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Sequence analysis of mouse mitochondrial chloramphenicol-resistant mutants
1Department of Radiation Therapy, University of Texas Medical Branch, Galveston 77550.
Abstract:
The nucleotide sequences of the 3' halves of the mitochondrial 16S rRNA genes from four independent mouse chloramphenicol-resistant (CAP-R) mutants were determined. Each contained a different, single base change that encodes the mutational phenotype. The mitochondrial rRNA gene from the SVA31 CAP-R mutant contains a G-to-A transition at nucleotide 2161 of the noncoding strand; the SVIS CAP-R mutant, a G-to-A transition at position 2375; the LA9 CAP-R mutant, an A-to-T transversion at position 2379; and the SVT2 CAP-R mutant, a T-to-C transition at position 2433. Three of these CAP-R mutants appear to be heteroplasmic as the mtDNA populations contain both wild-type and mutant copies of the rRNA gene. The SVIS CAP-R mutation has not been observed in other mammalian CAP-R mutants, although it occurs at a site homologous to one of the yeast mitochondrial CAP-R mutations. Based upon the locations of the mutated sites within the 16S rRNA, and their proximity to previously analyzed sites of mutations conferring increased inhibitor resistance, all these mutations occur within the ribosomal RNA peptidyltransferase domain. These results provide an explanation for the pleiotropic nature of mitochondrial CAP-R mutations in mammalian cells, particularly the observations that some of the mutant lines are partially respiration deficient.
Insights
Mitochondrial 16S rRNA gene mutations confer chloramphenicol resistance (CAP-R) in mice. These single base changes in the peptidyltransferase domain explain pleiotropic effects, including partial respiration deficiency in mutant cell lines.
Area of Science:
- Mitochondrial genetics
- Molecular biology
- Genomics
Background:
- Mitochondrial DNA (mtDNA) mutations can lead to drug resistance and cellular dysfunction.
- Chloramphenicol resistance (CAP-R) is a key phenotype studied in mitochondrial genetics.
- Understanding the molecular basis of CAP-R is crucial for deciphering mitochondrial gene function.
Purpose of the Study:
- To determine the specific nucleotide sequence changes in the mitochondrial 16S rRNA gene of mouse CAP-R mutants.
- To investigate the relationship between these mutations and the observed CAP-R phenotype.
- To elucidate the functional consequences of these mutations within the ribosomal RNA peptidyltransferase domain.
Main Methods:
- DNA sequencing of the 3' halves of mitochondrial 16S rRNA genes from four independent mouse CAP-R mutants.
- Analysis of nucleotide base changes and their locations within the rRNA gene.
- Assessment of heteroplasmy in mtDNA populations.
Main Results:
- Four distinct single-nucleotide changes were identified in the 16S rRNA genes of the CAP-R mutants.
- Mutations included G-to-A transitions, an A-to-T transversion, and a T-to-C transition at specific nucleotide positions.
- Three mutants exhibited heteroplasmy, with both wild-type and mutant mtDNA copies present.
- All identified mutations are located within the ribosomal RNA peptidyltransferase domain.
Conclusions:
- Specific single base changes in the mitochondrial 16S rRNA gene are responsible for chloramphenicol resistance in mice.
- These mutations, located in the peptidyltransferase domain, explain the pleiotropic effects observed in CAP-R mutants, including partial respiration deficiency.
- The findings provide molecular insights into mammalian mitochondrial drug resistance and its associated cellular phenotypes.