Direct quantification of mitochondrial DNA and its 4.9-kb common deletion without DNA purification

Quantitative analysis of mitochondrial DNA (mtDNA) and its common deletion (CD) are sensitive and early markers for mitochondrial mutations and suffering. However, the use of purified DNA can lead to quantification errors because of variable DNA extraction yields due to the significant differences in size and structure between genomic DNA (gDNA) and mtDNA. We report a real-time qPCR-based protocol directly on tissue lysate, without DNA extraction. This method, which allows both absolute and relative measure, increases the measuring accuracy of the mtDNA/gDNA ratio and leads to reliable and more reproducible results when measuring the deleted/total mtDNA ratio.     

Direct quantification of mitochondrial DNA and its 4.9-kb common deletion without DNA purification

We studied the extraction and analysis of integral membrane proteins possessing hydrophobic and hydrophilic domains and found that a nonionic detergent called MEGA-10, used in lysis buffers, had a superior extraction effect compared to most conventional detergents. A sodium dodecyl sulfate (SDS) concentration of >0.4% (w/v) in the sample buffer was crucial for those proteins to be clearly analyzed by electrophoresis and Western blotting. Furthermore, MEGA-10 had the tendency to maximally extract proteins around its critical micelle concentration (CMC) of 0.24% (w/v). These solutions can greatly assist functional investigations of membrane proteins in the proteomics era.     

Quantitation of a mitochondrial DNA deletion in Parkinson's disease.

A 5 kilobase deletion in mitochondrial DNA (mtDNA) has been reported to be responsible for the specific complex I deficiency in the substantia nigra (SN) of the Parkinson's disease (PD) brain. We have studied mitochondrial respiratory chain function in the SN from control and PD subjects, and analysed mtDNA, extracted from the same tissues, by Southern blot and the polymerase chain reaction (PCR). Quantitation of the levels of the deletion indicate that it does not contribute to the pathogenesis of PD nor to a complex I deficiency but seems likely to be an age-related observation.     

On the evolutionary origin of mitochondrial DNA

Current theories of mitochondrial evolution assume that this organelle evolved either from endosymbiotic bacterial-like organisms which invaded other cells or by a gradual sequestering of functional cytoplasmic units within membranes. In either case there has been relatively little discussion of the origin of mitochondrial DNA. Because of the marked similarity in the size, physical properties, replication and sensitivity to acridine dyes and ethidium bromide of both bacterial plasmid and mitochondrial DNA, it is proposed that the plasmid of an ancestral bacterial-like organism evolved into the mitochondrial DNA of eukaryotes. This hypothesis is consistent with either theory of the whole organelle but is easier to explain if mitochondria evolved within a prokaryote by invagination of the plasma membrane.     

Ancient DNA from a pre-Columbian Amerindian population

Ancient DNA was obtained from skeletal remains from the Norris Farms #36 cemetery, a pre-Columbian archeological site in central Illinois that dates to A. D. 1300. Four mitochondrial DNA (mtDNA) markers were analyzed that delineate the four primary mtDNA lineages found in contemporary Amerindian populations. mtDNA types were determined for 50 individuals; 49 belonged to one of these four lineages. One lineage occurred only in males, suggesting an immigration of maternally related males into this community. There was no significant spatial patterning of mtDNA lineages within the cemetery. This survey of ancient DNA variation in a preColumbian population supports the view that the initial colonization of the New World comprised just four primary mtDNA lineages. © 1993 Wiley-Liss, Inc.     

Founding Amerindian mitochondrial DNA lineages in ancient Maya from Xcaret, Quintana Roo

Ancient DNA from the bone remains of 25 out of 28 pre-Columbian individuals from the Late Classic-Postclassic Maya site of Xcaret, Quintana Roo, was recovered, and mitochondrial DNA (mtDNA) was amplified by using the polymerase chain reaction. The presence of the four founding Amerindian mtDNA lineages was investigated by restriction analysis and by direct sequencing in selected individuals. The mtDNA lineages A, B, and C were found in this population. Eighty-four percent of the individuals were lineage A, whereas lineages B and C were present at low frequencies, 4% and 8%, respectively. Lineage D was absent from our sample. One individual did not possess any of the four lineages. Six skeletons out of 7 dated from the Late Classic period were haplotype A, whereas 11 skeletons out of 16 dated from the Postclassic period were also haplotype A. The distribution of mtDNA lineages in the Xcaret population contrasts sharply with that found in ancient Maya from Copán, which lack lineages A and B. On the other hand, our results resemble more closely the frequencies of mtDNA lineages found in contemporary Maya from the Yucatán Peninsula and in other Native American contemporary populations of Mesoamerican origin.     

Cystic fibrosis: screening for a DNA deletion by field inversion gel electrophoresis

Summary We analyzed DNA of ten cystic fibrosis (CF) patients representing DNA of 19 different CF chromosomes and screened for deletions by means of field inversion gel electrophoresis (FIGE). No differences were detected after digestion of the DNA samples with the restriction enzymes Not I and Sfi I and hybridization with the probes MetH, MetD, J3.11, and 7C22. Thus the percentage of deletions occurring within the CF region and detectable with FIGE is less than 15.2% (95% confidence interval, N=19)     

Major mitochondrial DNA haplotype heterogeneity in highland and lowland Amerindian populations from Bolivia

This study provides the frequencies of four mitochondrial DNA (mtDNA) haplogroups of 233 native South Amerindians in eight populations living in the Beni Department of Bolivia, including six populations not previously studied. Linguistically, these populations belong to the three principal South Amerindian language stocks, Andean, Equatorial-Tucanoan, and Ge-Pano-Carib. Frequency analyses under geographic, historic, linguistic, and genetic configurations using the theta statistic of Weir (Weir 1990) and analysis of molecular variance (AMOVA) show similar results. Results are also similar when phenetic cluster is used. Aymara belongs almost exclusively to haplogroup B, Quechua- and Moseten-speaking tribes belong to haplogroups A and B, but the first tribe presents high frequencies of haplogroup B. Yuracare, Trinitario, and Ignaciano exhibit high frequencies of A, B, and C haplogroups, and the Movima present a large proportion of haplogroup C. There is some correspondence between mtDNA haplogroup frequencies and language affiliation and historical connections, but less so with geographic aspects. The present study provides a context for understanding the relationship between different Amerindian populations living in a multiethnic area of Bolivia.     

Animal mitochondrial DNA as a genetic marker in population and evolutionary biology

Animal mitochondrial DNA (mtDNA) is playing an increasingly important role as a genetic marker in population and evolutionary biology. The popularity of this molecule derives, in part, from the relative ease with which clearly homologous sequences can be isolated and compared. Simple sequence organization, maternal inheritance and absence of recombination make mtDNA an ideal marker for tracing maternal genealogies. Rapid rate of sequence divergence (at least in vertebrates) allows discrimination of recently diverged lineages. Studies of mtDNAs from a diversity of animal groups have revealed significant variation among taxa in mtDNA sequence dynamics, gene order and genome size. They have also provided important insights into population structure, geographic variation, zoogeography and phylogeny.     

Heteroplasmy in mice with deletion of a large coding region of mitochondrial DNA

Polymorphism of animal mitochondrial DNA (mtDNA) has been shown to involve point mutations and limited length variations affecting essentially noncoding regions. In two wild mice of the European subspecies Mus mus musculus we found a mitochondrial mutant with a very large deletion in a coding region. The deletion is 5 kbp long (31% of the mitochondrial chromosome) and encompasses six tRNA genes and seven protein genes. The two mice were heteroplasmic: they contained a mixture of normal mtDNA and the deletion mutant. Although the latter is functionally defective, it represents 78%-79% of the mtDNA molecules in our preparations from each animal.      

Detection of a specific mitochondrial DNA deletion in tissues of older humans.

Using PCR, we found that normal heart muscle and brain from adult human individuals contain low levels of a specific mitochondrial DNA deletion, previously found only in patients affected with certain types of neuromuscular disease. This deletion was not observed in fetal heart or brain. Experimental tests support the idea that the deletion exists in vivo in adult mitochondria and is not an in vitro artifact of PCR. Our data provide direct experimental support for the idea that accumulation of mitochondrial DNA deletions may be important in aging.     

Analysis of ancient DNA from a prehistoric Amerindian cemetery

The Norris Farms No. 36 cemetery in central Illinois has been the subject of considerable archaeological and genetic research. Both mitochondrial DNA (mtDNA) and nuclear DNA have been examined in this 700-year-old population. DNA preservation at the site was good, with about 70% of the samples producing mtDNA results and approximately 15% yielding nuclear DNA data. All four of the major Amerindian mtDNA haplogroups were found, in addition to a fifth haplogroup. Sequences of the first hypervariable region of the mtDNA control region revealed a high level of diversity in the Norris Farms population and confirmed that the fifth haplogroup associates with Mongolian sequences and hence is probably authentic. Other than a possible reduction in the number of rare mtDNA lineages in many populations, it does not appear as if European contact significantly altered patterns of Amerindian mtDNA variation, despite the large decrease in population size that occurred. For nuclear DNA analysis, a novel method for DNA-based sex identification that uses nucleotide differences between the X and Y copies of the amelogenin gene was developed and applied successfully in approximately 20 individuals. Despite the well-known problems of poor DNA preservation and the ever-present possibility of contamination with modern DNA, genetic analysis of the Norris Farms No. 36 population demonstrates that ancient DNA can be a fruitful source of new insights into prehistoric populations.     

Hybrid male sterility is caused by mitochondrial DNA deletion

Although it is known that the hybrid male mouse is sterile just like any other animal’s heterogametic sex, the reason why only the male germ cells are impaired has yet to be discovered. TdT-mediated dUTP nick end labeling assay using a confocal fluorescence microscope and DNA fragmentation assay of hybrid testis indicated destruction of the mitochondrial DNA (mtDNA) rather than the nuclear DNA. Previously we reported that maternal mtDNA inheritance is through selective sperm mtDNA elimination based on the sperm factor and two egg factors, and expression of these three factors was recognized in the hybrid testis. It was thereby assumed that mtDNA destruction caused by the expression of maternal mtDNA inheritance system in male germ cells is implicated in the hybrid male sterility of mice.     

Ageing-associated 5 kb deletion in human liver mitochondrial DNA

Using PCR technique and restriction mapping, we analyzed liver mitochondrial DNA (mtDNA) of 2 stillborn babies and 55 Chinese subjects from 27 to 86 years old and blood cell mtDNA from 20 subjects of various ages. An ageing-associated 4,977-bp deletion was detected between nucleotide position 8,469 and 13,447 (or between 8,482 and 13,460) in the liver mtDNA of older subjects. In the region containing the junction fragment, we observed a 13 bp repeat "ACCTCCCTCACCA". Moreover, the incidence of the deleted mtDNA of each of the study subjects was found to increase with age. The deletion was found in 5 out of 8 patients of the 31-40 age group and 9 out of 11 patients of the 41-50 age group, and in all the patients over 50 years old. The deletion was not observed in either the mtDNA of the liver of the stillbirth or the blood cells of subjects of all the age groups. These results support our previous contention that liver mitochondrial respiratory functions decline with age and the hypothesis that continuous accumulation of mitochondrial DNA mutation is an important contributor to ageing process.     

Possible migration routes into South America deduced from mitochondrial DNA studies in Colombian Amerindian populations

Mitochondrial DNA haplotype studies have been useful in unraveling the origins of Native Americans. Such studies are based on restriction site and intergenic deletion/insertion polymorphisms, which define four main haplotype groups common to Asian and American populations. Several studies have characterized these lineages in North, Central, and South American Amerindian, as well as Na Dene and Aleutian populations. Siberian, Central Asian, and Southeast Asian populations have also been analyzed, in the hope of fully depicting the route(s) of migration between Asia and America. Colombia, a key route of migration between North and South America, has until now not been studied. To resolve the current lack of information about Colombian Amerindian populations, we have investigated the presence of the founder haplogroups in 25 different ethnic groups from all over the country. The present research is part of an interdisciplinary program, Expedición Humana, fostered by the Universidad Javeriana and Dr. J. E. Bernal V. The results show the presence of the four founder A-D Amerindian lineages, with varied distributions in the different populations, as well as the presence of other haplotypes in frequencies ranging from 3% to 26%. These include some unique or private polymorphisms, and also indicate the probable presence of other Asian and a few non-Amerindian lineages. A spatial structure is apparent for haplogroups A and D, and to a lesser extent for haplogroup C. While haplogroup A and D frequencies in Colombian populations from the northwestern side of the Andes resemble those seen in Central American Amerindians more than those seen in South American populations, their frequencies on the southeastern side more closely resemble the bulk of South American frequencies so far reported, raising the question as to whether they reflect more than one migration route into South America. High frequencies of the B lineage are also characteristic of some populations. Our observations may be explained by historical events during the pre-Columbian dispersion of the first settlers and, later, by disruptions caused by the European colonization.     

Absolute quantitation of a heteroplasmic mitochondrial DNA deletion using a multiplex three-primer real-time PCR assay

Quantitation of wild-type and deleted mitochondrial DNA (mtDNA) coexisting within the same cell (a.k.a., heteroplasmy) is important in mitochondrial disease and aging. We report the development of a multiplex three-primer PCR assay that is capable of absolute quantitation of wild-type and deleted mtDNA simultaneously. Molecular beacons were designed to hybridize with either type of mtDNA molecule, allowing real-time detection during PCR amplification. The assay is specific and can detect down to six copies of mtDNA, making it suitable for single-cell analyses. The relative standard deviation in the threshold cycle number is approximately 0.6%. Heteroplasmy was quantitated in individual cytoplasmic hybrid cells (cybrids), containing a large mtDNA deletion, and bulk cell samples. Individual cybrid cells contained 100-2600 copies of wild-type mtDNA and 950-4700 copies of deleted mtDNA, and the percentage of heteroplasmy ranged from 43+/-16 to 95+/-16%. The average amount of total mtDNA was 3800+/-1600 copies/cybrid cell, and the average percentage of heteroplasmy correlated well with the bulk cell sample. The single-cell analysis also revealed that heteroplasmy in individual cells is highly heterogeneous. This assay will be useful for monitoring clonal expansions of mtDNA deletions and investigating the role of heteroplasmy in cell-to-cell heterogeneity in cellular models of mitochondrial disease and aging.     

A topoisomerase II cleavage site is associated with a novel mitochondrial DNA deletion

Mitochondrial myopathies and encephalopathies can be caused by nucleotide substitutions, deletions or duplications of the mitochondrial DNA (mtDNA). In one such disorder, Kearns-Sayre Syndrome (KSS), large-scale hetero-plasmic mtDNA deletions are often found. We describe a 14-year-old boy with clinical features of KSS, plus some additional features. Analysis of the entire mitochondrial genome by the polymerase chain reaction and Southern blotting revealed a 7864-bp mtDNA deletion, heteroplasmic in its tissue distribution. DNA sequencing established that the deletion was between nucleotides 6238 and 14103, and flanked by a 4-bp (TCCT) direct repeat sequence. Deletions between direct repeats have been hypothesised to occur by a slipped-mismatching or illegitimate recombination event, or following the DNA cleavage action of topoisomerase II. Analysis of the gene sequence in the region surrounding the mtDNA deletion breakpoint in this patient revealed the presence of putative vertebrate topoisomerase II sites. We suggest that direct repeat sequences, together with putative topoisomerase II sites, may predispose certain regions of the mitochondrial genome to deletions.     

Rapid screening of the entire mitochondrial DNA for low-level heteroplasmic mutations

Alterations of the mitochondrial DNA (mtDNA) are implicated in various pathological conditions. In this study, we used denaturing high performance liquid chromatography (DHPLC) as a method to rapidly screen the entire mtDNA for mutations. Overlapping DNA fragments, amplified by one single cycling protocol from frozen pre-formulated PCR mixes, were subjected to DHPLC analysis. Single DHPLC injections of fragments yielded straightforward interpretation of results with a detection limit down to 1% mtDNA heteroplasmy. Furthermore, collection and re-amplification of low degree heteroduplex peak-fractions allowed sequence analysis of mtDNA mutations down to the detection limit of the DHPLC method. In order to demonstrate that the method has diagnostic value, we analyzed and confirmed known mtDNA mutations in patient samples.     

Heteroplasmic mutation of mitochondrial DNA D-loop and 4977-bp deletion in human cancer cells during mitochondrial DNA depletion

Somatic mutations in mitochondrial DNA (mtDNA) have been demonstrated in various human cancers. Many cancers have high frequently of mtDNA with homoplasmic point mutations, and carry less frequently of mtDNA with large-scale deletions as compared with corresponding non-cancerous tissue. Moreover, most cancers harbor a decreased copy number of mtDNA than their corresponding non-cancerous tissue. However, it is unclear whether the process of decreasing in mtDNA content would be involved in an increase in the heteroplasmic level of somatic mtDNA point mutation, and/or involved in a decrease in the proportion of mtDNA with large-scale deletion in cancer cells. In this study, we provided evidence that the heteroplasmic levels of variations in cytidine number in np 303-309 poly C tract of mtDNA in three colon cancer cells were not changed during an ethidium bromide-induced mtDNA depleting process. In the mtDNA depleting process, the proportions of mtDNA with 4977-bp deletion in cybrid cells were not significantly altered. These results suggest that the decreasing process of mtDNA copy number per se may neither contribute to the shift of homoplasmic/heteroplasmic state of point mutation in mtDNA nor to the decrease in proportion of mtDNA with large-scale deletions in cancer cells. Mitochondrial genome instability and reduced mtDNA copy number may independently occur in human cancer.