Precise and simultaneous quantification of mitochondrial DNA heteroplasmy and copy number by digital PCR

Shoop, Wendy; Gorsuch, Cassandra L.; Bacman, Sandra R.; Moraes, Carlos T.

doi:10.1016/j.jbc.2022.102574

Cited by 11 publications

(5 citation statements)

References 33 publications

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“…PCR analysis showed that four independent Δ ifa38 deletion mutants, two Δ phs1 deletion mutants, and five Δ tsc13 deletion mutants were obtained (Figure S4e ). We further measured the HYG copy number in two independent deletion mutants of each gene by using nanoplate‐based digital PCR (dPCR), which has been widely used as an accurate and reliable quantitative method for absolute quantification of gene copy number (Murphy et al., 2023 ; Shoop et al., 2022 ). A single‐copy gene MGG_01944 of M. oryzae was employed in dPCR as the internal reference control to determine gene copy number.…”

Section: Resultsmentioning

confidence: 99%

“…We examined the targeted gene replacement in the M. oryzae deletion mutants on the nanoplate‐based QIAcuity digital PCR system according to previously reported dPCR method with slight modification (Murphy et al., 2023 ; Shoop et al., 2022 ). The PCR primers were designed for specific amplification of the hygromycin resistance gene HYG and the internal control single copy gene MGG_01944 (Table S3 ).…”

Section: Methodsmentioning

confidence: 99%

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A fatty acid elongase complex regulates cell membrane integrity and septin‐dependent host infection by the rice blast fungus

Su,

Xu,

Lei

et al. 2024

Molecular Plant Pathology

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Very‐long‐chain fatty acids (VLCFAs) regulate biophysical properties of cell membranes to determine growth and development of eukaryotes, such as the pathogenesis of the rice blast fungus Magnaporthe oryzae. The fatty acid elongase Elo1 regulates pathogenesis of M. oryzae by modulating VLCFA biosynthesis. However, it remains unknown whether and how Elo1 associates with other factors to regulate VLCFA biosynthesis in fungal pathogens. Here, we identified Ifa38, Phs1 and Tsc13 as interacting proteins of Elo1 by proximity labelling in M. oryzae. Elo1 associated with Ifa38, Phs1 and Tsc13 on the endoplasmic reticulum (ER) membrane to control VLCFA biosynthesis. Targeted gene deletion mutants Δifa38, Δphs1 and Δtsc13 were all similarly impaired as Δelo1 in vegetative growth, conidial morphology, stress responses in ER, cell wall and membrane. These deletion mutants also displayed severe damage in cell membrane integrity and failed to organize the septin ring that is essential for penetration peg formation and pathogenicity. Our study demonstrates that M. oryzae employs a fatty acid elongase complex to regulate VLCFAs for maintaining or remodelling cell membrane structure, which is important for septin‐mediated host penetration.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

A fatty acid elongase complex regulates cell membrane integrity and septin‐dependent host infection by the rice blast fungus

Su,

Xu,

Lei

et al. 2024

Molecular Plant Pathology

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“…mtDNA copy number was quantified as previously described using duplex direct PCR (dPCR), with one assay that amplified MT-ND2 and another that amplified 18S rDNA 81 . Primers are given in Supplementary Table 2 .…”

Section: Methodsmentioning

confidence: 99%

“…mtDNA heteroplasmy and copy number were quantified by duplex dPCR as previously described 81 . The calculated mtDNA copy number for each treated condition was normalized to control at each timepoint.…”

Section: Methodsmentioning

confidence: 99%

Efficient elimination of MELAS-associated m.3243G mutant mitochondrial DNA by an engineered mitoARCUS nuclease

Shoop,

Lape,

Trum

et al. 2023

Nat Metab

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Nuclease-mediated editing of heteroplasmic mitochondrial DNA (mtDNA) seeks to preferentially cleave and eliminate mutant mtDNA, leaving wild-type genomes to repopulate the cell and shift mtDNA heteroplasmy. Various technologies are available, but many suffer from limitations based on size and/or specificity. The use of ARCUS nucleases, derived from naturally occurring I-CreI, avoids these pitfalls due to their small size, single-component protein structure and high specificity resulting from a robust protein-engineering process. Here we describe the development of a mitochondrial-targeted ARCUS (mitoARCUS) nuclease designed to target one of the most common pathogenic mtDNA mutations, m.3243A>G. mitoARCUS robustly eliminated mutant mtDNA without cutting wild-type mtDNA, allowing for shifts in heteroplasmy and concomitant improvements in mitochondrial protein steady-state levels and respiration. In vivo efficacy was demonstrated using a m.3243A>G xenograft mouse model with mitoARCUS delivered systemically by adeno-associated virus. Together, these data support the development of mitoARCUS as an in vivo gene-editing therapeutic for m.3243A>G-associated diseases.

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“…Primers were selected based on literature data (Bai and Wong, 2005;Gu et al, 2013). The value of mtDNA copy number per cell that was used for analysis was calculated as a ratio of mtDNA and nDNA copies (Shoop et al, 2022).…”

Section: Quantitative Determination Of Mtdna Copiesmentioning

confidence: 99%

Mitochondrial DNA copy number in patients with systemic sclerosis

Bogatyreva,

Gerasimova,

Kirichenko

et al. 2023

Front. Mol. Biosci.

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Introduction: Systemic scleroderma (SSc) is a chronic autoimmune disease of inflammatory origin. Mitochondrial dysfunction is considered as an important mechanism in the pathogenesis of SSc. Currently mitochondrial DNA (mtDNA) copy number is used as a surrogate marker of mitochondrial dysfunction. Previous studies demonstrate that innate immune cells are important participants in inflammatory and fibrotic processes in SSc. The aim of the study was to evaluate the number of mtDNA copies in CD14+ monocytes and whole blood of patients with SSc in comparison with healthy individuals.Methods: Absolute mtDNA copy number was measured using digital PCR. It was found that the number of mtDNA copies in CD14+ monocytes was significantly higher in patients with SSc compared to control, while the number of mtDNA copies in the whole blood did not have significant differences.Results: The correlation analysis revealed an inverse association of mtDNA copy number with disease duration and the relationship between pro-inflammatory activation of CD14+ monocytes in terms of LPS-stimulated IL-6 secretion and mtDNA copy number. At the same time, basal and LPS-stimulated secretion of IL-6 by cultured CD+ monocytes were significantly higher in SSc group in comparison with control.Discussion: The study results suggest that increase of mtDNA copy number in CD14+ monocytes is a possible mechanism to maintain the reduced function of defective mitochondria in monocytes from patients with SSc associated with the development and progression of SSc.

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Precise and simultaneous quantification of mitochondrial DNA heteroplasmy and copy number by digital PCR

Cited by 11 publications

References 33 publications

A fatty acid elongase complex regulates cell membrane integrity and septin‐dependent host infection by the rice blast fungus

A fatty acid elongase complex regulates cell membrane integrity and septin‐dependent host infection by the rice blast fungus

Efficient elimination of MELAS-associated m.3243G mutant mitochondrial DNA by an engineered mitoARCUS nuclease

Mitochondrial DNA copy number in patients with systemic sclerosis

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