Mitochondrial DNA Heteroplasmy in Candida glabrata after Mitochondrial Transformation

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 ⌬atp6 mtDNA strains.

Mitochondrial diseases and the role of the yeast models

“…Recently, mitochondria of Candida glabrata were transformed with appearance of heteroplasmy. Deepening this observation may provide interesting information to control it (Zhou et al , 2010). Because Yarrowia lipolytica possesses the mitochondrial‐encoded subunits of complex I, it may be used in the future to model complex I human mitochondrial mutations.…”

The Maintenance of Mitochondrial DNA Integrity--Critical Analysis and Update

“…Homologous recombination (HR) in yeast and Chlamydomonas reinhardtii mitochondria is well established and is used as a tool for genetic manipulation of mtDNA in these organisms (Zhou et al 2010;Mileshina et al 2011). Recently, homologous recombination also was shown in plant mitochondria (Manchekar et al 2006;Davila et al 2011;Mileshina et al 2011).…”

DNA Delivery to Mitochondria: Sequence Specificity and Energy Enhancement

“…These degenerative disorders are currently incurable and would benefit from the development of gene replacement strategies. However, only yeast (Saccharomyces cerevisiae, Candida glabrata) and the unicellular alga Chlamydomonas reinhardtii are currently amenable to mitochondrial genetic transformation in intact cells (2,3). Conventional methodologies have failed so far to achieve transformation of mitochondria in plants or in mammalian cells.…”