A 3'-end proximal segment of most of the putative mRNAs encoded in the heavy strand of HeLa cell mtDNA has been partially sequences and aligned with the DNA sequence. In all cases, the 3'-end nucleotide of the individual mRNA coding sequences has been found to be immediately contiguous to a tRNA gene or another mRNA coding sequence. These and previous results indicate that the heavy (H) strand sequences coding for the rRNA, poly(A)-containing RNA and tRNA species form a continuum extending over almost the entire length of this strand. We propose that the H strand is transcribed into a single polycistronic RNA molecule, which is processed later into mature species by precise endonucleolytic cleavages which occur, in most cases, immediately before and after a tRNA sequence.

A variety of mtDNA mutations responsible for human diseases have been associated with molecular defects in the OXPHOS system. It has been proposed that mtDNA genetic alterations can also be responsible for sperm dysfunction. In addition, it was suggested that if sperm dysfunction is the main phenotypic consequence, these mutations could be fixed as stable mtDNA variants, because mtDNA is maternally inherited. To test this possibility, we have performed an extensive analysis of the distribution of mtDNA haplogroups in white men having fertility problems. We have found that asthenozoospermia, but not oligozoospermia, is associated with mtDNA haplogroups in whites. Thus, haplogroups H and T are significantly more abundant in nonasthenozoospermic and asthenozoospermic populations, respectively, and show significant differences in their OXPHOS performance.

Mitochondria are subcellular organelles, devoted mainly to energy production in the form of ATP, that contain their own genetic system. Mitochondrial DNA codifies a small, but essential number of polypeptides of the oxidative phosphorylation system. The mammalian mitochondrial genome is an example of extreme economy showing a compact gene organization. The coding sequences for two ribosomal RNAs (rRNAs), 22 transfer RNAs (tRNAs) and 13 polypeptides are contiguous and without introns. The tRNAs are regularly interspersed between the rRNA and protein‐coding genes, playing a crucial role in RNA maturation from the polycistronic transcripts. A single major non‐coding region, called the D‐loop region, contains the main regulatory sequences for transcription and replication initiation. This genetic organization has its precise correspondence in the mode of expression and distinctive structural features of the RNAs. The basic mechanisms of mitochondrial DNA transcription and replication and the main cis‐acting elements playing a role in both processes have been determined. Many trans‐acting factors involved in mitochondrial gene expression, including the RNA and DNA polymerases, have been cloned or identified. However, the regulatory mechanisms participating in mitochondrial gene expression are still poorly understood. The interest to complete this knowledge is increased by the involvement of mitochondria in human diseases, in basic processes such as heat production, Ca2+ homeostasis and apoptosis, and by their potential role in ageing and carcinogenesis.

Many epidemiologic studies have associated human mitochondrial haplogroups to rare mitochondrial diseases like Leber's hereditary optic neuropathy or to more common age-linked disorders such as Parkinson's disease. However, cellular, biochemical and molecular-genetic evidence that is able to explain these associations is very scarce. The etiology of multifactorial diseases is very difficult to sort out because such diseases are due to a combination of genetic and environmental factors that individually only contribute in small part to the development of the illness. Thus, the haplogroup-defining mutations might behave as susceptibility factors, but they could have only a small effect on oxidative phosphorylation (OXPHOS) function. Moreover, these effects would be highly dependent on the 'context' in which the genetic variant is acting. To homogenize this 'context' for mitochondrial DNA (mtDNA) mutations, a cellular approach is available that involves the use of what is known as 'cybrids'. By using this model, we demonstrate that mtDNA and mtRNA levels, mitochondrial protein synthesis, cytochrome oxidase activity and amount, normalized oxygen consumption, mitochondrial inner membrane potential and growth capacity are different in cybrids from the haplogroup H when compared with those of the haplogroup Uk. Thus, these inherited basal differences in OXPHOS capacity can help to explain why some individuals more quickly reach the bioenergetic threshold below which tissue symptoms appear and progress toward multifactorial disorders. Hence, some population genetic variants in mtDNA contribute to the genetic component of complex disorders. The existence of mtDNA-based OXPHOS differences opens possibilities for the existence of a new field, mitochondrial pharmacogenomics. New sequence accession nos: HM103354-HM103363.

Human mitochondrial transcription factor A, TFAM, is essential for mitochondrial DNA packaging and maintenance and also has a crucial role in transcription. Crystallographic analysis of TFAM in complex with an oligonucleotide containing the mitochondrial light strand promoter (LSP) revealed two high-mobility group (HMG) protein domains that, through different DNA recognition properties, intercalate residues at two inverted DNA motifs. This induced an overall DNA bend of ~180°, stabilized by the interdomain linker. This U-turn allows the TFAM C-terminal tail, which recruits the transcription machinery, to approach the initiation site, despite contacting a distant DNA sequence. We also ascertained that structured protein regions contacting DNA in the crystal were highly flexible in solution in the absence of DNA. Our data suggest that TFAM bends LSP to create an optimal DNA arrangement for transcriptional initiation while facilitating DNA compaction elsewhere in the genome.

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The 5'-end proximal sequences of all the putative mRNAs coded for by the heavy strand of HeLa cell mitochondrial DNA have been determined and aligned with the DNA sequence. All these mRNAs start directly at, or very near to, an AUG or AUA triplet, with the exception of one which starts at an AUU. The available evidence indicates that the terminal or subterminal AUGs and AUAs, and possibly also the terminal AUU, are initiator codons for the corresponding polypeptides. In most cases, the individual mRNA coding sequences are flanked on their 5' side by a tRNA gene, without any intervening nucleotide.

BackgroundThymine kinase 2 (TK2) is a mitochondrial matrix protein encoded in nuclear DNA and phosphorylates the pyrimidine nucleosides: thymidine and deoxycytidine. Autosomal recessive TK2 mutations cause a spectrum of disease from infantile onset to adult onset manifesting primarily as myopathy.ObjectiveTo perform a retrospective natural history study of a large cohort of patients with TK2 deficiency.MethodsThe study was conducted by 42 investigators across 31 academic medical centres.ResultsWe identified 92 patients with genetically confirmed diagnoses of TK2 deficiency: 67 from literature review and 25 unreported cases. Based on clinical and molecular genetics findings, we recognised three phenotypes with divergent survival: (1) infantile-onset myopathy (42.4%) with severe mitochondrial DNA (mtDNA) depletion, frequent neurological involvement and rapid progression to early mortality (median post-onset survival (POS) 1.00, CI 0.58 to 2.33 years); (2) childhood-onset myopathy (40.2%) with mtDNA depletion, moderate-to-severe progression of generalised weakness and median POS at least 13 years; and (3) late-onset myopathy (17.4%) with mild limb weakness at onset and slow progression to respiratory insufficiency with median POS of 23 years. Ophthalmoparesis and facial weakness are frequent in adults. Muscle biopsies show multiple mtDNA deletions often with mtDNA depletion.ConclusionsIn TK2 deficiency, age at onset, rate of weakness progression and POS are important variables that define three clinical subtypes. Nervous system involvement often complicates the clinical course of the infantile-onset form while extraocular muscle and facial involvement are characteristic of the late-onset form. Our observations provide essential information for planning future clinical trials in this disorder.