PCR-based cloning of the complete mouse mitochondrial genome and stable engineering in <Emphasis Type="Italic">Escherichia</Emphasis> <Emphasis Type="Italic">coli</Emphasis>

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.     

Direct cloning of full-length mouse mitochondrial DNA using a Bacillus subtilis genome vector

The complete mouse mitochondrial genome (16.3 kb) was directly cloned into a Bacillus subtilis genome (BGM) vector. Two DNA segments of 2.06 and 2.14 kb that flank the internal 12 kb of the mitochondrial DNA (mtDNA) were subcloned into an Escherichia coli plasmid. Subsequent integration of the plasmid at the cloning locus of the BGM vector yielded a derivative specific for the targeted cloning of the internal 12-kb mtDNA region. The BGM vector took up mtDNA purified from mouse liver and integrated it by homologous recombination at the two preinstalled mtDNA-flanking sequences. The complete cloned mtDNA in the BGM vector was converted to a covalently closed circular (ccc) plasmid form via gene conversion in B. subtilis. The mtDNA carried on this plasmid was then isolated and transferred to E. coli. DNA sequence fidelity and stability through the BGM vector-mediated cloning process were confirmed.     

Constancy of organellar genome copy numbers during leaf development and senescence in higher plants

In higher plants, plastid and mitochondrial genomes occur at high copy numbers per cell. Several recent publications have suggested that, in higher plants like Arabidopsis and maize, chloroplast DNA is virtually absent in mature and old leaves. This conclusion was mainly based on DAPI staining of isolated chloroplasts. If correct, the finding that chloroplasts in mature leaves lack DNA would change dramatically our understanding of gene expression, mRNA stability and protein stability in chloroplasts. In view of the wide implications that the disposal of chloroplast DNA during leaf development would have, we have reinvestigated the age dependency of genome copy numbers in chloroplasts and, in addition, tested for possible changes in mitochondrial genome copy number during plant development. Analyzing chloroplast and mitochondrial DNA amounts in Arabidopsis and tobacco plants, we find that organellar genome copy numbers remain remarkably constant during leaf development and are present in essentially unchanged numbers even in the senescing leaves. We conclude that, during leaf development, organellar gene expression in higher plants is not significantly regulated at the level of genome copy number and we discuss possible explanations for the failure to detect DNA in isolated chloroplasts stained with DAPI.     

Rescue of silent integrated polyoma genomes suggests homologous recombination between resident and transfected DNA fragments

Two defective polyoma virus genomes, deleted in the nucleotide sequences coding the N-termini of the tumor antigens, were introduced into Fisher 3T3 rat cells by DNA-mediated gene transfer (transfection). The resulting integrated genomes were incapable of conferring a transformed phenotype to the cells. However, after transfection of these lines with small polyoma fragments overlapping the deleted sequences, transformed clones were isolated. These clones were analyzed by Southern genomic blot hybridization and by isolation in E. coli of plasmids containing viral sequences excised following fusion with mouse polyoma growth-permissive cells. In all cases at least one intact copy of the early region of the polyoma genome was found. Furthermore, restriction sites adjacent to the initial inactive insertion remained unchanged in many of the transformed lines. These results show that functional restoration of the defective polyoma early region involves homologous recombination between the deleted viral genomes integrated in the cellular DNA and the transfecting viral fragments.     

Transferring isolated mitochondria into tissue culture cells

We have developed a new method for introducing large numbers of isolated mitochondria into tissue culture cells. Direct microinjection of mitochondria into typical mammalian cells has been found to be impractical due to the large size of mitochondria relative to microinjection needles. To circumvent this problem, we inject isolated mitochondria through appropriately sized microinjection needles into rodent oocytes or single-cell embryos, which are much larger than tissue culture cells, and then withdraw a ‘mitocytoplast’ cell fragment containing the injected mitochondria using a modified holding needle. These mitocytoplasts are then fused to recipient cells through viral-mediated membrane fusion and the injected mitochondria are transferred into the cytoplasm of the tissue culture cell. Since mouse oocytes contain large numbers of mouse mitochondria that repopulate recipient mouse cells along with the injected mitochondria, we used either gerbil single-cell embryos or rat oocytes to package injected mouse mitochondria. We found that the gerbil mitochondrial DNA (mtDNA) is not maintained in recipient rho0 mouse cells and that rat mtDNA initially replicated but was soon completely replaced by the injected mouse mtDNA, and so with both procedures mouse cells homoplasmic for the mouse mtDNA in the injected mitochondria were obtained.     

Autophagy determines mtDNA copy number dynamics during starvation

Derived from bacterial ancestors, mitochondria have maintained their own albeit strongly reduced genome, mitochondrial DNA (mtDNA), which encodes for a small and highly specialized set of genes. MtDNA exists in tens to thousands of copies packaged in numerous nucleoprotein complexes, termed nucleoids, distributed throughout the dynamic mitochondrial network. Our understanding of the mechanisms of how cells regulate the copy number of mitochondrial genomes has been limited. Here, we summarize and discuss our recent findings that Mip1/POLG (mitochondrial DNA polymerase gamma) critically controls mtDNA copy number by operating in 2 opposing modes, synthesis and, unexpectedly, degradation of mtDNA, when yeast cells face nutrient starvation. The balance of the 2 modes of Mip1/POLG and thus mtDNA copy number dynamics depends on the integrity of macroautophagy/autophagy, which sustains continuous synthesis and maintenance of mtDNA. In autophagy-deficient cells, a combination of nucleotide insufficiency and elevated mitochondrial ROS production impairs mtDNA synthesis and drives mtDNA degradation by the 3?-5?-exonuclease activity of Mip1/POLG resulting in mitochondrial genome depletion and irreversible respiratory deficiency.

Abbrivations: mtDNA: mitochondrial DNA; mtDCN: mitochondrial DNA copy number.     

qPCR-based mitochondrial DNA quantification: influence of template DNA fragmentation on accuracy

Real-time PCR (qPCR) is the method of choice for quantification of mitochondrial DNA (mtDNA) by relative comparison of a nuclear to a mitochondrial locus. Quantitative abnormal mtDNA content is indicative of mitochondrial disorders and mostly confines in a tissue-specific manner. Thus handling of degradation-prone bioptic material is inevitable. We established a serial qPCR assay based on increasing amplicon size to measure degradation status of any DNA sample. Using this approach we can exclude erroneous mtDNA quantification due to degraded samples (e.g. long post-exicision time, autolytic processus, freeze-thaw cycles) and ensure abnormal DNA content measurements (e.g. depletion) in non-degraded patient material. By preparation of degraded DNA under controlled conditions using sonification and DNaseI digestion we show that erroneous quantification is due to the different preservation qualities of the nuclear and the mitochondrial genome. This disparate degradation of the two genomes results in over- or underestimation of mtDNA copy number in degraded samples. Moreover, as analysis of defined archival tissue would allow to precise the molecular pathomechanism of mitochondrial disorders presenting with abnormal mtDNA content, we compared fresh frozen (FF) with formalin-fixed paraffin-embedded (FFPE) skeletal muscle tissue of the same sample. By extrapolation of measured decay constants for nuclear DNA (λnDNA) and mtDNA (λmtDNA) we present an approach to possibly correct measurements in degraded samples in the future. To our knowledge this is the first time different degradation impact of the two genomes is demonstrated and which evaluates systematically the impact of DNA degradation on quantification of mtDNA copy number.     

Introduction of chloramphenicol resistance into the modified mouse mitochondrial genome: cloning of unstable sequences by passage through yeast

Despite increasing awareness of the importance of the mitochondrial genome in human pathology, very few attempts have been made so far toward genetic engineering of mitochondrial DNA (mtDNA). One of the reasons for this slow progress is the difficulty of cloning mtDNA in Escherichia coli, a trait in common with repetitive or palindromic sequences, and some viral sequences. We have previously made a construct containing the entire mouse mitochondrial genome and a cDNA sequence coding for human ornithine transcarbamylase in a yeast/bacterial shuttle vector, which can be stably maintained in E. coli. We wished to modify this vector for mitochondrial gene therapy by the addition of mitochondrial chloramphenicol resistance, conferred by a point mutation in the 16S rRNA gene. Attempts to modify this construct by a straightforward cloning approach in E. coli proved unsuccessful. Two successful strategies for modification of large unstable constructs in both E. coli and the yeast Saccharomyces cerevisiae are compared here.     

Mitochondrial DNA of Marchantia polymorpha as a single circular form with no incorporation of foreign DNA.

A cosmid library and physical maps of mitochondrial DNA (mtDNA) from a liverwort, Marchantia polymorpha, were constructed using the cosmid clones. Electrophoresis profile and the physical maps indicated that the liverwort mtDNA was approximately 183 kb long, the smallest among plant mtDNAs, and that it consisted of a single circular molecule. Southern hybridization analysis showed that genes typical to the mitochondrial genome existed in a single copy, and also that there was no incorporation of chloroplast DNA fragments into the mitochondrial genome.     

Highly efficient DNA synthesis in isolated mitochondria from rat liver.

We have developed a highly efficient DNA-synthesizing system with isolated intact rat liver mitochondria. The ATP requirements for this in organello DNA synthesis are provided by endogenous synthesis in the presence of exogenous ADP and an oxidizable substrate. In this system, mitochondrial DNA synthesis strikingly proceeds at a constant rate for about 5 h at 37 degrees C. Gel electrophoresis, hybridization and restriction enzyme analyses show that intact mitochondria synthesize nucleic acids with a size of 16.5 kb, that correspond to mitochondrial DNA, and that both DNA strands are replicated. This in organello DNA synthesis requires the supply of dNTPs and decreases at high ADP concentration in the incubation medium.     

The structures and fidelity of replication of mouse mitochondrial DNA-pSC101 EcoRI recombinant plasmids grown in E. coli K12

Recombinant DNAs containing the E. coli plasmid pSC101 and mouse cell (LA9) mitochondrial DNA (mtDNA) were formed in vitro via ligation of DNA fragments from limit EcoRI endonuclease digests and were used to transform E. coli K12. Four structurally different recombinant plasmid DNAs from transformed clones were characterized. Two of these were analyzed extensively and the mtDNA portions compared with mtDNA from LA9 cells. No differences were detected in the physical or chemical properties examined, except that the E. coli mtDNA lacked the alkali lability characteristic of animal mtDNAs.Heteroduplexes between the LA9 portions of the recombinant plasmids and LA9 mtDNA were analyzed by absorbance melting. The melting temperatures were indistinguishable from reannealed LA9 mtDNA homoduplexes, indicating that single-base replication errors occur at a frequency of fewer than 1 nucleotide in 300. Electron microscopic analyses of plasmid-LA9 mtDNA heteroduplexes and a comparison of agarose gel electrophoresis of restriction endonuclease fragments also indicated no differences. These results were independent of the order or the relative orientation of the pSC101 and mtDNA fragments.A third EcoRI fragment in LA9 mtDNA, not found in an earlier study (Brown and Vinograd, 1974), has been positioned in the LA9, EcoRI map. This fragment contains 165±10 nucleotide pairs.     

The complete sequence of the mitochondrial genome of the African Penguin (Spheniscus demersus)

The complete mitochondrial genome of the African Penguin (Spheniscus demersus) was sequenced. The molecule was sequenced via next generation sequencing and primer walking. The size of the genome is 17,346 bp in length. Comparison with the mitochondrial DNA of two other penguin genomes that have so far been reported was conducted namely; Little blue penguin (Eudyptula minor) and the Rockhopper penguin (Eudyptes chrysocome). This analysis made it possible to identify common penguin mitochondrial DNA characteristics. The S. demersus mtDNA genome is very similar, both in composition and length to both the E. chrysocome and E. minor genomes. The gene content of the African penguin mitochondrial genome is typical of vertebrates and all three penguin species have the standard gene order originally identified in the chicken. The control region for S. demersus is located between tRNA-Glu and tRNA-Phe and all three species of penguins contain two sets of similar repeats with varying copy numbers towards the 3′ end of the control region, accounting for the size variance. This is the first report of the complete nucleotide sequence for the mitochondrial genome of the African penguin, S. demersus. These results can be subsequently used to provide information for penguin phylogenetic studies and insights into the evolution of genomes.     

Hexahydrohippuric Acid,a Newly Isolated Compound from the Cattle’s Urine

A cosmid library and physical maps of mitochondrial DNA (mtDNA) from a liverwort, Marchantia polymorpha, were constructed using the cosmid clones. Electrophoresis profile and the physical maps indicated that the liverwort mtDNA was approximately 183 kb long, the smallest among plant mtDNAs, and that it consisted of a single circular molecule. Southern hybridization analysis showed that genes typical to the mitochondrial genome existed in a single copy, and also that there was no incorporation of chloroplast DNA fragments into the mitochondrial genome.     

鸡马立克氏病病毒814株细菌人工染色体的构建

为构建全基因组鸡马立克氏病病毒814株感染性细菌人工染色体(bacterial artificial chromosome, BAC), 首先通过构建表达Eco-gpt(xanthine-guanine phosphoribosyl transferase, XGPRT, gpt)的哺乳动物细胞基因转移遗传选择标记(1.3 kb)和带有细菌人工染色体的基本功能基因序列的鸡马立克氏病病毒重组病毒转移载体pUAB-gpt-BAC11, 将重组病毒转移载体与鸡马立克氏病病毒细胞总DNA共转染鸡胚成纤维细胞, 在选择培养基中经过8轮加压筛选, 获得并纯化重组病毒; 将重组病毒细胞总DNA电转化大肠杆菌, 筛选共获得38个BAC分子克隆化病毒, 提取BAC-DNA转染鸡胚成纤维细胞以拯救重组病毒。结果表明, MDV-BAC2 DNA再次启动病毒感染, 拯救了重组鸡马立克氏病病毒。成功构建了鸡马立克氏病病毒814株基因组全长感染性细菌人工染色体, 为方便利用现代RED/ET基因重组系统对病毒进行反向遗传操作提供了技术平台; 同时为研究鸡马立克氏病病毒的基因功能和开发新型马立克氏病疫苗奠定了基础。     

Presequence-Independent Mitochondrial Import of DNA Ligase Facilitates Establishment of Cell Lines with Reduced mtDNA Copy Number

Due to the essential role played by mitochondrial DNA (mtDNA) in cellular physiology and bioenergetics, methods for establishing cell lines with altered mtDNA content are of considerable interest. Here, we report evidence for the existence in mammalian cells of a novel, low- efficiency, presequence-independent pathway for mitochondrial protein import, which facilitates mitochondrial uptake of such proteins as Chlorella virus ligase (ChVlig) and Escherichia coli LigA. Mouse cells engineered to depend on this pathway for mitochondrial import of the LigA protein for mtDNA maintenance had severely (up to >90%) reduced mtDNA content. These observations were used to establish a method for the generation of mouse cell lines with reduced mtDNA copy number by, first, transducing them with a retrovirus encoding LigA, and then inactivating in these transductants endogenous Lig3 with CRISPR-Cas9. Interestingly, mtDNA depletion to an average level of one copy per cell proceeds faster in cells engineered to maintain mtDNA at low copy number. This makes a low-mtDNA copy number phenotype resulting from dependence on mitochondrial import of DNA ligase through presequence-independent pathway potentially useful for rapidly shifting mtDNA heteroplasmy through partial mtDNA depletion.