MtDNA characterization

This complex consists of at least 25 separate polypeptides, seven of which are encoded by mtDNA. Its catalytic action is to transfer electrons from NADH to ubiquinone, thus replenishing NAD concentrations. Complex I deficiency has been described in myopathic syndromes, characterized by exercise intolerance and lactic acidemia. In at least some patients it has been demonstrated that the defect is tissue specific and a defect in nuclear DNA is assumed. Muscle biopsy findings in these patients are typical of those in many respiratory chain abnormalities. Instead of the even distribution of mitochondria seen in normal muscle fibers, mitochondria are seen in dense clusters, especially at the fiber periphery, giving rise to the ragged-red fiber (Figure 10). This appearance is a hallmark of many mitochondrial myopathies. 

Complex I deficiency due to mtDNA mutations (seven subunits of complex I are encoded by mtDNA) can be divided into encephalomyopathies and myopathies. The most important encephalomyopathy is Leber s hereditary optic neuropathy, characterized by acute or subacute loss of vision due to severe bilateral optic atrophy, with onset usually between 18 and 30 years and marked predominance in men. Three mutations (in ND1, ND4 and ND6)... 

Nucleoside analogues are drugs used to treat HIV and hepatitis. One such drug fialuridine and other drugs of this type have caused severe hepatic dysfunction. This dysfunction was characterized by fatty liver and fatal liver failure. Fialuridine caused fatal damage in 5 of 12 patients in early clinical trials. Fialuridine inhibits DNA polymerases. However, there is also DNA in the mitochondria [mitochondrial DNA (mtDNA)]. 

V3. van Goethem, G., Dermaut, B., Lofgren, A., Martin, J. J., and van Broeckhoven, C., Mutation of POLG is associated with progressive external ophthalmoplegia characterized by mtDNA deletions. Nat. Genet. 28, 211-212 (2001). 

Leber hereditary optic neuropathy (LHON) is the most common mitochondrial disease and the first linked to maternal inheritance through a mutation in the mtDNA. LHON is characterized by bilateral subacute loss of central vision caused by focal degeneration of the retinal ganglion cell layer and of the optic nerve. After initial symptoms, both eyes are usually affected within 6 months. Approximately 50% to 60% of males and only 8% to 32% of females who possess the mtDNA mutation will actually develop this optic neuropathy. Nuclear-encoded factors that affect mtDNA expression, mtDNA products, or mitochondrial metabolism may modify the phenotypic expression of LHON. Genetic coimseling in LHON is complicated in that the amount of mutant mtDNA transmitted by heteroplasmic females cannot be predicted, and testing cannot predict which individuals will develop visual symptoms. ... 

Fourteen mtDNA tRNA mutations have been associated with maternally inherited disease. Such mutations are typically associated with severe mitochondrial myopathies, characterized by ragged red skeletal muscle fibers upon Gomori trichrome staining and the accumulation of structurally abnormal mitochondria in muscle. Mutations in tRNAs exemplify the threshold effect whereby (due to replicative segragation) individuals may not exhibit clinical signs until the proportion of mutant mtDNA exceeds 80-90%. Myoclonic epilepsy and ragged red fiber (MERRF) disease, mitochondrial encephalomyo-pathy tactic acidosis (MELAS), as well as maternally inherited myopathy and cardiomyopathy (MMC) are well-characterized mitochondrial diseases. 

Large mtDNA deletions account for most cases of ocular myopathy and Pearson s marrow/pancreas syndrome. Ocular myopathy patients can exhibit a variety of clinical symptoms, from mild chronic progressive external ophthalmoplegia (CPEO) to Kearns-Sayre Syndrome (KSS). These diseases are characterized by an early onset of ophthalmoplegia, atypical retinitis pigmentosa, mitochondrial myopathy, and usually cerebellar syndrome and cardiac conduction abnormalities. More than 120 different mtDNA deletions have been identified from patients tissues. Partial duplications of mtDNA have been detected in ocular myopathy and Pearson s syndrome, however, duplications are much rarer than spontaneous deletions in patients with these conditions. Exactly how partial mtDNA duplications arise is unknown. 

The promoter sequences recognized by mitochondrial RNA polymerases include the transcription start site. These promoter sequences, which are rich in A residues, have been characterized in the mtDNA from yeast, plants, and animals. The circular, human mitochondrial genome contains two related 15-bp promoter sequences, one for the transcription of each strand. Each strand is transcribed in its entirety the long primary transcripts are then processed to yield mitochondrial mRNAs, rRNAs, and tRNAs. A small basic protein called mtTFl, which binds immediately upstream from the two mitochondrial promoters, greatly stimulates transcription. A homologous protein found in yeast mitochondria is required for maintenance of mtDNA and probably performs a similar function. 

Kearns-Sayre syndrome Onset before 20 years of age, characterized by opthalmoplegia, atypical retinitis pigmentosa, mitochondrial myopathy, and one of the following cardiac conduction defect, cerebellar syndrome, or elevated CSF proteins. Deletion of contiguous segments of tRNA and OXPHOS polypeptides, or duplication mutations consisting of tandemly arranged normal mtDNA and an mtDNA with a deletion mutation. 

This technique is very discriminating and not too expensive, but it is long and requires several complex manipulations. It is useful for the subtle characterization of a small number of sfiains. Inoculation effectiveness can also be verified by this method. To verify an inoculation, a sample is taken during or towards the end of alcoholic fermentation. In the laboratory, the lees are placed in a liquid medium culture. The mtDNA restriction profile of this total biomass and of the yeast starter strain are compared. If the restriction profile of the sample has no supernumerary bands with respect to the yeast starter strain profile, the yeast starter has been properly implanted, with an accuracy of 90%. In fact, in the case of a binary mixture, the minority strain must represent around 10% of the total population to be detected (Hallet et al., 1989). 

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