당뇨모델 조직내 미토콘드리아 DNA 복제수 감소

김영미,송지현,임선희,한복기,이홍규 국립보건연구원 1998 국립보건원보 Vol.35 No.-

제2형 당뇨병 환자 직계 자손의 인슐린 감수성과 말초혈액 mtDNA양의 관계

송지현,오지영,성연아,김영미,박경수,이홍규 국립보건연구원 2000 국립보건원보 Vol.37 No.-

Intramitochondrial transfer and engineering of mammalian mitochondrial genomes in yeast

Yoon, Young Geol,Koob, Michael D. Elsevier 2019 Mitochondrion Vol.46 No.-

<P><B>Abstract</B></P> <P>Mitochondrial genomes (mtDNA) depend on the nuclear genome with which they have evolved to provide essential replication functions and have been known to replicate as xenotransplants only in the cells of closely related species. We now report that complete mouse mitochondrial genomes can be stably transplanted into the mitochondrial network in yeast devoid of their own mtDNA. Our analyses of these xenomitochondrial yeast cells show that they are accurately replicating intact mouse mtDNA genomes without rearrangement and that these mtDNA genomes have the same overall topology as the mtDNA present in the mouse mitochondrial network (<I>i.e.</I>, circular monomers). Moreover, non-mtDNA replication and selection sequences required for maintaining the mitochondrial genomes in bacterial hosts are dispensable in these yeast mitochondria and could be efficiently and seamlessly removed by targeted homologous recombination within the mitochondria. These findings demonstrate that the yeast mtDNA replication system is capable of accurately replicating intact mammalian mtDNA genomes without sequence loss or rearrangement and that yeast mitochondria are a highly versatile host system for engineering complete mammalian mitochondrial genomes.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Entire mouse mtDNA can be stably transferred to the mitochondrial network in yeast devoid of their own mtDNA. </LI> <LI> The transferred mtDNA is accurately replicated with the same overall topology as the mtDNA present in the mouse mitochondria. </LI> <LI> The foreign non-mtDNA sequences could be seamlessly eliminated by targeted homologous recombination within the mitochondria. </LI> </UL> </P>

Chronic Progressive External Ophthalmoplegia에서의 미토콘드리아 유전자 결실

고경남(Kyung Nam Koh) 박수현(Su Yeon Park) 황희(Hee Hwang) 채종희(Jong Hee Chae) 최지은(JI Eun Choi) 김기중(Ki Joong Kim) 황용승(Yong Seung Hwang) 대한소아신경학회 2004 대한소아신경학회지 Vol.12 No.1

목적 : Chronic progressive external ophtahlmoplegia(CPEO)는 미토콘드리아 근병증의 흔한 표현형 중 하나로, 미토콘드리아 DNA(mtDNA)의 단일 결실, 다발성 결실, 점 돌연변이, 핵 운전자 이상 등의 원인이 알려져 있다. 저자들은 CPEO 환자에서 다기관 침범 여부와 mtDNA의 결실을 확인하기 위해 본 연구를 시행하였다. 방법 : 임상 소견에 근거하여 진단 받은 CPEO 환자 2명을 대상으로, 안과적 진찰과 신경학적 진찰을 시행하였다. 혈청 lactate와 갑상선 기능 검사를 시행하였고 근육 효소치를 측정하였으며, 근전도 검사, 근조직 검사, 심전도, 심초음과, 뇌 자기공명영상, 청력 검사를 시행하였다. 혈액과 근육에서 각각 mtDNA를 추출하여 PCR 증폭, RFLP assay, 직접 염기서열분석을 시행하였다. 결과 : 소아기 때부터 시작된 진행성 외안근 마비 외에 갑상선 기능저하증, 저신장증, 감각신경성 난청소견을 보였던 첫 번째 환자는 근육과 혈액의 mtDNA에서 7.591 kb의 대규모 단일 결실이 확인 되었다. 진행성 외안근 마비 증상만을 보인 두 번째 환자는 근육 mtDNA에서 6.191 kb의 대규모 단일 결실이 확인되었다. 결론 : 저자들은 다양한 임상 양상을 보인 2명의 CPEO 환자에서 mtDNA의 단일 결실을 확인하였다. 진행성 외안근 마비가 있는 환자에 대해서는 미토콘드리아 질환의 감별을 위한 근조직 검사 및 분자 유전학적 진단과 함께 여러 기관 침범 여부에 대한 적절한 검사와 추적 관찰이 필요할 것이다. Purpose : Chronic progressive external ophtahlmoplegia(CPEO) is a common phenotype of mitochondrial myopathy. CPEO has wide clinical spectrum with variable severity and can be divided into 3 groups; Kearns-Sayre syndrome, ophthalmoplegia plus and isolated CPEO. Single large-scale deletion, multiple deletions, point mutation of muscle mitochondrial DNA(mtDNA) and nuclear gene defect are associated with CPEO. We reviewed two cases of CPEO associated with the gene defect of mtDNA. Methods : mtDNA was extracted from muscle biopsy tissue and blood leukocytes. We carried out polymerase chain reaction(PCR), restriction fragment length polymorphism (RFLP) assay and automated sequencing of the mtDNA. Results : Case I presented with progressive external ophthalmoplegia, short stature, hypothyroidism and sensorineural hearing loss. A novel 7.6 kb-deletion was found in muscle and leukocyte mtDNA. Case 2 presented with isolated CPEO. A novel 6.2 kb-deletion was found in muscle mtDNA. Conclusion : We detected novel single large-scale deletion of mtDNA in 2 cases of CPEO with various clinical manifestations in our population. We have to investigate multi-organ involvement with regular follow-up for patients who present with progressive ophthalmoplegia

경남지역 수달( Lutra lutra)의 mitochondrial DNA D-loop지역과 microsatellite marker를 이용한 계통유전학적 유연관계 분석

Moon-Sung Park(박문성),Hyun-Tae Lim(임현태),Ki-Cheol Oh(오기철),Young-Rok Moon(문영록),Jong-Gap Kim(김종갑),Jin-Tae Jeon(전진태) 한국생명과학회 2011 생명과학회지 Vol.21 No.3

국내에 서식하는 수달의 경우 멸종 위기Ⅰ급 종으로 지정되어 국가적인 차원에서 관리하고 있는 보호종이다. 수달의 유전자원 보호 및 체계적인 관리를 위한 기초자료로 활용하기 위해 경남지역에 서식하는 수달의 계통유전학적 유연관계를 mtDNA D-loop 지역의 염기서열분석과 MS marker 분석을 통하여 실시하였다. 그 결과 mtDNA D-loop 지역의 676 bp 부분만 보았을 때 5개의 SNP가 확인되었으며, 6개의 haplotype이 추정되었다. 진주 인근 지역과 거제도 인근 지역에서 수집한 시료는 지역 내 유전적 거리가 지역 간의 유전적 거리보다는 가까운 것을 확인 할 수 있었고, 진주와 거제도 지역 간의 유전적 거리는 확연히 구분이 되었다. MrBays의 Bayesian Markov chain Monte Carlo 분석법을 이용하여 추정한 phylogeny 분석결과 뚜렷한 2개 그룹(진주와 거제/창녕 그룹)으로 분류 되었다. Parsimonious median-joining network [5] 분석의 결과 또한 2개의 뚜렷한 그룹으로 분류되어 phylogeny 분석결과와 일치하는 결과를 보였다. MS marker를 이용하여 추정한 유전적 거리지수를 활용하여 추정한 consensus tree의 결과 또한 크게 2개의 그룹으로 분류 되며, 첫 번째 그룹에는 거제도지역시료, 진주인근지역 시료 일부 그리고 창녕 우포늪에서 채취한 시료가 하나의 그룹으로 나뉘어 졌으며, 두 번째 그룹에는 진주인근 지역에서 채취한 시료만이 포함되어 하나의 그룹을 형성하여, mtDNA를 이용하여 분석한 것과 일부 다른 결과를 보였다. 이러한 결과의 차이는 모계를 추정하는 mtDNA와 상염색체 상의 MS marker의 특성에 기인한 것으로 보이나, 경상남도에 서식하는 수달을 크게 진주와 거제지역의 수달로 구분하는 것에는 유사한 결과를 보여 서식지 별 유전적 고정현상이 있음을 확인할 수 있었다. 하지만 좀 더 정확한 검증을 위해서는 수달의 full mtDNA 분석 및 국내에서 서식하는 수달에 적합한 MS marker발굴을 통한 대립유전자형을 분석하는 추가 연구가 필요하며, 전국 단위의 수달 시료를 확보하여 유전적 유연관계 분석을 실시한다면 한국 내 수달의 보전 및 보호에 도움이 될 것으로 사료되어 진다. The otter (Lutra lutra) in Korea is classified as a first grade endangered species and is managed under state control. We performed a phylogenetic analysis of the otter that inhabits the Changnyeong, Jinju, and Geoje areas in Gyeongsangnamdo, Korea using mtDNA and microsatellite (MS) markers. As a result of the analysis using the 676-bp D-loop sequence of mtDNA, six haplotypes were estimated from five single nucleotide polymorphisms. The genetic distance between the Jinju and Geoje areas was greater than distances within the areas, and the distance between Jinju and Geoje was especially clear. From the phylogenetic tree estimated using the Bayesian Markov chain Monte Carlo analysis by the MrBays program, two subgroups, one containing samples from Jinju and the other containing samples from the Changnyeong and Geoje areas were clearly identified. The result of a parsimonious median-joining network analysis also showed two clear subgroups, supporting the result of the phylogenetic analysis. On the other hand, in the consensus tree estimated using the genetic distances estimated from the genotypes of 13 MS markers, there were clear two subgroups, one containing samples from the Jinju, Geoje and Changnyeong areas and the other containing samples from only the Jinju area. The samples were not identically classified into each subgroup defined by mtDNA and MS markers. It could be inferred that the differential classification of samples by the two different marker systems was because of the different characteristics of the marker systems used, that is, the mtDNA was for detecting maternal lineage and the MS markers were for estimating autosomal genetic distances. Nonetheless, the results from the two marker systems showed that there has been a progressive genetic fixation according to the habitats of the otters. Further analyses using not only newly developed MS markers that will possess more analytical power but also the whole mtDNA are needed. Expansion of the phylogenetic analysis using otter samples collected from the major habitats in Korea should be helpful in scientifically and efficiently maintaining and preserving them.

Original Article: Biochemistry/Molecular Biology : Rapid Isolation of Mitochondrial DNA-Depleted Mammalian Cells by Ethidium Bromide and Dideoxycytidine Treatments

( Young Geol Yoon ),( Yoo Jin Oh ),( Young Hyun Yoo ) 한국응용생명화학회(구 한국농화학회) 2014 Journal of Applied Biological Chemistry (J. Appl. Vol.57 No.3

Mitochondrial DNA (mtDNA)-depleted (ρ0) cells areoften used as mtDNA recipients to study the interaction betweenthe nucleus and mitochondria in mammalian cells. Therefore, it iscrucial to obtain mtDNA-depleted cells with many differentnuclear backgrounds for the study. Here, we demonstrate a rapidand reliable method to isolate mammalian mtDNA-depleted cellsinvolving treatment with the antimitochondrial agents ethidiumbromide (EtBr) and 2``, 3``-dideoxycytidine (ddC). After a shortexposure to EtBr or ddC, followed by rapid clonal isolation, wewere able to generate viable mtDNA-depleted cells from mouseand human cells and were able to successfully repopulate themwith exogenous mitochondria from platelets isolated from mouseand human blood samples. These mtDNA-depleted cells can beused to characterize the nuclear mitochondrial interactions and tostudy mtDNA-associated defects in mammalian cells. Our methodof isolating mtDNA-depleted cells is practical and applicable to avariety of cell types.

Holstein의 유량과 유지방 생산에 미치는 Mitochondrial DNA D-loop 영역의 염기 서열 변이 효과

오재돈,공홍식,이학교,전광주 한국동물생명공학회(구 한국동물번식학회) 2005 Reproductive & developmental biology Vol.29 No.1

본 연구는 젖소(Holstein종)의 mtDNA D-loop 영역 염기변이 다형성과 경제형질간의 관련성을 분석하기 위하여 수행하였다. 젖소의 mtDNA D-loop 영역에서 단일염기의 치환에 의해 총 35개의 polymorphic site가 확인되었다. 그중 주요 Polymorphic site의 염기변이 빈도는 106, 169, 16057, 16231 및 16255 지역에서 높은 빈도의 염기치환이 검출되었다. 169 지역은 A가 G로 염기치환이 일어났고 그 빈도는 0.555로 높게 나타났으며 염기치환에 의한 산유량 효과는 1259.6 kg(P<0.05)로 나타났다. 유지방과의 관계를 분석한 결과 16118 지역은 A가 G로 치환되는 빈도가 0.02로 검출되었으며, 16135 지역은 T가 C로 치환되는 빈도가 0.02로 검출되었고, 16302 지역은 G가 A로 치환되는 빈도가 0.04로 검출되었다. 이 지역들이 유지방에 미치는 변이 효과는 - 156, - 118.15 및 - 67.77 kg(P<0.1)으로 나타났다. 본 연구에서 검출한 젖소 mtDNA내 D-loop 영역의 염기서열 변이 빈도와 유량, 유지방과의 연관성 분석 결과 등은 젖소집단의 유전적 변이성 추정과 좀 더 다양한 경제형질과의 관련성 분석으로 다양한 유전적 지표인자 발굴에 도움이 될 것이며 이를 통해 분자유전학적 기법을 이용한 젖소의 육종전략을 확립하는데 기초 자료가 되는 것은 물론 분자육종학적인 연구에 기초 자료로서 유용하게 활용할 수 있을 것으로 기대된다. This study was performed to analyze the sequence variation in mtDNA D-loop and their effects on milk and milk fat production in Holstein cows. The analyzed sequences were compared with previously published sequences from other cattle breeds (GenBank J01394). PCR was performed to amplify a total of 964 bp between nucleotide 15758 and 383 within D-loop region of mtDNA using specific primers. Thirty five polymorphic sites by nucleotide substitution were found in mtDNA. The frequencies of positions at 106, 169, 16057, 16231 and 16255 nt with high levels of sequence polymorphism were 0.090, 0.555, 0.055, 0.090 and 0.050, respectively. The substitution effect at 169 nt was found significant on milk production, and substitution effect at 16118, 16139 and 16302 nt was highly significant (p<0.1) on milk fat production. Polymorphism of mtDNA sequence in D-loop region might be useful for the analysis of cytoplasmic genetic variation and associations with the other economically important traits and maternal lineage analysis in Holstein cows.

미토콘드리아 DNA 데이터베이스의 통계학적 유용성 확인을 위한 파라미터 탐색

정종민,이지현,조소희,이숭덕 대한법의학회 2014 대한법의학회지 Vol.38 No.2

Recently, studies on mitochondrial DNA (mtDNA) have increased rapidly. Conventional parameters, such as diversity index, pairwise comparison, are used tointerpret and validate data on autosomal DNA; however, the use of these parametersto validate data from mitochondrial DNA databases (mtDNA DBs) needs to be verifiedbecause of the different transmission patterns of mtDNA. This study was done to verifythe use of these conventional parameters and to test the“ coverage concept”for anew parameter. The mtDNA DB is not very big; however, it is necessary to check howthe change in parameters corresponds to the DB size. For this, we artificiallyrearranged a Korean DB into several small sub-DBs of variable sizes. The resultsshow that the diversity in nucleotide variations and the different haplotype numbers donot vary as the size of DB increases. However, the “coverage”changed a lot. Thecoverage increased from 0.113 in a DB of 100 people to 0.260 in a DB of 653 people. Additionally, using the“ coverage concept”, we predicted how the total number of haplotypeschanged with variations in the sub-DB size and compared the predicted resultwith final result. In conclusion,“ coverage”, in addition to conventional statistical parameters,can be used to check the usability of an mtDNA DB. Finally, we tried to predictthe size of the whole mtDNA number in Korea using“ saturation concept”.

돼지 mtDNA D-loop 지역의 Large White 특이 중복현상 탐지

김재환(Jae-Hwan),한상현(Sang-Hyun Han),이성수(Sung-Soo Lee),고문석(Moon-Suk Ko),이정규(Jung-Gyu Lee),전진태(Jin-Tae Jeon),조인철(In-Cheol Cho) 한국생명과학회 2009 생명과학회지 Vol.19 No.4

돼지 6품종(Landrace, Duroc, Large White, 한국재래돼지, Berkshire, Hampshire)을 대상으로 기존에 보고된 서열을 바탕으로 제작한 primer를 이용하여 mtDNA D-loop 전체영역을 증폭하였다. 증폭된 PCR product를 cloning 및 DNA sequencing, 다중염기서열비교를 통하여 분석한 결과, mtDNA에서 heteroplasmy가 나타나는 D-loop 내 tandem repeat region 이후에 11-bp 중복이 존재하는 것을 확인하였다. 이런 중복현상은 일본재래돼지와 Duroc에서 보고되었지만, 이를 이용한 돼지 품종별 중복현상의 빈도 및 분포에 관한 연구는 이루어져있지 않다. 품종별 11-bp 중복현상을 분석하기 위해서 6품종을 대상으로 중복지역을 포함한 약 150 bp 절편을 증폭하였으며, PAGE 방법을 통하여 분석하였다. 그 결과 본 연구에서 사용한 품종들 중 모든 Large White에서 중복현상이 발생하는 것을 확인하였으며, Duroc인 경우 11.2% (9/80)에서 중복현상이 확인되었다. 반면에 Landrace, 한국재래돼지, Berkshire 및 Hampshire에서는 전혀 발견되지 않았다. 이런 결과로서, 11-bp 중복현상의 분석은 현재 구별이 불가능한 Landrace와 Large White를 구별할 수 있는 유용한 DNA marker로서 사용이 가능할 것이다. 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.

Mitochondrial genome and diverse inheritance patterns in Pleurotus pulmonarius

Li-Yun Ye,You-Jin Deng,Irum Mukhtar,Guo-Liang Meng,Yan-Jiao Song,Bing Cheng,Jin-bing Hao,Xiao-Ping Wu 한국미생물학회 2020 The journal of microbiology Vol.58 No.2

Pleurotus pulmonarius, a member of the Pleurotaceae family in Basidiomycota, is an edible, economically important mushroom in most Asian countries. In this study, the complete mitochondrial genomes (mtDNA) of three P. pulmonarius strains – two monokaryotic commercial (J1-13 and ZA3) and one wild (X1-15) – were sequenced and analyzed. In ZA3 and X1-15, the mtDNA molecule was found to be a single circle of 68,305 bp and 73,435 bp, respectively. Both strains contain 14 core protein-coding genes and two ribosomal RNA (rRNA) subunit genes. The ZA3 strain has 22 transfer RNA (tRNA) genes and nine introns: eight in cytochrome c oxidase subunit 1 (cox1), and one in the rRNA large subunit (rnl). Monokaryotic J1-13 and ZA3 mtDNAs were found to be similar in their structure. However, the wild strain X1-15 contains 25 tRNA genes and only seven introns in cox1. Open reading frames (ORFs) of ZA3/J1-13 and X1-15 encode LAGLIDADG, ribosomal protein S3, and DNA polymerase II. In addition, mtDNA inheritance in J1-13, ZA3, and X1-15 was also studied. Results showed that the mtDNA inheritance pattern was uniparental and closely related to dikaryotic hyphal location with respect to the parent. Results also show that mtDNA inheritance is influenced by both the parental nuclear genome and mitogenome in the zone of contact between two compatible parents. In summary, this analysis provides valuable information and a basis for further studies to improve our understanding of the inheritance of fungal mtDNA.