Papers by Young Geol Yoon
Mitochondrion, May 1, 2019
Mitochondrial genomes (mtDNA) depend on the nuclear genome with which they have evolved to provid... more 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.e., 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 a nd that yeast mitochondria are a highly versatile host system for engineering complete mammalian mitochondrial genomes.
Gene, Sep 1, 2011
Mitochondrial transcription factor A (Tfam) binds to and organizes mitochondrial DNA (mtDNA) geno... more Mitochondrial transcription factor A (Tfam) binds to and organizes mitochondrial DNA (mtDNA) genome into a mitochondrial nucleoid (mt-nucleoid) structure, which is necessary for mtDNA transcription and maintenance. Here, we demonstrate the mtDNA-organizing activity of mouse Tfam and its transcript isoform (Tfam iso), which has a smaller high-mobility group (HMG)-box1 domain, using a yeast model system that contains a deletion of the yeast homolog of mouse Tfam protein, Abf2p. When the mouse Tfam genes were introduced into the ABF2 locus of yeast genome, the corresponding mouse proteins, Tfam and Tfam iso , can functionally replace the yeast Abf2p and support mtDNA maintenance and mitochondrial biogenesis in yeast. Growth properties, mtDNA content and mitochondrial protein levels of genes encoded in the mtDNA were comparable in the strains expressing mouse proteins and the wild-type yeast strain, indicating that the proteins have robust mtDNA-maintaining and-expressing function in yeast mitochondria. These results imply that the mtDNA-organizing activities of the mouse mt-nucleoid proteins are structurally and evolutionary conserved, thus they can maintain the mtDNA of distantly related and distinctively different species, such as yeast.
Nucleic Acids Research, Sep 7, 2005
We have developed a method for transferring exogenous DNA molecules into isolated mammalian mitoc... more We have developed a method for transferring exogenous DNA molecules into isolated mammalian mitochondria using bacterial conjugation. In general, we accomplish this by (i) inserting an origin of DNA transfer (oriT) sequence into a DNA construct, (ii) transforming the construct into an appropriate Escherichia coli strain and then (iii) introducing the mobilizable DNA into mitochondria through conjugation. We tested this approach by transferring plasmid DNA containing a T7 promoter sequence into mitochondria that we had engineered to contain T7 RNA polymerase. After conjugation between E.coli and mitochondria, we detected robust levels of T7 transcription from the DNA constructs that had been transferred into the mitochondria. This approach for engineering DNA constructs in vitro and subsequent transfer into mitochondria by conjugation offers an attractive experimental system for studying many aspects of vertebrate mitochondrial gene expression and is a potential route for transforming mitochondrial networks within mammalian cells.
Anatomy & Cell Biology, 2010
Mitochondria are subcellular organelles composed of two discrete membranes in the cytoplasm of eu... more Mitochondria are subcellular organelles composed of two discrete membranes in the cytoplasm of eukaryotic cells. Th ey 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. Th e 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 signifi cant advances in discovering the mtDNA defects, however, there are currently no eff ective therapies for these clinically devastating diseases due to the lack of technology for introducing specifi c modifi cations into the mitochondrial genomes and for generating accurate mtDNA disease models. Th e 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 eff orts for introducing engineered mtDNA constructs into the mitochondria of living cells through bacterial conjugation.
Nucleic Acids Research, Mar 1, 2003
We have devised an ef®cient method for replicating and stably maintaining entire mitochondrial ge... more We have devised an ef®cient method for replicating and stably maintaining entire mitochondrial genomes in Escherichia coli and have shown that we can engineer these mitochondrial DNA (mtDNA) genome clones using standard molecular biological techniques. In general, we accomplish this by inserting an E.coli replication origin and selectable marker into isolated, circular mtDNA at random locations using an in vitro transposition reaction and then transforming the modi®ed genomes into E.coli. We tested this approach by cloning the 16.3 kb mouse mitochondrial genome and found that the resulting clones could be engineered and faithfully maintained when we used E.coli hosts that replicated them at moderately low copy numbers. When these recombinant mtDNAs were replicated at high copy numbers, however, mtDNA sequences were partially or fully deleted from the original clone. We successfully electroporated recombinant mouse mitochondrial genomes into isolated mouse mitochondria devoid of their own DNA and detected robust in organello RNA synthesis by RT±PCR. This approach for modifying mtDNA and subsequent in organello analysis of the recombinant genomes offers an attractive experimental system for studying many aspects of vertebrate mitochondrial gene expression and is a ®rst step towards true in vivo engineering of mammalian mitochondrial genomes.
Biotechnology Letters, Jun 30, 2009
We have devised a method for cloning an entire mammalian mitochondrial genome (mtDNA) in Escheric... more We have devised a method for cloning an entire mammalian mitochondrial genome (mtDNA) in Escherichia coli using PCR-based amplification and sequential ligation. Here we test this approach by cloning the complete mouse mtDNA. The mtDNA was divided into four to five fragments based on unique restriction enzyme sites and amplified by high-fidelity long-range DNA polymerase. The synthesized fragments were cloned individually to test their toxicity in the E. coli host and then combined sequentially into a vector containing the E. coli R6K origin of DNA replication. The synthetic complete mouse mtDNA clones were replicated stably and faithfully in E. coli when maintained at moderately low copy numbers per cell. The sequence integrity of the synthetic mouse mtDNA clones was confirmed by nucleotide sequencing; no mutations or rearrangements in the genome were found. This approach can facilitate the cloning of entire mammalian mitochondrial genomes in E. coli and assist in the introduction of desired modifications into the mitochondrial genome.
Mitochondrion, May 1, 2007
A detailed molecular understanding of mitochondrial fusion and fission in mammalian cells is rapi... more A detailed molecular understanding of mitochondrial fusion and fission in mammalian cells is rapidly emerging. In this report, we demonstrate for the first time cross-species mitochondrial fusion between distantly related species using green and red fluorescent proteins targeted to the mitochondrial matrix. We found that mouse mitochondria were able to efficiently fuse to unmodified mitochondria of human cells and that the contents of the mitochondrial matrix were completely mixed in less than 4 h. We also observed that mitochondria from the mtDNA-less (ρ 0) mouse cells can homogeneously fuse to the mitochondria of human cells. We were, however, unable to maintain human mitochondrial DNA in the mouse cells. These results indicate that mitochondrial fusion proteins in mouse and human cells have enough functional homology to mediate efficient cross-species mitochondrial fusion, but mouse nuclear and human mitochondrial genomes have not retained functional compatibility with one another.
Journal of Genetics and Genomics, Apr 1, 2011
Due to technical difficulties, the genetic transformation of mitochondria in mammalian cells is s... more Due to technical difficulties, the genetic transformation of mitochondria in mammalian cells is still a challenge. In this report, we described our attempts to transform mammalian mitochondria with an engineered mitochondrial genome based on selection using a drug resistance gene. Because the standard drug-resistant neomycin phosphotransferase confers resistance to high concentrations of G418 when targeted to the mitochondria, we generated a recoded neomycin resistance gene that uses the mammalian mitochondrial genetic code to direct the synthesis of this protein in the mitochondria, but not in the nucleus (mitochondrial version). We also generated a universal version of the recoded neomycin resistance gene that allows synthesis of the drug-resistant proteins both in the mitochondria and nucleus. When we transfected these recoded neomycin resistance genes that were incorporated into the mouse mitochondrial genome clones into mouse tissue culture cells by electroporation, no DNA constructs were delivered into the mitochondria. We found that the universal version of the recoded neomycin resistance gene was expressed in the nucleus and thus conferred drug resistance to G418 selection, while the synthetic mitochondrial version of the gene produced no background drug-resistant cells from nuclear transformation. These recoded synthetic drug-resistant genes could be a useful tool for selecting mitochondrial genetic transformants as a precise technology for mitochondrial transformation is developed.
<b>Copyright information:</b>Taken from "Transformation of isolated mammalian mi... more <b>Copyright information:</b>Taken from "Transformation of isolated mammalian mitochondria by bacterial conjugation"Nucleic Acids Research 2005;33(16):e139-e139.Published online 12 Sep 2005PMCID:PMC1201378.© The Author 2005. Published by Oxford University Press. All rights reserved S17-1 harboring pT7hpGFP (lanes 1 and 2) and pT7hpGFP+oriT (lanes 3 and 4), respectively, were used as the donors. All mating samples were treated with 200 µg/ml of ampicillin, 200 U of DNase I and 50 µg/ml of RNase A for 2 h at 37°C after 5 h mating in an agarose block. After isolating total RNA from the mating mixture, RT–PCR was carried out using -specific primers (see Materials and Methods). Expected band size of the RT–PCR products was 423 bp. Lane M, 1 kb plus DNA ladder (Life Technologies). Control lanes (lanes 1 and 3) in which reverse transcriptase (RT) was omitted are indicated by minus signs.
<b>Copyright information:</b>Taken from "Transformation of isolated mammalian mi... more <b>Copyright information:</b>Taken from "Transformation of isolated mammalian mitochondria by bacterial conjugation"Nucleic Acids Research 2005;33(16):e139-e139.Published online 12 Sep 2005PMCID:PMC1201378.© The Author 2005. Published by Oxford University Press. All rights reserved () Structures of control and mobilizing plasmids, pT7hpGFP and pT7hpGFP+oriT, respectively. The sequence was cloned in front of the promoter () and was designed to form a double-stranded hairpin-loop promoter after transferring the single-stranded plasmid DNA into the mitochondria by conjugation. Only the mobilizing plasmid contains the sequence. () A schematic representation for the formation of a double-stranded hairpin-loop promoter. Once a DNA strand is transferred to T7RNAP-mitochondria by conjugation, spontaneous annealing around the promoter region in the ssDNA will form a fully duplexed promoter inside the mitochondria. T7RNAP can recognize this duplexed sequence (,) and initiate transcription of an RNA molecule (red characters).
Journal of Microbiology and Biotechnology, 2020
Supplementary Figure S1. Growth phenotypes of the wild-type and Tfam yeast strains. (A and B) Gro... more Supplementary Figure S1. Growth phenotypes of the wild-type and Tfam yeast strains. (A and B) Growth of the MCC109 + wild-type and MCC109 Tfam + strains on synthetic minimal medium containing ethanol and glycerol (SEG). The colonies that grew on the SEG medium possessed active mitochondrial function, and the red color shown in each colony was due to the ade2-101 mutation. (C and D) Growth of the MCC109 + wild-type and MCC109 Tfam + strains on complete glucose medium (YPD). The MCC109 + wild-type cells grown on the YPD medium showed a red color and maintained active mitochondrial function by retaining the yeast mtDNA (C). The majority of MCC109 Tfam + cells grown on YPD medium turned white and lost mitochondrial function by losing the yeast mtDNA (D).
Mitochondrion, 2019
Mitochondrial genomes (mtDNA) depend on the nuclear genome with which they have evolved to provid... more 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.e., 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 a nd that yeast mitochondria are a highly versatile host system for engineering complete mammalian mitochondrial genomes.
International journal of oncology, 2012
The Bcl-2 protein is known to exert not only anti-apoptotic but also anti-autophagic activities. ... more The Bcl-2 protein is known to exert not only anti-apoptotic but also anti-autophagic activities. Numerous studies have demonstrated that etoposide, which is one of the most widely used cancer chemotherapy agents, induces apoptotic cell death. However, the exact molecular mechanism leading to cell death by etoposide remains to be resolved. This study aimed to dissect the mode of cell death induced by etoposide in Hep3B hepatoma cells. Furthermore, this study was conducted to examine whether etoposide overcomes the resistance conferred by Bcl-2 in Hep3B hepatoma cells. We observed that Hep3B cells treated with etoposide show not only apoptotic but autophagic phenotypes. Autophagy inhibition by 3-methyladenine (3MA) and caspase inhibition by zVAD-fmk effectively decreased autophagic and apoptotic phenotypes, respectively. However, either zVAD-fmk or 3MA only partially prevented cell death. These data indicate that etoposide concomitantly induces autophagic cell death and apoptosis in H...
International Journal of Oncology, 2011
Since resveratrol is not a potent cytotoxic compound when compared with other chemotherapeutic ag... more Since resveratrol is not a potent cytotoxic compound when compared with other chemotherapeutic agents, several previous studies have been performed to obtain synthetic analogs of resveratrol with potent activity. Our previous study demonstrated that the resveratrol analog HS-1793 showed stronger antitumor activity than resveratrol in various cancer cells. We examined the antitumor activity exerted by HS-1793 in prostate cancer cells, and we observed that HS-1793 acts as a polyploidy inducer. Noticeably, multinucleation and polyploidization were induced in most LNCaP cells treated with HS-1793 at the dose causing a slight decline in cell viability. However, the induction of multinucleation and polyploidization was much lower in PC-3 prostate cancer cells treated with the same dose of HS-1793. Western blot and RT-PCR analyses showed that the expression of Aurora B was almost undetectable in LNCaP cells, but it was highly expressed in PC-3 cells. Further, silencing of Aurora B sensitized PC-3 cells to HS-1793-induced multi-nucleation. These results indicate that expression of Aurora B determines multinucleation in prostate cancer cells treated with HS-1793. Additional assays using multiple cancer cell lines show that the population of multinucleated cells induced by HS-1793 treatment is inversely proportional to Aurora B expression. We further elicited that the HS-1793-induced polyploid LNCaP cells are vulnerable to downregulation of Bcl-xL. Since the polyploidization in LNCaP induced by HS-1793 does not appear to cause definite commitment to apoptosis, the termination of polyploid cells by inhibition of Bcl-xL could provide an advantageous means to improve chemotherapeutic efficacy of HS-1793.
Anatomy & Cell Biology, 2010
Mitochondria are subcellular organelles composed of two discrete membranes in the cytoplasm of eu... more Mitochondria are subcellular organelles composed of two discrete membranes in the cytoplasm of eukaryotic cells. Th ey 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. Th e 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 signifi cant advances in discovering the mtDNA defects, however, there are currently no eff ective therapies for these clinically devastating diseases due to the lack of technology for introducing specifi c modifi cations into the mitochondrial genomes and for generating accurate mtDNA disease models. Th e 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 eff orts for introducing engineered mtDNA constructs into the mitochondria of living cells through bacterial conjugation.
Journal of Applied Biological Chemistry, 2014
Mitochondrial DNA (mtDNA)-depleted (ρ 0) cells are often used as mtDNA recipients to study the in... more Mitochondrial DNA (mtDNA)-depleted (ρ 0) cells are often used as mtDNA recipients to study the interaction between the nucleus and mitochondria in mammalian cells. Therefore, it is crucial to obtain mtDNA-depleted cells with many different nuclear backgrounds for the study. Here, we demonstrate a rapid and reliable method to isolate mammalian mtDNA-depleted cells involving treatment with the antimitochondrial agents ethidium bromide (EtBr) and 2',3'-dideoxycytidine (ddC). After a short exposure to EtBr or ddC, followed by rapid clonal isolation, we were able to generate viable mtDNA-depleted cells from mouse and human cells and were able to successfully repopulate them with exogenous mitochondria from platelets isolated from mouse and human blood samples. These mtDNA-depleted cells can be used to characterize the nuclear mitochondrial interactions and to study mtDNA-associated defects in mammalian cells. Our method of isolating mtDNA-depleted cells is practical and applicable to a variety of cell types.
Toxicology and Applied Pharmacology, 2013
Previous studies have reported that a Gamitrinib variant containing triphenylphosphonium (G-TPP) ... more Previous studies have reported that a Gamitrinib variant containing triphenylphosphonium (G-TPP) binds to mitochondrial Hsp90 and rapidly inhibits its activity, thus inducing the apoptotic pathway in the cells. Accordingly, G-TPP shows a potential as a promising drug for the treatment of cancer. A cell can die from different types of cell death such as apoptosis, necrosis, necroptosis, and autophagic cell death. In this study, we further investigated the mechanisms and modes of cell death in the G-TPP-treated Hep3B and U937 cell lines. We discovered that G-TPP kills the U937 cells through the apoptotic pathway and the overexpression of Bcl-2 significantly inhibits U937 cell death to G-TPP. We further discovered that G-TPP kills the Hep3B cells by activating necroptosis in combination with the partial activation of caspase-dependent apoptosis. Importantly, G-TPP overcomes the apoptosis resistance conferred by Bcl-2 in Hep3B cells via necroptosis. We also observed that G-TPP induces compensatory autophagy in the Hep3B cell line. We further found that whereas there is a Bcl-2-Beclin 1 interaction in response to G-TPP, silencing the beclin 1 gene failed to block LC3-II accumulation in the Hep3B cells, indicating that G-TPP triggers Beclin 1-independent protective autophagy in Hep3B cells. Taken together, these data reveal that G-TPP induces cell death through a combination of death pathways, including necroptosis and apoptosis, and overcomes the apoptosis resistance conferred by Bcl-2 in Hep3B cells via necroptosis. These findings are important for the therapeutic exploitation of necroptosis as an alternative cell death program to bypass the resistance to apoptosis.
Nucleic Acids Research, 2003
We have devised an ef®cient method for replicating and stably maintaining entire mitochondrial ge... more We have devised an ef®cient method for replicating and stably maintaining entire mitochondrial genomes in Escherichia coli and have shown that we can engineer these mitochondrial DNA (mtDNA) genome clones using standard molecular biological techniques. In general, we accomplish this by inserting an E.coli replication origin and selectable marker into isolated, circular mtDNA at random locations using an in vitro transposition reaction and then transforming the modi®ed genomes into E.coli. We tested this approach by cloning the 16.3 kb mouse mitochondrial genome and found that the resulting clones could be engineered and faithfully maintained when we used E.coli hosts that replicated them at moderately low copy numbers. When these recombinant mtDNAs were replicated at high copy numbers, however, mtDNA sequences were partially or fully deleted from the original clone. We successfully electroporated recombinant mouse mitochondrial genomes into isolated mouse mitochondria devoid of their own DNA and detected robust in organello RNA synthesis by RT±PCR. This approach for modifying mtDNA and subsequent in organello analysis of the recombinant genomes offers an attractive experimental system for studying many aspects of vertebrate mitochondrial gene expression and is a ®rst step towards true in vivo engineering of mammalian mitochondrial genomes.
Nucleic Acids Research, 2005
We have developed a method for transferring exogenous DNA molecules into isolated mammalian mitoc... more We have developed a method for transferring exogenous DNA molecules into isolated mammalian mitochondria using bacterial conjugation. In general, we accomplish this by (i) inserting an origin of DNA transfer (oriT) sequence into a DNA construct, (ii) transforming the construct into an appropriate Escherichia coli strain and then (iii) introducing the mobilizable DNA into mitochondria through conjugation. We tested this approach by transferring plasmid DNA containing a T7 promoter sequence into mitochondria that we had engineered to contain T7 RNA polymerase. After conjugation between E.coli and mitochondria, we detected robust levels of T7 transcription from the DNA constructs that had been transferred into the mitochondria. This approach for engineering DNA constructs in vitro and subsequent transfer into mitochondria by conjugation offers an attractive experimental system for studying many aspects of vertebrate mitochondrial gene expression and is a potential route for transforming mitochondrial networks within mammalian cells.
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Papers by Young Geol Yoon