Mitochondrial DNA Template Research Articles

Extensive Heteroplasmy and Evidence for Fragmentation in theCallinectes sapidusMitochondrial Genome

Heteroplasmy is the presence of multiple genotypes in an individual and is commonly associated with mitochondria where thousands of copies of the genome can be present in a single organelle. Even in low frequencies, it can cause severe illness or death and strong selective pressures usually result in a low occurrence. Despite this, cloning and sequencing of a portion of the open reading frames of the nad2, nad4, and coI mitochondrial loci in the blue crab Callinectes sapidus (Rathbun, 1896) revealed abnormally high levels of heteroplasmy at all loci with as many as 24 haplotypes in a single individual and the dominant haplotype accounting for as little as 43.9% of the total sequences. A minimum spanning network comparing these individuals at each locus demonstrated an overlap of haplotypes between the female and themegalopa with almost no overlap of themale sequences. Illumina sequencing ofmuscle and testes froma single individual usingmultiple differentDNA-based amplificationmethods as well as cDNA amplification confirmed heteroplasmic circular mitochondrial DNA templates with portions of the reads containing unmapped highly repetitive regions at specific sequence positions in both tissue types. Thus, the blue crab appears to possess a highly degenerate and possibly fragmented mitochondrial genome that is present in germline tissue and linearly inherited. A general lack of fragmentation present in the cDNA sequences as well as site-specific sequence changes argues for mitochondrial editing as a way of maintaining sequence integrity. This is the first evidence for mitochondrial editing in a crustacean and a potential explanation for observations of extremely high population diversity when using blue crab mitochondrial markers.

Analysis of enzyme kinetic data for mtDNA replication

A significant amount of experimental data on the reaction kinetics for the mitochondrial DNA polymerase gamma exist, but interpreting that data is difficult due to the complex nature of the function of the polymerase. In order to model how these measured kinetics values for polymerase gamma affect the final function of the polymerase, the replication of an entire strand of mitochondrial DNA, we implement a stochastic simulation of the series of reaction events that the polymerase carries out. These reactions include the correct and incorrect polymerization events, exonuclease events which may remove both incorrectly and correctly matched base pairs, and the disassociation of the polymerase from the mitochondrial DNA template. We also describe other reactions which may be included, such as the addition of nucleoside analog tri-phosphates as substrates. The simulation analysis of the kinetics data is implemented through a standard Gillespie algorithm. We describe the methods necessary to define, code and test this algorithm, as well as describing the hardware and software options that are available.

Non-DNA-Templated Addition of Nucleotides to the 3′ End of RNAs by the Mitochondrial RNA Polymerase of Physarum polycephalum

Mitochondrial gene expression is necessary for proper mitochondrial biogenesis. Genes on the mitochondrial DNA are transcribed by a dedicated mitochondrial RNA polymerase (mtRNAP) that is encoded in the nucleus and imported into mitochondria. In the myxomycete Physarum polycephalum, nucleotides that are not specified by the mitochondrial DNA templates are inserted into some RNAs, a process called RNA editing. This is an essential step in the expression of these RNAs, as the insertion of the nontemplated nucleotides creates open reading frames for the production of proteins from mRNAs or produces required secondary structure in rRNAs and tRNAs. The nontemplated nucleotide is added to the 3' end of the RNA as the RNA is being synthesized during mitochondrial transcription. Because RNA editing is cotranscriptional, the mtRNAP is implicated in RNA editing as well as transcription. We have cloned the cDNA for the mtRNAP of Physarum and have expressed the mtRNAP in Escherichia coli. We have used in vitro transcription assays based on the Physarum mtRNAP to identify a novel activity associated with the mtRNAP in which non-DNA-templated nucleotides are added to the 3' end of RNAs. Any of the four ribonucleoside triphosphates (rNTPs) can act as precursors for this process, and this novel activity is observed when only one rNTP is supplied, a condition under which transcription does not occur. The implications of this activity for the mechanism of RNA editing are discussed.

Bridging PNAs can bind preferentially to a deleted mitochondrial DNA template but replication by mitochondrial DNA polymerase γ in vitro is not impaired

Mutations in mitochondrial DNA (mtDNA) are an important cause of neurological and other human pathologies. In the vast majority of cases, supportive care only is available. Mutated and wild-type mtDNAs often coexist in the same cell. A strategy for treatment has been proposed whereby replication of mutated mtDNA is inhibited by selective hybridisation of a nucleic acid derivative, allowing propagation of the wild-type genome and correction of the associated respiratory chain defect. Peptide nucleic acid molecules (PNAs) can be designed to selectively target pathogenic mtDNA with single point mutations. Molecules harbouring deletions present a complex problem. Deletions often occur between two short repeat sequences (4–13 residues), one of which is retained in the deleted molecule. With the more common large repeats, it is therefore difficult to design an antigenomic molecule that will bind selectively under physiological conditions. Following limited success with antigenomic oligodeoxynucleotides (ODNs), we have repeated these studies with a series of bridging PNAs. Molecules complementary to the sequence flanking either side of the 13 bp ‘common deletion’ were synthesised. The PNAs demonstrated markedly greater affinity for the delete than to the wild-type template. In runoff assays using Klenow fragment, these PNAs selectively inhibited replication of the delete template. However, no selective inhibition was observed when a polymerase γ-containing mitochondrial fraction was used.

Linked oligodeoxynucleotides show binding cooperativity and can selectively impair replication of deleted mitochondrial DNA templates.

Mutations in mitochondrial DNA (mtDNA) cause a spectrum of human pathologies, which predominantly affect skeletal muscle and the central nervous system. In patients, mutated and wild-type mtDNAs often co-exist in the same cell (mtDNA heteroplasmy). In the absence of pharmacological therapy, a genetic strategy for treatment has been proposed whereby replication of mutated mtDNA is inhibited by selective hybridisation of a nucleic acid derivative to the single-stranded replication intermediate, allowing propagation of the wild-type genome and correction of the associated respiratory chain defect. Previous studies have shown the efficacy of this anti-genomic approach in vitro, targeting pathogenic mtDNA templates with only a single point mutation. Pathogenic molecules harbouring deletions, however, present a more difficult problem. Deletions often occur at the site of two short repeat sequences (4-13 residues), only one of which is retained in the deleted molecule. With the more common larger repeats it is therefore difficult to design an anti-genomic molecule that will bind selectively across the breakpoint of the deleted mtDNA. To address this problem, we have used linker-substituted oligodeoxynucleotides to bridge the repeated residues. We show that molecules can be designed to bind more tightly to the deleted as compared to the wild-type mtDNA template, consistent with the nucleotide sequence on either side of the linker co-operating to increase binding affinity. Furthermore, these bridging molecules are capable of sequence-dependent partial inhibition of replication in vitro.

The effects of age and physical activity on cardiac mitochondrial DNA template availability

The purpose of this study was to examine the effects of aging and physical activityon heart mtDNA template availability. Young, mature and aged C57BL6 mice were used in this study with half of the animals having access to exercise wheels. Cardiac DNA was isolated and the mitochondrial DNA content was assessed by slot blots with a specific mtDNA probe. Mitochondrial DNA template availability increased in mouse cardiac tissue with senescence. This was reflected in increases in mitochondrial DNA content per milligram of cardiac tissue and mitochondrial DNA relative to nuclear DNA ratios. Oxidative capacity, as determined by citrate synthase activity, was not significantly decreased with aging. Compromises to the erLergy transducing pathway due to aging responses in cardiac tissue may have led to higher replicative rates of mtDNA to induce more mitochondrial specific proteins. The mature mice responded positively to the voluntary chronic physical activity displaying increased mitochondrial DNA to nuclear DNA ratios and elevated citrate synthase activities. Thus the increasing metabolic demand put on the mitochondrial system was associated with elevated mtDNA template levels in these animals. In contrast, the senescent animals’ mtDNA template content was unaffected by the physical activity. This lack of a relationship in the senescent animals is most likely due to low running velocities and therefore relative workloads in this age group, but this suggestion remains to be confirmed.

Occurrence of a Particular Base Substitution (3243 A to G) in Mitochondrial DNA of Tissues of Ageing Humans

Allele-specific polymerase chain reaction has been used to demonstrate the occurrence, during the human ageing process, of the mutation A to G at nucleotide position 3243 on mitochondrial DNA. An oligonucleotide with a 3′-end specific to this mutation was designed to selectively amplify DNA bearing this mutation under annealing temperature conditions that efficiently discriminate between normal and mutant mitochondrial DNA templates. Seven out of 38 adult tissues tested were found to contain this mutation, whereas the mutation could not be detected in any of the 16 infant tissues analyzed. The present demonstration of an age-related point mutation adds to the growing list of mutations in human mitochondrial DNA, till now almost exclusively large deletions, that accumulate in human tissues as a function of age. The method described here has general application in the detection of other age-related point mutations in human mitochondrial DNA.

Template sequences required for transcription of Xenopus laevis mitochondrial DNA from two bidirectional promoters.

Previous work from our laboratory has shown that transcription of Xenopus laevis mitochondrial DNA initiates both in vivo and in vitro from bidirectional promoters located between the gene for tRNA(Phe) and the 5' termini of displacement loop DNA strands. A consensus sequence matching the octanucleotide ACGTTATA surrounds each transcription start site. In the present study, we used in vitro mutagenesis to define sequences required for specific transcription in vitro. First, cloned mitochondrial DNA templates generated by deletion mutagenesis were transcribed in vitro to define the limits of functional promoters. The bidirectional promoter located approximately 33 nucleotides upstream from the gene for tRNA(Phe), termed promoter 1, was studied in greatest detail. The results confirmed the hypothesis that the consensus octanucleotide sequence surrounding each start site is an essential promoter element. A duplex 18-base-pair oligonucleotide encoding the symmetrical promoter 1 region was synthesized and cloned in a plasmid vector. This synthetic oligonucleotide was sufficient to support bidirectional transcription. Point mutations within this oligonucleotide were used to identify critical residues within the consensus sequence.

DNA synthesis by a membrane-DNA complex from rat liver mitochondria

A membrane-DNA complex isolated from Sarkosyl lysates of inner membrane-matrix preparations from rat liver mitochondria has been shown to carry out the synthesis of mitochondrial DNA in vitro . The incorporation of ( 3 H)dTTP is completely dependent upon Mg 2+ , the endogenous mitochondrial DNA template, and four deoxynucleoside triphosphates. A number of inhibitors of DNA synthesis completely suppress the incorporation by the membrane-DNA complex. The incorporation of ( 3 H)dTTP takes place into the same types of replicative intermediates of mtDNA which are labeled when incorporation is allowed to take place in intact mitochondria in vitro or in vivo .

Purification and Properties of Deoxyribonucleic Acid Polymerase from Rat Liver Mitochondria

Abstract These studies report the isolation of a DNA polymerase from rat liver mitochondria. The enzyme has been purified 22-fold by differential ultracentrifugation, ammonium sulfate precipitation, and column chromatography on DEAE-cellulose. The partially purified enzyme manifests all of the requirements shown by the known DNA polymerases of mammalian nuclei and bacteria. The enzyme has an optimal pH of 7.5, and requires Mg++ (12 mm) and the presence of all four deoxyribonucleoside triphosphates. The reaction is inhibited by pyrophosphate and by the addition of deoxyribonuclease but not by ribonuclease. Enzyme purified in this manner is free of nuclear DNA polymerase, terminal addition enzyme, and DNase. The enzyme shows an absolute requirement for added DNA template which can be furnished rather specifically by native, double-stranded, circular, mitochondrial DNA. Furthermore, the labeled DNA was shown to be mitochondrial in nature by virtue of the fact that it had the same buoyant density in CsCl as unlabeled, highly purified, mitochondrial DNA and the fact that it was renaturable after heat denaturation, a property shown by mitochondrial DNA but not by mammalian nuclear DNA. The product of the reaction was shown to be a double stranded replica of the mitochondrial DNA template on the basis of autocatalytic synthesis by the enzyme with the use of nonsaturating levels of mitochondrial DNA template. A 3.5-fold synthesis of new DNA was achieved.