2.2. The mitochondrial genome
Mitochondria have been essential for the evolution of animals. It is generally believed that the energy-converting organelles of eucaryotes evolved from procaryotes that were engulfed by primitive eucaryotic cells and developed a symbiotic relationship with them about 1.5 x 109 years ago. This would explain why mitochondria have their own DNA (mtDNA), which codes for some of their proteins. Since their initial uptake by a host cell, these organelles have lost much of their own genome and have become heavily dependent on genes in the nucleus.
Extant mammalian mtDNAs have retained only 13 polypeptide genes, all of which encode essential components of OXPHOS. MtDNA also encodes the 12S and 16S rRNA genes and the 22 tRNA genes required for mitochondrial protein synthesis. The remaining mitochondrial OXPHOS proteins, the metabolic enzymes, the DNA and RNA polymerases, the ribosomal proteins and the mtDNA regulatory factors are all encoded by nuclear genes, synthesized in the cytosol and then imported into the organelle (Shoffner & Wallace 1995, Wallace et al. 1997a).
The transfer of mtDNA sequences to the nucleus is a continuous process (Wallace 1997, Hirano et al. 1997), but not all of the mtDNA genes transferred to the nucleus are functional. The presence of hundreds of mtDNA-like sequences or pseudogenes in the human nuclear genome has been well documented (Tsuzuki et al. 1983, Shay & Werbin 1992).
Furthermore, the genetic code in human mitochondria has come to differ from that used in the nucleus, and thus mtDNA genes are no longer intelligible to the nucleocytosolic system (Wallace 1982). UGA is read as tryptophan rather than ‘stop’, AGA and AGG as ‘stop’ rather than arginine, AUA as methionine rather than isoleucine, and AUA or AUU is sometimes used as an initiation codon instead of AUG (Anderson et al. 1981, Montoya et al. 1981).
2.2.1. Organization of the human mitochondrial genome
The human mitochondrial genome is 16,569 base pairs (bp) in length (Anderson et al. 1981), a closed, circular molecule (Figure 2) located within the mitochondrial matrix and present in thousands of copies per cell. Mitochondrial DNA has two strands, a guanine-rich heavy (H) strand and a cytosine-rich light (L) strand. The heavy strand contains 12 of the 13 polypeptide-encoding genes, 14 of the 22 tRNA-encoding genes and both rRNA-encoding genes. Introns are absent in mtDNA, and all of the coding sequences are contiguous (Anderson et al. 1981, Wallace et al. 1992, Zeviani et al. 1998). The only non-coding segment of mtDNA is the displacement loop (D-loop), a region of 1121 bp that contains the origin of replication of the H-strand (OH) and the promoters for L and H-strand transcription. The mtDNA is replicated from two origins. DNA replication is initiated at OH using an RNA primer generated from the L-strand transcript. H-strand synthesis proceeds two-thirds of the way around the mtDNA, displacing the parental H-strand until it reaches the L-strand origin (OL), situated in a cluster of five tRNA genes. Once exposed on the displaced H-strand, OL folds a stem-loop structure and L-strand synthesis is initiated and proceeds back along the H-strand template. Consequently, mtDNA replication is bidirectional but asynchronous (Clayton 1982). MtDNA transcription is initiated from two promoters in the D-loop, PL and PH. Transcription from both promoters proceeds around the mtDNA circle, creating a polycistronic RNA. The tRNA genes which punctuate the larger rRNA and mRNA sequences then fold within the transcript and are cleaved out. The mRNAs and rRNAs liberated are post-transcriptionally polyadenylated and the tRNAs are modified and the 3’ terminal CCA added (Attardi et al. 1982, Attardi & Montoya 1983, Clayton 1984, Wallace 1993, Taanman 1999).
Figure 2. The human mitochondrial genome encodes 13 subunits of respiratory chain complexes: seven subunits (ND 1–6 and 4L) of complex I, cytochrome b (Cyt b) of complex III, the COX I–III subunits of cytochrome oxidase or complex IV, and the ATPase 6 and 8 subunits of FOF1 ATP synthase. MtDNA also encodes 12S and 16S rRNA genes and 22 tRNA genes. The abbreviated amino acid names indicate the corresponding amino acid tRNA genes. The outer strand is heavy-chain DNA and the inner one light-chain DNA. OH and OL are the replication origins of the light and heavy chain, respectively, while PH and PL indicate the transcription sites.
2.2.2. Special features of mitochondrial genetics
The cytoplasmic location of mtDNA and the high copy number contribute to certain unique features of mitochondrial genetics. First, mtDNA is maternally inherited. Second, mtDNA genes have a much higher mutation rate than nuclear DNA genes. Third, mitochondria undergo replicative segregation at cell division. Fourth, many of the pathogenic mtDNA mutations are heteroplasmic. For expression of a disease it is required that a certain threshold level of mutant mtDNA should be exceeded. Fifth, somatic mtDNA mutations accumulate in post-mitotic tissues with age, reducing the ATP generating capacity.
2.2.2.1. Maternal inheritance
MtDNA is maternally inherited. The mammalian egg contains about 100,000 molecules of mtDNA, while the sperm contains of the order of 100–1500 mtDNAs (Chen et al. 1995b, Manfredi et al. 1997). Sperm mitochondria enter the egg during fertilization but they appear to be lost early in embryogenesis, soon after fertilization, between the two-cell and four-cell stages (Manfredi et al. 1997). Paternal mtDNA could not be detected in human neonates born after in vitro fertilization by intracytoplasmic sperm injection (Danan et al. 1999). This could be due either to destruction of sperm mitochondria or to impaired replication of sperm mtDNA in the cells (Manfredi et al. 1997). However, the presence of paternal mtDNA has been shown at the blastocyst stage in some abnormal (polyploid) human embryos generated by in vitro fertilization and intracytoplasmic sperm injection techniques (St John et al. 2000).
2.2.2.2. High mutation rate
Mitochondria seem to lack an efficient DNA repair system (Bogenhagen 1999). Moreover, protective proteins such as histones are missing and mtDNA is physically associated with the inner mitochondrial membrane, where highly mutagenic oxygen radicals are generated as by-products of OXPHOS (Richter 1988). Furthermore, abnormal mitochondrial metabolism may accelerate the rate of mtDNA mutation (Lightowlers et al. 1997). These unique features are probably the cause of the about 10 to 17 times faster accumulation of polymorphisms in mtDNA than in nuclear DNA (Neckelmann et al. 1987, Wallace et al. 1997a). The hypervariable sequences in the D-loop evolve even more rapidly than the coding regions (Howell et al. 1996).
2.2.2.3. Replicative segregation
Each cell has hundreds of mitochondria, each containing 2 to 10 copies of mtDNA molecules. Normally all mtDNAs in a cell are identical, a condition known as homoplasmy. At cell division, the mitochondria and their genomes are randomly distributed to the daughter cells, a process known as replicative segregation. When a mutation arises in mtDNA, it creates an intracellular mixture of mutant and normal molecules, a condition known as heteroplasmy. Despite the high mtDNA copy number in mature oocytes and the relatively small number of cell divisions in the female germline, mtDNA sequence variants segregate rapidly between generations (Poulton et al. 1998). This has been attributed to a genetic bottleneck.
A major component of the mtDNA bottleneck occurs by the time that oocytes are mature (Jenuth et al. 1996, Marchington et al. 1998). Jenuth et al. (1996) have estimated that in oogenesis the effective number of segregating units for mtDNA is approximately 200 in mice. The model used by Jenuth et al. (1996) assumes that the variance in genotypic ratios of the progeny or developing oocytes is caused by an identical random-sampling event that occurs during each of the 15 or so cell divisions during the later stages of oogenesis (i.e. repeated selection), in contrast to a more dramatic reduction in segregating units during a briefer period (i.e. single selection). On the basis of single selection model it is estimated that the most probable bottleneck size is 1–31 segregating units in humans (Marchington et al. 1998).
Both estimations of the number of segregating units are far below the number of mtDNA molecules in a cell, suggesting that there is first a restriction in the quantity of mtDNA to be transmitted, followed by amplification, and thereby constituting a genetic bottleneck (Poulton et al. 1998).
2.2.2.4. Threshold effect
Many but not all pathogenic mtDNA mutations are heteroplasmic. The phenotype is normal until a critical proportion of mutant mtDNA is present within the tissue and the threshold for genotype expression is exceeded (Wallace et al. 1997a). This threshold varies for different types of mtDNA mutation and is about 60% for deleted mtDNA (Hayashi et al. 1991). For the mutation 8344A>G, which causes the syndrome of myoclonic epilepsy and ragged-red fibers, the threshold level is about 85% mutated DNA (Chomyn 1998). Once this is exceeded, large changes in the phenotype can be observed with minor increases in the proportion of the mutant mtDNA.
Different phenotypes associated with the same genotype are determined mainly by the localized concentration and distribution of the mutation in affected tissues (Petruzzella et al. 1994). Furthermore, different tissues have different dependences on oxidative phosphorylation for normal function. Organs with the highest ATP requirements and the lowest regenerative capacities, such as the brain, heart and skeletal muscle, are the most sensitive to the effects of pathogenic mtDNA mutations (Wallace 1994, 1995).
2.2.2.5. Age-related somatic mtDNA mutations
Oxygen free radicals damage mtDNA, causing oxidative modifications of DNA bases, base substitutions and rearrangements. The cumulative accumulation of these somatic mutations during life may cause a bioenergetic deficit leading to cell death, or apoptosis, and normal ageing (Trounce et al. 1989, Simonetti et al. 1992, Ozawa 1995). In addition to the ageing or senescence process somatic mtDNA mutations may be important for determining the onset and progression of mtDNA diseases. Most inherited mutations are insufficient to suppress mitochondrial OXPHOS below the expression threshold and thus it is the accumulation of somatic mutations in postmitotic tissues that exacerbates the inherited OXPHOS defect and ultimately leads to phenotypic expression (Wallace et al. 1992, Wallace 1995).
Oxidative stress has been implicated in the pathogenesis of neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease. There is much evidence of increased oxidative stress and free radical damage in the substantia nigra in patients with Parkinson’s disease, and there is also evidence for a defect in mitochondrial energy production, and especially reduced complex I activity, in the substantia nigra (Schapira 1999a).