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PubMed2004

Alteration of the copy number and deletion of mitochondrial DNA in human hepatocellular carcinoma.

Somatic mutations in mitochondrial DNA (mtDNA) have been detected in hepatocellular carcinoma (HCC). However, it remains unclear whether mtDNA copy number and mitochondrial biogenesis are altered in HCC. In this study, we found that mtDNA copy number and the content of mitochondrial respiratory proteins were reduced in HCCs as compared with the corresponding non-tumorous livers. MtDNA copy number was significantly reduced in female HCC but not in male HCC. Expression of the peroxisome proliferator-activated receptor gamma coactivator-1 was significantly repressed in HCCs (P<0.005), while the expression of the mitochondrial single-strand DNA-binding protein was upregulated, indicating that the regulation of mitochondria biogenesis is disturbed in HCC. Moreover, 22% of HCCs carried a somatic mutation in the mtDNA D-loop region. The non-tumorous liver of the HCC patients with a long-term alcohol-drinking history contained reduced mtDNA copy number (P<0.05) and higher level of the 4977 bp-deleted mtDNA (P<0.05) as compared with non-alcohol patients. Our results suggest that reduced mtDNA copy number, impaired mitochondrial biogenesis and somatic mutations in mtDNA are important events during carcinogenesis of HCC, and the differential alterations in mtDNA of male and female HCC may contribute to the differences in the clinical manifestation between female and male HCC patients.

Alcohol Drinking↗
PubMed1993

Content of mutant mitochondrial DNA and organ dysfunction in a patient with a MELAS subgroup of mitochondrial encephalomyopathies.

A point mutation of mitochondrial tRNALeu(UUR) gene is responsible for a MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes) subgroup of mitochondrial encephalomyopathies. In most cases, the mutant mitochondrial DNA (mtDNA) coexists with normal mtDNA in a heteroplasmic manner. In order to quantify the content of mutant mtDNA, we developed a quantitative method of PCR. Using this method, the distribution of the mutant mtDNA was examined in 32 different tissues among 18 autopsied organs from a patient with MELAS, who had shown hypophyseal dysfunction. The percentage of the mutant mtDNA at nucleotide number 3243 in each tissue was ranged between 22% and 95%. The content of the mutant mtDNA was at the highest (95%) in the hypophysis and higher in the cerebral cortex than in the white matter. This study shows a possible correlation of tissue dysfunction with accumulation of the mutant mtDNA within the brain.

Adult↗
PubMed2004

Quantitative analysis of total mitochondrial DNA: competitive polymerase chain reaction versus real-time polymerase chain reaction.

An efficient and effective method for quantification of small amounts of nucleic acids contained within a sample specimen would be an important diagnostic tool for determining the content of mitochondrial DNA (mtDNA) in situations where the depletion thereof may be a contributing factor to the exhibited pathology phenotype. This study compares two quantification assays for calculating the total mtDNA molecule number per nanogram of total genomic DNA isolated from human blood, through the amplification of a 613-bp region on the mtDNA molecule. In one case, the mtDNA copy number was calculated by standard competitive polymerase chain reaction (PCR) technique that involves co-amplification of target DNA with various dilutions of a nonhomologous internal competitor that has the same primer binding sites as the target sequence, and subsequent determination of an equivalence point of target and competitor concentrations. In the second method, the calculation of copy number involved extrapolation from the fluorescence versus copy number standard curve generated by real-time PCR using various dilutions of the target amplicon sequence. While the mtDNA copy number was comparable using the two methods (4.92 +/- 1.01 x 10(4) molecules/ng total genomic DNA using competitive PCR vs 4.90 +/- 0.84 x 10(4) molecules/ng total genomic DNA using real-time PCR), both inter- and intraexperimental variance were significantly lower using the real-time PCR analysis. On the basis of reproducibility, assay complexity, and overall efficiency, including the time requirement and number of PCR reactions necessary for the analysis of a single sample, we recommend the real-time PCR quantification method described here, as its versatility and effectiveness will undoubtedly be of great use in various kinds of research related to mitochondrial DNA damage- and depletion-associated disorders.

DNA, Mitochondrial↗
PubMed1995

Increased genetic diversity in mitochondrial genes is correlated with the evolution of parasitism in the Hymenoptera.

A higher AT content and rate of mtDNA sequence divergence was found in parasitic wasps (Apocrita) compared with nonparasitic wasps (Symphyta). The compositional bias was reflected in extreme codon bias for a cytochrome oxidase I protein coding gene fragment as well as in the types of amino acid substitutions that have occurred during the evolution of this gene fragment. In some instances, compositional bias influenced the definition of a conservative amino acid change. The increased rate of mtDNA sequence evolution probably arose during the early Jurassic, coincident with the first appearance of parasitic wasps in the fossil record. Our results suggest a causal link between the rate of sequence divergence and the parasitic lifestyle.

Animals↗
PubMed1992

Genome organization of mitochondrial DNA from the non-saccharomycete yeast Arxula adeninivorans LS3.

Mitochondrial (mt) DNA of the asexual ascomycetous yeast Arxula adeninivorans LS3 was isolated and characterized. The mtDNA has a GC content of 30.3 mol%. It is circular and its size, as estimated by restriction analysis performed with nine endonucleases, was 35.5 kbp. Using mt gene-probes from Saccharomyces cerevisiae six structural genes (cob, cox1, cox2, oli1, oli2, and 21S rRNA) were located on the mitochondrial genome of A. adeninivorans. The comparison between the mt genomes of A. adeninivorans and other yeasts showed differences in genome organization.

Ascomycota↗
PubMed1999

The mitochondrial genome of Arabidopsis is composed of both native and immigrant information.

Plants contain large mitochondrial genomes, which are several times as complex as those in animals, fungi or algae. However, genome size is not correlated with information content. The mitochondrial genome (mtDNA) of Arabidopsis specifies only 58 genes in 367 kb, whereas the 184 kb mtDNA in the liverwort Marchantia polymorpha codes for 66 genes, and the 58 kb genome in the green alga Prototheca wickerhamii encodes 63 genes. In Arabidopsis' mtDNA, genes for subunits of complex II, for several ribosomal proteins and for 16 tRNAs are missing, some of which have been transferred recently to the nuclear genome. Numerous integrated fragments originate from alien genomes, including 16 sequence stretches of plastid origin, 41 fragments of nuclear (retro)transposons and two fragments of fungal viruses. These immigrant sequences suggest that the large size of plant mitochondrial genomes is caused by secondary expansion as a result of integration and propagation, and is thus a derived trait established during the evolution of land plants.

Journal Article↗
PubMed1999

1-Methyl-4-phenylpyridinium ion (MPP+) selectively inhibits the replication of mitochondrial DNA.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine is known to cause Parkinsonism in its neurotoxic form, 1-methyl-4-phenylpyridinium ion (MPP+). We have previously reported that MPP+ decreases the content of mitochondrial DNA (mtDNA) independently of the inhibition of complex I in human cells [Miyako, K., Kai, Y., Irie, T., Takeshige, K., and Kang, D. (1997) J. Biol. Chem. 272, 9605-9608]. Here we study the mechanism causing the decrease in mtDNA. MPP+ inhibits the incorporation of 5-bromo-2'-deoxyuridine into mtDNA but not into nuclear DNA, indicating that MPP+ inhibits the replication of mtDNA but not that of the nuclear genome. The replication of mtDNA is initiated by the synthesis of the heavy strand switched from the transcription of the light strand. MPP+ decreases the nascent heavy strands per mtDNA and increases the transcript of the ND6 gene, encoded on light strand, per mtDNA. The amount of mitochondrial transcription factor A is not decreased. These data suggest that the transcription is not inhibited and therefore the transition from transcription to replication of mtDNA is lowered in the MPP+-treated cells. Electron microscopy shows that the number of mitochondria is not decreased in the MPP+-treated cells, suggesting that MPP+ does not affect the overall biogenesis of mitochondria. Thus, MPP+ selectively inhibits the replication of mtDNA.

1-Methyl-4-phenylpyridinium↗
PubMed2002

Depletion of mitochondrial DNA in the liver of an infant with neonatal giant cell hepatitis.

A boy presented with lactic acidosis, hepatomegaly, hypoglycemia, generalised icterus, and muscle hypotonia in the first weeks of life. At the age of 2 months, neonatal giant cell hepatitis was diagnosed by light microscopy. Electron microscopy of the liver revealed an accumulation of abnormal mitochondria and steatosis. Skeletal muscle was normal on both light and electron microscopy. At the age of 5 months, the patient died of liver failure. Biochemical studies of the respiratory chain enzymes in muscle showed that cytochrome-c oxidase (complex IV) and succinate-cytochrome-c oxidoreductase (complex II + III) activities were (just) below the control range. When related to citrate synthase activity, however, complex IV and complex II + III activities were normal. Complex I activity was within the control range. The content of mitochondrial DNA (mtDNA) was severely reduced in the liver (17% to 18% of control values). Ultracytochemistry and immunocytochemistry of cytochrome-c oxidase demonstrated a mosaic pattern of normal and defective liver cells. In defective cells, a reduced amount of the mtDNA-encoded subunits II-III and the nuclear DNA-encoded subunits Vab was found. Cells of the biliary system were spared. Immunohistochemistry of mtDNA replication factors revealed normal expression of DNA polymerase gamma. The mitochondrial single-stranded binding protein (mtSSB) was absent in some abnormal hepatocytes, whereas the mitochondrial transcription factor A (mtTFA) was deficient in all abnormal hepatocytes. In conclusion, depletion of mtDNA may present as giant cell hepatitis. mtTFA and to a lesser degree mtSSB are reduced in mtDNA depletion of the liver and may, therefore, be of pathogenetic importance. The primary defect, however, is still unknown.

Citrate (si)-Synthase↗
PubMed2001

Mitochondria-to-nucleus stress signaling induces phenotypic changes, tumor progression and cell invasion.

Recently we showed that partial depletion of mitochondrial DNA (genetic stress) or treatment with mitochondrial-specific inhibitors (metabolic stress) induced a stress signaling that was associated with increased cytoplasmic-free Ca(2+) [Ca(2+)](c). In the present study we show that the mitochondria-to-nucleus stress signaling induces invasive phenotypes in otherwise non-invasive C2C12 myoblasts and human pulmonary carcinoma A549 cells. Tumor-specific markers cathepsin L and transforming growth factor beta (TGFbeta) are overexpressed in cells subjected to mitochondrial genetic as well as metabolic stress. C2C12 myoblasts subjected to stress showed 4- to 6-fold higher invasion through reconstituted Matrigel membrane as well as rat tracheal xenotransplants in Scid mice. Activation of Ca(2+)-dependent protein kinase C (PKC) under both genetic and metabolic stress conditions was associated with increased cathepsin L gene expression, which contributes to increased invasive property of cells. Reverted cells with approximately 70% of control cell mtDNA exhibited marker mRNA contents, cell morphology and invasive property closer to control cells. These results provide insights into a new pathway by which mitochondrial DNA and membrane damage can contribute to tumor progression and metastasis.

Animals↗
PubMed1978

Uniparental inheritance of mitochondrial genes in yeast: dependence on input bias of mitochondrial DNA and preliminary investigations of the mechanism.

In Saccharomyces cerevisiae, previous studies on the inheritance of mitochondrial genes controlling antibiotic resistance have shown that some crosses produce a substantial number of uniparental zygotes, which transmit to their diploid progeny mitochondrial alleles from only one parent. In this paper, we show that uniparental zygotes are formed especially when one parent (majority parent) contributes substantially more mitochondrial DNA molecules to the zygote than does the other (minority) parent. Cellular contents of mitochondrial DNA (mtDNA) are increased in these experiments by treatment with cycloheximide, alpha-factor, or the uvsp5 nuclear mutation. In such a biased cross, some zygotes are uniparental for mitochondrial alleles from the majority parent, and the frequency of such zygotes increases with increasing bias. In two- and three-factor crosses the cap1, ery1, and oli1 loci behave coordinately, rather than independently; minority markers tend to be transmitted or lost as a unit, suggesting that the uniparental mechanism acts on entire mtDNA molecules rather than on individual loci. This rules out the possibility that uniparental inheritance can be explained by the conversion of minority markers to the majority alleles during recombination. Exceptions to the coordinate behavior of different loci can be explained by marker rescue via recombination. Uniparental inheritance is largely independent of the position of buds on the zygote. We conclude that it is due to the failure of minority markers to replicate in some zygotes, possibly involving the rapid enzymatic destruction of such markers. We have considered two general classes of mechanisms: (1) random selection of molecules for replication, as for example by competition for replicating sites on a membrane; and (2) differential marking of mtDNA molecules in the two parents, possibly by modification enzymes, followed by a mechanism that "counts" molecules and replicates only the majority type. These classes of models are distinguished genetically by the fact that the first predicts that the output frequency of a given allele among the progeny of a large number of zygotes will approximately equal the average input frequency of that allele, while the second class predicts that any input bias will be amplified in the output. The data suggest that bias amplification does occur. We hypothesize that maternal inheritance of mitochondrial or chloroplast genes in many organisms may depend upon a biased input of organelle DNA molecules, which usually favors the maternal parent, followed by failure of the minority (paternal) molecules to replicate in many or all zygotes.

Alleles↗
PubMedDate not supplied

Hepatoma HepG2 cells as a model for in vitro studies on mitochondrial toxicity of antiviral drugs: which correlation with the patient?

Currently, drugs have been synthesised that can significantly delay the course of several viral infections, including those provoked by HBV, HCV or HIV, but that display consistent side effects, including toxicity for organelles such as mitochondria. Several in vitro models and techniques have been developed to analyse the effects of such compounds. HepG2 cells (from human hepatoma) are an excellent model to investigate mitochondrial (mt) toxicity because of their high content of organelles and mtDNA, and actually different investigators are indeed using such cells. Studies in vitro on cell lines are relatively easy, but it is necessary to be careful in the interpretation of data, which are usually obtained on continuously growing, tumour cells, quite different from normal, resting, non-neoplastic cells collected from a patient. Direct analysis of drug-induced mt damage in patients is extremely more complex than that performed using in vitro models because of the difficulty to obtain adequate cells or to have discrete amounts of biological material, the status of the patient at the moment of cell collection, the use of an adequate assay and its correct execution, and finally the possibility to find sex- and age-matched healthy controls as source of reference parameters.

Antiviral Agents↗
PubMed1992

Mitochondrial DNA alterations as ageing-associated molecular events.

Mitochondrial DNA (mtDNA) is a naked double-stranded circular extrachromosomal genetic element continuously exposed to the matrix that contains great amounts of reactive oxygen species and free radicals. The age-dependent decline in the capability and capacity of mitochondria to dispose these oxy-radicals will render mtDNA more vulnerable to mutations during the ageing process. During the past 3 years, more than 10 different types of deletions have been identified in the mtDNA of various tissues of old humans. Some of them were found only in a certain tissue but some others appeared in more than one organ or tissue. The 4977-bp deletion is the most prevalent and abundant one among these deletions. Skeletal muscle is the target tissue of most ageing-associated mtDNA deletions and has often been found to carry multiple deletions. The onset age of the various deletions in mtDNA varies greatly with individual and type of the deletion. The 4977-bp deletion has been independently demonstrated to occur in the mtDNA of various tissues of the human in the early third decade of life. However, the 7436-bp deletion was only detected in the heart mtDNA of human subjects in their late thirties. The others appeared only in older humans over 40 years old. No apparent sex difference was found in the onset age of these ageing-associated mtDNA deletions. The various ageing-associated deletions could be classified into two groups. Most of the deletions belong to the first group, in which the 5'- and 3'-end breakpoints of the deletion are flanked by 4-bp or longer direct repeats. The deletion in the second group occurs less frequently and shows no distinct repeat sequences flanking the deletion sites. These two groups of mtDNA deletions may occur by different mechanisms. The first group is most probably caused by internal recombination or slippage mispairing during replication of mtDNA by the D-loop mechanism. The deleted mtDNA and the deleted DNA fragment may be further degraded or escape from the mitochondria and get translocated into the nucleus. The latter route has been substantiated by many observations of inserted mtDNA sequences in the nuclear DNA. Thus, the fragments of migrating mtDNA may change the information content and expression level of certain nuclear genes and thereby promote the ageing process or cause cancer. Similar ageing-associated alterations of mtDNA have also been observed in aged animals and plants. I suggest that mtDNA deletions and other mutations to be discovered are molecular events generally associated with the ageing process.

Aging↗
PubMed2006

The complete mitochondrial DNA sequence of the green alga Oltmannsiellopsis viridis: evolutionary trends of the mitochondrial genome in the Ulvophyceae.

The mitochondrial genome displays a highly plastic architecture in the green algal division comprising the classes Prasinophyceae, Trebouxiophyceae, Ulvophyceae, and Chlorophyceae (Chlorophyta). The compact mitochondrial DNAs (mtDNAs) of Nephroselmis (Prasinophyceae) and Prototheca (Trebouxiophyceae) encode about 60 genes and have been ascribed an 'ancestral' pattern of evolution, whereas those of chlorophycean green algae are much more reduced in gene content and size. Although the mtDNA of the early-diverging ulvophyte Pseudendoclonium contains 57 conserved genes, it differs from 'ancestral' chlorophyte mtDNAs by its unusually large size (96 kb) and long intergenic spacers. To gain insights into the evolutionary trends of mtDNA in the Ulvophyceae, we have determined the complete mtDNA sequence of Oltmannsiellopsis viridis, an ulvophyte belonging to a distinct, early-diverging lineage. This 56,761 bp genome harbours 54 conserved genes, numerous repeated sequences, and only three introns. From our comparative analyses with Pseudendoclonium mtDNA, we infer that the mitochondrial genome of the last common ancestor of the two ulvophytes closely resembled that of the trebouxiophyte Prototheca in terms of gene content and gene density. Our results also provide strong evidence for the intracellular, interorganellar transfer of a group I intron and for two distinct events of intercellular, horizontal DNA transfer.

Base Pairing↗
PubMed2006

The complete nucleotide sequence of the mitochondrial genome of Tetraodon nigroviridis.

The fresh water pufferfish Tetraodon nigroviridis is a model organism for studying evolution of genome and gene functions, but its mitochondrial genome (mtDNA) sequence is still not available. We determined the complete nucleotide sequence of its mtDNA using shotgun sequencing. The T. nigroviridis mtDNA was 16,462 bp, and contained 13 protein coding genes, 22 tRNAs, 2 rRNAs and a major non-coding region. The gene order was identical to the common type of vertebrate mtDNA, whereas the G + C content in the sense strand was 46.9%, much higher than most other fish species. One hundred and three SNPs were detected in the control region of the mtDNA of 35 individuals, a majority of SNPs were detected in the 5' end of the control region. A phylogenetic study including 21 fish species was performed on concatenated amino acid sequences of 12 protein coding genes, and revealed that the T. nigroviridis was clustered with Fugu rubripes into a group. The complete mtDNA sequence and SNPs in its control region will be useful in studying fish evolution, in differentiating different Tetraodon species and in analyzing genetic diversity within T. nigroviridis.

Animals↗
PubMed1978

Physicochemical characterization of mitochondrial DNA from soybean.

Mitochondrial DNA (mtDNA) of soybean (Glycine max L.) was isolated and its buoyant density was contrasted with that of nuclear (nDNA) and chloroplast (ctDNA) DNA. Each of the three DNAs banded at a single, characteristic buoyant density when centrifuged to equilibrium in a CsCl gradient. Buoyant densities were 1.694 g/cm(3) for nDNA and 1.706 g/cm(3) for mtDNA. These values correspond to G-C contents of 34.7 and 46.9%, respectively. Covalently closed, circular mtDNA molecules were isolated from soybean hypocotyls by ethidium bromide-cesium chloride density gradient centrifugation. Considerable variation in mtDNA circle size was observed by electron microscopy. There were seven apparent size classes with mean lengths of 5.9 mum (class 1), 10 mum (class 2), 12.9 mum (class 3), 16.6 mum (class 4), 20.4 mum (class 5), 24.5 mum (class 6), and 29.9 mum (class 7). In addition, minicircles were observed in all preparations. Partially denatured, circular mtDNA molecules with at least one representative from six of the seven observed size classes were mapped. In class 4, there appear to be at least three distinct denaturation patterns, indicating heterogeneity within this class. It is proposed that the mitochondrial genome of soybean is distributed among the different size circular molecules, several copies of the genome are contained within these classes and that the majority of the various size molecules may be a result of recombination events between circular molecules.

Journal Article↗
PubMed1997

Cloning and characterization of cDNAs encoding chicken mitogen-activated protein kinase kinase type 2, MEK2: downregulation of MEK2 in response to inhibition of mitochondrial DNA expression.

The present work was initiated with the aim of identifying nuclear genes whose expression is sensitive to the mitochondrial DNA (mtDNA) status of transformed chicken DU24 cells. We cloned and sequenced cDNAs for the mitogen-activated protein kinase kinase type 2, MEK2, a protein involved in the mitogenic growth factor signal transduction pathway in vertebrates. Sequence comparisons between the chicken protein and its mammalian counterparts indicated that MEK2 proteins are highly conserved among vertebrates. Southern blot analysis of endonuclease-digested genomic DNA from primary chick embryo fibroblasts (CEF) suggested that MEK2 is a single-copy gene in this vertebrate species. The steady-state level of MEK2 transcripts is decreased in DUS3 mtDNA-less (rho0) cells developed by long-term exposure of DU24 rho+ cells to ethidium bromide (EtdBr). Run-on in vitro transcription assays and mRNA stability studies indicated that the decrease in MEK2 mRNA content is associated with post-transcriptional regulation. In parental DU24 cells, MEK2 mRNA content decreased after inhibition of mtDNA transcription by EtdBr and inhibition of translation on mitoribosomes by chloramphenicol (CAM). Cytoplasmic hybrids (cybrids) constructed by fusion of chicken rho0 cells with enucleated parental cells and CEF recovered a basal level of MEK2 expression. The MEK2 protein content is decreased in DUS3 rho0 cells and in parental DU24 rho+ cells treated with EtdBr and CAM for 6 days, while that of MEK1, a closely related kinase, remained unchanged. On the basis of these observations, we propose that mitochondria participate in the mitogenic signal transduction pathway in chicken cells through regulation of MEK2 expression.

Amino Acid Sequence↗
PubMed1998

Bioenergetic consequences of accumulating the common 4977-bp mitochondrial DNA deletion.

Mutations and deletions in mitochondrial DNA (mtDNA) lead to a number of human diseases characterized by neuromuscular degeneration. Accumulation of truncated mtDNA molecules (delta-mtDNA) lacking a specific 4977-bp fragment, the common deletion, leads to three related mtDNA diseases: Pearson's syndrome; Kearns-Sayre syndrome; and chronic progressive external ophthalmoplegia (CPEO). In addition, the proportion of delta-mtDNA present increases with age in a range of tissues. Consequently, there is considerable interest in the effects of the accumulation of delta-mtDNA on cell function. The 4977-bp deletion affects genes encoding 7 polypeptide components of the mitochondrial respiratory chain, and 5 of the 22 tRNAs necessary for mitochondrial protein synthesis. To determine how the accumulation of delta-mtDNA affects oxidative phosphorylation we constructed a series of cybrids by fusing a human osteosarcoma cell line depleted of mtDNA (rho0) with enucleated skin fibroblasts from a CPEO patient. The ensuing cybrids contained 0-86% delta-mtDNA and all had volumes, protein contents, plasma-membrane potentials and mitochondrial contents similar to those of the parental cell line. The bioenergetic consequences of accumulating delta-mtDNA were assessed by measuring the mitochondrial membrane potential, rate of ATP synthesis and ATP/ADP ratio. In cybrids containing less than 50-55% delta-mtDNA, these bioenergetic functions were equivalent to those of cybrids with intact mtDNA. However, once the proportion of delta-mtDNA exceeded this threshold, the mitochondrial membrane potential, rate of ATP synthesis, and cellular ATP/ADP ratio decreased. These bioenergetic deficits will contribute to the cellular pathology associated with the accumulation of delta-mtDNA in the target tissues of patients with mtDNA diseases.

Adenosine Diphosphate↗
PubMed2006

Differential mitochondrial DNA and gene expression in inherited retinal dysplasia in miniature Schnauzer dogs.

PURPOSE: To investigate the molecular basis of inherited retinal dysplasia in miniature Schnauzers. METHODS: Retina and retinal pigment epithelial tissues were collected from canine subjects at the age of 3 weeks. Total RNA isolated from these tissues was reverse transcribed to make representative cDNA pools that were compared for differences in gene expression by using a subtractive hybridization technique referred to as representational difference analysis (RDA). Expression differences identified by RDA were confirmed and quantified by real-time reverse-transcription PCR. Mitochondrial morphology from leukocytes and skeletal muscle of normal and affected miniature Schnauzers was examined by transmission electron microscopy. RESULTS: RDA screening of retinal pigment epithelial cDNA identified differences in mRNA transcript coding for two mitochondrial (mt) proteins--cytochrome oxidase subunit 1 and NADH dehydrogenase subunit 6--in affected dogs. Contrary to expectations, these identified sequences did not contain mutations. Based on the implication of mt-DNA-encoded proteins by the RDA experiments we used real-time PCR to compare the relative amounts of mt-DNA template in white blood cells from normal and affected dogs. White blood cells of affected dogs contained less than 30% of the normal amount of two specific mtDNA sequences, compared with the content of the nuclear-encoded glyceraldehyde-3-phosphate dehydrogenase (GA-3-PDH) reference gene. Retina and RPE tissue from affected dogs had reduced mRNA transcript levels for the two mitochondrial genes detected in the RDA experiment. Transcript levels for another mtDNA-encoded gene as well as the nuclear-encoded mitochondrial Tfam transcription factor were reduced in these tissues in affected dogs. Mitochondria from affected dogs were reduced in number and size and were unusually electron dense. CONCLUSIONS: Reduced levels of nuclear and mitochondrial transcripts in the retina and RPE of miniature Schnauzers affected with retinal dysplasia suggest that the pathogenesis of the disorder may arise from a lowered energy supply to the retina and RPE.

Animals↗