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Sequence analysis of mitochondrial DNA in a mouse cell line resistant to chloramphenicol and oligomycin
Abstract:
A mouse L-cell line, designated 111-OB3, is described which is resistant to two drugs, chloramphenicol and oligomycin. The cells contain two types of mitochondrial DNA molecules, in roughly equal proportions, which differ in that one is cleaved by endonuclease EcoRI at a novel site within the coding sequence for subunit 6 of the mitochondrial ATPase (ATPase-6). Sequence analysis reveals that the cleavage site was created by a single transversion which predicts a replacement of valine in the wild-type ATPase-6 by glutamic acid. The replacement occurs in a hydrophobic amino acid sequence which is highly conserved in mouse, human, and bovine proteins. The position of the replacement is similar to a substitution observed in one class of yeast mutants resistant to oligomycin. Both of the mitochondrial DNA molecules in 111-OB3 also have a single nucleotide change in the gene encoding the large (16S) rRNA. These observations are consistent with the hypothesis that oligomycin resistance in mammalian cells can be cytoplasmically determined and can result from alterations in ATPase-6. The appearance of the mutation before selection in oligomycin suggests a model for the origin of mitochondrial mutations in mammalian cells.
Insights
Mitochondrial DNA mutations in mouse cells confer drug resistance. A novel mutation in the ATPase-6 gene, identified before drug selection, suggests a new model for mitochondrial mutation origins.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Mitochondrial DNA (mtDNA) mutations are implicated in various diseases.
- Understanding the mechanisms of mtDNA mutation and drug resistance is crucial.
Purpose of the Study:
- To characterize a drug-resistant mouse cell line (111-OB3).
- To investigate the genetic basis of chloramphenicol and oligomycin resistance in mammalian cells.
- To explore the origins of mitochondrial mutations.
Main Methods:
- Isolation and characterization of a drug-resistant mouse L-cell line (111-OB3).
- Analysis of mitochondrial DNA (mtDNA) using endonuclease EcoRI cleavage.
- DNA sequencing to identify mutations in ATPase-6 and 16S rRNA genes.
Main Results:
- The 111-OB3 cell line exhibits resistance to chloramphenicol and oligomycin.
- Two mtDNA types were found, with one containing a novel EcoRI site in the ATPase-6 gene.
- Sequencing revealed a valine to glutamic acid substitution in ATPase-6 and a nucleotide change in 16S rRNA.
- The ATPase-6 mutation occurred prior to oligomycin selection.
Conclusions:
- Oligomycin resistance in mammalian cells can be cytoplasmically inherited and linked to ATPase-6 alterations.
- The findings support a model for the origin of mitochondrial mutations in mammalian cells, potentially arising before selective pressure.