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Mitochondrial DNA heteroplasmy in Candida glabrata after mitochondrial transformation
Jingwen Zhou1, Liming Liu, Jian Chen
1School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi 214122, China.
Abstract:
Genetic manipulation of mitochondrial DNA (mtDNA) is the most direct method for investigating mtDNA, but until now, this has been achieved only in the diploid yeast Saccharomyces cerevisiae. In this study, the ATP6 gene on mtDNA of the haploid yeast Candida glabrata (Torulopsis glabrata) was deleted by biolistic transformation of DNA fragments with a recoded ARG8(m) mitochondrial genetic marker, flanked by homologous arms to the ATP6 gene. Transformants were identified by arginine prototrophy. However, in the transformants, the original mtDNA was not lost spontaneously, even under arginine selective pressure. Moreover, the mtDNA transformants selectively lost the transformed mtDNA under aerobic conditions. The mtDNA heteroplasmy in the transformants was characterized by PCR, quantitative PCR, and Southern blotting, showing that the heteroplasmy was relatively stable in the absence of arginine. Aerobic conditions facilitated the loss of the original mtDNA, and anaerobic conditions favored loss of the transformed mtDNA. Moreover, detailed investigations showed that increases in reactive oxygen species in mitochondria lacking ATP6, along with their equal cell division, played important roles in determining the dynamics of heteroplasmy. Based on our analysis of mtDNA heteroplasmy in C. glabrata, we were able to generate homoplasmic Deltaatp6 mtDNA strains.
Insights
Researchers successfully manipulated mitochondrial DNA (mtDNA) in the haploid yeast Candida glabrata, creating strains with deleted ATP6 genes. This breakthrough enables new investigations into mtDNA function and genetics.
Area of Science:
- Mitochondrial genetics
- Yeast molecular biology
- Cellular respiration
Background:
- Investigating mitochondrial DNA (mtDNA) function typically requires genetic manipulation, a technique previously limited to diploid yeast.
- Haploid yeast like Candida glabrata offer a simpler model for genetic studies but have lacked efficient mtDNA manipulation methods.
Purpose of the Study:
- To develop a method for genetic manipulation of mtDNA in the haploid yeast Candida glabrata.
- To investigate the dynamics of mtDNA heteroplasmy under varying conditions.
- To generate homoplasmic strains with specific mtDNA deletions for further research.
Main Methods:
- Biolistic transformation of Candida glabrata with DNA fragments containing a recoded ARG8(m) marker and homologous arms to the ATP6 gene.
- Selection of transformants based on arginine prototrophy.
- Characterization of mtDNA heteroplasmy using PCR, quantitative PCR, and Southern blotting.
- Analysis of mtDNA dynamics under aerobic and anaerobic conditions.
Main Results:
- Successful deletion of the ATP6 gene in Candida glabrata mtDNA was achieved.
- Spontaneous loss of original mtDNA did not occur readily, even under selective pressure.
- Aerobic conditions promoted the loss of wild-type mtDNA, while anaerobic conditions favored the loss of transformed mtDNA.
- Increased reactive oxygen species in mitochondria lacking ATP6 were identified as a key factor in heteroplasmy dynamics.
Conclusions:
- A novel method for genetic manipulation of mtDNA in haploid yeast Candida glabrata was established.
- Environmental conditions significantly influence mtDNA heteroplasmy dynamics, with implications for mitochondrial function.
- The study successfully generated homoplasmic Deltaatp6 mtDNA strains, paving the way for detailed functional analysis.
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