Targeting peptide nucleic acid (PNA) oligomers to mitochondria within cells by conjugation to lipophilic cations: implications for mitochondrial DNA replication, expression and disease

The selective manipulation of mitochondrial DNA (mtDNA) replication and expression within mammalian cells has proven difficult. One promising approach is to use peptide nucleic acid (PNA) oligomers, nucleic acid analogues that bind selectively to complementary DNA or RNA sequences inhibiting replication and translation. However, the potential of PNAs is restricted by the difficulties of delivering them to mitochondria within cells. To overcome this problem we conjugated a PNA 11mer to a lipophilic phosphonium cation. Such cations are taken up by mitochondria through the lipid bilayer driven by the membrane potential across the inner membrane. As anticipated, phosphonium–PNA (ph–PNA) conjugates of 3.4–4 kDa were imported into both isolated mitochondria and mitochondria within human cells in culture. This was confirmed by using an ion-selective electrode to measure uptake of the ph–PNA conjugates; by cell fractionation in conjunction with immunoblotting; by confocal microscopy; by immunogold-electron microscopy; and by crosslinking ph–PNA conjugates to mitochondrial matrix proteins. In all cases dissipating the mitochondrial membrane potential with an uncoupler prevented ph–PNA uptake. The ph–PNA conjugate selectively inhibited the in vitro replication of DNA containing the A8344G point mutation that causes the human mtDNA disease ‘myoclonic epilepsy and ragged red fibres’ (MERRF) but not the wild-type sequence that differs at a single nucleotide position. Therefore these modified PNA oligomers retain their selective binding to DNA and the lipophilic cation delivers them to mitochondria within cells. When MERRF cells were incubated with the ph–PNA conjugate the ratio of MERRF to wild-type mtDNA was unaffected, even though the ph–PNA content of the mitochondria was sufficient to inhibit MERRF mtDNA replication in a cell-free system. This unexpected finding suggests that nucleic acid derivatives cannot bind their complementary sequences during mtDNA replication. In summary, we have developed a new strategy for targeting PNA oligomers to mitochondria and used it to determine the effects of PNA on mutated mtDNA replication in cells. This work presents new approaches for the manipulation of mtDNA replication and expression, and will assist in the development of therapies for mtDNA diseases.

Human brain contains high levels of heteroplasmy in the noncoding regions of mitochondrial DNA.

We have analyzed the level of intraindividual sequence variability (heteroplasmy) of mtDNA in human brain by denaturing gradient gel electrophoresis and sequencing. Single base substitutions, as well as insertions or deletions of single bases, were numerous in the noncoding control region (D-loop), and 35-45% of the molecules from a single tissue showed sequence differences. By contrast, heteroplasmy in coding regions was not detected. The lower level of heteroplasmy in the coding regions is indicative of selection against deleterious mutations. Similar levels of heteroplasmy were found in two brain regions from the same individual, while no heteroplasmy was detected in blood. Thus, heteroplasmy seems to be more frequent in nonmitotic tissues. We observed a 7.7-fold increase in the frequency of deletions/insertions and a 2.2-fold increase in the overall frequency of heteroplasmic mutations in two individuals aged 96 and 99, relative to an individual aged 28. Our results show that intraindividual sequence variability occurs at a high frequency in the noncoding regions of normal human brain and indicate that small insertions and deletions might accumulate with age at a lower rate than large rearrangements.

Genes encoding the same three subunits of respiratory complex II are present in the mitochondrial DNA of two phylogenetically distant eukaryotes.

Although mitochondrial DNA is known to encode a limited number (<20) of the polypeptide components of respiratory complexes I, III, IV, and V, genes for components of complex II [succinate dehydrogenase (ubiquinone); succinate:ubiquinone oxidoreductase, EC 1.3.5.1] are conspicuously lacking in mitochondrial genomes so far characterized. Here we show that the same three subunits of complex II are encoded in the mitochondrial DNA of two phylogenetically distant eukaryotes, Porphyra purpurea (a photosynthetic red alga) and Reclinomonas americana (a heterotrophic zooflagellate). These complex II genes, sdh2, sdh3, and sdh4, are homologs, respectively, of Escherichia coli sdhB, sdhC, and sdhD. In E. coli, sdhB encodes the iron-sulfur subunit of succinate dehydrogenase (SDH), whereas sdhC and sdhD specify, respectively, apocytochrome b558 and a hydrophobic 13-kDa polypeptide, which together anchor SDH to the inner mitochondrial membrane. Amino acid sequence similarities indicate that sdh2, sdh3, and sdh4 were originally encoded in the protomitochondrial genome and have subsequently been transferred to the nuclear genome in most eukaryotes. The data presented here are consistent with the view that mitochondria constitute a monophyletic lineage.

Phylogenetic resolution within the Elephantidae using fossil DNA sequence from the American mastodon (Mammut americanum) as an outgroup.

DNA was extracted from the extinct American mastodon, the extinct woolly mammoth, and the modern Asian and African elephants to test the traditional morphologically based phylogeny within Elephantidae. Phylogenetic analyses of the aligned sequences of the mitochondrial gene cytochrome b support a monophyletic Asian elephant-woolly mammoth clade when the American mastodon is used as an outgroup. Previous molecular studies were unable to resolve the relationships of the woolly mammoth, Asian elephant, and African elephant because the sequences appear to have evolved at heterogeneous rates and inappropriate outgroups were used for analysis. The results demonstrate the usefulness of fossil molecular data from appropriate sister taxa for resolving phylogenies of highly derived or early radiating lineages.

Nuclear and mitochondrial DNA markers in traceability of retail beef samples

Several characteristics are important in a traceability system of animal products, such as age at slaughter, breed composition, besides information of the productive chain. In general, the certification agent records information about the animals and the system which it came from, although cannot guarantee that the slaughtering, meat processing and distribution are error proof. Besides, there is a differential price, at least at the international market, based on sex and breed composition of the animals. Genetic markers allow identification of characteristics controlled in the beef cattle traceability program, as sex and breed composition, in order to correctly identify and appraise the final product for the consumer. The hypothesis of this study was that the majority beef samples retailed in the local market originate from female with a great participation of zebu breeds. Therefore, the objective of this work was to characterize retail beef samples with DNA markers that identify cattle sex and breed composition. Within 10 beef shops localized in Pirassununga, SP, Brazil, 61 samples were collected, all were genotyped as harboring Bos taurus mitochondrial DNA and 18 were positive for the Y chromosome amplification (male). For the marker sat1711b-Msp I the frequency of the allele A was 0.278 and for the marker Lhr-Hha I the frequency of the allele T was 0.417. The results of sat1711b-Msp I and Lhr-Hha I allelic frequencies are suggestive that the proportion of indicus genome compared with the taurine genome in the market meat is smaller than the observed in the Nellore breed. The procedure described in this study identified sex and subspecies characteristics of beef meat samples, with potential application in meat products certification in special as an auxiliary tool in beef cattle traceability programs.

Species determination of Brazilian mammals implicated in the epidemiology of rabies based on the control region of mitochondrial DNA

Identification of animals that are decomposing or have been run over or burnt and cannot be visually identified is a problem in the surveillance and control of infectious diseases. Many of these animals are wild and represent a valuable source of information for epidemiologic research as they may be carriers of an infectious agent. This article discusses the results obtained using a method for identifying mammals genetically by sequencing their mitochondrial DNA control region. Fourteen species were analyzed and identified. These included the main reservoirs and transmitters of rabies virus, namely, canids, chiroptera and primates. The results prove that this method of genetic identification is both efficient and simple and that it can be used in the surveillance of infectious diseases which includes mammals in their epidemiologic cycle, such as rabies.

CONTRASTING PHYLOGEOGRAPHIC PATTERNS IN MITOCHONDRIAL DNA AND MICROSATELLITES: EVIDENCE OF FEMALE PHILOPATRY AND MALE-BIASED GENE FLOW AMONG REGIONAL POPULATIONS OF THE BLUE-AND-YELLOW MACAW (PSITTACIFORMES: ARA ARARAUNA) IN BRAZIL

Comparing the patterns of population differentiation among genetic markers with different modes of inheritance call provide insights into patterns of sex-biased dispersal and gene flow. The blue-and-yellow Macaw (Ara ararauna) is a Neotropical parrot with a broad geographic distribution ill South America. However, little is known about the natural history and current status Of remaining wild populations, including levels of genetic variability. The progressive decline and possible fragmentation of populations may endanger this species in the near future. We analyzed mitochondrial DNA (mtDNA) control-region sequences and six microsatellite 106 Of Blue-and-yellow Macaws sampled throughout their geographic range ill Brazil to describe population genetic Structure, to make inferences about historical demography and dispersal behavior, and to provide insight for conservation efforts. Analyses of population genetic structure based on mtDNA showed evidence of two major populations ill western and eastern Brazil that share a few low-frequency haplotypes. This phylogeographic pattern seems to have originated by the historical isolation of Blue-and-yellow Macaw populations similar to 374,000 years ago and has been maintained by restricted gene flow and female philopatry. By contrast, variation ill biparentally inherited microsatellites was not structured geographically, Male-biased dispersal and female philopatry best explain the different patterns observed in these two markers. Because females disperse less than males, the two regional populations with well-differentiated mtDNA haplogroups should be considered two different management units for conservation purposes. Received 4 November 2007 accepted 10 December 2008.

No relationship found between point heteroplasmy in mitochondrial DNA control region and age range, sex and haplogroup in human hairs

The analysis of heteroplasmy (presence of more than one type of mitochondrial DNA in an individual) is used as a tool in human identification studies, anthropology, and most currently in studies that relate heteroplasmy with longevity. The frequency of heteroplasmy and its correlation with age has been analyzed using different tissues such as blood, muscle, heart, bone and brain and in different regions of mitochondrial DNA, but this analysis had never been performed using hair samples. In this study, samples of hair were sequenced in order to ascertain whether the presence or not of heteroplasmy varied according to age, sex and origin of haplogroup individuals. The samples were grouped by age (3 groups), gender (male and female) and haplogroup of origin (European, African and Native American), and analyzed using the chi-square statistical test (chi(2)). Based in statistical results obtained, we conclude that there is no relationship between heteroplasmy and sex, age and haplogroup origin using hair samples.

Geographic variation of sex pheromone and mitochondrial DNA in Diatraea saccharalis (Fab., 1794) (Lepidoptera: Crambidae)

(9Z,11E)-hexadecadienal and (Z11)-hexadecenal, the main sex pheromone components of the sugarcane borer, Diatraea saccharalis, were identified and quantified from four Brazilian and one Colombian populations using GC-EAD, GC-MS and GC analyses. Three different ratios were observed, 9:1,6:1, and 3:1. The pheromone concentration for the major component, (9Z,11E)-hexadecadienal, varied from 6.8 ng/gland to 21.9 ng/gland and from 1.7 ng/gland to 6.5 to the minor component, (Z11)-hexadecenal. The 25 D. saccharalis cytochrome oxidase II sequences that were analyzed showed low intra-specific variation and represented only 11 haplotypes, with the most frequent being the one represented by specimens from Sao Paulo, Parana, and Pernambuco states. Specimens from Colombia showed the highest genetic divergence from the others haplotypes studied. Data on the genetic variability among specimens, more than their geographic proximity, were in agreement with data obtained from analyses of the pheromone extracts. Our data demonstrate a variation in pheromone composition and a covariation in haplotypes of the D. saccharalis populations studied. (C) 2010 Elsevier Ltd. All rights reserved.

Heteroplasmy in Hair: Study of Mitochondrial DNA Third Hypervariable Region in Hair and Blood Samples

Mitochondrial DNA (mtDNA) analysis has proved useful for forensic identification especially in cases where nuclear DNA is not available, such as with hair evidence. Heteroplasmy, the presence of more than one type of mtDNA in one individual, is a common situation often reported in the first and second mtDNA hypervariable regions (HV1/HV2), particularly in hair samples. However, there is no data about heteroplasmy frequency in the third mtDNA hypervariable region (HV3). To investigate possible heteroplasmy hotspots, HV3 from hair and blood samples of 100 individuals were sequenced and compared. No point heteroplasmy was observed, but length heteroplasmy was, both in C-stretch and CA repeat. To observe which CA ""alleles"" were present in each tissue, PCR products were cloned and re-sequenced. However, no variation among CA alleles was observed. Regarding forensic practice, we conclude that point heteroplasmy in HV3 is not as frequent as in the HV1/HV2.

Mitochondrial DNA Variability Among Eight Tikuna Villages: Evidence for an Intratribal Genetic Heterogeneity Pattern

To study the genetic structure of the Tikuna tribe, four major Native American mitochondrial DNA (mtDNA) founder haplogroups were analyzed in 187 Amerindians from eight Tikuna villages located in the Brazilian Amazon. The central position of these villages in the continent makes them relevant for attempts to reconstruct population movements in South America. In this geographic region, there is particular concern regarding the genetic structure of the Tikuna tribe, formerly designated ""enigmatic"" due to its remarkable degree of intratribal homogeneity and the scarcity of private protein variants. In spite of its large population size and geographic distribution, the Tikuna tribe presents marked genetic and linguistic isolation. All individuals presented indigenous mtDNA haplogroups. An intratribal genetic heterogeneity pattern characterized by two highly homogeneous Tikuna groups that differ considerably from each other was observed. Such a finding was unexpected, since the Tikuna tribe is characterized by a social system that favors intratribal exogamy and patrilocality that would lead to a higher female migration rate and homogenization of the mtDNA gene pool. Demographic explosions and religious events, which significantly changed the sizes and compositions of many Tikuna villages, may be reflected in the genetic results presented here. Am J Phys Anthropol 140:526-531,2009. (C) 2009 Wiley-Liss, Inc

Mitochondrial DNA depletion and its correlation with TFAM, TFB1M, TFB2M and POLG in human diffusely infiltrating astrocytomas

Mitochondrial DNA (mtDNA) alterations and their clinical and pathological implications have been analyzed in several human malignancies. A marked decrease in mtDNA copy number along with the increase in malignancy was observed in diffusely infiltrating astrocytomas (24 WHO grade II, 18 grade III, and 78 grade IV or GBM) compared to non-neoplastic brain tissues, being mostly depleted in GBM. Although high relative gene expression levels of mtDNA replication regulators (mitochondrial polymerase catalytic subunit (POLG), transcription factors A (TFAM), B1 (TFB1M) and B2 (TFB2M)) were detected, it cannot successfully revert the mtDNA depletion observed in our samples. On the other hand, a strong correlation among the expression levels of mitochondrial transcription factors corroborates with the TFAM role in the direct control of TFB1M and TFB2M during initiation of mtDNA replication. POLG expression was related to decreased mtDNA copy number, and its overexpression associated with TFAM expression levels also have an impact on long-term survival among GBM patients, interpreted as a potential predictive factor for better prognosis. (C) 2010 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

Ultrastructure and small-subunit ribosomal DNA sequence of Henneguya lesteri n. sp (Myxosporea), a parasite of sand whiting Sillago analis (Sillaginidae) from the coast of Queensland, Australia

Henneguya lesteri n. sp, (Myxosporea) is described from sand whiting, Sillago analis, from the southern Queensland coast of Australia. H. lesteri displays a preference for the pseudobranchs and is typically positioned along the afferent blood vessels, displacing the adjoining lamellae and disrupting their normal array, The plasmodia appeared as whitish-hyaline, elliptical cysts (mean dimensions 230 x 410 mum) attached to the oral mucosa lining of the hyoid arch on the inner surface of the operculum. Infections of the gills were also found, in which the plasmodia were spherical, averaged 240 x 240 mum in size and were located on the inner hemibranch margin. The parasites lodged in the gill filament crypts and generated a mild hyperplastic response of the branchial epithelium, In histological sections, the plasmodium wall and adjoining ectoplasm appeared as a finely granulated, weakly eosinophilic layer, Ultrastructurally, this section of the host-parasite interface contained an intricate complex of pinocytotic channels. H. lesteri is polysporic, disporoblastic and pansporoblast forming. Sporogenesis is asynchronous, with the earliest developmental stages aligned predominantly along the plasmodium periphery, and maturing sporoblasts and spores toward the center. Ultrastructural details of sporoblast and spore development are in agreement with previously described myxosporeans. The mature spore is drop-shaped, length (mean) 9.1 mum, width 4.7 mum, thickness 2.5 mum, and comprises 2 polar capsules positioned closely together, a binucleated sporoplasm and a caudal process of 12.6 mum. The polar capsules are elongated, 3.2 x 1.6 mum, with 4 turns of the polar filament. Mean length of the everted filament is 23.2 mum, Few studies have analyzed the 18S gene-of marine Myxosporea. In fact, H. lesteri is the first marine species of Henneguya to be characterized at the molecular level: we determined 1966 bp of the small-subunit (18S) rDNA, The results indicated that differences between this and the hitherto studied freshwater Henneguya species are greater than differences among the freshwater Henneguya species.