Basic mtDNA Biology

Human mtDNA is a circular double-stranded molecule that is 16,569 bp long (other sequenced mammalian mitochondrial genomes have similar lengths; see Figure 41.1).

It carries 37 genes, of which two encode rRNAs, 22 encode tRNAs, and 13 encode polypeptides. All 13 mitochondrially encoded polypeptides represent components of the electron transfer chain (ETC). Of these, seven are subunits of complex I (NADH dehydrogenase), three are subunits of complex IV (cytochrome c oxidase), two are subunits of complex V (ATP synthase), and cytochrome b (a subunit of complex III). The inheritance of mtDNA is almost exclusively maternal, although instances of paternal inheritance have been reported in humans, mice, and birds. mtDNA is present in one to several thousand copies per cell and is encapsulated into mitochondria between 1 and 11 copies per mitochondrion, with the mean being two genomes per organelle. Mitochondria are dynamic structures that, in different cell types and under different physiological conditions, can assume a variety of conformations, the two extremes being reticular (all mitochondria in a cell are fused to form a network) and particular (each mitochondrion exists as a separate entity). In both conformations, mitochondria perpetually undergo the processes of fission and fusion. Therefore, the earlier definitions of reticulate and particulate mitochondrial conformations are relative terms referring to a snapshot of the mitochondrial network in a cell. Nevertheless, these terms are useful as they describe the prevalence of either mitochondrial fission (particulate conformation) or fusion (reticulate conformation) in a given cell under given physiological conditions. In this light, the average number of mtDNA copies per mitochondrion may simply reflect the extent of mitochondrial fragmentation under the assay conditions, which is defined by two factors: a) the mitochondrial conformation inside the cell, and b) the extent of mitochondrial fragmentation during isolation for the analysis of mtDNA content.

The two mtDNA strands can be separated by denaturing CsCl gradient centrifugation. Most of the information is encoded in the heavy (purine-rich) strand

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Figure 41.1 The map of human mitochondrial DNA. OH and OL, origins of heavy and light strand replication, respectively; ND1-ND6 NADH dehydrogenase (ETC complex I) subunits 1 through 6; Coxl-Cox3, cytochrome oxidase subunits 1 through 3 (ETC complex IV), ATP6 and ATP8, subunits 6 and 8 of mitochondrial ATPase (complex V), Cyt b, cytochrome b (complex III).

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Figure 41.1 The map of human mitochondrial DNA. OH and OL, origins of heavy and light strand replication, respectively; ND1-ND6 NADH dehydrogenase (ETC complex I) subunits 1 through 6; Coxl-Cox3, cytochrome oxidase subunits 1 through 3 (ETC complex IV), ATP6 and ATP8, subunits 6 and 8 of mitochondrial ATPase (complex V), Cyt b, cytochrome b (complex III).

(2 rRNA, 14 tRNA, and 12 polypeptides). The light (pyrimidine-rich) strand contains genetic information for only one polypeptide and eight tRNAs. Mitochondrial genes have no introns, and intergenic sequences are absent or limited to a few bases. Some genes overlap, and, in some instances, termination codons are not encoded but are generated post-transcriptionally by polyadenylation (Ojala et al., 1981). mtDNA is totally dependent upon proteins encoded in the nuclear genome for its maintenance and transcription. In fact, although the mitochondrial proteome consists of an estimated 1500 polypeptides, only 13 of those are encoded by mtDNA. The mitochondrial proteome is dynamic; that is, both the precise polypeptide composition and the relative abundance of a given polypeptide may vary in mitochondrial proteomes of different tissues as well as in the same tissue over time. mtDNA replication is conducted by the heterodimeric DNA polymerase gamma. The current paradigm states that replication of mammalian mtDNA continues throughout the lifespan of an organism in both proliferating and post-mitotic cells. It occurs bidirection-ally, initiated at two spatially and temporally distinct origins of replication, OH and OL, for the heavy and light strand origins of replication, respectively (for review, see Taanman, 1999). However, this paradigm recently has been challenged, and evidence has been presented for bidirectional 0-like replication initiated over a fairly wide zone (Yang et al., 2002). With the discovery of mitochondrial diseases caused by mutations of mtDNA, it has been found that wild-type (WT, normal) and mutated mtDNA may coexist in the same cell, a condition called heteroplasmy.

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