if the mutation affects the transport of other amino acids or cellular processes.
Main Methods:
- Selection of L-lysine resistant variants from CHO-K1 cell cultures.
- Screening of surviving colonies for altered lysine uptake.
- Detailed analysis of a selected clone (CH-Kr) for amino acid transport kinetics (Vmax, Km).
- Measurement of intracellular amino acid pools, internal sodium concentration, and hexose import.
Main Results:
- Clone CH-Kr exhibited a 50% reduction in Vmax for lysine uptake compared to wild-type CHO cells, with no change in Km.
- Intracellular lysine pools were significantly reduced in CH-Kr cells.
- Transport and pools of non-y+ substrate amino acids were also reduced.
- Internal sodium concentration decreased, while hexose import increased in CH-Kr cells.
Conclusions:
- The mutation in CH-Kr cells is likely pleiotropic, impacting multiple amino acid transport systems.
- This finding suggests a potential link between lysine transport and broader cellular nutrient import mechanisms.
- The characterized variant provides a valuable tool for studying amino acid transport regulation.