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Sequence and gene organization of mouse mitochondrial DNA
Abstract
The complete sequence of the 16,295 bp mouse L cell mitochondrial DNA genome has been determined. Genes for the 12S and 16S ribosomal RNAs; 22 tRNAs; cytochrome c oxidase subunits I, II and III; ATPase subunit 6; cytochrome b; and eight unidentified proteins have been located. The genome displays exceptional economy of organization, with tRNA genes interspersed between rRNA and protein-coding genes with zero or few noncoding nucleotides between coding sequences. Only two significant portions of the genome, the 879 nucleotide displacement-loop region containing the origin of heavy-strand replication and the 32 nucleotide origin of light-strand replication, do not encode a functional RNA species. All of the remaining nucleotide sequence serves as a defined coding function, with the exception of 32 nucleotides, of which 18 occur at the 5' ends of open reading frames. Mouse mitochondrial DNA is unique in that the translational start codon is AUN, with any of the four nucleotides in the third position, whereas the only translational stop codon is the orthodox UAA. The mouse mitochondrial DNA genome is highly homologous in overall sequence and in gene organization to human mitochondrial DNA, with the descending order of conserved regions being tRNA genes; origin of light-strand replication; rRNA genes; known protein-coding genes; unidentified protein-coding genes; displacement-loop region.
Supplementary resources (11)
Nucleotide Sequence
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... Universal primers designed by Irwin et al. (1991: L14724/H15915) were used to redesign primers specific to Phenacomys (numbers corresponding to Mus 14348-15202, Bibb et al. 1981): RTV-F: 59-CGAGACGTAAAYTATGGCTGA-39; RTV-R 59-GAGGCT-ACTTGTCCAATTATGA-39. PCR amplification was checked by visualization with ethidium bromide on 1% agarose gels. ...
... We then calibrated our molecular clock using the Mus-Rattus split of 14 million years ago (mya) to estimate time since coalescence within Phenacomys (Jacobs and Pilbeam 1980). Cytb sequences of Mus musculus and Rattus norvegicus used for molecular clock were obtained from Genbank (NC001569- Bibb et al. 1981;NC00165-Gadaleta et al. 1989, respectively). Due to computational time constraints using TrNþGþclock, the longicaudus portion of the Phenacomys data set was pruned to include only 5 of the most common haplotypes. ...
Taxonomic relationships among red tree voles (Phenacomys longicaudus longicaudus, P. l. silvicola), the Sonoma tree vole (P. pomo), the white-footed vole (P. albipes), and the heather vole (P. intermedius) were examined using 664 base pairs of the mitochondrial cytochrome b gene. Results indicate specific differences among red tree voles, Sonoma tree voles, white-footed voles, and heather voles, but no clear difference between the 2 Oregon subspecies of red tree voles (P. l. longicaudus and P. l. silvicola). Our data further indicated a close relationship between tree voles and albipes, validating inclusion of albipes in the subgenus Arborimus. These 3 congeners shared a closer relationship to P. intermedius than to other arvicolids. A moderate association between pomo and albipes was indicated by maximum parsimony and neighbor-joining phylogenetic analyses. Molecular clock estimates suggest a Pleistocene radiation of the Arborimus clade, which is concordant with pulses of diversification observed in other murid rodents. The generic rank of Arborimus is subject to interpretation of data.
... We also included the neuroblastoma cell line N2a as a representative mouse cancer cell line. Consistent with previous reports in mice [23,36,37] and also with the human data described above, we could observe strong initiation signals on the H strand represented by two clusters of peaks corresponding to the OH and Ori-b regions in the NCR (Figures 5, S3 and S4), and For each sample, the copy number was measured by two qPCR assays, each using one indicated primer pair designed against the mitochondrial genome and one against the nuclear genome (Supplementary Table S1, Section 4). Error bars indicate SD of 9 measurements (shown by dots): 3 technical replicates for each of the 3 biological replicates. ...
... We also included the neuroblastoma cell line N2a as a representative mouse cancer cell line. Consistent with previous reports in mice [23,36,37] and also with the human data described above, we could observe strong initiation signals on the H strand represented by two clusters of peaks corresponding to the OH and Ori-b regions in the NCR (Figures 5, S3 and S4), and on the L strand in the OL annotated region (Figures 5 and S3) in all mouse samples. Since, to our knowledge, the mouse replication initiation sites have not been as comprehensively studied as the human ones, we could not compare our mouse results with the previously published data at a fine level as we have done for the human origins. ...
The proper replication of mitochondrial DNA is key to the maintenance of this crucial organelle. Multiple studies aimed at understanding the mechanisms of replication of the mitochondrial genome have been conducted in the past several decades; however, while highly informative, they were conducted using relatively low-sensitivity techniques. Here, we established a high-throughput approach based on next-generation sequencing to identify replication start sites with nucleotide-level resolution and applied it to the genome of mitochondria from different human and mouse cell types. We found complex and highly reproducible patterns of mitochondrial initiation sites, both previously annotated and newly discovered in this work, that showed differences among different cell types and species. These results suggest that the patterns of the replication initiation sites are dynamic and might reflect, in some yet unknown ways, the complexities of mitochondrial and cellular physiology. Overall, this work suggests that much remains unknown about the details of mitochondrial DNA replication in different biological states, and the method established here opens up a new avenue in the study of the replication of mitochondrial and potentially other genomes.
... Multiple identical copies of mitochondria are often found in each cell [14]. Figure 1 shows a schematic representation of some mitochondria genetic maps including human (a) [15], horse (b) [16], pig (c) [17], and mouse (d) [18]. In this review, we have described the full range of applications of mitochondrial Fig. 1 The schematic structure of the human (a) [15], horse (b) [19], pig (c) [17], and mouse (d) [18] ...
... Figure 1 shows a schematic representation of some mitochondria genetic maps including human (a) [15], horse (b) [16], pig (c) [17], and mouse (d) [18]. In this review, we have described the full range of applications of mitochondrial Fig. 1 The schematic structure of the human (a) [15], horse (b) [19], pig (c) [17], and mouse (d) [18] ...
... Multiple identical copies of mitochondria are often found in each cell [11]. Figure 1 shows a schematic representation of some mitochondria genetic maps including human (a) [6], horse (b) [103], pig (c) [135], and mouse (d) [13]. In this review, we have described the full range of applications of mitochondrial Fig. 1 The schematic structure of the human (a) [6], horse (b) [16], pig (c) [135], and mouse (d) [13] mitochondrial DNA genome genes for diagnostic techniques in the animal investigation, including medicine, biology, wildlife, and food industry. ...
... Figure 1 shows a schematic representation of some mitochondria genetic maps including human (a) [6], horse (b) [103], pig (c) [135], and mouse (d) [13]. In this review, we have described the full range of applications of mitochondrial Fig. 1 The schematic structure of the human (a) [6], horse (b) [16], pig (c) [135], and mouse (d) [13] mitochondrial DNA genome genes for diagnostic techniques in the animal investigation, including medicine, biology, wildlife, and food industry. ...
Species identification is one of the critical challenges, which researchers in the animal investigation are facing under various objective scopes, such as cell culture in medicine, the ancient remainder in archaeology, traditional medicine conservation, ecology, biodiversity studies, wildlife criminal science, and food industry. Recently, various strategies have been developed, but specifically, genome-based methods for the identification of species are preferred over protein-based experiments. The reasons include but are not limited to sample damaging and deficiency or closely related species. Although novel approaches, such as next-generation sequencing (NGS), barcoding, loop-mediated isothermal amplification (LAMP), and droplet digital PCR (ddPCR) have been developed, still traditional methods of RFLP, Nested PCR, Multiplex PCR, and Real-time PCR are in wide use. Most of the above-mentioned methods have utilized conserved mitochondrial genomic regions such as cytochrome c oxidase subunit I (COI), Ribosomal DNA (rDNA), D-Loop, NADH, and cytochrome b for species identification. It was accepted that species identification from a small amount of the target sample is still a challenge. Hence, this presentation proposes a diverse application of high mitochondrion gene copy number per cell as an effective way for species identification with a small amount of targeted gene. Furthermore, a relatively high mutation rate can be found in this method in comparison to nuclear DNA, which helps to determine closely related animal species. This review discusses a broad range of species identification applications by the mitochondrial gene-based strategies, which can provide an exciting perspective for future research in animal investigation.
... The human and mouse mtDNA were fully sequenced for the first time in 1981 (Anderson et al., 1981, Bibb et al., 1981. Based on the sequence, at that time, researchers were able to identify the genes for the 12S and 16S ribosomal RNAs, 22 ...
... However, 8 unidentified proteins have been detected, dubbed unidentified reading frames (URFs). These URFs were later identified as the 7 subunits of NADH dehydrogenase (CI) and the remaining subunit of ATP synthase (CV), ATPase subunit 8 (Bibb et al., 1981, Chomyn et al., 1985, Macreadie et al., 1983. The mammalian mtDNA sequence reveals extreme economy and does not contain introns. ...
Mitochondria are highly dynamic organelles found in most eukaryotic cells, with a fundamental role in the generation of cellular energy through oxidative phosphorylation (OXPHOS). Critical for their function, mitochondria have retained their own genome the mitochondrial DNA, mtDNA. In mammals, replication of mtDNA is ensured by the DNA polymerase POLγ, which is composed by one catalytic subunit POLγA and two accessory subunits POLγB. Mutations in the nuclear-encoded POLG gene, coding for POLγA, are a common cause of human disease leading to a spectrum of disorders characterised by mtDNA instability, thus compromising mitochondrial function. Despite being relatively frequent, the molecular pathogenesis of POLG-related diseases is poorly understood and efficient treatments are missing, partly due to the lack of relevant in vivo models. Here, I describe the generation of two mouse models: 1) the PolgA449T/A449T mouse, which reproduces the A467T change, the most common human recessive mutation of POLG and 2) the PolgWT/Y933C mouse, which reproduces the Y955C change, the most common human dominant mutation of POLG. I focused on the use of the PolgA449T/A449T mouse and complementary in vitro techniques to provide insights into the molecular pathogenic mechanism of this POLG mutation. I describe the data showing that the mouse A449T mutation impairs DNA binding and mtDNA synthesis activities of POLγ, leading to a stalling phenotype. Most importantly, the A449T mutation also strongly impairs interaction with POLγB, the accessory subunit of the POLγ holoenzyme. This allows the free POLγA to become a substrate for LONP1 protease degradation, leading to dramatically reduced levels of POLγA in A449T mouse tissues, with consequences for the pathogenesis of the disease. In the second part of the dissertation, I explore a gene therapy approach for mitochondrial diseases associated with mutations in nuclear-encoded genes. In particular, I test the use of a novel adeno-associated virus (AAV) capsid (PHP.B) as a gene therapy platform to ameliorate the neurological symptoms of a pre-clinical mouse model of mitochondrial disease, the Ndufs4 knockout (Ndufs4-/-) mouse. A single injection with AAV-PHP.B to express the human NDUFS4 in Ndufs4-/- mice, improved lifespan, body weight gain, motor coordination and several molecular and histological features of the brain. These data provide promising proof-of-concept for the use of AAV-mediated gene therapy as a therapeutic option for the number of patients with, currently incurable, mitochondrial disease.
... The sequencing of the human and mouse mitochondrial genomes ignited the field of organelle genomics 40 years ago [1,2]. These compact genomes might have given the initial impression that organelle chromosomes provide very little genetic fodder for the scientific community to analyze. ...
40 years ago, organelle genomes were assumed to be streamlined and, perhaps, unexciting remnants of their prokaryotic past. However, the field of organelle genomics has exposed an unparallel diversity in genome architecture (i.e. genome size, structure, and content). The transcription of these eccentric genomes can be just as elaborate – organelle genomes are pervasively transcribed into a plethora of RNA types. However, while organelle protein-coding genes are known to produce polycistronic transcripts that undergo heavy posttranscriptional processing, the nature of organelle noncoding transcriptomes is still poorly resolved. Here, we review how wet-lab experiments and second-generation sequencing data (i.e. short reads) have been useful to determine certain types of organelle RNAs, particularly noncoding RNAs. We then explain how third-generation (long-read) RNA-Seq data represent the new frontier in organelle transcriptomics. We show that public repositories (e.g. NCBI SRA) already contain enough data for inter-phyla comparative studies and argue that organelle biologists can benefit from such data. We discuss the prospects of using publicly available sequencing data for organelle-focused studies and examine the challenges of such an approach. We highlight that the lack of a comprehensive database dedicated to organelle genomics/transcriptomics is a major impediment to the development of a field with implications in basic and applied science.
... Mitochondria contain their own genome 2 (mtDNA/chrM), usually circular in topology and highly compact, especially in metazoans. The mammalian chrM is around 16-17 kbp in size and encodes for 13 proteins (all of them components of electron transport chains), 22 tRNAs and two rRNAs 3,4 . It has a peculiar compared to the nuclear genome organization and is replicated, transcribed and regulated by its own dedicated set of information processing factors. ...
In most eukaryotes, mitochondrial organelles contain their own genome, usually circular, which is the remnant of the genome of the ancestral bacterial endosymbiont that gave rise to modern mitochondria. Mitochondrial genomes are dramatically reduced in their gene content due to the process of endosymbiotic gene transfer to the nucleus; as a result most mitochondrial proteins are encoded in the nucleus and imported into mitochondria. This includes the components of the dedicated mitochondrial transcription and replication systems and regulatory factors, which are entirely distinct from the information processing systems in the nucleus. However, since the 1990s several nuclear transcription factors have been reported to act in mitochondria, and previously we identified 8 human and 3 mouse transcription factors (TFs) with strong localized enrichment over the mitochondrial genome using ChIP-seq (Chromatin Immunoprecipitation) datasets from the second phase of the ENCODE (Encyclopedia of DNA Elements) Project Consortium. Here, we analyze the greatly expanded in the intervening decade ENCODE compendium of TF ChIP-seq datasets (a total of 6,153 ChIP experiments for 942 proteins, of which 763 are sequence-specific TFs) combined with interpretative deep learning models of TF occupancy to create a comprehensive compendium of nuclear TFs that show evidence of association with the mitochondrial genome. We find some evidence for chrM occupancy for 50 nuclear TFs and two other proteins, with bZIP TFs emerging as most likely to be playing a role in mitochondria. However, we also observe that in cases where the same TF has been assayed with multiple antibodies and ChIP protocols, evidence for its chrM occupancy is not always reproducible. In the light of these findings, we discuss the evidential criteria for establishing chrM occupancy and reevaluate the overall compendium of putative mitochondrial-acting nuclear TFs.
... Most mitochondrial genes are encoded in nuclear DNA and are under the transcriptional control of PGC1a and NRF1 [17]. However, mouse mtDNA encodes two ribosomal RNAs, 22 transference RNAs, and 13 mitochondrial proteins [54]. These proteins are involved in the mitochondrial electron transfer chain and are polycistronically transcribed under TFAM control [55]. ...
The presence of arsenic (As) and fluoride (F⁻) in drinking water is of concern due to the enormous number of individuals exposed to this condition worldwide. Studies in cultured cells and animal models have shown that As- or F-induced hepatotoxicity is primarily associated with redox disturbance and altered mitochondrial homeostasis. To explore the hepatotoxic effects of chronic combined exposure to As and F⁻ in drinking water, pregnant CD-1 mice were exposed to 2 mg/L As (sodium arsenite) and/or 25 mg/L F⁻ (sodium fluoride). The male offspring continued the exposure treatment up to 30 (P30) or 90 (P90) postnatal days. GSH levels, cysteine synthesis enzyme activities, and cysteine transporter levels were investigated in liver homogenates, as well as the expression of biomarkers of ferroptosis and mitochondrial biogenesis-related proteins. Serum transaminase levels and Hematoxylin–Eosin and Masson trichrome-stained liver tissue slices were examined. Combined exposure at P30 significantly reduced GSH levels and the mitochondrial transcription factor A (TFAM) expression while increasing lipid peroxidation, free Fe ²⁺, p53 expression, and serum ALT activity. At P90, the upregulation of cysteine uptake and synthesis was associated with a recovery of GSH levels. Nevertheless, the downregulation of TFAM continued and was now associated with a downstream inhibition of the expression of MT-CO2 and reduced levels of mtDNA and fibrotic liver damage. Our experimental approach using human-relevant doses gives evidence of the increased risk for early liver damage associated with elevated levels of As and F⁻ in the diet during intrauterine and postnatal period.
... Moreover, six missense variants were identified, including A6 A8701G (Ala to Thr) and A8860G (Thr to Ala), ND3 A10398G (Thr to Ala), ND6 C14668T (Tyr to Asn), CytB C14766T (Thr to Ile) and A15326G (Thr to Ala). We further used phylogenetic conservation analysis including mouse, 35 bovine 36 and Xenopus laevis 37 to evaluate the potential pathogenicity. We found that, except for the m.A5826G and m.C14668T mutations (Figures 2 and 3), other mutations were not well conserved and may not be involved in the pathogenesis of T2DM. ...
Background
Mutations in mitochondrial tRNA (mt-tRNA) could be the origin of some type 2 diabetes mellitus (T2DM) cases, but the mechanism remained largely unknown.
Aim
The aim of this study was to assess the impact of a novel mitochondrial tRNACys/tRNATyr A5826G mutation on the development and progression of T2DM.
Methods
A four-generation Han Chinese family with maternally inherited diabetes underwent clinical, genetic and biochemical analyses. The mitochondrial DNA (mtDNA) mutations of three matrilineal relatives were screened by PCR-Sanger sequencing. Furthermore, to see whether m.A5826G mutations affected mitochondrial functions, the cybrid cell lines were derived from three subjects with m.A5826G mutation and three controls without this mutation. ATP was evaluated by luminescent cell viability assay, mitochondrial membrane potential (MMP), and reactive oxygen species (ROS) were determined by flow cytometry. The student’s two-tailed, unpaired t-test was used to assess the statistical significance between the control and mutant results.
Results
The age at onset of diabetes in this pedigree varied from 40 to 63 years, with an average of 54 years. Mutational analysis of mitochondrial genomes revealed the presence of a novel m.A5826G mutation. Interestingly, the m.A5826G mutation occurred at the conjunction between tRNACys and tRNATyr, a very conserved position that was critical for tRNAs processing and functions. Using trans-mitochondrial cybrid cells, we found that mutant cells carrying the m.A5826G showed approximately 36.5% and 22.4% reductions in ATP and MMP, respectively. By contrast, mitochondrial ROS levels increased approximately 33.3%, as compared with the wild type cells.
Conclusion
A novel m.A5826G mutation was identified in a pedigree with T2DM, and this mutation would lead to mitochondrial dysfunction. Thus, the genetic spectrum of mitochondrial diabetes was expanded by including m.A5826G mutation in tRNACys/tRNATyr, our study provided novel insight into the molecular pathogenesis, early diagnosis, prevention and clinical treatment for mitochondrial diabetes.
... 761 bp of ABPa intron 2 sequences were gathered from Bonhomme et al. (2004) and GENBANK for one specimen of each species. Additionally, complete mitochondrial D-loop sequences were obtained for 12 specimens (11 cypriacus and 1 domesticus) using primers defined at positions H41 and L15372 of the domesticus DNA reference sequence (Bibb, et al., 1981). Only one sequence in five is common to this study and the previous one (Bonhomme et al. 2004). ...
The house mouse (Mus musculus domesticus) and the short-tailed mouse of the eastern Mediterranean area (M. macedonicus) were thought to live sympatrically on Cyprus Island. Recently, a phylogenetic survey has shown that the non-commensal mouse of Cyprus was an unknown sister species of European wild mice. Here, we describe this new species of the genus Mus (Rodentia, Mammalia), namely Mus cypriacus sp. n., based on 19 specimens trapped in the southern part of Cyprus. These animals were first compared to Eurasian species of mice using both molecular genetics (complete D-loop sequences and nuclear gene intron) and cytogenetics to state on its systematic status. Then classical and geometric morphometric analyses on both cranial and dental characters have been performed to compare Mus cypriacus with circum-Mediterranean species and provide diagnositic morphological characters. Genetic data strongly support a sister species relationship of the new species to M. macedonicus, the closest mainland taxon. Morphometric analyses provide satisfying criteria for diagnosis of this species relative to other Mediterranean species. The most obvious phenotypic characteristics are its long tail and the allometric gigantism and shape robustness of its cranial and dental characters compared to other Mediterranean mice. The molecular clock and the history of the murine settlement on Cyprus are congruent and suggest that the common ancestor of M. cypriacus and M. macedonicus arrived on Cyprus during the Middle Pleistocene by a founder event on natural raft. The remoteness of Cyprus through time has prevented introgression from the mainland gene pool, and favoured phenotypic adaptation to competition release, leading to the allopatric speciation of M. cypriacus.
... Given the mitochondrial genome's characteristics such as (i) strict maternal inheritance (Gyllensten et al. 1985); (ii) relatively straightforward structure (Bibb et al. 1981); (iii) optimal size and evolutionary rate, as well as different conservativeness in each gene, etc. (Vawter and Brown 1986); and (iv) it is widespread application as a molecular marker in insect phylogenetic studies, particularly within Lepidoptera (Gray 1989, Niu et al. 2001, Daizhen et al. 2008, Dai et al. 2016. We attempted to extract DNA from different NIS samples of T. aureus in this study and analyzed the effects of source, preservation, and preserved duration on the DNA quality. ...
The butterfly genus of Teinopalpus, endemic to Asia, embodies a distinct species of mountain-dwelling butterflies with specific habitat requirements. These species are rare in the wild and hold high conservation and research value. Similar to other protected species, the genetic analysis of the rare Teinopalpus aureus poses challenges due to the complexity of sampling. In this study, we successfully extracted DNA and amplified mitochondrial genomic DNA from various noninvasive sources such as larval feces, larval exuviae, larval head capsules, pupal exuviaes, and filamentous gland secretions, all integral parts of butterfly metamorphosis. This was conducted as part of a research initiative focused on the artificial conservation of T. aureus population in Jinggang Shan Nature Reserve. Our findings illustrated the successful extraction of DNA from multiple noninvasive sources, achieved through modified DNA extraction methodologies. Although the DNA concentration obtained from noninvasive samples was lower than that from muscle tissues of newly dead larvae during rearing, all samples met the requirements for PCR amplification and sequencing, yielding complete circular sequences. These sequences are pivotal for both interspecific and intraspecific genetic relationship analysis. Our methods can be extended to other insects, especially scarce species.
... The key initial step in the field of mitochondrial heterogeneity was the reconstruction of electron micrographs that revealed mitochondrial networks in rat hepatocytes in 1974, 41 and this discovery was reproduced in several cell types, including human endothelial cells and astrocytes, 42 demonstrating that mitochondrial morphology varies in different cells. In the 1980s, the field was advanced with sequencing of the mouse mitochondrial genome, providing a molecular framework for understanding mtDNA heterogeneity, 43 culminating in a summary of the extreme genetic variation within mtDNA in 2021. 44 During the 2000s, studies began to focus on the association between the heterogeneity of mitochondrial proteins, noncoding RNAs (ncRNAs), lipids and the cause of mitochondrial-related disease. ...
As key organelles involved in cellular metabolism, mitochondria frequently undergo adaptive changes in morphology, components and functions in response to various environmental stresses and cellular demands. Previous studies of mitochondria research have gradually evolved, from focusing on morphological change analysis to systematic multiomics, thereby revealing the mitochondrial variation between cells or within the mitochondrial population within a single cell. The phenomenon of mitochondrial variation features is defined as mitochondrial heterogeneity. Moreover, mitochondrial heterogeneity has been reported to influence a variety of physiological processes, including tissue homeostasis, tissue repair, immunoregulation, and tumor progression. Here, we comprehensively review the mitochondrial heterogeneity in different tissues under pathological states, involving variant features of mitochondrial DNA, RNA, protein and lipid components. Then, the mechanisms that contribute to mitochondrial heterogeneity are also summarized, such as the mutation of the mitochondrial genome and the import of mitochondrial proteins that result in the heterogeneity of mitochondrial DNA and protein components. Additionally, multiple perspectives are investigated to better comprehend the mysteries of mitochondrial heterogeneity between cells. Finally, we summarize the prospective mitochondrial heterogeneity-targeting therapies in terms of alleviating mitochondrial oxidative damage, reducing mitochondrial carbon stress and enhancing mitochondrial biogenesis to relieve various pathological conditions. The possibility of recent technological advances in targeted mitochondrial gene editing is also discussed.
... 16,17 Each serves as a danger signal for the immune system and can function as drivers of inflammation. 16,18 Extracellular mtDNA bind to Toll-like receptor 9 (TLR9), which, in macrophages causes the production of proinflammatory cytokines. 19 Proinflammatory cytokines have been shown to promote the generation of ROS in the mitochondria. ...
Background:
Physical stressors can cause a physiological response that can contribute to an increase in mitochondrial dysfunction and mtDNA damage. People living with HIV (PWH) are more likely to suffer from chronic pain and may be more susceptible to mitochondrial dysfunction following exposure to a stressor. We used Quantitative Sensory Testing (QST) as an acute painful stressor in order to investigate whether PWH with/without chronic pain show differential mitochondrial physiological responses.
Methods:
The current study included PWH with (n=26), and without (n=29), chronic pain. Participants completed a single session that lasted approximately 180 minutes, including QST. Blood was taken prior to and following the QST battery for assays measuring mtDNA damage, mtDNA copy number, and mtDNA damage-associated molecular pattern (DAMP) levels (i.e. ND1 and ND6).
Results:
We examined differences between those with and without pain on various indicators of mitochondrial reactivity following exposure to QST. However, only ND6 and mtDNA damage were shown to be statistically significant between pain groups.
Conclusion:
PWH with chronic pain showed greater mitochondrial reactivity to laboratory stressors. Consequently, PWH and chronic pain may be more susceptible to conditions in which mitochondrial damage/dysfunction play a central role, such as cognitive decline.
... To distinguish the two strands, one is named "heavy" (the sense, which is purine rich), and the other is named "light" (the antisense, which contains high amounts of pyrimidine). The mtDNA has 37 genes, which encode 13 polypeptides involved into the oxidative phosphorylation, 2 rRNAs and 22 tRNAs (11). Third, similar to genomic DNA, the mtDNA assumes a secondary structure that regulates the genes transcription and the synthesis of new mtDNA molecules (12). ...
Mitochondria are cellular organelles which generate adenosine triphosphate (ATP) molecules for the maintenance of cellular energy through the oxidative phosphorylation. They also regulate a variety of cellular processes including apoptosis and metabolism. Of interest, the inner part of mitochondria—the mitochondrial matrix—contains a circular molecule of DNA (mtDNA) characterised by its own transcriptional machinery. As with genomic DNA, mtDNA may also undergo nucleotide mutations that have been shown to be responsible for mitochondrial dysfunction. During physiological aging, the mitochondrial membrane potential declines and associates with enhanced mitophagy to avoid the accumulation of damaged organelles. Moreover, if the dysfunctional mitochondria are not properly cleared, this could lead to cellular dysfunction and subsequent development of several comorbidities such as cardiovascular diseases (CVDs), diabetes, respiratory and cardiovascular diseases as well as inflammatory disorders and psychiatric diseases. As reported for genomic DNA, mtDNA is also amenable to chemical modifications, namely DNA methylation. Changes in mtDNA methylation have shown to be associated with altered transcriptional programs and mitochondrial dysfunction during aging. In addition, other epigenetic signals have been observed in mitochondria, in particular the interaction between mtDNA methylation and non-coding RNAs. Mitoepigenetic modifications are also involved in the pathogenesis of CVDs where oxygen chain disruption, mitochondrial fission, and ROS formation alter cardiac energy metabolism leading to hypertrophy, hypertension, heart failure and ischemia/reperfusion injury. In the present review, we summarize current evidence on the growing importance of epigenetic changes as modulator of mitochondrial function in aging. A better understanding of the mitochondrial epigenetic landscape may pave the way for personalized therapies to prevent age-related diseases.
... In addition, 13 missense mutations were identified, including ND1 C3497T (Ala to Val), ND2 A4833G (Thr to Ala) and C5178A (Leu to Met), A8 C8414T (Leu to Phe), A6 A8701G (Thr to Ala) and A8860G (Thr to Ala), ND3 A10398G (Thr to Ala), ND4 G11696A (Val to Ile) and A12026G (Ile to Val), ND5 G13928C (Ser to Thr), Cyt b C14766T (Thr to Ile), A15326G (Thr to Ala), and A15851G (Ile to Val). These variants in RNAs and polypeptides were further evaluated using phylogenetic analysis and sequences from other 16 vertebrates, including mouse [29], bovine [30], and Xenopus laevis [31]. We noticed that except for the ND4 G11696A, tRNA Ala C5601T, and tRNA Cys T5813C mutations (Figures 2 and 3), others were not well conserved, suggesting that they may be involved in the pathogenesis of T2DM (Table 3). ...
Type 2 diabetes mellitus (T2DM) is a common endocrine disorder which remains a large challenge for clinicians. Previous studies have suggested that mitochondrial dysfunction plays an active role in T2DM progression, but a detailed mechanism is still elusive. In the current study, two Han Chinese families with maternally inherited T2DM were evaluated using clinical, genetic, molecular, and biochemical analyses. The mitochondrial genomes were PCR amplified and sequenced. Phylogenetic and bioinformatic analyses were used to assess the potential pathogenicity of mitochondrial DNA (mtDNA) mutations. Interestingly, the matrilineal relatives of these pedigrees exhibited variable severity of T2DM, in particular, the age at onset of T2DM varied from 26 to 65 years, with an average of 49 years. Sequence analysis revealed the presence of ND4 G11696A mutation, which resulted in the substitution of an isoleucine for valine at amino acid (AA) position 312. Indeed, this mutation was present in homoplasmy only in the maternal lineage, not in other members of these families, as well as 200 controls. Furthermore, the m.C5601T in the tRNAAla and novel m.T5813C in the tRNACys, showing high evolutional conservation, may contribute to the phenotypic expression of ND4 G11696A mutation. In addition, biochemical analysis revealed that cells with ND4 G11696A mutation exhibited higher levels of reactive oxygen species (ROS) productions than the controls. In contrast, the levels of mitochondrial membrane potential (MMP), ATP, mtDNA copy number (mtDNA-CN), Complex I activity, and NAD+/NADH ratio significantly decreased in cell lines carrying the m.G11696A and tRNA mutations, suggesting that these mutations affected the respiratory chain function and led to mitochondrial dysfunction that was involved in T2DM. Thus, our study broadened the clinical phenotypes of m.G11696A mutation.
... Moreover, recent studies suggest that mitochondrial fission is related to mitochondrial DNA (mtDNA) replication and segregation [21,22]. The mitochondrial genome encodes 37 genes including tRNA, rRNA, and 13 subunits of the OXPHOS complex I, III, IV, and V [23]. Mutations or reduced content of mtDNA are frequently associated with several metabolic diseases [24]. ...
FTY720 is an FDA-approved sphingosine derivative drug for the treatment of multiple sclerosis. This compound blocks lymphocyte egress from lymphoid organs and autoimmunity through sphingosine 1-phosphate (S1P) receptor blockage. Drug repurposing of FTY720 has revealed improvements in glucose metabolism and metabolic diseases. Studies also demonstrate that preconditioning with this compound preserves the ATP levels during cardiac ischemia in rats. The molecular mechanisms by which FTY720 promotes metabolism are not well understood. Here, we demonstrate that nanomolar concentrations of the phosphorylated form of FTY720 (FTY720-P), the active ligand of S1P receptor (S1PR), activates mitochondrial respiration and the mitochondrial ATP production rate in AC16 human cardiomyocyte cells. Additionally, FTY720-P increases the number of mitochondrial nucleoids, promotes mitochondrial morphology alterations, and induces activation of STAT3, a transcription factor that promotes mitochondrial function. Notably, the effect of FTY720-P on mitochondrial function was suppressed in the presence of a STAT3 inhibitor. In summary, our results suggest that FTY720 promotes the activation of mitochondrial function, in part, through a STAT3 action.
... DNA was transferred to Hybond-N+ membranes (Amersham) and UV crosslinked for blotting. mtDNA was detected using the pAM1 plasmid, 65 after radiolabeling with the Prime-IT II Random Primer Labeling kit (Agilent Technologies). 18S probes was used as the loading control for nuclear DNA. ...
Mitochondrial activity differs markedly between organs, but it is not known how and when this arises. Here we show that cell lineage-specific expression profiles involving essential mitochondrial genes emerge at an early stage in mouse development, including tissue-specific isoforms present before organ formation. However, the nuclear transcriptional signatures were not independent of organelle function. Genetically disrupting intra-mitochondrial protein synthesis with two different mtDNA mutations induced cell lineage-specific compensatory responses, including molecular pathways not previously implicated in organellar maintenance. We saw downregulation of genes whose expression is known to exacerbate the effects of exogenous mitochondrial toxins, indicating a transcriptional adaptation to mitochondrial dysfunction during embryonic development. The compensatory pathways were both tissue and mutation specific and under the control of transcription factors which promote organelle resilience. These are likely to contribute to the tissue specificity which characterizes human mitochondrial diseases and are potential targets for organ-directed treatments.
... The mitochondrial genome is represented by a covalently closed circular double-stranded molecule with a typical length of 16,569 base pairs in humans. mtDNA encodes 37 genes, including 13 polypeptide components of the OxPhos system, 2 rRNAs, and 22 tRNAs (Anderson et al., 1981;Bibb, Van Etten, Wright, Walberg, & Clayton, 1981). Since the discovery of the role of mtDNA mutations in human disease, aging, and cellular bioenergetics, there has been ongoing interest in the study of interactions between the mitochondrial and nuclear genomes and epigenomes. ...
To cope with DNA damage, mitochondria have developed a pathway whereby severely damaged or unrepairable mitochondrial DNA (mtDNA) molecules can be discarded and degraded, after which new molecules are synthesized using intact templates. In this unit, we describe a method that harnesses this pathway to eliminate mtDNA from mammalian cells by transiently overexpressing the Y147A mutant of human uracil‐N‐glycosylase (mUNG1) in mitochondria. We also provide alternate protocols for mtDNA elimination using either combined treatment with ethidium bromide (EtBr) and dideoxycytidine (ddC) or clustered regulatory interspersed short palindromic repeat (CRISPR)‐Cas9‐mediated knockout of TFAM or other genes essential for mtDNA replication. Support protocols detail approaches for several processes: (1) genotyping ρ0 cells of human, mouse, and rat origin by polymerase chain reaction (PCR); (2) quantification of mtDNA by quantitative PCR (qPCR); (3) preparation of calibrator plasmids for mtDNA quantification; and (4) quantification of mtDNA by direct droplet digital PCR (dddPCR). © 2023 Wiley Periodicals LLC. Basic Protocol: Inducing mtDNA loss with mUNG1 Alternate Protocol 1: Generation of ρ0 cells by mtDNA depletion with EtBr and ddC Alternate Protocol 2: Generation of ρ0 cells by knocking out genes critical for mtDNA replication Support Protocol 1: Genotyping ρ0 cells by DirectPCR Support Protocol 2: Determination of mtDNA copy number by qPCR Support Protocol 3: Preparation of calibrator plasmid for qPCR Support Protocol 4: Determination of mtCN by direct droplet digital PCR (dddPCR)
... As molecular markers, the highest conserved genes are the first choice, such as the cytochrome c oxidase subunits I-III (COI-III), cytochrome b (Cytb), and control (D-loop) regions [8][9][10]. They have the characteristics of a simple molecular structure, strict maternal inheritance, no recombination, no common sequence with the nuclear genome, fast evolution rate, and multiple copies [11][12][13][14][15]. Compared with other genes in mtDNA, these gene regions can provide enough sequence information to search for suitable genetic variation and are amenable to amplification and sequencing. ...
In order to study the genetic structure and population geographic distribution pattern of coastal mussel populations in the coast of China, mitochondrial DNA (COI and Cytb genes) were used to analyze the genetic diversity, genetic structure, and population history dynamics of Mytilus unguiculatus in the East China Sea and the Yellow Sea. We detected high levels of genetic diversity in seven populations of M. unguiculatus. A total of 34 haplotypes of COI genes and 29 haplotypes of Cytb were obtained. The haplotype diversity of COI ranged from around 0.77 to 0.93 (Cytb: 0.83~0.91). The nucleotide diversity of COI ranged from around 0.0044 to 0.0064 (Cytb: 0.0049~0.0063). The coefficient of genetic differentiation (FST) of COI ranged from around 0.031 to 0.080, and Cytb ranged from around 0.028 to 0.039. Analysis of molecular variance (AMOVA) and a phylogenetic tree showed that the genetic structure was relatively weak, and there was no clear population differentiation. The neutrality test results showed that Tajima’s D value and Fu’s Fs value were not significant, and no significant population demographic events, including population expansion or population bottleneck, were detected in the samples. The Bayesian skyline graph analysis showed that the effective population size has been relatively stable for nearly 10,000 years, without any large population fluctuations. It was speculated that the seven populations in the present study should belong to the same group. This study provides a comprehensive survey of the genetic characteristics of M. unguiculatus, filling the gaps among related studies. It provides theoretical support and material accumulation for seed selection and breeding, genetic resources’ protection, and breeding management in the future.
... We thus reasoned that such biases may confound our results. Prior to correcting for such biases, it is important to note that the heavy and light strand terminology of the mtDNA is a source of confusion and is largely based on the mtDNA nomenclature of mice 49 and human 50 : the heavy strand has a high G + T content as compared to the light strand and is the so-called 'coding' strand 51 . However, that is not the case for other phyla, such as nematodes, in which all genes are located in a single strand, and arthropods which do not have any reported coding strand asymmetry 52 . ...
Mitochondrial DNA (mtDNA) harbors essential genes in most metazoans, yet the regulatory impact of the multiple evolutionary mtDNA rearrangements has been overlooked. Here, by analyzing mtDNAs from ~8000 metazoans we found high gene content conservation (especially of protein and rRNA genes), and codon preferences for mtDNA-encoded tRNAs across most metazoans. In contrast, mtDNA gene order (MGO) was selectively constrained within but not between phyla, yet certain gene stretches (ATP8-ATP6, ND4-ND4L) were highly conserved across metazoans. Since certain metazoans with different MGOs diverge in mtDNA transcription, we hypothesized that evolutionary mtDNA rearrangements affected mtDNA transcriptional patterns. As a first step to test this hypothesis, we analyzed available RNA-seq data from 53 metazoans. Since polycistron mtDNA transcripts constitute a small fraction of the steady-state RNA, we enriched for polycistronic boundaries by calculating RNA-seq read densities across junctions between gene couples encoded either by the same strand (SSJ) or by different strands (DSJ). We found that organisms whose mtDNA is organized in alternating reverse-strand/forward-strand gene blocks (mostly arthropods), displayed significantly reduced DSJ read counts, in contrast to organisms whose mtDNA genes are preferentially encoded by one strand (all chordates). Our findings suggest that mtDNA rearrangements are selectively constrained and likely impact mtDNA regulation.
... Mitochondria are the powerhouse and metabolic center of mammalian cells and the only organelles other than the nucleus contain their own DNA. Mitochondrial DNA (mtDNA) encodes 13 peptides, 22 transfer RNAs (tRNAs), and two ribosomal RNAs (rRNAs), which are critical for mitochondrial metabolism, respiration, transcription, and translation (Anderson et al., 1981;Bayona-Bafaluy et al., 2008;Bibb et al., 1981;van der Bliek et al., 2017). The mtDNA mutations are the main cause of many serious diseases in humans, the prevalence of which is 5-15 cases per 100,000 in childhood (< 16 years old) and 9.6 cases per 100,000 in adults (Russell et al., 2020). ...
Mitochondria are the only organelles other than the nucleus harboring their DNA in mammalian cells. The mitochondrial DNA (mtDNA) mutation is the cause of many serious diseases, which still lack effective therapies. Our previous report showed that mtDNA mutation exacerbates female reproductive aging. However, the regulation of mtDNA mutation on stem cells is still unknown. Herein, we generated embryonic stem cells (ESCs) harboring massive mtDNA mutations without affecting genomic DNA to study the role of mtDNA mutation in pluripotency. The mtDNA mutation impacted the balance of pluripotency and totipotency of ESCs with a metabolism modulation as the down-regulation of oxidative phosphorylation in energy production, followed by a high level of ROS production by mitochondria. This work would shed light on the investigation and treatment of mtDNA mutant diseases.
... In addition, ten missense mutations were found in this study, including ND2 4491G>A (p.V8I), 5178C>A (p.L237M), COII 7976G>A (p.G131S), A8 8414C>T (p.L17F), 8584G>A (p.A20T), A6 8701A>G (p.T59A), 8860A>G (p.T112A), ND3 10398A>G (p.T112A), CytB 14766C>T (p.T7I) and 15326A>G (p.T194A). These mtDNA variants were further assessed by evolutionary conservation analysis in four species: human [28], bovine [42], mouse [43] and Xenopus laevis [44]. We found that aside from the m.1555A>G and m.4394C>T mutations (Figure 3), other variants were not well conserved. ...
The mitochondrial 1555A>G mutation plays a critical role in aminoglycoside-induced and non-syndromic hearing loss (AINSHL). Previous studies have suggested that mitochondrial secondary variants may modulate the clinical expression of m.1555A>G-induced deafness, but the molecular mechanism has remained largely undetermined. In this study, we investigated the contribution of a deafness-associated tRNAGln 4394C>T mutation to the clinical expression of the m.1555A>G mutation. Interestingly, a three-generation family with both the m.1555A>G and m.4394C>T mutations exhibited a higher penetrance of hearing loss than another family harboring only the m.1555A>G mutation. At the molecular level, the m.4394C>T mutation resides within a very conserved nucleotide of tRNAGln, which forms a new base-pairing (7T-66A) and may affect tRNA structure and function. Using trans-mitochondrial cybrid cells derived from three subjects with both the m.1555A>G and m.4394C>T mutations, three patients with only the m.1555A>G mutation and three control subjects without these primary mutations, we observed that cells with both the m.1555A>G and m.4394C>T mutations exhibited more severely impaired mitochondrial functions than those with only the m.1555A>G mutation. Furthermore, a marked decrease in mitochondrial RNA transcripts and respiratory chain enzymes was observed in cells harboring both the m.1555A>G and m.4394C>T mutations. Thus, our data suggest that the m.4394C>T mutation may play a synergistic role in the m.1555A>G mutation, enhancing mitochondrial dysfunctions and contributing to a high penetrance of hearing loss in families with both mtDNA pathogenic mutations.
... Moreover, ten missense mutations were identified: ND1 C3497T (Ala to Val) and T4216C (Tyr to His), ND2 G4491A (Val to Ile), A8 C8414T (Leu to Phe), A6 A8701G (Thr to Ala) and A8860G (Thr to Ala), ND3 A10398G (Thr to Ala), ND5 G13928C (Ser to Thr), CytB C14766T (Thr to Ile) and A15326G (Thr to Ala). These variants in RNAs and polypeptides were further evaluated by phylogenetic analysis and sequences from other 16 vertebrates, including mouse (Bibb et al. 1981), bovine (Gadaleta et al. 1989) and Xenopus laevis (Roe et al. 1985). We found that except for the m.A15901G and m.C15926T mutations (Figure 2), other variants were not well conserved. ...
Mutations in mitochondrial DNA (mtDNA), especially in mitochondrial tRNA (mt-tRNAs) genes, play important roles in maternally inherited type 2 diabetes mellitus (T2DM), but the molecular mechanism remains unclear. In this study, two families with maternally transmitted T2DM are underwent clinical, genetic and molecular assessments. The mtDNA mutations are screened by direct sequencing. Furthermore, the phylogenetic conservation analysis and pathogenicity scoring system were used to evaluate the pathogenic status of mt-tRNA mutations. Interestingly, matrilineal relatives exhibit variable severity of DM, in particular, the age at onset of DM varies from 39 to 60 years, with an average of 50 years. Screening for the entire mitochondrial genomes identifies the existence of tRNAThr A15901G and C15926T mutations, as well as 59 variants belonging to mtDNA haplogroups D2 and C4c. Notably, the m.A15901G mutation is located at D-arm of tRNAThr, whereas the m.C15926T mutation resides in the anticodon loop of tRNAThr, both of these positions are well conserved and critical for tRNA functions. Thus, the m.A15901G and m.C15926T mutations may impair mitochondrial translation and lead to mitochondrial dysfunctions. However, the fail to identify any other functional variants indicate that mitochondrial haplogroup may not play a role in T2DM. Hence, tRNAThr A15901G and C15926T may be the novel mutations associated with T2DM.
... We verified the off-target mutations by analyzing the full sequence of mtDNA extracted from liver tissues of mito-mice tRNA Leu (Supplementary Table S4). Among those, T9461C in ND3, G9348A in CO3, and 9821 insA in tRNA Arg have been reported as genetic mtDNA polymorphisms caused by mouse strain differences (Supplementary Table S4) (37); C4794T in ND2 and T12048C in ND5 have been previously reported as genetic mtDNA polymorphisms in the mouse LA9 cell line (Supplementary Table S4) (38). Because mito-mice tRNA Leu(UUR)2748 have a mixed genetic background including C57BL/6, CBA/J, and C3H/An and these mutations have been previously reported, it is most likely that these mutations are derived from the mixed genetic background and not from ENUmediated mutagenesis. ...
Mitochondrial tRNAs are indispensable for the intra-mitochondrial translation of genes related to respiratory subunits, and mutations in mitochondrial tRNA genes have been identified in various disease patients. However, the molecular mechanism underlying pathogenesis remains unclear due to the lack of animal models. Here, we established a mouse model, designated 'mito-mice tRNALeu(UUR)2748', that carries a pathogenic A2748G mutation in the tRNALeu(UUR) gene of mitochondrial DNA (mtDNA). The A2748G mutation is orthologous to the human A3302G mutation found in patients with mitochondrial diseases and diabetes. A2748G mtDNA was maternally inherited, equally distributed among tissues in individual mice, and its abundance did not change with age. At the molecular level, A2748G mutation is associated with aberrant processing of precursor mRNA containing tRNALeu(UUR) and mt-ND1, leading to a marked decrease in the steady-levels of ND1 protein and Complex I activity in tissues. Mito-mice tRNALeu(UUR)2748 with ≥50% A2748G mtDNA exhibited age-dependent metabolic defects including hyperglycemia, insulin insensitivity, and hepatic steatosis, resembling symptoms of patients carrying the A3302G mutation. This work demonstrates a valuable mouse model with an inheritable pathological A2748G mutation in mt-tRNALeu(UUR) that shows metabolic syndrome-like phenotypes at high heteroplasmy level. Furthermore, our findings provide molecular basis for understanding A3302G mutation-mediated mitochondrial disorders.
... The maternally inherited mitochondrial genome (Giles et al., 1980), otherwise known as mtDNA, resides in each of the large numbers of mitochondria present within many cell types. It is a double-stranded circular genome that ranges from 16.2 kb (mice) to almost 16.7 kb (pigs) in size across mammalian species (Anderson et al., 1981;Bibb et al., 1981;Ursing & Arnason, 1998) (Figure 1a). It encodes 13 of the greater than 90 subunits of the electron transfer chain, which generates ATP through oxidative phosphorylation (OXPHOS), and is essential for cells with high energy requirements, such as neurons, and heart and skeletal muscle cells (Moyes et al., 1998). ...
The mitochondrial genome resides in the mitochondria present in nearly all cell types. The porcine (Sus scrofa) mitochondrial genome is circa 16.7 kb in size and exists in the multimeric format in cells. Individual cell types have different numbers of mitochondrial DNA (mtDNA) copy number based on their requirements for ATP produced by oxidative phosphorylation. The oocyte has the largest number of mtDNA of any cell type. During oogenesis, the oocyte sets mtDNA copy number in order that sufficient copies are available to support subsequent developmental events. It also initiates a program of epigenetic patterning that regulates, for example, DNA methylation levels of the nuclear genome. Once fertilized, the nuclear and mitochondrial genomes establish synchrony to ensure that the embryo and fetus can complete each developmental milestone. However, altering the oocyte's mtDNA copy number by mitochondrial supplementation can affect the programming and gene expression profiles of the developing embryo and, in oocytes deficient of mtDNA, it appears to have a positive impact on the embryo development rates and gene expression profiles. Furthermore, mtDNA haplotypes, which define common maternal origins, appear to affect developmental outcomes and certain reproductive traits. Nevertheless, the manipulation of the mitochondrial content of an oocyte might have a developmental advantage.
... The structure of the vertebrate mitogenome typically consists of 13 protein-coding genes (PCGs), 22 tRNA genes (tRNAs), and two rRNA genes (rRNAs) (Boore, 1999). In the vertebrate mitogenome, the order of these genes is conserved and generally unaltered (Bibb et al., 1981;Roe et al., 1985). The mitochondrial genome is inherited maternally in most animals and therefore has a very low rate of recombination (Brown et al., 1979). ...
Complete mitochondrial genomes (mitogenomes) can provide valuable information for phylogenetic relationships, gene rearrangement, and molecular evolution. Here, we report the mitochondrial whole genomes of three Garra species and explore the mechanisms of rearrangements that occur in their mitochondrial genomes. The lengths of the mitogenomes’ sequences of Garra dengba, Garra tibetana, and Garra yajiangensis were 16,876, 16,861, and 16,835, respectively. They contained 13 protein-coding genes, two ribosomal RNAs, 22 transfer RNA genes, and two identical control regions (CRs). The mitochondrial genomes of three Garra species were rearranged compared to other fish mitochondrial genomes. The tRNA-Thr, tRNA-Pro and CR (T-P-CR) genes undergo replication followed by random loss of the tRNA-Thr and tRNA-Pro genes to form tRNA-Thr, CR1, tRNA-Pro and CR2 (T-CR-P-CR). Tandem duplication and random loss best explain this mitochondrial gene rearrangement. These results provide a foundation for future characterization of the mitochondrial gene arrangement of Labeoninae and further phylogenetic studies.
... The first mitochondrial genome was sequenced entirely in 1981 (Bibb et al. 1981;Anderson et al. 1981), whereas the chloroplastic genome of Marchantia polymorpha and tobacco was entirely sequenced five years later in 1986 (Shinozaki et al. 1986;Ohyama et al. 1986). Because of their evolutionary histories, mitochondrial and chloroplastic genomes share high sequence similarities. ...
Plant organelles like chloroplasts and mitochondria are essential organelles serving critical functions like photosynthesis and respiration, respectively, in plants. While most of the processes and the components required by the functioning of these organelles are contributed by nuclear DNA, they have few of their own components encoded by their respective genome. Mitochondrial and chloroplast genomes give a real insight into the evolution of land plants, as evident by several studies. Few studies have successfully conducted gene transfer technology into these organelles’ genomes. Although extensive research on plant organelle genome is yet to be done, recent research has shown the probability of these organelles as a target of genome engineering. From targeting individual genes of their genome to incorporating new genes from other species, they hold promises to produce improved traits. Packaging of their genome, which varies significantly in various hierarchies of land and primitive plants, has also been studied in few plant species. This chapter summarizes the current studies and findings in the study of the organellar genome concerning their structure, organization, distribution, regulatory mechanism, and gene transfer technologies. This chapter provides an updated account of the evolution of these organelle genomes.
... Quantitative densitometry of these data indicate that ~ 35% of the hybridization signal for the retinal mtDNA/RNA sample is contributed by the DNA and 65% by RNA. Mitochondrial RNome is represented by the intrinsic transcriptome encoded by the mitochondrial genome, including 2 ribosomal RNAs, 22 tRNAs, 13 essential protein subunits in the OXPHOS pathway, small noncoding RNAs and the extrinsic nuclear-encoded RNA importome of some noncoding RNAs that participate in regulation of gene expression [30][31][32] . RNA molecules are known to be less protected against ROS under oxidative stress conditions and, therefore more susceptible to oxidation than DNA, which when damaged can be repaired by cellular DNA repair mechanisms 33,34 . ...
This study examines retinas from a rat glaucoma model for oxidized nucleosides 8OHdG and 8OHG, biomarkers for oxidative damage of DNA and RNA, respectively. Immunohistochemical data indicate a predominant localization of 8OHdG/8OHG in retinal ganglion cells (RGCs). The levels for these oxidized DNA/RNA products were 3.2 and 2.8 fold higher at 1 and 2 weeks after intraocular pressure elevation compared to control retinas, respectively. 8OHdG/8OHG were almost exclusively associated with mitochondrial DNA/RNA: ~ 65% of 8OHdG/8OHG were associated with RNA isolated from mitochondrial fraction and ~ 35% with DNA. Furthermore, we analyzed retinas of the rd10 mouse, a model for retinitis pigmentosa, with severe degeneration of photoreceptors to determine whether high levels of 8OHdG/8OHG staining intensity in RGCs of control animals is related to the high level of mitochondrial oxidative phosphorylation necessary to support light-evoked RGC activity. No significant difference in 8OHdG/8OHG staining intensity between control and rd10 mouse retinas was observed. The results of this study suggest that high levels of 8OHdG/8OHG in RGCs of wild-type animals may lead to cell damage and progressive loss of RGCs observed during normal aging, whereas ocular hypertension-induced increase in the level of oxidatively damaged mitochondrial DNA/RNA could contribute to glaucomatous neurodegeneration.
... A major contributor to unstable noncoding RNA products is antisense transcripts, i.e., RNAs transcribed from the strand opposite the sense strand of a protein-coding gene. Originally identified in bacteria (Spiegelman et al., 1972), antisense transcripts were soon discovered in eukaryotes as well (Anderson et al., 1981;Bibb et al., 1981). Since its discovery, antisense transcription has been detected opposite the vast majority of annotated genes in yeast (Xu et al., 2011), arising initially as a natural consequence of open chromatin regions (Jin et al., 2017). ...
Understanding the complex network that regulates transcription elongation requires the quantitative analysis of RNA polymerase II (Pol II) activity in a wide variety of regulatory environments. We performed native elongating transcript sequencing (NET-seq) in 41 strains of S. cerevisiae lacking known elongation regulators, including RNA processing factors, transcription elongation factors, chromatin modifiers, and remodelers. We found that the opposing effects of these factors balance transcription elongation and antisense transcription. Different sets of factors tightly regulate Pol II progression across gene bodies so that Pol II density peaks at key points of RNA processing. These regulators control where Pol II pauses with each obscuring large numbers of potential pause sites that are primarily determined by DNA sequence and shape. Antisense transcription varies highly across the regulatory landscapes analyzed, but antisense transcription in itself does not affect sense transcription at the same locus. Our findings collectively show that a diverse array of factors regulate transcription elongation by precisely balancing Pol II activity.
... For amplification of~600 bp long 16S rRNA gene fragments, universal primers were used (Table S3), as described in [46]. The targeted regions included Domains III, IV, and partly II (position 837-1416 at 16S rRNA gene), located between positions 1913 and 2507 of the murine mtDNA [47]. For archived samples, we applied additional semi-nested PCR, which produced 480 bp long fragments. ...
Simple Summary
Cryptic species, hidden by morphological uniformity, represent a significant part of the diversity in some taxonomic groups, and pose a real challenge for conservation planning. Here, we explore cryptic speciation in blind mole rats of the genus Nannospalax—comprehensively studied mammals for many unusual features (cancer resistance, longevity, etc.). Intensive chromosomal changes are one of these peculiarities. In the European N. leucodon species complex, 25 lineages with different karyotypes have been described, comprising undetected/cryptic species. As some of them are endangered, taxonomic revision is urgent for conservation purposes. Using 36–60-year-old archived teeth samples and newly captured animals, we analysed the nucleotide polymorphism of two mitochondrial gene sequences among 17 out of 25 chromosomal forms—the highest number studied so far—and provided molecular genetic records for 5 of them for the first time. Eleven chromosomal forms were separated into distinct clades in phylogenetic trees. High evolutionary divergence values among several chromosomal forms overlapped with those acquired for higher taxonomic categories. By integrating the results of previous karyological analyses and crossbreeding experiments that revealed complete reproductive isolation of seven chromosomal forms, with our new findings, we propose conservation strategies to preserve their genetic diversity.
Abstract
We explored the cryptic speciation of the Nannospalax leucodon species complex, characterised by intense karyotype evolution and reduced phenotypic variability that has produced different lineages, out of which 25 are described as chromosomal forms (CFs), so many cryptic species remain unnoticed. Although some of them should be classified as threatened, they lack the official nomenclature necessary to be involved in conservation strategies. Reproductive isolation between seven CFs has previously been demonstrated. To investigate the amount and dynamics of genetic discrepancy that follows chromosomal changes, infer speciation levels, and obtain phylogenetic patterns, we analysed mitochondrial 16S rRNA and MT-CYTB nucleotide polymorphism among 17 CFs—the highest number studied so far. Phylogenetic trees delineated 11 CFs as separate clades. Evolutionary divergence values overlapped with acknowledged higher taxonomic categories, or sometimes exceeded them. The fact that CFs with higher 2n are evolutionary older corresponds to the fusion hypothesis of Nannospalax karyotype evolution. To participate in conservation strategies, N. leucodon classification should follow the biological species concept, and proposed cryptic species should be formally named, despite a lack of classical morphometric discrepancy. We draw attention towards the syrmiensis and montanosyrmiensis CFs, estimated to be endangered/critically endangered, and emphasise the need for detailed monitoring and population survey for other cryptic species.
... Important sequence elements within the NCR are the three conserved sequence blocks (CSB1-3), located between LSP and O H , and the termination-associated sequence (TAS) located at the 3' end. Initiation of mtDNA transcription from both strands generates polycistronic transcripts that are processed at the mt-tRNA junctions to yield individual RNA molecules (Anderson et al. 1981;Bibb et al. 1981;Crews et al. 1979;Montoya et al. 1982;Ojala et al. 1981). The cleavage of the polycistronic mitochondrial transcripts is performed by the mitochondrial RNase P and the mitochondrial RNase Z ELAC2 (Dubrovsky et al. 2004;Holzmann et al. 2008;Takaku et al. 2003). ...
Mitochondria are central hubs for cellular metabolism, coordinating a variety of metabolic reactions crucial for human health. Mitochondria provide most of the cellular energy via their oxidative phosphorylation (OXPHOS) system, which requires the coordinated expression of genes encoded by both the nuclear (nDNA) and mitochondrial genomes (mtDNA). Transcription of mtDNA is not only essential for the biogenesis of the OXPHOS system, but also generates RNA primers necessary to initiate mtDNA replication. Like the prokaryotic system, mitochondria have no membrane-based compartmentalization to separate the different steps of mtDNA maintenance and expression and depend entirely on nDNA-encoded factors imported into the organelle. Our understanding of mitochondrial transcription in mammalian cells has largely progressed, but the mechanisms regulating mtDNA gene expression are still poorly understood despite their profound importance for human disease. Here, we review mechanisms of mitochondrial gene expression with a focus on the recent findings in the field of mammalian mtDNA transcription and disease phenotypes caused by defects in proteins involved in this process.
... The complete mitogenomes of M. tamariscinus, B. przewalskii, and R. opimus were 16,393 bp, 16,357 bp, and 16,352 bp in length, respectively, and three mitogenomes have been deposited in the GenBank database with accession number KT834971 (for M. tamariscinus), KT834972 (for B. przewalskii), and MK359635 (for R. opimus). Here, three gerbil mitogenomes contained 13 PCGs, 2 rRNA (12S rRNA and 16S rRNA), and 22 tRNA genes and one control region (D-loop) ( Fig. 1; Supplementary Tables S4, S5 and S6) was similar to other mammals in terms of gene quantity, orientation, and organization structure without any gene insertion, deletion, and rearrangement (Bibb et al. 1981;Jiang et al. 2012;Ding et al. 2016b). The 22 tRNA genes were interspersed among rRNAs and PCGs, with few gaps between them, and some genes even overlapped with each other (Fig. 1). ...
To enrich the mitogenomic database of Gerbillinae (Rodentia: Muridae), mitogenomes of three gerbils from different genera, Meriones tamariscinus (16,393 bp), Brachiones przewalskii (16,357 bp), and Rhombomys opimus (16,352 bp), were elaborated and compared with those of other gerbils in the present study. The three gerbil mitogenomes consisted of 2 ribosomal RNA genes, 13 protein-coding genes (PCGs), 22 transfer RNA genes, and one control region. Here, gerbil mitogenomes have shown unique characteristics in terms of base composition, codon usage, non-coding region, and the replication origin of the light strand. There was no significant correlation between the nucleotide percentage of G + C and the phylogenetic status in gerbils, and between the GC content of PCGs and the leucine count. Phylogenetic relationships of the subfamily Gerbillinae were reconstructed by 7 gerbils that represented four genera based on concatenated mitochondrial DNA data using both Bayesian Inference and Maximum Likelihood. The phylogenetic analysis indicated that M. tamariscinus was phylogenetically distant from the genus Meriones, but has a close relationship with R. opimus. B. przewalskii was closely related to the genus Meriones rather than that of R. opimus.
... PolG is the major polymerase of mitochondrial DNA and responsible for replication and correction of mtDNA, to guarantee the expression of respiratory complexes related proteins synthesized by mtDNA and maintain mitochondrial function [1][2][3]. Some previous studies indicated the quantity and quality of PolG could affect mitochondrial function, such as mitophagy, mitochondrial fusion and division, and mitochondria biogenesis [4][5][6]. ...
DNA polymerase gamma (PolG) is the major polymerase of mitochondrial DNA (mtDNA) and essential for stabilizing mitochondrial function. Vascular calcification (VC) is common senescence related degenerative pathology phenomenon in the end-stage of multiple chronic diseases. Mitochondrial dysfunction was often observed in calcified vessels, but the function and mechanism of PolG in the calcification process was still unknown. The present study found PolGD257A/D257A mice presented more severe calcification of aortas than wild type (WT) mice with vitamin D3 (Vit D3) treatment, and this phenomenon was also confirmed in vitro. Mechanistically, PolG could enhance the recruitment and interaction of p53 in calcification condition to recover mitochondrial function and eventually to resist calcification. Meanwhile, we found the mutant PolG (D257A) failed to achieve the same rescue effects, suggesting the 3'-5' exonuclease activity guarantee the enhanced interaction of p53 and PolG in response to calcification stimulation. Thus, we believed that it was PolG, not mutant PolG, could maintain mitochondrial function and attenuate calcification in vitro and in vivo. And PolG could be a novel potential therapeutic target against calcification, providing a novel insight to clinical treatment.
... The myth of a "typical" mitochondrial genome (mtDNA) is a rock-hard belief in the field of genetics, at least for the animal kingdom [1]. The first complete mitochondrial genomes were published in the 1980s [2][3][4][5]; since then, thousands of mtDNAs have been studied, and it is now well evident that only a few features (if any) are conserved among eukaryotic mtDNAs. Nonetheless, mtDNA has demonstrated, and it is everyday demonstrating, its suitability as a phylogenetic marker, ranging from population to phylum scale. ...
The myth of a “typical” mitochondrial genome (mtDNA) is a rock-hard belief in the field of genetics, at least for the animal kingdom [...]
... Mitochondria contain their own DNA (mitochondrial DNA, mtDNA), which in mammals encodes 13 subunits of the oxidative phosphorylation (OXPHOS) system, as well as mitochondrial ribosomal RNAs (rRNAs) and transfer RNAs (tRNAs) (Anderson et al, 1981;Bibb et al, 1981). Expression of mtDNA is required for proper OXPHOS function, and its disruption causes a variety of diseases, including metabolic disorders and neurodegeneration (Chinnery, 2015;Gustafsson et al, 2016;Kauppila et al, 2017). ...
Cancer cells depend on mitochondria to sustain their increased metabolic need and mitochondria therefore constitute possible targets for cancer treatment. We recently developed small-molecule inhibitors of mitochondrial transcription (IMTs) that selectively impair mitochondrial gene expression. IMTs have potent antitumor properties in vitro and in vivo, without affecting normal tissues. Because therapy-induced resistance is a major constraint to successful cancer therapy, we investigated mechanisms conferring resistance to IMTs. We employed a CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats)-(CRISP-associated protein 9) whole-genome screen to determine pathways conferring resistance to acute IMT1 treatment. Loss of genes belonging to von Hippel-Lindau (VHL) and mammalian target of rapamycin complex 1 (mTORC1) pathways caused resistance to acute IMT1 treatment and the relevance of these pathways was confirmed by chemical modulation. We also generated cells resistant to chronic IMT treatment to understand responses to persistent mitochondrial gene expression impairment. We report that IMT1-acquired resistance occurs through a compensatory increase of mitochondrial DNA (mtDNA) expression and cellular metabolites. We found that mitochondrial transcription factor A (TFAM) downregulation and inhibition of mitochondrial translation impaired survival of resistant cells. The identified susceptibility and resistance mechanisms to IMTs may be relevant for different types of mitochondria-targeted therapies.
Purpose
This study aimed to examine the frequencies of mt-tRNAGlu variants in 180 pediatric patients with non-syndromic hearing loss (NSHL) and 100 controls.
Methods
Sanger sequencing was performed to screen for mt-tRNAGlu variants. These mitochondrial DNA (mtDNA) pathogenic mutations were further assessed using phylogenetic conservation and haplogroup analyses. We also traced the origins of the family history of probands carrying potential pathogenic mtDNA mutations. Mitochondrial functions including mtDNA content, ATP and reactive oxygen species (ROS) were examined in cells derived from patients carrying the mt-tRNAGlu A14692G and CO1/tRNASer(UCN) G7444A variants and controls.
Results
We identified four possible pathogenic variants: m.T14709C, m.A14683G, m.A14692G and m.A14693G, which were found in NSHL patients but not in controls. Genetic counseling suggested that one child with the m.A14692G variant had a family history of NSHL. Sequence analysis of mtDNA suggested the presence of the CO1/tRNASer(UCN) G7444A and mt-tRNAGlu A14692G variants. Molecular analysis suggested that, compared with the controls, patients with these variants exhibited much lower mtDNA copy numbers, ATP production, whereas ROS levels increased (p<0.05 for all), suggesting that the m.A14692G and m.G7444A variants led to mitochondrial dysfunction.
Conclusion
mt-tRNAGlu variants are important risk factors for NSHL.
One of the major challenges that remain in the fields of aging and lifespan determination concerns the precise roles that reactive oxygen species (ROS) play in these processes. ROS, including superoxide and hydrogen peroxide, are constantly generated as byproducts of aerobic metabolism, as well as in response to endogenous and exogenous cues. While ROS accumulation and oxidative damage were long considered to constitute some of the main causes of age-associated decline, more recent studies reveal a signaling role in the aging process. In fact, accumulation of ROS, in a spatiotemporal manner, can trigger beneficial cellular responses that promote longevity and healthy aging. In this review, we discuss the importance of timing and compartmentalization of external and internal ROS perturbations in organismal lifespan and the role of redox regulated pathways.
Background
Mitochondrial DNA (mtDNA) mutations are associated with essential hypertension (EH), but the molecular mechanism remains largely unknown.
Objective
The aim of this study is to explore the association between mtDNA mutations and EH.
Methods
Two maternally inherited families with EH are underwent clinical, genetic and biochemical assessments. mtDNA mutations are screened by PCR-Sanger sequencing and phylogenetic, and bioinformatics analyses are performed to evaluate the pathogenicity of mtDNA mutations. We also generate cytoplasmic hybrid (cybrid) cell lines to analysis mitochondrial functions.
Results
Matrilineal relatives exhibit variable degree of clinical phenotypes. Molecular analysis reveals the presence of m.A14693G and m.A14696G mutations in two pedigrees. Notably, the m.A14693G mutation occurs at position 54 in the TψC loop of tRNAGlu, a position which is critical for post-transcriptionally modification of tRNAGlu. While the m.A14696G mutation creates a novel base-pairing (51C-64G). Bioinformatic analysis shows that these mutations alter tRNAGlu secondary structure. Additionally, patients with tRNAGlu mutations exhibit markedly decreased in mtDNA copy number, mitochondrial membrane potential (MMP) and ATP, whereas the levels of reactive oxygen species (ROS) increase significantly.
Conclusion
The m.A14696G and m.A14693G mutations lead to failure in tRNAGlu metabolism and cause mitochondrial dysfunction that is responsible for EH.
The mitochondrial genome structure of a teleostean group is generally considered to be conservative. However, two types of gene arrangements have been identified in the mitogenomes of Anguilliformes. In this study, we report the complete mitochondrial genome of Ariosoma meeki (Anguilliformes (Congridae)). For this research, first, the mitochondrial genome structure and composition were analyzed. As opposed to the typical gene arrangement pattern in other Anguilliformes species, the mitogenome of A. meeki has undergone gene rearrangement. The ND6 and the conjoint tRNA-Glu genes were translocated to the location between the tRNA-Thr and tRNA-Pro genes, and a duplicated D-loop region was translocated to move upstream of the ND6 gene. Second, comparative genomic analysis was carried out between the mitogenomes of A. meeki and Ariosoma shiroanago. The gene arrangement between them was found to be highly consistent, against the published A. meeki mitogenomes. Third, we reproduced the possible evolutionary process of gene rearrangement in Ariosoma mitogenomes and attributed such an occurrence to tandem repeat and random loss events. Fourth, a phylogenetic analysis of Anguilliformes was conducted, and the clustering results supported the non-monophyly hypothesis regarding the Congridae. This study is expected to provide a new perspective on the A. meeki mitogenome and lay the foundation for the further exploration of gene rearrangement mechanisms.
Background:
Major depressive disorder (MDD) is the most frequent reason of disabled people in the world, as reported by the World Health Organization. However, the diagnosis of MDD is mainly based on clinical symptoms.
Case summary:
The clinical, genetic, and molecular characteristics of two Chinese families with MDD are described in this study. There were variable ages of onset and severity in depression among the families. Both Chinese families had a very low pre-valence of MDD. The mitochondrial genomes of these pedigrees were sequenced and indicated a homoplasmic T3394C (Y30H) mutation, with the polymorphism located at a highly conserved tyrosine at position 30 of ND1. The analysis also revealed unique sets of mitochondrial DNA (mtDNA) polymorphisms orig-inating from haplogroups M9a3 and M9a.
Conclusion:
This finding of the T3394C mutation in two unrelated depressed patients provides strong evidence that this mutation may have a part in the etiology of MDD. However, In these two Chinese families having the T3394C mutation, no functional mtDNA mutation was observed. Therefore, T3394C mutations are related with MDD, and the phenotypic manifestation of these mutations may be affected by changes in nuclear genes or environmental factors.
Background: Analyses of mitogenome structure and its evolution have provided new insights of species evolution and helped to improve in situ and ex situ conservation strategies. Although the characterization of snakes mitogenomes have been improved, the access of neotropical species molecules is still scarce, such as the case of Bothrops insularis. B. insularis is a Brazilian critically endangered snake which genomic characterization could improve information related to its evolutionary history and conservation strategies delimitation. Here we characterize for the first time the mitogenome of B. insularis, compare it with other mitogenomes available for the genus Bothrops, and used those genomes to recover the putative phylogenetic context in which the species evolved. \
Results: B. insularis mitogenome is a circular molecule with 17,523 bp length, encompassing 13 protein-coding genes, 22 tRNA, two rRNA, two control regions, one region of the light strand origin replication, a duplicate tRNA-Phe, and a non-coding region. Within the genus Bothrops mitogenomes diverge due to the presence of tRNA duplications and non-coding regions. Despite the divergences found in the mitogenome nucleotide composition and structure, evidence of positive selection was not observed in B. insularis. Comparisons among 129 snake species allowed us to identify 18 mitotypes, which originated from rearrangement processes within three tRNA clusters: the WAN-Ol-CY; the CR regions and adjacent tRNA; and the S2D cluster. These processes might have occurred in the family (Mitogenome 3B and variants), subfamily (Mitogenome 3D and variants), and species-specific levels.
Conclusion: Our results provide the first description of B. insularis mitogenome, which reinforce its evolutionary significant unit status, in agreement with previous ecological, genetic, and evolutionary data. Moreover, we report a higher diversity of gene order and structure within snake mitogenomes which brings another question to be investigated: mitotypes could be correlated to habits or habitats?
In the course of its short history, mitochondrial DNA (mtDNA) has made a long journey from obscurity to the forefront of research on major biological processes. mtDNA alterations have been found in all major disease groups, and their significance remains the subject of intense research. Despite remarkable progress, our understanding of the major aspects of mtDNA biology, such as its replication, damage, repair, transcription, maintenance, etc., is frustratingly limited. The path to better understanding mtDNA and its role in cells, however, remains torturous and not without errors, which sometimes leave a long trail of controversy behind them. This review aims to provide a brief summary of our current knowledge of mtDNA and highlight some of the controversies that require attention from the mitochondrial research community.
We present two methods for enhancing the efficiency of mitochondrial DNA (mtDNA) editing in mice with DddA-derived cytosine base editors (DdCBEs). First, we fused DdCBEs to a nuclear export signal (DdCBE-NES) to avoid off-target C-to-T conversions in the nuclear genome and improve editing efficiency in mtDNA. Second, mtDNA-targeted TALENs (mitoTALENs) are co-injected into mouse embryos to cleave unedited mtDNA. We generated a mouse model with the m.G12918A mutation in the MT-ND5 gene, associated with mitochondrial genetic disorders in humans. The mutant mice show hunched appearances, damaged mitochondria in kidney and brown adipose tissues, and hippocampal atrophy, resulting in premature death.
Background:
Mutations in mitochondrial DNA (mtDNA) are associated with type 2 diabetes mellitus (T2DM). In particular, m.A3243G is the most common T2DM-related mtDNA mutation in many families worldwide. However, the clinical features and pathophysiology of m.A3243G-induced T2DM are largely undefined.
Methods:
Two pedigrees with maternally inherited T2DM were underwent clinical, molecular and biochemical assessments. The mtDNA genes were PCR amplified and sequenced. Mitochondrial adenosine triphosphate (ATP) and reactive oxygen species (ROS) were measured in polymononuclear leukocytes derived from three patients with both the m.A3243G and m.T14502C mutations, three patients with only the m.A3243G mutation and three controls without these mutations. Moreover, GJB2, GJB3 and GJB6 mutations were screened by PCR-Sanger sequencing.
Results:
Members of the two pedigrees manifestated variable clinical phenotypes including diabetes and hearing and vision impairments. The age at onset of T2DM varied from 31 to 66 years, with an average of 41 years. Mutational analysis of mitochondrial genomes indicated the presence of the m.A3243G mutation in both pedigrees. Matrilineal relatives in one of the pedigrees harbored the coexisting of m.A3243G and m.T14502C mutations. Remarkably, the m.T14502C mutation, which causes the substitution of a conserved isoleucine for valine at position 58 in ND6 mRNA, may affect the mitochondrial respiratory chain functions. Biochemical analysis revealed that cell lines bearing both the m.A3243G and m.T14502C mutations exhibited greater reductions in ATP levels and increased ROS production compared with those carrying only the m.A3243G mutation. However, we did not find any mutations in the GJB2, GJB3 and GJB6 genes.
Conclusion:
Our study indicated that mitochondrial diabetes is associated with the tRNALeu(UUR) A3243G and ND6 T14502C mutations.
Where differences have been reported between tumor and normal mitochondrial DNA (mtDNA), they have generally involved limited modifications of the genome (Tairaet al., Nucleic Acids Res.
11:1635, 1983; Shay and Werbin,Mutat. Res.
186:149, 1987). However, Corralet al. (Nucleic Acids Res.
16:10935, 1988;17:5191, 1989) observed recombination between cytochrome oxidase subunit I (COI) and NADH dehydrogenase subunit 6 (ND6), two genes normally on opposite sides of the circular mitochondrial genome. In rat hepatoma mtDNA COI and ND6 were reported to be separated by only 230 base pairs (Corralet al., 1988, 1989). We have performed RFLP analysis on mtDNA from normal rat livers and rat hepatomas, using COI and ND6 probes. Additional experiments compared end-labeled DNA fragments produced byEcoRI andHindIII digestion of mtDNA. These studies failed to provide any evidence for genetic recombination in rat hepatoma mtDNA, even in the same cell line used by Corralet al. Rather, they support the conclusion that mtDNA from tumor and normal tissues exhibits a low degree of heterogeneity.
Objectives:
Mutations in mitochondrial tRNA (mt-tRNA) are the important causes for maternally inherited hypertension, however, the pathophysiology of mt-tRNA mutations in clinical expression of hypertension remains poorly understood.
Material and methods:
In this study, we report the molecular features of a Han Chinese pedigree with maternally transmitted essential hypertension. The entire mitochondrial genomes are PCR amplified and sequenced, Moreover, phylogenetic analysis, haplogroup analysis, as well as pathogenicity scoring system are used to assess the potential roles for mtDNA mutations.
Results:
Strikingly, among ten matrilineal relatives, three of them suffer from variable degree of hypertension at different age at onset. Sequence analysis of the complete mitochondrial genomes suggests the presence of three possible pathogenic mtDNA mutations: tRNAAsp T7561C, tRNAHis C12153T and A12172G, together with a set of variants belonging to East Asian mitochondrial haplogroup M7a. Interestingly, the T7561C mutation occurs at position 44 in the variable region of tRNAAsp, while the C12153T and A12172G mutations are localized at extremely conserved nucleotides in the D-arm and anticodon stem of tRNAHis gene, respectively, which are critical for tRNA steady-state level and function.
Conclusions:
Mitochondrial T7561C, C12153T and A12172G mutations may lead to the failure in tRNAs metabolism, and cause mitochondrial dysfunction that is responsible for hypertension. However, the homoplasmy form of mt-tRNA mutations, incomplete penetrance of hypertension suggest that T7561C, C12153T and A12172G mutations are insufficient to produce the clinical phenotype, hence, other risk factors such as environmental factors, nuclear genes and epigenetic modifications may contribute to the phenotypic manifestation of maternally inherited hypertension in this Chinese pedigree.
Background
Sequence alternations in mitochondrial genomes, especially in genes encoding mitochondrial tRNA (mt‐tRNA), were the important contributors to nonsyndromic hearing loss (NSHL); however, the molecular mechanisms remained largely undetermined.
Methods
A maternally transmitted Chinese pedigree with NSHL underwent clinical, genetic, and biochemical assessment. PCR and direct sequence analyses were performed to detect mitochondrial DNA (mtDNA), GJB2, and SLC26A4 gene mutations from matrilineal relatives of this family. Mitochondrial functions including mitochondrial membrane potential (MMP), ATP, and ROS were evaluated in polymononuclear leukocytes (PMNs) derived from three deaf patients and three controls from this pedigree.
Results
Four of nine matrilineal relatives developed hearing loss at the variable age of onset. Two putative pathogenic mutations, m.5601C>T in tRNAAla and m.12311T>C in tRNALeu(CUN), were identified via PCR‐Sanger sequencing, as well as 34 variants that belonged to mtDNA haplogroup G2b2. Intriguingly, m.5601C>T mutation resided at very conserved nucleotide in the TψC loop of tRNAAla (position 59), while the T‐to‐C substitution at position 12311 located at position 48 in the variable stem of tRNALeu(CUN) and was believed to alter the aminoacylation and the steady‐state level of tRNA. Biochemical analysis revealed the impairment of mitochondrial functions including the significant reductions of ATP and MMP, whereas markedly increased ROS levels were found in PMNs derived from NSHL patients with m.5601C>T and m.12311T>C mutations. However, we did not detect any mutations in GJB2 and SLC26A4 genes.
Conclusion
Our data indicated that mt‐tRNAAla m.5601C>T and tRNALeu(CUN) 12311T>C mutations were associated with NSHL.
IntroductionMutations/variants in mitochondrial genomes are found to be associated with type 2 diabetes mellitus (T2DM), but the pathophysiology of this disease remains largely unknown.AimThe aim of this study is to investigate the relationship between mitochondrial DNA (mtDNA) variants and T2DM.MethodologyA maternally inherited T2DM pedigree is underwent clinical, genetic, and molecular assessment. Moreover, the complete mitochondrial genomes of the matrilineal relatives of this family are PCR amplified and sequenced. We also utilize the phylogenetic conservation analysis, haplogroup classification, and the pathogenicity scoring system to determine the T2DM-associated potential pathogenic mtDNA variants.ResultFour of seven matrilineal relatives of this pedigree suffered from T2DM with variable ages of onset. Screening for the entire mtDNA genes of matrilineal members reveals co-existence of ND5 T12338C and tRNAAla T5587C variants, as well as 21 genetic polymorphisms which belong to East Asian haplogroup F2. Interestingly, the T12338C variant causes the alternation of first amino acid Met to Thr, shortened two amino acids of ND5 protein. Furthermore, T5587C variant is located at position 73 in the 3’end of mt-tRNAAla and may have structural and functional consequences.Conclusions
The co-occurrence of ND5 T12338C and tRNAAla T5587C variants may impair the mitochondrial function, which are associated with the development of T2DM in this family.
Background
Mitochondrial dysfunctions caused by mitochondrial DNA (mtDNA) pathogenic mutations play putative roles in type 2 diabetes mellitus (T2DM) progression. But the underlying mechanism remains poorly understood.
Methods
A large Chinese family with maternally inherited diabetes and deafness (MIDD) underwent clinical, genetic, and molecular assessment. PCR and sequence analysis are carried out to detect mtDNA variants in affected family members, in addition, phylogenetic conservation analysis, haplogroup classification, and pathogenicity scoring system are performed. Moreover, the GJB2, GJB3, GJB6, and TRMU genes mutations are screened by PCR‐Sanger sequencing.
Results
Six of 18 matrilineal subjects manifested different clinical phenotypes of diabetes. The average age at onset of diabetic patients is 52 years. Screening for the entire mitochondrial genomes suggests the co‐existence of two possibly pathogenic mutations: tRNATrp A5514G and tRNASer(AGY) C12237T, which belongs to East Asia haplogroup G2a. By molecular level, m.A5514G mutation resides at acceptor stem of tRNATrp (position 3), which is critical for steady‐state level of tRNATrp. Conversely, m.C12237T mutation occurs in the variable region of tRNASer(AGY) (position 31), which creates a novel base‐pairing (11A‐31T). Thus, the mitochondrial dysfunctions caused by tRNATrp A5514G and tRNASer(AGY) C12237T mutations, may be associated with T2DM in this pedigree. But we do not find any functional mutations in those nuclear genes.
Conclusion
Our findings suggest that m.A5514G and m.C12337T mutations are associated with T2DM, screening for mt‐tRNA mutations is useful for molecular diagnosis and prevention of mitochondrial diabetes.
Mitochondrial DNA can be isotopically labeled to the virtual exclusion of nuclear DNA labeling in cell lines lacking the major soluble thymidine kinase (EC 2.7.1.21) but retaining a mitochondrial thymidine kinase. This characteristic provides a means for the selective assay of mitochondrial DNA during isolation and a determination of the cellular content of mitochondrial DNA. The simplest of the three isolation procedures compared in this study is shown to yield approximately two-thirds of the total mitochondrial DNA in the tissue culture cells examined. Mouse L cells containing predominantly a 10⁷ dalton closed circular mitochondrial DNA species have 1100 ± 250 molecules per cell. A second line of mouse L cells which has mitochondrial DNA of molecular weight 2 x 10⁷ contains approximately 900 molecules per cell. HeLa cells have at least four times the mitochondrial DNA mass per mitochondrial volume as L cells.
The complete sequence of the 16,569-base pair human mitochondrial genome is presented. The genes for the 12S and 16S rRNAs, 22 tRNAs, cytochrome c oxidase subunits I, II and III, ATPase subunit 6, cytochrome b and eight other predicted protein coding genes have been located. The sequence shows extreme economy in that the genes have none or only a few noncoding bases between them, and in many cases the termination codons are not coded in the DNA but are created post-transcriptionally by polyadenylation of the mRNAs.
Heat-treated samples of human mitochondrial DNA (mtDNA) exhibited a set of three low molecular weight DNA bands in addition to the major mtDNA band when electrophoresed in polyacrylamide gels. These DNA components were seen only after heat treatment or after relaxation of the mtDNA with a restriction endonuclease. The three components were single stranded and had sizes of 550, 585, and 629 nucleotides, close to the size (600 nucleotides) estimated from contour length measurements for the 7S DNA from the D loop of human mtDNA. Hybridization of the components with restriction endonuclease fragments of known position in the mtDNA confirmed this identification. Digestion of each 7S DNA component with the restriction endonuclease Hae III produced three fragments, two of which were identical in size among the components and the third of which varied. This third fragment, shown to be from the 5' end of each component, differed in length by approximately 35 nucleotides among the components. These results suggest that human 7S mtDNA synthesis is terminated at a distinct position and that it is either initiated at one of three possible sites in the same mtDNA or that the mtDNA population consists of three subpopulations, each differing from the others by the presence or absence of a nucleotide sequence immediately adjacent to the origin of replication.
Transfer RNA (tRNA) molecules have been labeled with 32P at the 5' end and subjected to S1 nuclease digestion. The products were analyzed by high-resolution gel electrophoresis. Three initiator tRNAs and six chain-elongating tRNAs were examined. S1 nuclease cleaved Escherichia coli tRNAfMet, yeast tRNAfMet, and mammalian tRNAfMet at the same two positions in the anticodon loop. In contrast, S1 nuclease cleaved the anticodon loop of E. coli tRNAmMet, yeast tRNAmMet, yeast tRNAPhe, Schizosaccharomyces pombe tRNAPhe, E. coli tRNA2Glu, and E. coli tRNATrp (su+) at four positions generally, except where a modified nucleotide in the wobble position inhibited the enzyme. The marked contrast between these cleavage patterns suggests a different conformation for the anticodon loops of these two classes of tRNA molecules. It is suggested that the specialized conformation in the anticodon loop of initiator tRNAs may be due to a special sequence of GC base pairs in the adjoining anticodon stem.
Initiation complexes formed by E. coli ribosomes in the presence of 32P-labeled A protein initiator region from R17 bacteriophage Rna have been treated with colicin E3 and disassembled by exposure to 1% sodium dodecyl sulfate. Electrophoresis on 9% polyacrylamide gels reveals a dissociable complex containing the 30-nucleotide-long messenger fragment and the 50-nucleotide-long colicin fragment, which arises from the 3' terminus of the 16S RNA. The complex is a pure RNA-RNA hybird; it is apparently maintained by a seven-base complementarity between the two RNA fragments. Detection of this mRNA-rRNA complex strongly supports the hypothesis that during the initiation step of protein biosynthesis the 3' end of 16S RNA base pairs with the polypurine stretch common to initiator regions in E. coli and bacteriophage mRNAs. The implications of our findings with respect to the molecular mechanism of initiation site selection and mRNA binding to ribosomes, the role of rRNA in ribosome function, and species specificity in translation are explored.
With a stepwise degradation and terminal labeling procedure the 3'-terminal sequence of E. coli 16S ribosomal RNA is shown to be Pyd-A-C-C-U-C-C-U-U-A(OH). It is suggested that this region of the RNA is able to interact with mRNA and that the 3'-terminal U-U-A(OH) is involved in the termination of protein synthesis through base-pairing with terminator codons. The sequence A-C-C-U-C-C could recognize a conserved sequence found in the ribosome binding sites of various coliphage mRNAs; it may thus be involved in the formation of the mRNA.30S subunit complex.
The 3-angstrom electron density map of crystalline yeast phenylalanine transfer RNA has provided us with a complete three-dimensional model which defines the positions of all of the nucleotide residues in the moleclule. The overall features of the molecule are virtually the same as those seen at a resolution of 4 angstroms except that many additional details of tertiary structure are now visualized. Ten types of hydrogen bonding are identified which define the specificity of tertiary interactions. The molecule is also stabilized by considerable stacking of the planar purines and pyrimidines. This tertiary structure explains, in a simple and direct fashion, chemical modification studies of transfer RNA. Since most of the tertiary interactions involve nucleotides which are common to all transfer RNA 's, it is likely that this three-dimensional structure provides a basic pattern of folding which may help to clarify the three-dimensional structure of all transfer RNA's.
The map positions in mouse mitochondrial DNA of the two ribosomal RNA genes and adjacent genes coding several small transcripts
have been determined precisely by application of a procedure in which DNA-RNA hybrids have been subjected to digestion by
Sl nuclease under conditions of varying severity. Digestion of the DNA-RNA hybrids with Sl nuclease yielded a series of species
which were shown to contain ribcaomal RNA molecules together with adjacent transcripts hybridized conjointly to a continuous
segment of mitochondrial DNA. There is one small transcript about 60 bases long whose gene adjoins the sequences coding the
5′-end of the small ribosomal RNA (950 bases) and which lies approximately 200 nucleotides from the D-loop origin of heavy
strand mitochondrial DNA synthesis. An 80-base transcript lies between the small and large ribosomal RNA genes, and genes
for two further short transcripts (each about 80 bases in length) abut the sequences coding the 3′-end of the large ribosomal
RNA $$$500 bases). The ability to isolate a discrete DNA-RNA hybrid species approximately 2700 base pairs in length containing
all these transcripts suggests that there can be few nucleotides in this region of mouse mitochondrial DNA which are not represented
as stable RNA species.
A unique transfer DNA has been identified in human and bovine mitochondria that lacks the “dihydrouridine” loop and stem structure.
This tRNA ie mitochondrially coded as shown by DNA sequence analysis of the human and bovine mitochondrial DNA. Sequence analysis
of the INA shows that it is post-transcriptionally modified by the addition of CCA at the 3′ terminus and that at least one
base is modified. As predicted by its anticodon (GCU, corresponding to the serine codons AGU/C) this tRNA can be aminoacylated
with serine when purified mitochondria are incubated in a medium containing 3H-serine.
The primary structure of a 3S serine tRNA from beef heart mitochondria has been determined using our new two-dimensional “read-off”
sequencing method (Tanaka, Y., Dyer, T.A. and Brownlee, G.G. (1980) Nucleic Acids Ree. 8, 1259–1272). When arranged in the “cloverleaf” form it shows unique features since (i) it completely lacks the dihydrouridine
arm, (ii) it has an extended “Tγ” loop, but lacks the T and γ residues, and (iii) it has only one minor base, N6(N-threonylcarbamoyl)adenosine, next to the anticodon.
Analysis of an almost complete mammalian mitochondrial DNA sequence has identified 23 possible tRNA genes and we speculate here that these are sufficient to translate all the codons of the mitochondrial genetic code. This number is much smaller than the minimum of 31 required by the wobble hypothesis. For each of the eight genetic code boxes with four codons for one amino acid we find a single specific tRNA gene with T in the first (wobble) position of the anticodon. We suggest that these tRNAs with U in the wobble position can recognize all four codons in these genetic code boxes either by a "two out of three" base interaction or by U.N wobble.
The cloned 18 S ribosomal RNA gene from Saccharomyces cerevisiae have been sequenced, using the Maxam - Gilbert procedure. From this data the complete sequence of 1789 nucleotides of the
18 S RNA was deduced. Extensive homology with many eucaryotic as well as E.coli ribosomal small subunit rRNA (S-rRNA) has
been observed in the 3′-end region of the rRNA molecule. Comparison of the yeast 18 S rRNA sequences with partial sequence
data, available for rRNAs of the other eucaryotes provides strong evidence that a substantial portion of the 18 S RNA sequence
has been conserved in evolution.
Chloramphenicol resistance in mammalian cells is cytoplasmically inherited. In yeast, a similar phenotype is caused by mutations in the mitochondrial DNA (mtDNA), and sequencing of carefully constructed strains has identified nucleotide monosubstitutions in the 3' region of the large (21S) rRNA gene which correlate with the antibiotic resistance. We have sequenced the corresponding section of mammalian mtDNA from chloramphenicol-resistant cell lines for comparison with the wild-type sequence. Differences between the sequences occur at positions similar to those altered in the yeast mutants, in a highly conserved region of the large (16S) rRNA gene.
The nucleotide sequence of ribosomal DNA coding for 16S rRNA from Zea mays chloroplast has been determined. A comparison with the 16S rRNA sequence from Escherichia coli reveals strong homology and thereby demonstrates the prokaryotic nature of chloroplast ribosomes from a higher plant.
The amino acid sequence of polypeptide II from beef heart cytochrome c oxidase is described. Comparision of this primary structure with those of azurins, plastocyanins and stellacyanins reveals clear homologies among them. Thus subunit II of the oxidase is a member of this copper protein family. The sequence homology indicates a copper binding site consisting of two invariant histidines and two sulfur-containing amino acids. Thus subunit II is like a blue copper protein with type I copper.
Comparison of the human mitochrondial DNA sequence of the cytochrome oxidase subunit II gene and the sequence of the corresponding beef heart protein shows that UGA is used as a tryptophan codon and not as a termination codon and suggests that AUA may be a methionine and not an isoleucine codon. The cytochrome oxidase II gene is contiguous at its 5' end with a tRNAAsp gene and there are only 25 bases at its 3' end before a tRNALys gene. These tRNA'S are different from all other known tRNA sequences.
The single-stranded mitochondrial DNA (mtDNA) displacement-loop initiation sequence (7S mtDNA) is hydrogen-bonded at the origin of replication in animal cell mtDNA. Analysis of 7S mtDNA from several cell sources indicates that this initiation sequence exists as a family of fragments of relatively discrete lengths. mtDNA from both mouse L cells and mouse liver has four major sizes of 7S mtDNA fragments, ranging from 500 to 580 nucleotides in length. The 5′-end region of each of these species is the same; thus, the size heterogeneity is due primarily to differences in length at the 3′-end of these molecules. By contrast, 7S mtDNA from both human KB cells and human liver exists in three major forms, ranging from 555 to 615 nucleotides in length, due to differences at both terminal regions. The mtDNA initiation sequence from Xenopus laevis oocytes also exists in at least two forms, 1350 and 1510 nucleotides in length. Thus, the maintenance of multiple forms of mtDNA initiation sequence appears to be a general phenomenon of animal cells, although the precise mechanism of synthesis or processing of these forms is variable.
The sequence of 42 nucleotides at the 5′-end of 7S mtDNA from mouse L cells has been determined and found to be rich in dGuo and dThd residues, with no apparent palindromes or potential secondary structures. We thus present sequence information on the replication origin of mtDNA, as defined by the naturally occurring 7S mtDNA.
An alternative method for codon reading, whereby only the first two codon nucleotides are recognized by the anticodon, is discussed and the experimental evidence for this "two of three" reading method is reviewed. Misreading of codons by the "two out of three" method could pose a significant threat to the fidelity of protein synthesis unless the genetic code is organized in such a way as to prevent this method from being used when it might compromise translational fidelity. Inspection of the genetic code shows that it is arranged in such a way that the "two out of three" reading method can be used without translational errors.
At least three different transfer RNAs are produced by in vitro processing of 30S ribosomal RNA which accumulates in RNAase III- strains of E. coli. Two of these tRNAs, tRNAGlu2 and tRNAIle1, have previously been shown to be "spacer tRNAs"--that is, genes for their synthesis are located in rRNA transcription units between the cistrons for 16S and 23S rRNAs (Lund et al., 1976). The third tRNA whose sequences are contained in 30S rRNA is tRNAAla1B. In addition to the tRNAs, 5S rRNA and several other 4S fragments are produced. Some of these 4S fragments may represent additional spacer tRNAs. One fragment, about 70 nucleotides long, arises from the 5' end of the 17S precursor of 16S rRNA. Four or five other tRNAs are hydrogen-bonded to 30S rRNA as we prepare it; one or more of these tRNAs may also be a spacer tRNA. The enzymes that process tRNAs out of 30S rRNA are associated with ribosomes, but can be removed from them by washing in 0.2 M NH4Cl; the enzymes required for 5S rRNA processing remain bound to the 0.2 M NH4Cl-washed ribosomes. Treatment of 30S rRNA with purified RNAase III produces 6-8S fragments which contain the sequences of tRNAGlu2, tRNAAla1B and 5S rRNA.
Incubation of isolated rat liver mitochondria with radioactive amino acids resulted in the charging of tRNAs for arginine,
asparagine, leucine, lysine, methionine, proline and valine. The aminoacyl-tRNAs were shown to be distinct from their cytosolic
counterparts by chromatography on RPC-5. By electrophoresis on urea polyacrylamide slab gels it was found that all these mitochondrial
aminoacyl-tRNAs were about 70–76 nucleotides long. The unique mitochondrial asparaginyl- and prolyl-tRNAs, not previously
identified in mammalian cells, were shown to hybridize to mtDNA. Mitochondrial leucyl-tRNA separated into 3 peaks on RPC-5
and the first species was shown to be different than a combination of the other two by molecular size and partial RNase T1 digestion patterns. Each was coded by a separate gene on mtDNA as shown by partial additivity of hybridization. Separate
genes for mitochondrial tRNAmMet and tRNAfMet separated by RPC-5 chromatography, were also demonstrated. These results bring to 21 the number of individual tRNAs coded
by mammalian mtDNA.
The major form of mouse L-cell mitochondrial DNA contains a small displacement loop at the replication origin, created by synthesis of a 550 to 670-nucleotide portion of the heavy strand. These short heavy-strand segments remain hydrogen-bonded to the parental light strand and are collectively termed 7 S mitochondrial DNA. The unique location of these 7 S mitochondrial DNAs at the heavy-strand origin suggests that they may function as primers in the synthesis of full-length heavy strands. Ribonucleotides have been detected at the 5′-end of some of these molecules, which are most likely remnants of primer RNAs. Using 5′-end labeling in vitro, we have determined that these ribonucleotides occur at several discrete positions along the nucleotide sequence of the origin region, which suggests that there may be variability in the precise initiation point of RNA priming or in the location of the switchover from RNA priming to DNA synthesis. The length of 5′-end RNA was estimated by alkali treatment of mitochondrial DNA prior to end labeling. A range of one to ten ribonucleotides was hydrolyzed from the 5′-end of some 7 S mitochondrial DNA strands. This is the first evidence of RNA priming at a eukaryotic cell DNA replication origin.
In the novel replication mechanism of closed circular mouse L-cell mitochondrial DNA synthesis one strand of the duplex (the heavy-strand) is initiated at a defined origin and proceeds unidirectionally. Synthesis of the complementary light-strand is initiated at a different origin, located approximately two-thirds genome length from the heavy-strand origin, and also proceeds unidirectionally. The initiation of light-strand synthesis does not occur until synthesis of the heavy-strand has extended past the light-strand origin region. One intriguing consequence of this asynchrony is that the heavy-strand origin functions in a DNA duplex, while the light-strand origin functions as a single-stranded template. In order to obtain the precise location of the light-strand origin we have isolated replicative molecules in which light-strand synthesis has begun and subjected them to digestion by a combination of the single-strand specific nuclease S1 and various restriction cndonucleases. By comparison of the sizes of the duplex fragments thus generated with those produced by cleavage of non-replicating molecules cleaved with the same enzymes we have located the 5′-end of daughter light-strands at a position 55 to 90 nucleotides from a HpaI cleavage site 0.67 genome length from the heavy-strand origin. The nucleotide sequence of a 318-base region surrounding this site, determined by chemical sequencing techniques, possesses the symmetry required for the formation of three hairpin loops. The most striking of these has a stem consisting of 12 consecutive basepairs and a 13-base loop. In the heavy-strand template, this loop contains 11 consecutive thymidine nucleotides. This light-strand origin region has been found to possess a remarkable degree of homology with several other prokaryotic and eukaryotic origin-related sequences, particularly those of the øX174 A region and the simian virus 40 EcoRII G fragment.
The number of mitochondrial DNA molecules in a cell population doubles at the same rate as the cell generation time. This could occur by a random selection of molecules for replication or by a process that ensures the replication of each individual molecule in the cell. We have investigated the rate at which mouse L cell mitochondrial DNA molecules labeled with 3H-thymidine during one round of replication are reselected for a second round of replication. Mouse L cells were labeled with 3H-thymidine for 2 hr, chased for various periods of time and then labeled with 5-bromodeoxyuridine for 4 hr immediately before mitochondrial DNA isolation. A constant fraction of 3H-thymidine-labeled mitochondrial DNA incorporated 5-bromodeoxyuridine after chase intervals ranging from 1.5-22 hr. This result demonstrates that mitochondrial DNA molecules replicated in a short time interval are randomly selected for later rounds of replication, and that replication of mitochondrial DNA continues throughout the cell cycle in mouse L cells.
The first closed circular product of mouse L-cell mitochondrial DNA synthesis is a zero superhelix density molecule. Both of the asynchronously synthesized mitochondrial DNA daughter molecules pass through the zero superhelix density state. These molecules have a mean lifetime of approximately one hour before conversion to supercoiled molecules containing approximately 100 superhelical turns. A low frequency of intermediates in the conversion of these two closed circular forms is demonstrable by agarose gel electrophoresis. The degree of sensitivity to alkali has been used to demonstrate that newly replicated mitochondrial DNA has the same content of ribonucleotides as mass-labeled mitochondrial DNA.
Pulse-chase radioactive labeling experiments using thymidine kinase-plus mouse LA9 cells have shown that the 7 S mitochondrial DNA initiation sequence of mitochondrial DNA is synthesized and turned over at a faster rate than previously determined. These pulse-chase labeling experiments have also determined that the replication time of mouse LA9 cell mitochondrial DNA is one hour. The halflife of pulse-labeled 7 S mitochondrial DNA initiation sequences is approximately 70 minutes. This turnover is so rapid that at least 95% of the mitochondrial DNA initiation sequences synthesized are lost to turnover without acting as primers for expansion synthesis of the mitochondrial DNA heavy strand. The mechanism of 7 S mitochondrial DNA turnover does not lead to significant accumulation of free 7 S mitochondrial DNA single-strands within mitochondria. Resynthesis of the 7 S mitochondrial DNA initiation sequence is sufficiently rapid that the majority of mitochondrial DNA molecules are maintained as displacement loop molecules. Approximately 20% of all nucleotides polymerized into mitochondrial DNA are incorporated into the 7 S initiation sequences. The size of newly synthesized 7 S mitochondrial DNA strands varies from about 500 to 620 nucleotides. Several size classes are resolved by polyacrylamide/urea gel electrophoresis and each class has approximately the same turnover rate.Mouse LD cells maintain their mitochondrial DNA genomes as unicircular, head-to-tail dimers. Since a significant fraction of these unicircular dimers contain only one displacement loop, the size of the initiation sequence in such molecules should be twice as long if synthesis of the strand is limited by the free energy of superhelix formation. An identical array of size classes of 7 S strands is obtained from this cell line as compared to mouse LA9 cells. This indicates that the extent of 7 S mitochondrial DNA synthesis is most likely determined by a nucleotide sequence specific event.
Nine transcripts complementary to mouse L cell mitochondrial DNA have been detected, sized and mapped to restriction fragments using the method of Berk and Sharp (1977). RNA isolated from L cell mitochondria was hybridized to 32P-labeled, cloned L cell mitochondrial DNA restriction fragments in 70% formamide under conditions 5 degrees C above the melting temperature of the DNA-DNA duplex, but approximately 15 degrees C below the melting temperature of the RNA-DNA duplex. The heteroduplexed material was then treated with the single-strand-specific nuclease S1, whick cleaves the single-stranded DNA not protected by RNA-DNA duplex formation into oligonucleotides and leaves intact 32P-labeled, single-stranded DNA replicas complementary to the transcripts. The single-stranded DNA replicas were then resolved and sized by alkaline agarose gel electrophoresis. Hybridization to strand-separated, 32P-labeled L cell mitochondria DNA restriction fragments under the same conditions showed that all nine transcripts hybridized exclusively to the heavy strand (H strand) of restriction fragments isolated as the dense strand from alkaline CsCl gradients, indicating that all stable transcripts 300 bases or longer detected by this technique originate from genes on the H strand. The two most abundant transcripts homologous to mitochondrial DNA map adjacent to the origin of replication. This result is consistent with map positions assigned to the large and small mitochondrial ribosomal RNAs isolated from Xenopus laevis and HeLa cells. Six of the other seven transcripts map continuously in approximately 40% of the genome. Only one transcript of 950 bases maps in the first quadrant of the genome as defined by the origin and direction of mitochondrial DNA replication, and it does not lie within the D loop region. The genetic function of the remaining 75% of this region of the genome is yet to be determined.
The amino acid specificity of the tRNA species coded for by HeLa cell mitochondrial DNA has been investigated by carrying out hybridizations between amino acid-tRNA complexes labeled in the amino acid and separated mitochondrial DNA strands.The results indicate that there are in HeLa cell mitochondria at least 17 distinct tRNA species hybridizable with mitochondrial DNA, which are specific for 16 amino acids. For 14 of the 16 amino acids, amino-acyl-tRNA synthetase activities distinct from the cytoplasmic ones have been detected in mitochondria. The remaining four amino acids (asparagine, glutamine, histidine and proline) have consistently failed to charge to any detectable extent mitochondrial tRNA species hybridizable with mitochondrial DNA.No obvious relationship appears to exist between the amino acids incorporated into tRNAs hybridizable to mitochondrial DNA and the previously observed pattern of chloramphenicol-sensitive amino acid incorporation by HeLa cell mitochondria.
The sequence A-A-U-A-A-A is present in six different purified messenger RNA molecules (specifically the alpha-and beta-globulin mRNAs of rabbit and human, the immunoglobulin light chain mRNA of mouse (MOPC 21) and the ovalbumin mRNA of chicken) about 20 residues away from the 3'-terminal poly (A) sequence. In addition, a large selection of the 3' non-coding regions of rabbit and human globulin mRNAs (both the alpha and beta globin mRNAs) are 85% homologous, demonstrating that this region is significantly conserved in evolution.
Pulse-chase studies were performed, and the topology and specific activity of pulso-label in closed circular molecules determined, to investigate the mechanism by which newly segregated open circular daughter molecules are converted to the major stable form of mitochondrial DNA, D-mtDNA. These and previous results suggest the following model: newly segregated daughter molecules are first converted to closed circular molecules with a superhelix density of approximately zero. Watson-Crick turns are then removed by an unwinding mechanism to produce a stable intermediate with a superhelix density of ≈−0·03. Initiation of heavy-strand synthesis and polymerization of 450 nucleotides then occurs without further unwinding of the parental strands to yield the D-mtDNA molecule.
The specific activity of pulse-label in catenated dimer mitochondrial DNA is 0·71 and 0·96 of that of monomer mitochondrial DNA after 60 and 150 minutes of chase, respectively. The topology of the interlocked monomers in pulse-labeled catenated dimers indicates that catenanes are not formed by aberrant segregation of daughter molecules. No linear intermediates in the segregation and closure of daughter molecules were detected. The simplest interpretation of these results is that monomers and catenated oligomers are in rapid equilibrium.
Examination of in vivo long-labeled, pulse-labeled and pulse-chase-labeled mitochondrial DNA has corroborated and extended the basic elements of the displacement model of replication. Mitochondrial DNA molecules are shown to replicate an average of once per cell doubling in exponentially growing cultures. Analysis of the separate strands of partially replicated molecules indicates that replication is highly asynchronous with heavy-strand synthesis preceding light-strand synthesis. Native and denatured pulse-labeled replicating molecules exhibit sedimentation properties predicted by the displacement model of replication. Pulse-label incorporated into molecules isolated in the lower band region of ethidium bromide/cesium chloride gradients is found primarily in heavy daughter strands. Pulse-label incorporated into molecules isolated in the upper band region is found primarily in light daughter strands. The results of a series of pulse-chase experiments indicate that the complete process of replication requires approximately 120 minutes. Both daughter molecules are shown to segregate in an open circular form. They are then converted to closed circular molecules having a superhelix density near zero. After closure, the 7 S heavy-strand initation sequence is synthesized, and this process is accompanied by nicking, unwinding and closing of at least one of the parental strands resulting in the formation of the D-loop structure. The 7 S heavy-strand initiation sequence of the D-loop structure is not stable and turns over with a half-life of 7·9 hours. We suggest that all in vivo forms of parental closed circular mitochondrial DNA have superhelix densities of near zero, and that the previously observed superhelix density of closed circular mitochondrial DNA, σ~ −0·02, results from the loss of the 7 S heavy-strand initiation sequence from D-loop mitochondrial DNA molecules during isolation.
It is suggested that while the standard base pairs may be used rather strictly in the first two positions of the triplet, there may be some wobble in the pairing of the third base. This hypothesis is explored systematically, and it is shown that such a wobble could explain the general nature of the degeneracy of the genetic code.
This chapter focuses on determination of fragment order through partial digests and multiple enzyme digests. The chapter describes the basic principles of two techniques for fragment ordering: analysis of partial digestion products and multiple enzyme digestion. Ordering DNA fragments by partial endonuclease digestion is analogous to a sequencing technique for RNA. Multiple enzyme digestion for ordering DNA fragments employs an approach used for sequencing proteins and RNA. For DNA, the cleavage products of one endonuclease are characterized with respect to size and are then digested with a second endonuclease. Analysis of the resultant double-digestion products establishes the relationship between the cleavage sites of the two enzymes. The chapter presents a model study that develops a physical map of the SV40 genome. The chapter describes the procedures for digestion of DNA with endonuclease and for analysis of cleavage products. Ordering fragments through analysis of partial digestion products is illustrated for the two sets of SV40 DNA fragments produced by cleavage with HincII and Hind lII. Taq I and Barn H1 are used in multiple enzyme digestions with HincII and Hind lII to generate a complex physical map that includes the cleavage sites for all four enzymes.
Bacteriophage T4 codes for eight tRNAs, whose genes are tightly clustered between genes e and 57. The clustering of the transfer RNA genes suggested that these tRNAs are synthesized in a single transcriptional unit. Transcriptional experiments in vitro and experiments using the λ-T4 hybrid in vivo have supported this notion and located a T4 tRNA promoter.Detailed biochemical pathways of parts of the T4 tRNA processing are known. Maturation of the 3′ end precedes that of the 5′ end, and the 3′ ends of three dimeric precursors are matured in different ways by tRNA nucleotidyltransferase and ribonuclease BN. All T4 tRNAs require RNAase P for their 5′ maturation. Monomeric and dimeric precursors accumulate in the absence of RNAase P.How these monomeric and dimeric precursors are synthesized from their primary transcript, however, is poorly understood. Using the λ-T4 hybrid, carrying tRNA genes, and a fine restriction map of the tRNA gene cluster, we have determined the DNA sequence of the tRNA gene cluster, hoping that understanding of gene organization may suggest how the tRNAs are processed from their primary transcript. The DNA sequence of the tRNA gene cluster indicates that the monomeric and dimeric precursors are generated from their primary transcript by single endonucleolytic cleavages. We propose a model for the maturation of T4 tRNAs by predicting such an endonuclease (or endonucleases). The proposed endonucleolytic activities exist in the host. This model answers various questions about processing and reveals the unique features of T4 tRNA processing.
Animal mitochondrial DNA (mtDNA) maintains a displacement loop (D loop) at the heavy strand origin of replication. These D loops represent sharply limited synthesis of heavy strands and provide a unique opportunity to examine the termination of DNA synthesis. Direct sizing at the nucleotide level indicates that the 3' ends of D-loop strands of human and mouse mtDNA are discrete and map within three to five nucleotides on the complementary template strand. In the case of human mtDNA, there is a single trinucleotide stop point 51-53 nucleotides downstream from a 15-nucleotide template sequence (3'T-A-A-C-C-C-A-A-A-A-A-T-A-C-A 5') which is repeated four times in the mouse mtDNA D-loop region 3'(T-A-A-Py-Py-A-A-A-T-T-A-C-A 5'). The stop points of the five major mouse D-loop strands are 24-63 nucleotides downstream from the four repeated template sequences. These results suggest that the arrest of D-loop strand elongation is an event determined by template sequence.
The complete DNA sequence of the rRNA genes of mouse L cell mtDNA provides a basis for the examination of the nucleotide sequence of this region in a mutant mouse cell line that is resistant to chloramphenicol, a known inhibitor of mitochondrial protein synthesis. Resistance to chloramphenicol (CAPr) is conferred by a cytoplasmic determinant that is linked to mtDNA restriction endonuclease site polymorphisms. We have determined the sequence of a 212-nucleotide region of mtDNA from a CAPr mouse cell line that encodes a portion of the 1582-nucleotide large rRNA. This sequence is located 107-318 nucleotides from the 5' end of the heavy strand coding sequence, which corresponds to the 3' end of the rRNA. There is a single nucleotide difference in the large rRNA gene from CAPr cells, an A-to-G transition 243 nucleotides from the 5' end of the coding sequence. This single transition is located within a region of 10 nucleotides tht is otherwise completely homologous to human and yeast mitochondrial large rRNAs and Escherichia coli 23S rRNA and is positioned immediately adjacent to a single nucleotide transversion known to occur in a yeast CAPr mutant. This characterization of a mammalian mitochondrial mutant at the nucleotide level directly demonstrates that a mutant phenotype may result from a single mtDNA nucleotide change in an animal cell.
The nucleotide sequence of an E. coli isoleucine tRNA (tRNAIle minor) specific for the codon AUA was determined by postlabeling procedures using only 2.5 micrograms (0.05 A260 unit) of the material. The sequence was pG-G-C-C-C-C-U-s4U-A-G-C-U-C-A-G-U-Gm-G-D-D-A-G-A-G-C-A-A-G-C-G-A-C-U-N+-A-U-t6A-A-psi-C-G-C-U-U-G-m7G-acp3U-C-G-C-U-G-G-T-psi-C-A-A-G-U-C-C-A-G-C-A-G-G-G-G-C-C-A-C-C-AOH. The nucleotide sequences in the regions of the D arm and T psi C arm of tRNAIle minor were quite similar to the corresponding regions of tRNAIle major. However, the sequences in the CCA stem and anticodon stem of tRNAIle minor were different from those of tRNAIle major. The overall homology between the two isoleucine tRNAs was 68%. E. coli tRNALys, tRNAMet, tRNAValIIA and tRNAArg also have relatively high sequence homology with tRNAIle minor.
The nucleotide sequence of 23S rDNA from Zea mays chloroplasts has been determined. Alignment with 23S rDNA from E.coli reveals 71 percent homology when maize 4.5S rDNA is included as an equivalent of the 31 end of E.coli 23S rDNA. Among the conserved sequences are sites for base modification, chloramphenicol sensitivity and ribosomal subunit
interaction. A proposal for the base pairs formed between 16S and 23S rRNAs during the 3OS/5OS subunit interaction is presented.
The alignment of maize 23S rDNA with that of E.coli reveals three small insertion sequences of 25/65 and 78 base pairs, whereas maize 16S rDNA shows only deletions when compared
with the E.coli species.
The 3'-terminal sequence of the large ribosomal subunit ("17S") RNA of hamster mitochondria has been examined by means of oligonucleotide fingerprint analysis of 3' end-labeled samples. Patterns from partial acid or alkaline hydrolysates indicated marked heterogeneity and suggested an array of termini that included GGUUAOH, AnAOH and AnGOH (where n is about 10). Patterns from samples subjected to exhaustive digestion with ribonucleases T1 plus A, or with each separately, confirmed these inferences, and strikingly demonstrated the existence of oligoadenylated families of 3'-terminal sequences. Under the assumption that the oligoadenylate was added posttranscriptionally, these results indicated that the 3'-terminal transcribed moieties consist of variants of YAGGUUOH. Counting from the last U, we inferred that many such sequences end at U1 and G4, while smaller numbers end at G3 and A5; and that major sites of oligoadenylation occur at U1 and G4, and a minor site occurs at A5. This novel combination of imprecise termination of transcription or "sizing" of transcripts, and variable addition of adenylate residues, is discussed with regard to the mechanism of expression of the mammalian mitochondrial ribosomal RNA genes.
A 3'-end proximal segment of most of the putative mRNAs encoded in the heavy strand of HeLa cell mtDNA has been partially sequences and aligned with the DNA sequence. In all cases, the 3'-end nucleotide of the individual mRNA coding sequences has been found to be immediately contiguous to a tRNA gene or another mRNA coding sequence. These and previous results indicate that the heavy (H) strand sequences coding for the rRNA, poly(A)-containing RNA and tRNA species form a continuum extending over almost the entire length of this strand. We propose that the H strand is transcribed into a single polycistronic RNA molecule, which is processed later into mature species by precise endonucleolytic cleavages which occur, in most cases, immediately before and after a tRNA sequence.
The 5'-end proximal sequences of all the putative mRNAs coded for by the heavy strand of HeLa cell mitochondrial DNA have been determined and aligned with the DNA sequence. All these mRNAs start directly at, or very near to, an AUG or AUA triplet, with the exception of one which starts at an AUU. The available evidence indicates that the terminal or subterminal AUGs and AUAs, and possibly also the terminal AUU, are initiator codons for the corresponding polypeptides. In most cases, the individual mRNA coding sequences are flanked on their 5' side by a tRNA gene, without any intervening nucleotide.
The mouse L-cell line LD maintains its mitochondrial DNA genome in the form of a head-to-tail unicircular dimer of the monomeric 16,000 base-pair species. This situation permits a comparison of the mechanism of replication of this dimeric molecule with our previous studies of replication of monomeric mouse L-cell mitochondrial DNA. Whereas monomeric mitochondrial DNA requires about one hour for a round of replication, the dimeric molecule requires almost three hours. Denaturing agarose gel electrophoretic analyses of replicative intermediates reveals several discrete size classes of partially replicated daughter strands of dimeric mitochondrial DNA. This suggests that replication occurs with specific discontinuities in the rate of daughter strand synthesis. The strand specificity of these daughter strands was determined by hybridization with 32P-labeled DNA representing either the heavy or light strand mitochondrial DNA sequence. The sizes and strand specificities of these discrete daughter strands indicate that the same set of control sequences is functional in both dimer and monomer mitochondrial DNA replication.Immediately following a round of replication, the majority of dimeric mitochondrial DNA molecules contain displacement loops, as assessed by their sensitivity to nicking within the displaced DNA strand by single-strand DNA specific S1 nuclease under conditions which leave supercoiled DNA intact. This result is in contrast with the conformation of newly replicated monomeric mitochondrial DNA molecules, which lack both superhelical turns and displacement loops. This indicates that dimeric mitochondrial DNA proceeds through a different series of post-replicative processing steps than does monomeric mitochondrial DNA. We postulate that intermediates at late stages of dimeric mitochondrial DNA replication contain displacement loops which remain intact following closure of the full-length daughter strands.
The 5′ end proximal regions of the two HeLa cell mitochondrial rRNAs (16S and 12S) have been sequenced by partial enzymatic digestions of the 5′ end 32P-labeled RNAs followed by electrophoretic fractionation of the products on polyacrylamide/urea gels. Likewise, a 600 nucleotide mitochondrial DNA (mit-DNA) fragment, previously designated Δ8aHae, that contains the 5′ end of the 12S rRNA, has been sequenced by the method of Maxam and Gilbert. The first 71 nucleotides of the 12S rRNA and the DNA coding sequence have been aligned and found to be colinear. This observation extends to the 5′ end proximal segment of the 12S rRNA gene the conclusion of earlier experiments, indicating the absence of intervening sequences in the body of the small rRNA gene. A comparison of the 12S rRNA coding sequence determined here (286 nucleotides) with that of an E. coli 16S rRNA gene has revealed significant homologies. Previous electron microscopic analysis of hybrids between the heavy (H) strand of mit-DNA and ferritin-labeled mitochondrial 4S RNAs had shown the presence of a 4S RNA gene near the 5′ end of the 12S rRNA coding sequence. In the present work, a search of the DNA sequence for a cloverleaf structure has indeed revealed the occurrence of a tRNAPhe gene. The unexpected finding, however, has been that the 3′ end of this gene is contiguous to the 5′ end of the 12S rRNA coding sequence without any intervening nucleotides.
The complete DNA sequence of the ribosomal RNA region of mouse L cell mitochondrial DNA has been determined. Genes for the small (12S) and large (16S) rRNAs have been precisely located by direct sequencing of the termini of the two mature rRNAs. A comparison of the lengths (956 and 1582 nucleotides) and terminal sequences of the mature rRNAs with the DNA coding sequences indicates that mouse mt rRNAs are not spliced. Computer analysis of the complete DNA sequence has identified three potential transfer RNA genes. A gene for phenylalanine tRNA is located immediately adjacent to the 5' end of the 12S rRNA gene, a valine tRNA gene occupies the entire region between the 12S and 16S rRNA genes and a leucine tRNA gene is located immediately adjacent to the 3' end of the 16S gene. Hybridization of 32P-labeled, tRNA-sized mtRNA to selected DNA restriction endonuclease fragments from the rRNA region confirms the existence of small, abundant mtRNAs transcribed from these DNA sequences. All three tRNA genes and both rRNA genes are transcribed from the heavy strand of mtDNA. The mt rRNA sequences exhibit notable homologies to other rRNAs and, in particular, to those of E. coli. Within the 3' terminal 50 nucleotides, the mouse mt 12S rRNA contains a potential 10 bp hairpin structure and a sequence of 15 consecutive nucleotides common to the RNA of the small ribosomal subunit in all systems, but does not contain the mRNA binding site (ACCUCC) found in E. coli and corn chloroplast rRNAs. The mt tRNA genes do not have the 3' terminal CCA sequence encoded in the DNA, nor do they contain any intervening sequences. Two of the three tRNSa would lack many features which are known to be strictly conserved in all other nonorganelle tRNAs which have been sequenced. The fact that all the genes in this region are directly contiguous with at most one intervening nucleotide suggests that the entire region is transcribed into a polycistronic precursor RNA which is processed by endonucleolytic cleavages. The organization of the genes of the rRNA operon of mouse mtDNA, when compared to the organization of rRNA and tRNA genes in bacterial or eucaryotic nuclear genomes, provides evidence for the endosymbiotic hypothesis of the biogenesis of mammalian mitochondria.
A detailed transcription map of HeLa cell mitochondrial DNA (mtDNA) has been constructed by using the S1 protection technique to localize precisely the sequences coding for the ribosomal RNA (rRNA) and poly(A)-containing species on the physical map of the DNA. This transcription map has been correlated with the positions of the tRNA genes derived from the mtDNA sequence. It has been shown that, with the exception of the D loop and another small segment near the origin of replication, the mtDNA sequences are completely saturated by the rRNAs, poly(A)-containing RNAs and tRNA coded for by the two strands. No evidence for intervening sequences has been found. The sequences coding for the individual poly(A)-containing RNA and rRNA species appear to be immediately contiguous on one side, and most frequently on both sides, to tRNA coding sequences. Furthermore, the H strand sequences coding for the two rRNAs, the poly(A)-containing RNAs and the tRNAs appear to be adjacent to each other, extending from coordinate 2/100 to coordinate 95/100 of the genome relative to the origin taken as 0/100. The results are consistent with a model of transcription of the H strand in the form of a single molecule which is processed into mature RNA species by precise endonucleolytic cleavages, occurring in almost all cases immediately before and after a tRNA sequence. The tRNA sequences may play an important role as recognition signals in the processing of the primary transcripts.
Expression of the mouse mitochondrial DNA genome Cold Spring Harbor Labo-ratory). in press Initiator tRNAs have a unique anticodon loop conformation
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The nucleotide sequence of a small (3S) seryl-tRNA (anticodon GCU) from beef heart mitochondria.
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- Brownlee G.G.
Expression of the mouse mitochondrial DNA genome.
- Van Etten R.A.
- Michael N.L.
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- Clayton D.A.