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Direct selection of mutations in the human mitochondrial tRNAThr gene: reversion of an 'uncloneable' phenotype
1Joseph Gottstein Memorial Cancer Research Laboratory, Department of Pathology SM-30, University of Washington, Seattle 98195.
Abstract:
Several regions of the human mitochondrial genome are refractory to cloning in plasmid and bacteriophage DNA vectors. For example, recovery of recombinant M13 clones containing a 462 basepair MboI-Kpn I restriction fragment that spans nucleotide positions 15591 to 16053 of HeLa cell mitochondrial DNA was as much as 100-fold lower than the recovery of M13 clones containing other regions of the human mitochondrial genome. All of 50 recombinant M13 clones containing this 'uncloneable' fragment had one or more changes in nucleotide sequence. Each clone contained at least one alteration in two nucleotide positions within the tRNAThr gene that encode portions of the anticodon loop and D-stem of the HeLa mitochondrial tRNAThr. These results imply that the HeLa mitochondrial tRNAThr gene is responsible for the 'uncloneable' phenotype of this region of human mitochondrial (mt) DNA. A total of 61 nucleotide sequence alterations were identified in 50 independent clones containing the HeLa mt tRNAThr gene. 56 mutations were single-base substitutions; 5 were deletions. Approximately 80% of the base substitution mutations were A:T----G:C transitions. A preference for A:T----G:C transition mutations also characterizes polymorphic base substitution variants in the mitochondrial DNA of unrelated individuals. This similarity suggests that human mitochondrial DNA sequence variation within and between individuals may have a common origin.
Insights
The human mitochondrial tRNAThr gene causes cloning difficulties, leading to sequence alterations. These mutations, primarily A:T to G:C transitions, mirror natural DNA variations, suggesting a shared origin for both phenomena.
Area of Science:
- Molecular Biology
- Genetics
- Genomics
Background:
- Certain human mitochondrial DNA regions are difficult to clone using standard vectors.
- Previous studies noted challenges in replicating specific mitochondrial DNA fragments.
Purpose of the Study:
- To investigate the reasons behind the 'uncloneable' phenotype of a specific human mitochondrial DNA region.
- To identify sequence alterations in cloned mitochondrial DNA fragments and their potential implications.
Main Methods:
- Utilized M13 bacteriophage vectors for cloning human mitochondrial DNA fragments.
- Sequenced 50 independent recombinant M13 clones containing a 462 bp fragment from the mitochondrial tRNAThr gene.
- Analyzed nucleotide sequence alterations, including substitutions and deletions.
Main Results:
- Recovery of clones with the target mitochondrial DNA fragment was significantly lower (up to 100-fold).
- All 50 clones exhibited nucleotide sequence changes, primarily within the tRNAThr gene.
- Identified 61 alterations, with 56 single-base substitutions (80% A:T----G:C transitions) and 5 deletions.
Conclusions:
- The human mitochondrial tRNAThr gene is responsible for the observed 'uncloneable' phenotype.
- The high frequency of A:T----G:C transitions in cloned DNA mirrors those in natural mitochondrial DNA variation.
- This suggests a common mechanism underlies both experimental cloning artifacts and endogenous mitochondrial DNA sequence diversity.