• Journal of Korean Medical Science
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• v.33 no.52
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• pp.337.1-337.14
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• 2018

Background: Mitochondrial heteroplasmy, the co-existence of different mitochondrial polymorphisms within an individual, has various forensic and clinical implications. But there is still no guideline on the application of massively parallel sequencing (MPS) in heteroplasmy detection. We present here some critical issues that should be considered in heteroplasmy studies using MPS. Methods: Among five samples with known innate heteroplasmies, two pairs of mixture were generated for artificial heteroplasmies with target minor allele frequencies (MAFs) ranging from 50% to 1%. Each sample was amplified by two-amplicon method and sequenced by Ion Torrent system. The outcomes of two different analysis tools, Torrent Suite Variant Caller (TVC) and mtDNA-Server (mDS), were compared. Results: All the innate heteroplasmies were detected correctly by both analysis tools. Average MAFs of artificial heteroplasmies correlated well to the target values. The detection rates were almost 90% for high-level heteroplasmies, but decreased for low-level heteroplasmies. TVC generally showed lower detection rates than mDS, which seems to be due to their own computation algorithms which drop out some reference-dominant heteroplasmies. Meanwhile, mDS reported several unintended low-level heteroplasmies which were suggested as nuclear mitochondrial DNA sequences. The average coverage depth of each sample placed on the same chip showed considerable variation. The increase of coverage depth had no effect on the detection rates. Conclusion: In addition to the general accuracy of the MPS application on detecting heteroplasmy, our study indicates that the understanding of the nature of mitochondrial DNA and analysis algorithm would be crucial for appropriate interpretation of MPS results.

• Journal of Animal Reproduction and Biotechnology
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• v.35 no.2
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• pp.198-206
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• 2020

Food and agricultural production sector, especially livestock production is vital for Mongolia's economic and social development. Domestic sheep play key roles for Mongolians, providing food (meat, milk) and raw materials (wool, sheepskin), but genetic diversity, origin of sheep populations in Mongolia have not been well studied. Studies of population genetic diversity is important research field in conservation and restoration of animal breeds and genetic resources. Therefore, this study aimed to investigate genetic characteristics and estimate origin through the analysis of mitochondrial DNA control region D-loop and Cytochrome b of Mongolian indigenous sheep (Mongolian native, Orkhon and Altanbulag) and one Europe sheep (Suffolk). As a result of there were found, 220 SNPs (Single nucleotide polymorphism) in the D-loop region, 28 SNPs in the Cytochrome B region, furthermore, 77 Haplotypes. The nucleotide diversity was only found in D-loop region (n = 0.0184). Phylogenetic analysis showed that 3 (A, B, and C) of 5 haplogroups of sheep have been identified in our research. Haplogroup C was only found in Mongolian indigenous sheep. Haplogroup D and E were not observed. As a result of haplogroups, haplogroup A was dominant (n = 46 of 94 sheeps), followed by haplogroup B (n = 36) and haplogroup C (n = 12). Sequence analysis showed that T deletion, insertion and heteroplasmy in D-loop region occurred at a high rate in Mongolian indigenous sheep population (T insertion = 47, T deletion = 83). The heteroplasmy, which has never been found in Mongolian sheep, has been newly discovered in this study. As a result, the Mongolian sheep varieties, which mainly derived from Asia, were in hybridization with European sheep varieties.

• Proceedings of the KSAR Conference
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• 2002.06a
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• pp.40-40
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• 2002

Nuclear transfer (NT) has the potential to produce large number of identical progeny and would greatly benefit ongoing research efforts, Cloned animals produced by NT, however, may not be genetically identical to the donor cell. In NT procedures, nucleus genes originate from donor cell, and mitochondrial genes originate from recipient oocytes. (omitted)

• Journal of Genetic Medicine
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• v.10 no.1
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• pp.20-26
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• 2013

Mitochondrion is an intracellular organelle with its own genome. Its function in cellular metabolism is indispensable that mitochondrial dysfunction gives rise to multisystemic failure. The manifestation is most prominent with tissues of high energy demand such as muscle and nerve. Mitochondrial myopathies occur not only by mutations in mitochondrial genome, but also by defects in nuclear genes or secondarily by toxic insult on mitochondrial replication. Currently curative treatment modality does not exist and symptomatic treatment remains mainstay. Administration of L-arginine holds great promise according to the recent reports. Advances in mitochondrial RNA import might enable a new therapeutic strategy.

• Journal of Life Science
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• v.9 no.1
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• pp.1-4
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• 1999

Large-scale deletions of mitochondrial DNA(mtDNA) have been documented in patients with mitochondrial myopathies and seem to be especially frequent in patients with Kearns-Sayre syndrome (KSS). About one third of all patients shows a 4,977 bp deletion, known as the "common deletion", that removes a segment of DNA that includes several genes encoding for respiratory chain subunits. In this disorder, the population of deleted mtDNA molecules coexists with population of normal, wild-type full length mtDNAs, a situation known as heteroplasmy. We have performed polymerase chain reaction (PCR) on paraffin-embedded muscle tissues from two korean KSS patients. The PCR analysis revealed the existence of two amplified fragments, the deleted fragments, the deleted fragment of 123 bp characteristic for common deletion and the wild-type fragment of 152 bp.of 152 bp.

• BMB Reports
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• v.53 no.1
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• pp.3-9
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• 2020

The mitochondrial genome encodes 13 proteins that are components of the oxidative phosphorylation system (OXPHOS), suggesting that precise regulation of these genes is crucial for maintaining OXPHOS functions, including ATP production, calcium buffering, cell signaling, ROS production, and apoptosis. Furthermore, heteroplasmy or mis-regulation of gene expression in mitochondria frequently is associated with human mitochondrial diseases. Thus, various approaches have been developed to investigate the roles of genes encoded by the mitochondrial genome. In this review, we will discuss a wide range of techniques available for investigating the mitochondrial genome, mitochondrial transcription, and mitochondrial translation, which provide a useful guide to understanding mitochondrial gene expression.

• Journal of Animal Science and Technology
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• v.63 no.2
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• pp.281-294
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• 2021

Although somatic cell nuclear transfer (SCNT) is frequently employed to produce cloned animals in laboratories, this technique is expensive and inefficient. Therefore, the handmade cloning (HMC) technique has been suggested to simplify and advance the cloning process, however, HMC wastes many oocytes and leads to mitochondrial heteroplasmy. To solve these problems, we propose a modified handmade cloning (mHMC) technique that uses simple laboratory equipment, i.e., a Pasteur pipette and an alcohol lamp, applying it to porcine embryo cloning. To validate the application of mHMC to pig cloning, embryos produced through SCNT and mHMC are compared using multiple methods, such as enucleation efficiency, oxidative stress, embryo developmental competence, and gene expression. The results show no significant differences between techniques except in the enucleation efficiency. The 8-cell and 16-cell embryo developmental competence and Oct4 expression levels exhibit significant differences. However, the blastocyst rate is not significantly different between mHMC and SCNT. This study verifies that cloned embryos derived from the two techniques exhibit similar generation and developmental competence. Thus, we suggest that mHMC could replace SCNT for simpler and cheaper porcine cloning.

• Journal of Genetic Medicine
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• v.1 no.1
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• pp.39-43
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• 1997

The mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) is inherited maternally, in which the MTTL1*MELAS 3243 mutation has been most commonly found as a heteroplasmy of A to G point mutation in the $tRNA^{Leu(UUR)}$ gene. The MTTL1*MELAS 3271 mutation is known to be the second common mutation, though clinical features of both mutations are not remarkably different. Recently, a variety of minor mutations have been reported in patients with MELAS. In this study, major efforts have been made to investigate the allele frequency of major three mutations including MTTL1*MELAS 3243, 3252, 3271 in 10 Korean families with MELAS probands. The PCR and subsequent direct sequencing of the PCR product in the regions spanning these three mutation sites were employed to identify the mutation in each proband. All family members have been screened for the presence of these three mutations by PCR-RFLP assay using Apa I, Acc I and Bfr I restriction enzymes. The MTTL1*MELAS 3243 mutation was most commonly found (7 out of 10 families tested) followed by the MTTL1*MELAS 3271 which was identified in 1 out of 10 families. In the remaining 2 families none of three mutations were found, indicating the presence of either nuclear mutation or yet unidentified mitochondrial DNA mutation in these families.

• Journal of Animal Science and Technology
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• v.65 no.4
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• pp.767-778
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• 2023

The aim of the research is to identify that porcine oocytes can function as recipients for interspecies cloning and have the ability to develop to blastocysts. Furthermore each mitochondrial DNA (mtDNA) in interspecises cloned embryos was analyzed. For the study, mouse-porcine and porcine-porcine cloned embryos were produced with mouse fetal fibroblasts (MFF) and porcine fetal fibroblasts (PFF), respectively, introduced as donor cells into enucleated porcine oocytes. The developmental rate and cell numbers of blastocysts between intraspecies porcine-porcine and interspecies mouse-porcine cloned embryos were compared and real-time polymerase chain reaction (PCR) was performed for the estimate of mouse and porcine mtDNA copy number in mouse-porcine cloned embryos at different stages.There was no significant difference in the developmental rate or total blastocyst number between mouse-porcine cloned embryos and porcine-porcine cloned embryos (11.1 ± 0.9%, 25 ± 3.5 vs. 10.1 ± 1.2%, 24 ± 6.3). In mouse-porcine reconstructed embryos, the copy numbers of mouse somatic cell-derived mtDNA decreased between the 1-cell and blastocyst stages, whereas the copy number of porcine oocyte-derived mtDNA significantly increased during this period, as assessed by real-time PCR analysis. In our real-time PCR analysis, we improved the standard curve construction-based method to analyze the level of mtDNA between mouse donor cells and porcine oocytes using the copy number of mouse beta-actin DNA as a standard. Our findings suggest that mouse-porcine cloned embryos have the ability to develop to blastocysts in vitro and exhibit mitochondrial heteroplasmy from the 1-cell to blastocyst stages and the mouse-derived mitochondria can be gradually replaced with those of the porcine oocyte in the early developmental stages of mouse-porcine cloned embryos.

• Journal of Life Science
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• v.19 no.4
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• pp.467-471
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• 2009

The entire D-loop region of the porcine mitochondrial DNA (mtDNA) was amplified from six pig breeds (Landrace, Duroc, Large White, Korean native pig, Berkshire, and Hampshire) using a primer set designed on the basis of reported porcine mtDNA sequences. From analyses through cloning, DNA sequencing and multiple sequence alignment, an 11-bp (TAAAACACTTA) duplication was observed after known tandem repetition in the D-loop region, which promoted hetroplasmy in mtDNA. Although the existence of the 11-bp duplication has been previously reported in Duroc and Japanese native pigs, there have not been any attempts to know the characteristics of this duplication in other breeds so far. A 150 bp fragment containing the 11-duplication was amplified and typed by polyacrylamide gel electrophoresis (PAGE). All Large Whites had two duplication units and Duroc showed heteromorphic patterns, 11.2% (9/80) of the animals had the 11-bp duplication in total. On the other hand, Landrace, Berkshire, Hampshire and Korean native pigs were non-duplicated. This result showed that the 11-bp duplication could be used as a breed-specific DNA marker for distinguishing pure Landrace and Large White breeds.