Circular Mitochondrial DNA Research Articles

Endemic Radiation of African Moonfish, Selene dorsalis (Gill 1863), in the Eastern Atlantic: Mitogenomic Characterization and Phylogenetic Implications of Carangids (Teleostei: Carangiformes).

This study offers an in-depth analysis of the mitochondrial genome of Selene dorsalis (Gill 1863), a species native to the Eastern Atlantic Ocean. The circular mitochondrial DNA molecule measures 16,541 base pairs and comprises 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, two ribosomal RNA genes, and a control region (CR). The nucleotide composition exhibits a notable adenine-thymine (AT) bias, accounting for 53.13%, which aligns with other species in the Carangidae family. Most PCGs initiate with the ATG codon, with the exception of Cytochrome C oxidase subunit I, which starts with GTG. Analysis of relative synonymous codon usage reveals that leucine and serine are the most prevalent amino acids in the mitochondrial genome of S. dorsalis and its congeners (S. vomer and S. setapinnis). All tRNAs display the typical cloverleaf structure, though tRNA Serine (S1) lacks a dihydrouracil arm. Pairwise comparisons of synonymous and nonsynonymous substitutions for all PCGs yielded values below '1', indicating strong purifying selection. The CR spans 847 bp, representing 5.12% of the mitochondrial genome, and is characterized by high AT content (62.81%). It is situated between tRNA-Pro (TGG) and tRNA-Phe (GAA). The CR contains conserved sequence blocks, with CSB-1 being the longest at 22 bp and CSB-D the shortest at 18 bp. Phylogenetic analysis, using Bayesian and Maximum-likelihood trees constructed from concatenated PCGs across 72 species, successfully differentiates S. dorsalis from other carangids. This study also explores how ocean currents and gyres might influence lineage diversification and parapatric speciation of Selene species between the Atlantic and Pacific Oceans. These results highlight the importance of the mitochondrial genome in elucidating the structural organization and evolutionary dynamics of S. dorsalis and its relatives within marine ecosystems.

Inhibiting Mitochondrial RNA Degradosome Complex SUV3 and PNPT1 Increases dsRNA in the Cytoplasm, Triggers Viral Mimicry Response and Sensitizes AML Cells to Immune Mediated Killing

Eukaryotic cells have two separate genomes; nuclear DNA organized in chromosomes, and circular mitochondrial DNA located within mitochondria. Mitochondrial DNA is comprised of a double-stranded circular genome that is 16.6 kB in length, lacks introns, and encodes two rRNAs, 22 t-RNAs and 13 of the 90 proteins in the mitochondrial respiratory chain. To maintain homeostasis, mitochondria possess RNA degradation machinery to regulate mitochondrial RNA turnover. The ATP-dependent helicase, SUV3 (encoded by the gene SUPV3L1) and the exonuclease PNPase (encoded by the gene PNPT1) function in a complex to degrade mitochondrial dsRNA. We identified PNPT1 and SUPV3L1 through an unbiased in-silico screen using bioinformatics platform to identify novel targets in AML. Our analysis revealed a strong correlation between these genes and ontologies of exogenous dsRNA, response to viruses, and RNA catabolic processes. PNPase and SUV3 protein were increased in 7/7 AML patient samples and 10/10 of AML cell lines compared to the normal hematopoietic cells by immunoblotting. Analysis of the TARGET AML dataset revealed that AML patients with increased expression of SUPV3L1 (p = 0.051, p= 0.045) and PNPT1 (p = 0.0013, p = 0.018) had decreased overall survival and event-free survival respectively. We knocked down or knocked out PNPT1 and SUPV3L1 with shRNA or sgRNA in AML cells, to study the importance of these genes in AML. Genetic knockdown or knockout of PNPT1 or SUPV3L1 decreased the growth and viability of OCI-AML2, TEX, K562, U937, NB4 and 8227 leukemia cells. Moreover, SUPV3L1 & PNPT1 ranked top 5.2% and 7.4% of essential genes in 26 leukemia cell lines in CRISPR screens and 2.7% and 4.9% in RNAi screens (https://depmap.org/portal). Knockdown of PNPT1 & SUPV3L1 also reduced the clonogenic growth of AML cells. Demonstrating the functional importance of PNPT1 & SUPV3L1 on leukemia-initiating cells in vivo, genetic knockdown of PNPT1 and SUPV3L1 significantly reduced engraftment of TEX cells into the marrow of immune-deficient mice. Finally, primary AML cells with SUPV3L1 knockdown had reduced engraftment into the marrow of immune-deficient mice. Whereas, the knockdown of SUPV3L did not alter the primary engraftment of CD34+ enriched cord blood cells. Mechanistically, knockdown of PNPT1 and SUPV3L1 in OCI-AML2 cells increased levels of cytoplasmic dsRNA 3-4 fold compared to control. Knockdown of PNPT1 and SUPV3L1 also increased cytoplasmic dsRNA in 143B cells, but not Rho(0) 143B cells that lack mitochondrial DNA, demonstrating a mitochondrial source for the increased dsRNA. Increased cytoplasmic dsRNA can mimic viral infection and trigger a type 1 Interferon response. Knockdown of PNPT1 or SUPV3L1 increased expression of genes (INFgR1, ICAM, IRF7 & JAK/STAT) associated with a type 1 interferon response compared to control. We also observed that PNPT1 and SUPV3L1 knockdown in AML cells sensitized the leukemic cells to T-cell mediated killing upon co-culturing with double negative T cells. In addition, using an immunocompetent Balbc mouse model, we demonstrated endogenous T cells were required to observe the greatest reduction in tumor burden after SUPV3L1 knockdown. In summary, the mitochondrial RNA degradosome complex SUPV3L1 and PNPT1 are overexpressed in AML and are essential for AML cells and stem/progenitors. These enzymes regulate the levels of mitochondrial dsRNA and their inhibition leads to a viral mimicry response and heightened sensitivity to immune-mediated killing.

Abstract 4833: Inhibiting the mitochondrial RNA degradosome complex SUV3 and PNPase increases dsRNA in the cytoplasm, triggers a viral mimicry response and kills AML cells and progenitors

Abstract Eukaryotic cells have two separate genomes; nuclear chromosomal DNA and circular mitochondrial DNA. Mitochondrial DNA lacks introns, and encodes 2 rRNAs, 22 t-RNAs and 13 of the 90 proteins that constitute the mitochondrial respiratory chain. To maintain homeostasis, mitochondrial RNA degradation machinery regulates RNA turnover. ATP-dependent helicase, SUV3 (gene SUPV3L1), and exonuclease PNPase (gene PNPT1) function as a complex to degrade mitochondrial dsRNA. By immunoblotting, PNPase and SUV3 proteins were increased in 7/7 AML patient samples and 13/13 of AML cell lines, compared to the normal hematopoietic cells. Analysis of the TARGET AML dataset revealed AML patients with increased expression of SUPV3L1 (p = 0.051, p= 0.045) and PNPT1 (p = 0.0013, p = 0.018) had decreased overall and event free survival. Genetic knockdown or knockout using shRNA or sgRNA against PNPT1 or SUPV3L1 decreased growth and viability of OCI-AML2, TEX, K562, U937, NB4 and OCI-AML 8227 cells. Furthermore, SUPV3L1 & PNPT1 ranked in the top 5.2% and 7.4% of essential genes in 26 leukemia cell lines in CRISPR screens and 2.7% and 4.9% in RNAi screens (depmap.org). Knockdown of PNPT1 & SUPV3L1 also reduced the clonogenic growth of OCI-AML2, TEX and U937 cells and significantly reduced engraftment of TEX cells into the marrow of immune deficient mice, demonstrating the functional importance on leukemia initiating cells in vivo. SUPV3L1 knockdown in primary AML cells reduced engraftment in marrow of immune deficient mice. Bioinformatics analysis to detect processes associated with PNPT1 and SUPV3L1, we identified associations with Response to exogenous dsRNA, Response to virus, and RNA catabolic process ontologies. Consistent with this, we observed knockdown of PNPT1 or SUPV3L1 increased expression of genes (INFgR1, ICAM, IRF7 & JAK/STAT) suggesting an interferon response. As PNPT1 and SUPV3L1 degrade mitochondrial dsRNA, we measured levels of dsRNA after knockdown of these genes. Knockdown of PNPT1 and SUPV3L1 in OCI-AML2 cells increased levels of cytoplasmic/mitochondrial dsRNA 3-4 fold compared to control. Knockdown of PNPT1 and SUPV3L1 also increased dsRNA in 143B cells, but not Rho (0) 143B cells that lack mitochondrial DNA. Upregulation of inflammatory genes leads to viral mimicry and can increase sensitivity to immune mediated killing. We observed enhanced sensitivity to Double Negative T (DNT) cells mediated killing in PNPT1 and SUPV3L1 knockdown OCI-AML2 cells compared to control cells. In summary, RNA degradosome complex proteins SUPV3L1 and PNPT1 are overexpressed in AML, and are essential for the survival of AML cells and AML stem/progenitors. These enzymes regulate levels of mitochondrial dsRNA, and their inhibition leads to increased cytoplasmic dsRNA triggering a viral mimicry response and enhanced sensitivity to immune-mediated killing. Citation Format: Geethu Emily Thomas, Kazem Nouri, Jong Bok Lee, Rose Hurren, Yongran Yan, Neil MacLean, Yulia Jitkova, Li Ma, Xiao Ming Wang, Chaitra Sarathy, Andrea Arruda, Mark D. Minden, Li Zhang, Vito Spadavecchio, Aaron Schimmer. Inhibiting the mitochondrial RNA degradosome complex SUV3 and PNPase increases dsRNA in the cytoplasm, triggers a viral mimicry response and kills AML cells and progenitors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4833.

The complete mitochondrial genome of Cuspidaria undata (Bivalvia, Anomalodesmata, Cuspidariidae)

The mitochondrial genome of Cuspidaria undata (Verrill, 1884) was sequenced in full using Illumina HiSeq 2500. The circular mitochondrial DNA (mtDNA) was 16,266 bp in size, encoded 37 genes, and contained 13 protein-coding genes (PCGs), 2 rRNAs and 22 tRNAs. The gene order of the 13 PCGs in this species exhibited extensive rearrangement and differences in comparison to other Cuspidariidae, indicating that gene order is not conserved within this family. Phylogenetic analysis based on 13 PCGs and 2 rRNAs recovered a monophyletic Cuspidariidae.

Mitochondrial Replication Assay (MIRA) for Efficient in situ Quantification of Nascent mtDNA and Protein Interactions with Nascent mtDNA (mitoSIRF).

Mitochondria play decisive roles in bioenergetics and intracellular communication. These organelles contain a circular mitochondrial DNA (mtDNA) genome that is duplicated within one to two hours by a mitochondrial replisome, independently from the nuclear replisome. mtDNA stability is regulated in part at the level of mtDNA replication. Consequently, mutations in mitochondrial replisome components result in mtDNA instability and are associated with diverse disease phenotypes, including premature aging, aberrant cellular energetics, and developmental defects. The mechanisms ensuring mtDNA replication stability are not completely understood. Thus, there remains a need to develop tools to specifically and quantifiably examine mtDNA replication. To date, methods for labeling mtDNA have relied on prolonged exposures of 5'-bromo-2'-deoxyuridine (BrdU) or 5'-ethynyl-2'-deoxyuridine (EdU). However, labeling with these nucleoside analogs for a sufficiently short time in order to monitor nascent mtDNA replication, such as under two hours, does not produce signals suited for efficient or accurate quantitative analysis. The assay system described here, termed Mitochondrial Replication Assay (MIRA), utilizes proximity ligation assay (PLA) combined with EdU-coupled Click-IT chemistry to address this limitation, thereby enabling sensitive and quantitative analysis of nascent in situ mtDNA replication with single-cell resolution. This method can be further paired with conventional immunofluorescence (IF) for multi-parameter cell analysis. By enabling monitoring nascent mtDNA prior to the complete replication of the entire mtDNA genome, this new assay system allowed the discovery of a new mitochondrial stability pathway, mtDNA fork protection. Moreover, a modification in primary antibodies application allows the adaptation of our previously described in situ protein Interactions with nascent DNA Replication Forks (SIRF) for the detection of proteins of interest to nascent mtDNA replication forks on a single molecule level (mitoSIRF). Graphical overview Schematic overview of Mitochondrial Replication Assay (MIRA). 5'-ethynyl-2'-deoxyuridine (EdU; green) incorporated in DNA is tagged with biotin (blue) using Click-IT chemistry. Subsequent proximity ligation assay (PLA, pink circles) using antibodies against biotin allows the fluorescent tagging of the nascent EdU and amplification of the signal sufficient for visualization by standard immunofluorescence. PLA signals outside the nucleus denote mitochondrial DNA (mtDNA) signals. Ab, antibody. In in situ protein interactions with nascent DNA replication forks (mitoSIRF), one of the primary antibodies is directed against a protein of interest, while the other detects nascent biotinylated EdU, thus enabling in situ protein interactions with nascent mtDNA.

Feed Your Mitochondria and Shape Your Body!

If your body were a train, what would give it the power to move? The food you eat, which is burned by the train’s engine. Do you know how many engines your body has? Thousands of millions! They are called mitochondria. Mitochondria are tiny structures contained within the cells of your body that burn food, providing energy. The power of each mitochondrion is due to its own gearwheel: the circular mitochondrial DNA. There are many gears in each mitochondrion. Together, they regulate the mitochondrion’s ability to produce energy. Obesity modifies both the abundance and the function of the mitochondrial DNA, altering the ability of the body to efficiently produce energy, with some differences between males and females. Eating healthy foods not only makes you slim, but also boosts your mitochondria and makes you powerful!

Inhibiting the Mitochondrial RNA Degradosome Complex SUV3 and Pnpase Increases dsRNA in the Cytoplasm, Triggers a Viral Mimicry Response and Kills AML Cells and Progenitors

Eukaryotic cells have two separate genomes; nuclear DNA organized in chromosomes, and the circular mitochondrial DNA located within mitochondria. Mitochondrial DNA is comprised of a double-stranded circular genome that is 16.6 kB in length, lacks introns, and encodes two rRNAs, 22 t-RNAs and 13 of the 90 proteins in the mitochondrial respiratory chain. To maintain homeostasis, mitochondria possess RNA degradation machinery to regulate mitochondrial RNA turnover. The ATP-dependent helicase, SUV3 (encoded by the gene SUPV3L1), and the exonuclease PNPase (encoded by the gene PNPT1) function in complex to degrade mitochondrial dsRNA. We identified PNPT1 and SUPV3L1 through an unbiased in silico screen using the ITX machine learning platform to identify novel targets in AML. Further simulations revealed inhibition of these genes could lead to significant enrichment of Response to exogenous dsRNA, Response to virus, and RNA catabolic process ontologies. By immunoblotting PNPase and SUV3 protein were increased in 7/7 AML patient samples and 13/13 of AML cell lines compared to the normal hematopoietic cells. Analysis of the TARGET AML dataset revealed that AML patients with increased expression of SUPV3L1 (p = 0.051, p= 0.045) and PNPT1 (p = 0.0013, p = 0.018) had decreased overall survival and event free survival respectively. To study the importance of PNPT1 and SUPV3L1 in AML, we knocked down or knocked out these genes with shRNA or sgRNA in AML cells. Genetic knockdown or knock out of PNPT1 or SUPV3L1 decreased growth and viability of OCI-AML2, TEX, K562, U937, NB4 and OCI-AML-8227 leukemia cells. Moreover, SUPV3L1 & PNPT1 ranked top 5.2% and 7.4% of essential genes in 26 leukemia cell lines in CRISPR screens and 2.7% and 4.9% in RNAi screens (https://depmap.org/portal). Knockdown of PNPT1 & SUPV3L1 also reduced the clonogenic growth of OCI-AML2, TEX and U937 cells. Demonstrating the functional importance of PNPT1 & SUPV3L1 on leukemia initiating cells in vivo, genetic knockdown of PNPT1 and SUPV3L1 significantly reduced engraftment of TEX cells into the marrow of immune deficient mice. Finally, primary AML cells with SUPV3L1 knockdown had reduced engraftment into the marrow of immune deficient mice. Mechanistically, knockdown of PNPT1 and SUPV3L1 in AML2 cells increased levels of cytoplasmic dsRNA 3-4 fold compared to control. Knockdown of PNPT1 and SUPV3L1 also increased cytoplasmic dsRNA in 143B cells, but not Rho(0) 143B cells that lack mitochondrial DNA. Increased cytoplasmic dsRNA can mimic viral infection and trigger a type 1 Interferon response. Knockdown of PNPT1 or SUPV3L1 increased expression of genes (INFgR1, ICAM, IRF7 & JAK/STAT) associated with a type 1 interferon response compared to control. In summary, the RNA degradosome complex proteins SUPV3L1 and PNPT1 are overexpressed in AML and are essential for AML cells and stem/progenitors. These enzymes regulate the levels of mitochondrial dsRNA and their inhibition leads to increased cytoplasmic dsRNA and triggers a viral mimicry response.

Targeted removal of mitochondrial DNA from mouse and human extrachromosomal circular DNA with CRISPR-Cas9

Extrachromosomal circular DNA (eccDNA) of chromosomal origin is common in eukaryotic cells. Amplification of oncogenes on large eccDNA (ecDNA) can drive biological processes such as tumorigenesis, and identification of eccDNA by sequencing after removal of chromosomal DNA is therefore important for understanding their impact on the expressed phenotype. However, the circular mitochondrial DNA (mtDNA) might challenge the detection of eccDNA because the average somatic cell has hundreds of copies of mtDNA. Here we show that 61.2–99.5% of reads from eccDNA-enriched samples correspond to mtDNA in mouse tissues. We have developed a method to selectively remove mtDNA from total circular DNA by CRISPR/Cas9 guided cleavage of mtDNA with one single-guide RNA (sgRNA) or two sgRNAs followed by exonuclease degradation of the linearized mtDNA. Sequencing revealed that mtDNA reads were 85.9% ± 12.6% removed from eccDNA of 9 investigated mouse tissues. CRISPR/Cas9 cleavage also efficiently removed mtDNA from a human HeLa cell line and colorectal cancer samples. We identified up to 14 times more, and also larger eccDNA in CRISPR/Cas9 treated colorectal cancer samples than in untreated samples. We foresee that the method can be applied to effectively remove mtDNA from any eukaryotic species to obtain higher eccDNA yields.

Mitochondrial augmentation of CD34+ cells from healthy donors and patients with mitochondrial DNA disorders confers functional benefit

Mitochondria are cellular organelles critical for numerous cellular processes and harboring their own circular mitochondrial DNA (mtDNA). Most mtDNA associated disorders (either deletions, mutations, or depletion) lead to multisystemic disease, often severe at a young age, with no disease-modifying therapies. Mitochondria have a capacity to enter eukaryotic cells and to be transported between cells. We describe a method of ex vivo augmentation of hematopoietic stem and progenitor cells (HSPCs) with normal exogenous mitochondria, termed mitochondrial augmentation therapy (MAT). Here, we show that MAT is feasible and dose dependent, and improves mitochondrial content and oxygen consumption of healthy and diseased HSPCs. Ex vivo mitochondrial augmentation of HSPCs from a patient with a mtDNA disorder leads to superior human engraftment in a non-conditioned NSGS mouse model. Using a syngeneic mouse model of accumulating mitochondrial dysfunction (Polg), we show durable engraftment in non-conditioned animals, with in vivo transfer of mitochondria to recipient hematopoietic cells. Taken together, this study supports MAT as a potential disease-modifying therapy for mtDNA disorders.

Genome sequencing of the neotype strain CBS 554.65 reveals the MAT1\u20132 locus of Aspergillus niger

BackgroundAspergillus niger is a ubiquitous filamentous fungus widely employed as a cell factory thanks to its abilities to produce a wide range of organic acids and enzymes. Its genome was one of the first Aspergillus genomes to be sequenced in 2007, due to its economic importance and its role as model organism to study fungal fermentation. Nowadays, the genome sequences of more than 20 A. niger strains are available. These, however, do not include the neotype strain CBS 554.65.ResultsThe genome of CBS 554.65 was sequenced with PacBio. A high-quality nuclear genome sequence consisting of 17 contigs with a N50 value of 4.07 Mbp was obtained. The assembly covered all the 8 centromeric regions of the chromosomes. In addition, a complete circular mitochondrial DNA assembly was obtained. Bioinformatic analyses revealed the presence of a MAT1-2-1 gene in this genome, contrary to the most commonly used A. niger strains, such as ATCC 1015 and CBS 513.88, which contain a MAT1-1-1 gene. A nucleotide alignment showed a different orientation of the MAT1–1 locus of ATCC 1015 compared to the MAT1–2 locus of CBS 554.65, relative to conserved genes flanking the MAT locus. Within 24 newly sequenced isolates of A. niger half of them had a MAT1–1 locus and the other half a MAT1–2 locus. The genomic organization of the MAT1–2 locus in CBS 554.65 is similar to other Aspergillus species. In contrast, the region comprising the MAT1–1 locus is flipped in all sequenced strains of A. niger.ConclusionsThis study, besides providing a high-quality genome sequence of an important A. niger strain, suggests the occurrence of genetic flipping or switching events at the MAT1–1 locus of A. niger. These results provide new insights in the mating system of A. niger and could contribute to the investigation and potential discovery of sexuality in this species long thought to be asexual.

The complete mitochondrial genome of Populus rotundifolia var. duclouxiana

The complete sequence of the circular mitochondrial (mt) DNA of Populus rotundifolia var. duclouxiana is presented in this study. The size of the P. rotundifolia var. duclouxiana mt genome was 775,549 bp and the GC content of the genome was 44.8%. The P. rotundifolia var. duclouxiana mt genome encoded 58 genes, including 33 protein-coding genes, 22 tRNA genes, and three rRNA genes. Among the 58 genes, three genes contained one intron, one gene contained three introns, and four genes contained four introns. Phylogenetic analysis using seven complete mt genomes of Salicaceae supported that P. rotundifolia var. duclouxiana is most closely related to P. davidiana.

The complete mitochondrial genome and phylogenetic analysis of Neorhodomela munita

Neorhodomela munita (Perestenko) Masuda 1982 is distributed in the coastal areas of Shandong and Liaoning in China, and also in Japan. In this study, the complete nucleotide sequence of the circular mitochondrial DNA of the red alga Neorhodomela munita has been determined. The complete mitochondrial DNA sequence of Neorhodomela munita was 25,318 bp in length with an overall GC content of 25.1% and encoded 23 protein-coding genes, two ribosomal RNAs and 24 transfer RNAs. Phylogenetic tree showed that Neorhodomela munita clustered together with Choreocolax polysiphoniae. The phylogenetic analysis may provide a better understanding of the evolution of the Rhodophyta species.

Genome analysis of Candida subhashii reveals its hybrid nature and dual mitochondrial genome conformations.

Candida subhashii belongs to the CUG-Ser clade, a group of phylogenetically closely related yeast species that includes some human opportunistic pathogens, such as Candida albicans. Despite being present in the environment, C. subhashii was initially described as the causative agent of a case of peritonitis. Considering the relevance of whole-genome sequencing and analysis for our understanding of genome evolution and pathogenicity, we sequenced, assembled and annotated the genome of C. subhashii type strain. Our results show that C. subhashii presents a highly heterozygous genome and other signatures that point to a hybrid ancestry. The presence of functional pathways for assimilation of hydroxyaromatic compounds goes in line with the affiliation of this yeast with soil microbial communities involved in lignin decomposition. Furthermore, we observed that different clones of this strain may present circular or linear mitochondrial DNA. Re-sequencing and comparison of strains with differential mitochondrial genome topology revealed five candidate genes potentially associated with this conformational change: MSK1, SSZ1, ALG5, MRPL9 and OYE32.

Determination of Oxidative Phosphorylation Complexes Activities.

Mitochondria possess a genome that codes for proteins, in the same fashion as the nuclear genome. However, the small, circular mitochondrial DNA (mtDNA) molecule has a reduced base pair content, for it can only code for 2 rRNA, 22 tRNA molecules, and 13 proteins, all of them part of the mitochondrial respiratory chain. As such, all of the other mitochondrial components derive from nuclear genome. This separation leads to a requirement for a well-tuned coordination between both genomes, in order to produce fully functional mitochondria. A vast number of pathologies have been demonstrated to involve, to some extent, alterations in mitochondrial function that, no doubt, can be caused by alterations to the respiratory chain activity. As such, several methods and techniques have been developed to assess both content and function of mitochondrial proteins, in order to help understand mitochondrial involvement on the pathogenesis of disease. In this chapter, we will address some of these methods, with the main focus being on isolated mitochondria.

Purification of Functional Platelet Mitochondria Using a Discontinuous Percoll Gradient.

The isolation of mitochondria is gaining importance in experimental and clinical laboratory settings. Of interest, mitochondria and mitochondrial components (i.e., circular mitochondrial DNA, N-formylated peptides, cardiolipin) have been involved in several human inflammatory pathologies, such as cancer, Alzheimer's disease, Parkinson's disease, and rheumatoid arthritis. While several mitochondrial isolation methods have been previously published, these techniques are aimed at yielding mitochondria from cell types other than platelets. In addition, little information is known on the number of platelet-derived microvesicles that can contaminate the mitochondrial preparation or even the overall quality as well as functional and structural integrity of mitochondria. Here we describe a purification method, using a discontinuous Percoll gradient, yielding mitochondria of high purity and integrity from human platelets.

Mitogenome-wide comparison and phylogeny reveal group I intron dynamics and intraspecific diversification within the phytopathogen Corynespora cassiicola

Corynespora cassiicola, the causal agent of an extensive range of plant diseases worldwide, is a momentous fungus with diverse lifestyles and rich in intraspecies variations. In the present study, a total of 56 mitochondrial genomes of C. cassiicola were assembled (except two available online) and analyzed, of which 16 mitogenomes were newly sequenced here. All these circular mitochondrial DNA (mtDNA) molecules, ranging from 39,223 bp to 45,786 bp in length, comprised the same set of 13 core protein-coding genes (PCGs), two rRNAs and 27 tRNAs arranged in identical order. Across the above conserved genes, nad3 had the largest genetic distance between different isolates and was possibly subjected to positive selection pressure. Comparative mitogenomic analysis indicated that seven group I (IB, IC1, and IC2) introns with a length range of 1013–1876 bp were differentially inserted in three core PCGs (cox1, nad1, and nad5), resulting in the varied mitogenome sizes among C. cassiicola isolates. In combination with dynamic distribution of the introns, a well-supported mitogenome-wide phylogeny of the 56 C. cassiicola isolates revealed eight phylogenetic groups, which only had weak correlations with host range and toxin class. Different groups of isolates exhibited obvious differences in length and GC content of some genes, while a degree of variance in codon usage and tRNA structure was also observed. This research served as the first report on mitogenomic comparisons within C. cassiicola, and could provide new insights into its intraspecific microevolution and genetic diversity.

Mitochondrial DNA copy number in human disease: the more the better?

Most of the genetic information has been lost or transferred to the nucleus during the evolution of mitochondria. Nevertheless, mitochondria have retained their own genome that is essential for oxidative phosphorylation (OXPHOS). In mammals, a gene‐dense circular mitochondrial DNA (mtDNA) of about 16.5 kb encodes 13 proteins, which constitute only 1% of the mitochondrial proteome. Mammalian mtDNA is present in thousands of copies per cell and mutations often affect only a fraction of them. Most pathogenic human mtDNA mutations are recessive and only cause OXPHOS defects if present above a certain critical threshold. However, emerging evidence strongly suggests that the proportion of mutated mtDNA copies is not the only determinant of disease but that also the absolute copy number matters. In this review, we critically discuss current knowledge of the role of mtDNA copy number regulation in various types of human diseases, including mitochondrial disorders, neurodegenerative disorders and cancer, and during ageing. We also provide an overview of new exciting therapeutic strategies to directly manipulate mtDNA to restore OXPHOS in mitochondrial diseases.

A new method for long-read sequencing of animal mitochondrial genomes: application to the identification of equine mitochondrial DNA variants

BackgroundMitochondrial DNA is remarkably polymorphic. This is why animal geneticists survey mitochondrial genomes variations for fundamental and applied purposes. We present here an approach to sequence whole mitochondrial genomes using nanopore long-read sequencing. Our method relies on the selective elimination of nuclear DNA using an exonuclease treatment and on the amplification of circular mitochondrial DNA using a multiple displacement amplification step.ResultsWe optimized each preparative step to obtain a 100 million-fold enrichment of horse mitochondrial DNA relative to nuclear DNA. We sequenced these amplified mitochondrial DNA using nanopore sequencing technology and obtained mitochondrial DNA reads that represented up to half of the sequencing output. The sequence reads were 2.3 kb of mean length and provided an even coverage of the mitochondrial genome. Long-reads spanning half or more of the whole mtDNA provided a coverage that varied between 118X and 488X. We evaluated SNPs identified using these long-reads by Sanger sequencing as ground truth and found a precision of 100.0%; a recall of 93.1% and a F1-score of 0.964 using the Twilight horse mtDNA reference. The choice of the mtDNA reference impacted variant calling efficiency with F1-scores varying between 0.947 and 0.964.ConclusionsOur method to amplify mtDNA and to sequence it using the nanopore technology is usable for mitochondrial DNA variant analysis. With minor modifications, this approach could easily be applied to other large circular DNA molecules.

The complete mitochondrial genome of chlorella sp. Dachan (Chlorellaceae: Chlorella)

The Chlorella sp. Dachan belongs to the genus Chlorella. Chlorella sp is widely distributed in the fresh water area with the largest number of species. It is easy to cultivate and propagate and it can use photosynthesis with high application value. In this study, the complete nucleotide sequence of the circular mitochondrial (mt) DNA of the chlorophyte alga C. sp. Dachan has been determined (55,450 base-pairs, A + T content 71.69%). The genes identified encode three subunits of the cytochome oxidase, apocytochrome b, seven subunits of the NADH dehydrogenase complex (nad1 3 4 5 7 9 and 4 L), three ATPase subunits (atp1, atp6, atp9), two ribosomal RNAs small subunit (srn) and large subunit (lrn) RNA), 27 tRNAs, 2 ribosome proteins. The complete mitochondrial genome sequence will provide new molecular biology information to further understand the genetic diversity of the Chlorella sp. Dachan and to protect this population.

Mitonuclear genomics and aging.

Our cells operate based on two distinct genomes that are enclosed in the nucleus and mitochondria. The mitochondrial genome presumably originates from endosymbiotic bacteria. With time, a large portion of the original genes in the bacterial genome is considered to have been lost or transferred to the nuclear genome, leaving a reduced 16.5Kb circular mitochondrial DNA (mtDNA). Traditionally only 37 genes, including 13 proteins, were thought to be encoded within mtDNA, its genetic repertoire is expanding with the identification of mitochondrial-derived peptides (MDPs). The biology of aging has been largely unveiled to be regulated by genes that are encoded in the nuclear genome, whereas the mitochondrial genome remained more cryptic. However, recent studies position mitochondria and mtDNA as an important counterpart to the nuclear genome, whereby the two organelles constantly regulate each other. Thus, the genomic network that regulates lifespan and/or healthspan is likely constituted by two unique, yet co-evolved, genomes. Here, we will discuss aspects of mitochondrial biology, especially mitochondrial communication that may add substantial momentum to aging research by accounting for both mitonuclear genomes to more comprehensively and inclusively map the genetic and molecular networks that govern aging and age-related diseases.