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Isolation and characterization of 5-fluorotryptophan-resistant mutants with altered L-tryptophan transport
Abstract:
Mutants of A9 mouse fibroblast, resistant to the killing effect of 0.4 mM 5-flurotryptophan (5-FT), have altered L-tryptophan transport properties. The resistant phenotype is stable for at least 90 generations of growth in MEM. A fluctuation test indicated that clones resistant to 0.4 mM 5-FT occurred spontaneously. An average mutation rate was estimated at 1.6 X 10(-6). Treatment with N-methyl-N'-nitro-N-nitrosoguanidine increased the frequency of these clones by at least 100-fold. These results indicate that the resistant clones arose as a result of a mutation. All the resistant mutant tested accumulate less 5-FT at near steady-state conditions than the wild type. Lineweaver-Burk plots of initial rates of tryptophan uptake yield a biphasic curve suggesting that tryptophan is transported by two transport systems. Kinetic constants determined by a computer program indicate that both proposed transport systems were modified in each of two 5-FT resistant mutants.
Insights
Mutant mouse cells resistant to 5-flurotryptophan (5-FT) show altered L-tryptophan transport. These genetic mutations affect both identified tryptophan transport systems, impacting cellular uptake.
Area of Science:
- Cell Biology
- Genetics
- Biochemistry
Background:
- L-tryptophan is an essential amino acid crucial for protein synthesis and cellular function.
- Amino acid transport systems are vital for nutrient uptake and cellular homeostasis.
- 5-flurotryptophan (5-FT) is a toxic analog of L-tryptophan used to select for cells with altered amino acid transport.
Purpose of the Study:
- To investigate the genetic basis of resistance to 5-flurotryptophan (5-FT) in mouse fibroblast cells.
- To characterize the alterations in L-tryptophan transport properties associated with 5-FT resistance.
- To determine the kinetic parameters of tryptophan transport in wild-type and mutant cells.
Main Methods:
- Selection of 5-flurotryptophan (5-FT) resistant mutants from A9 mouse fibroblast cell lines.
- Fluctuation analysis to estimate spontaneous mutation rates.
- Treatment with N-methyl-N'-nitro-N-nitrosoguanidine (mutagen) to assess mutation frequency.
- Measurement of 5-FT and L-tryptophan uptake kinetics using Lineweaver-Burk plots and computer analysis.
Main Results:
- Stable 5-flurotryptophan (5-FT) resistant mutants were isolated, exhibiting altered L-tryptophan transport.
- The spontaneous mutation rate for 5-FT resistance was estimated at 1.6 X 10(-6), with a >100-fold increase after mutagen treatment.
- Resistant mutants showed reduced accumulation of 5-FT and modifications in both identified L-tryptophan transport systems.
- Kinetic analysis revealed alterations in the transport parameters for both systems in 5-FT resistant mutants.
Conclusions:
- The resistant phenotype arises from spontaneous mutations affecting L-tryptophan transport.
- Mouse fibroblast cells possess at least two distinct L-tryptophan transport systems.
- Mutations conferring 5-FT resistance alter the kinetic properties of both identified L-tryptophan transport systems.