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Evidence for structural gene alterations affecting aminoacyl-tRNA synthetases in CHO cell mutants and revertants
Abstract:
Aminoacyl-tRNA synthetase (aaRS) activities in extracts of mutant strains of the Chinese hamster ovary line (CHO) were examined for alterations in thermal stability. Mutants having low activity for MetRS, AsnRS, or GlnRS contained aaRSs that were inactivated much more rapidly upon heating than those from wild-type cells. Revertant lines, isolated from cultures of these mutants (Asn-5, Met-2, and Gln-2) after treatment with nitrosoguanidine or ethyl methanesulfonate, had thermolabilities intermediate between mutant and wild-type, and consistently had higher activities than the mutants. With a modified in vivo aminoacylation procedure, two previously exceptional mutants. Arg-1 and His-1, showed pronounced reductions in the amount of arginyl-tRNA or histidyl-tRNA, respectively, under restrictive conditions, compared to wild type. Revertants of Arg-1 (like the mutant itself) had no measurable ArgRS in vitro activity (less than 0.4% of wild type) although in vivo aminoacylation in the one revertant tested was partially restored. These data provide evidence that the forward mutations have occurred in the structural genes of the aaRSs and that most of the reversions are probably the result of second-site point mutations in the aaRS genes.
Insights
Mutant aminoacyl-tRNA synthetases (aaRS) showed reduced thermal stability. Revertant strains exhibited intermediate stability, suggesting mutations occurred in aaRS structural genes.
Area of Science:
- Biochemistry
- Molecular Biology
- Genetics
Background:
- Aminoacyl-tRNA synthetases (aaRS) are crucial enzymes for protein synthesis.
- Mutant Chinese hamster ovary (CHO) cell lines were used to study aaRS function.
- Thermal stability assays are valuable for assessing enzyme integrity and function.
Purpose of the Study:
- To investigate thermal stability alterations in aaRS from mutant CHO cell lines.
- To characterize revertant strains and understand mutation/reversion mechanisms.
- To determine if mutations affect aaRS structural genes.
Main Methods:
- Enzyme activity assays on aaRS from wild-type and mutant CHO cells.
- Thermal inactivation experiments to measure enzyme stability.
- In vivo aminoacylation procedures to assess tRNA charging.
- Analysis of revertant lines generated after chemical mutagenesis.
Main Results:
- Mutant aaRS (MetRS, AsnRS, GlnRS) exhibited significantly decreased thermal stability compared to wild-type.
- Revertant lines showed intermediate thermal stability and partially restored enzyme activity.
- Mutants Arg-1 and His-1 displayed reduced in vivo aminoacylation of cognate tRNAs.
- Revertants of Arg-1 showed restored in vivo aminoacylation despite absent in vitro ArgRS activity.
Conclusions:
- Forward mutations likely occurred within the structural genes of specific aaRS.
- Reversions are probably due to second-site point mutations within the same aaRS genes.
- These findings support the role of aaRS structural genes in enzyme stability and function.