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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>
Yoon, Young Hoon,Kim, Doo Soo,Kim, MinJung,Park, Min Sang,Lee, Young-Chul,Kim, Kwang Ho,Kim, Il Tae,Hur, Jaehyun,Lee, Seung Geol Elsevier 2018 ELECTROCHIMICA ACTA Vol.266 No.-
<P><B>Abstract</B></P> <P>Bimetallic compound, composed of two different metal elements, has emerged as an important class of electrode system. Amorphous carbon materials are widely used in anodes to reduce the internal resistance of electrodes. Therefore, SnSe bimetallic compound uniformly dispersed in acetylene black as a carbon-support has been fabricated for lithium ion batteries by high energy mechanical milling (HEMM) process under argon atmosphere. The SnSe-C composite retains a reversible capacity of 564 mAh g<SUP>−1</SUP> with a coulombic efficiency of 99.8%, at a current rate of 100 mA g<SUP>−1</SUP> after 50 cycles. In the high rate capability test, the SnSe-C composite exhibits the charge capacity of 530 mAh g<SUP>−1</SUP> at 5000 mA g<SUP>−1</SUP> charge rate. Electrochemical impedance spectroscopy (EIS) results indicate that SnSe-C composite shows small increase of surface resistance than that of plain SnSe composite. The enhanced cycle stability of SnSe-C composite can be attributed to the amorphous carbon additive that offers high electrical conductivity as well as a buffer matrix that prevents the volume change during cycling.</P>
Re-engineering the mitochondrial genomes in mammalian cells
Young Geol Yoon,Michael D Koob,Young Hyun Yoo 대한해부학회 2010 Anatomy & Cell Biology Vol.43 No.2
Mitochondria are subcellular organelles composed of two discrete membranes in the cytoplasm of eukaryotic cells. They have long been recognized as the generators of energy for the cell and also have been known to associate with several metabolic pathways that are crucial for cellular function. Mitochondria have their own genome, mitochondrial DNA (mtDNA), that is completely separated and independent from the much larger nuclear genome, and even have their own system for making proteins from the genes in this mtDNA genome. The human mtDNA is a small (~16.5 kb) circular DNA and defects in this genome can cause a wide range of inherited human diseases. Despite of the significant advances in discovering the mtDNA defects, however, there are currently no effective therapies for these clinically devastating diseases due to the lack of technology for introducing specific modifications into the mitochondrial genomes and for generating accurate mtDNA disease models. The ability to engineer the mitochondrial genomes would provide a powerful tool to create mutants with which many crucial experiments can be performed in the basic mammalian mitochondrial genetic studies as well as in the treatment of human mtDNA diseases. In this review we summarize the current approaches associated with the correction of mtDNA mutations in cells and describe our own efforts for introducing engineered mtDNA constructs into the mitochondria of living cells through bacterial conjugation.
Yoon, Young Geol,Kim, Jung Min,Kim, Sun Chang,Choi, Ja Young,Lee, Jun Hyoung 생화학분자생물학회 1970 BMB Reports Vol.34 No.4
Excision and amplification of pre-determined, large genomic segments (taken directly from the genome of a natural host, which provides an alternative to conventional cloning in foreign vectors and hosts) was explored in human cells. In this approach, we devised a procedure for excising a large segment of human genomic DNA, the iNOS gene, by using the Cre/loxP system of bacteriophage Pl and amplifying the excised circles with the EBNA-1/oriP system of the Epstein-Barr virus. Two loxP sequences, each of which serves as a recognition site for recombinase Cre, were integrated unidirectionally into the 5'-UTR and 3'-UTR regions of the iNOS gene, together with an oriP sequence for conditional replication. The traps-acting genes cre and EBNA-1, which were under the control of a tetracycline responsive P_(hcmv^*-1) promoter, were also inserted into the 5'-UTR and 3'-UTR regions of the iNOS gene, respectively, by homologous recombination. The strain carrying the inserted elements was stably maintained until the excision and amplification functions were triggered by the induction of cre and EBNA-1. Upon induction by doxycycline, Cre excised the iNOS gene that was flanked by two ZoxP sites and circularized it. The circularized iNOS gene was then amplified by the EBNA-1/oriP-system. With this procedure, approximately a 45.8-kb iNOS genomic fragment of human chromosome 17 was excised and successfully amplified in human cells. Our procedure can be used effectively for the sequencing of unclonable genes, the functional analysis of unknown genes, and gene therapy
( 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.
Yoon, Young-Geol,Choi, Ja-Young,Kim, Jung-Min,Lee, Jun-Hyoung,Kim, Sun-Chang Korean Society for Biochemistry and Molecular Biol 2001 Journal of biochemistry and molecular biology Vol.34 No.4
Excision and amplification of pre-determined, large genomic segments (taken directly from the genome of a natural host, which provides an alternative to conventional cloning in foreign vectors and hosts) was explored in human cells. In this approach, we devised a procedure for excising a large segment of human genomic DNA, the iNOS gene, by using the Cre/loxP system of bacteriophage P1 and amplifying the excised circles with the EBNA-1/oriP system of the Epstein-Barr virus. Two loxP sequences, each of which serves as a recognition site for recombinase Cre, were integrated unidirectionally into the 5'-UTR and 3'-UTR regions of the iNOS gene, together with an oriP sequence for conditional replication. The traps-acting genes cre and EBNA-1, which were under the control of a tetracycline responsive $P_{hcmv^*-1}$ promoter, were also inserted into the 5'-UTR and 3'-UTR regions of the iNOS gene, respectively, by homologous recombination. The strain carrying the inserted elements was stably maintained until the excision and amplification functions were triggered by the induction of cre and EBNA-1. Upon induction by doxycycline, Cre excised the iNOS gene that was flanked by two ZoxP sites and circularized it. The circularized iNOS gene was then amplified by the EBNA-1/oriP-system. With this procedure, approximately a 45.8-kb iNOS genomic fragment of human chromosome 17 was excised and successfully amplified in human cells. Our procedure can be used effectively for the sequencing of unclonable genes, the functional analysis of unknown genes, and gene therapy.