Multi-copy nuclear pseudogenes of mitochondrial DNA reveal recent acute genetic changes in the human genome

Four nuclear pseudogenes homologous to the 10031–10195-bp region of the human mitochondrial genome were detected by constant denaturant capillary electrophoresis. Among them, one pseudogene is present as at least five copies in each cell, in accordance with our previous observations of multi-copy mitochondrial DNA pseudogenes. The presence of multiple identical copies of pseudogenes suggests that the human genome underwent a series of genetic changes, including gene amplifications, very recently in evolutionary history, i.e., within the last 390000 years.     

Gene organization of the Peking duck mitochondrial genome

Summary The gene organization of the Peking duck mitochondrial (mt)DNA has been deduced through heterologous hybridization using different cloned fragments of the chicken or Japanese quail mitochondrial genome as probes. As in the chicken, and other gallinaceous birds, the Peking duck mtDNA displays a novel gene order which differs from that of other vertebrates by the unusual localization of the tRNA^Glu and ND6 genes next to the displacement (D) loop region of the molecule. The position of these genes with respect to the mitochondrial D-loop region, the cytochrome oxidase subunits I, II and III, the NADH dehydrogenase subunit I and the ribosomal (r) RNAs, was confirmed by the partial nucleotide sequence of cloned mtDNA fragments.     

Linear mitochondrial genome organization in vivo in the genus Pythium

Pulsed-field gel electrophoresis (PFGE) of isolates of Pythium oligandrum with linear mitochondrial genomes revealed a distinct band in ethidium bromide-stained gels similar in size to values estimated by restriction mapping of mitochondrial DNA (mtDNA). Southern analysis confirmed that these bands were mtDNA and indicated that linear genomes were present in unit-length size as well as multimers. Isolates of this species with circular mtDNA restriction maps also had low levels of linear mono- and multimers. visualized by Southern analysis of PFGE gels. Examination of 17 additional species revealed similar results; three species had distinct linear mtDNA bands in ethidium bromide-stained gels while the remainder had linear mono- and multi-mers in lower amounts detected only by Southern analysis. Sequence analysis of an isolate of P. oligandrum with a primarily circular mitochondrial genomic map and a low amount of linear molecules revealed that the small unique region of the circular map (which corresponded to the terminal region of linear genomes) was flanked by palindromic intrastrand complementary sequences separated by a unique 194-bp sequence. Sequences with similarity to ATPase9 coding regions from other organisms were located adjacent to this region. Sequences with similarity to mitochondrial origins of replication and autonomously replicating sequences were also located in this region: their potential involvement in the generation of linear molecules is discussed.     

Transmission of the human mitochondrial genome

The segregation and transmission of mitochondrial genomes in humans are complicated processes, but are particularly important for understanding the inheritance and clinical abnormalities of mitochondrial disorders. This review describes three aspects of mitochondrial genetics. First, that the segregation and transmission of mitochondrial (mt)DNA molecules are likely to be determined by their physical association within the organelles and by the dynamics of mitochondrial structure and subcellular organization. Second, that the transmission of heteroplasmic mtDNA sequence changes from one generation to the next often involves rapid shifts in allele frequency. For >20 years, the standard explanation has been that there is a developmental bottleneck in which, at some stage of oogenesis, there is a reduction in the effective number of mitochondrial units of inheritance. The third aspect is that ongoing analyses of the segregation and transmission of pathogenic mtDNA mutations indicate the operation of multiple genetic processes. Thus, the segregation and transmission of mtDNA mutations occurs predominantly, but not exclusively, under conditions of random genetic drift. However, there is also evidence for bias due to incomplete ascertainment of pedigrees and for negative selection of pathogenic mutations in rapidly dividing somatic tissues such as the white blood cell population.     

Frequent somatic transfer of mitochondrial DNA into the nuclear genome of human cancer cells

Mitochondrial genomes are separated from the nuclear genome for most of the cell cycle by the nuclear double membrane, intervening cytoplasm, and the mitochondrial double membrane. Despite these physical barriers, we show that somatically acquired mitochondrial-nuclear genome fusion sequences are present in cancer cells. Most occur in conjunction with intranuclear genomic rearrangements, and the features of the fusion fragments indicate that nonhomologous end joining and/or replication-dependent DNA double-strand break repair are the dominant mechanisms involved. Remarkably, mitochondrial-nuclear genome fusions occur at a similar rate per base pair of DNA as interchromosomal nuclear rearrangements, indicating the presence of a high frequency of contact between mitochondrial and nuclear DNA in some somatic cells. Transmission of mitochondrial DNA to the nuclear genome occurs in neoplastically transformed cells, but we do not exclude the possibility that some mitochondrial-nuclear DNA fusions observed in cancer occurred years earlier in normal somatic cells.Somatically acquired structural rearrangements are common features of the nuclear genomes of cancer cells. These may range from simple chromosomal rearrangements (Campbell et al. 2008) to more complex, compound patterns, such as chromothripsis (Stephens et al. 2011) and chromoplexy (Baca et al. 2013), or mobilization of transposable elements (Lee et al. 2012; Tubio et al. 2014). Intrachromosomal rearrangements are generally more common than interchromosomal rearrangements, indicating a higher likelihood of joining a double-strand break in a chromosome to another break in the same chromosome despite the availability of a much larger quantity of nuclear DNA from other chromosomes (Stephens et al. 2009).In addition to the nuclear genome, human cells have a few hundred to a few thousand mitochondria, each carrying one or a few copies of the 16,569-bp-long circular mtDNA (Smeitink et al. 2001; Friedman and Nunnari 2014; Ju et al. 2014). During endosymbiotic co-evolution, most of the genetic information present in the ancestral mitochondrion has transferred to the nuclear genome (Gray et al. 1999; Adams and Palmer 2003; Timmis et al. 2004). An apparent burst of mtDNA transfer occurred during primate evolution ∼54 million years ago (Gherman et al. 2007) and occasional, probably more recent, transfer in humans has been observed in the germline (Turner et al. 2003; Goldin et al. 2004; Chen et al. 2005; Millar et al. 2010; Dayama et al. 2014). Although mtDNA nuclear transfer in a HeLa cell line derivative, and thus occurring in vitro, has been reported (Shay et al. 1991), de novo nuclear transfer of mtDNA in animal somatic tissues has not previously been comprehensively studied to our knowledge. To investigate the possibility of somatic mitochondrial-nuclear DNA fusion, we analyzed next-generation paired-end DNA whole-genome sequencing data from 559 primary cancers, 28 cancer cell lines (referred as 587 cancer whole genome below) and normal DNAs from the same individuals (Supplemental Table 1).     

The complete mitochondrial genome sequence of the hornwort Phaeoceros laevis: retention of many ancient pseudogenes and conservative evolution of mitochondrial genomes in hornworts

Plants have large and complex mitochondrial genomes in comparison to other eukaryotes. In bryophytes, the mitochondrial genomes exhibit a mixed mode of conservative and dynamic evolution. Here, we sequenced the complete mitochondrial genome from hornwort Phaeoceros laevis, to investigate the level of conservation in mitochondrial genome evolution within hornworts. The circular molecule consists of 209,482 base pairs and represents the largest known mitochondrial genome of bryophytes. It contains 30 protein genes, 3 rRNA genes, and 21 tRNA genes, with 34 cis-spliced group II introns disrupting 16 protein genes. There are 11 pseudogenes in this genome, and nine of them are shared with the other fully sequenced hornwort chondriome from Megaceros aenigmaticus, a distant relative of P. laevis. These pseudogenes were likely formed during an early stage of hornwort evolution. The two hornwort chondriomes differ by four inversions and translocations, seven genes, and four introns in the genome structure and organization. At the sequence level, they are very similar, with the identity values ranging mostly from 80 to 95% in intergenic spacers, introns, and exons. These data indicate that mitochondrial genome evolution in hornworts is less conservative than in liverworts, but has not reached the dynamic level as seen in seed plants.     

Somatic mutations of the mitochondrial genome in human breast cancers

Somatic mutations in mitochondrial DNA (mtDNA) have been identified in various tumors, including breast cancer. However, their clinicopathological impact on breast cancer still remains unclear. In this study, we re-sequenced the entire mtDNA from breast cancer samples together with paired non-tumorous breast tissues from 58 Taiwanese patients. We identified 19 somatic mutations in the mtDNA coding region of 16 breast cancers. Out of these mutations, 12 of the 19 mutations (63%) are missense or frame-shift mutations that have the potential to cause mitochondrial dysfunction. In combination with our previously study on the D-loop region of mtDNA, we found that 47% (27/58) of the breast cancers harbored somatic mtDNA mutations. Among a total of 40 somatic mutations, 53% (21/40) were located in the D-loop region of the mtDNA, 5% (2/40) were in the ribosomal RNA genes, 5% (2/40) were in the tRNA genes, and 38% (15/40) occurred in mRNA genes. The occurrence of these somatic mtDNA mutations is associated with an older onset age (≥ 50-year old, P = 0.039), a higher TNM stage (P = 0.027), and a higher histological grade (P = 0.012). Multiple logistic regression analysis revealed that an older onset age (P = 0.029) and a higher histological grade (P = 0.006) are significantly correlated with patients having somatic mutations in the mtDNA in their breast cancer sample. In conclusion, our results suggest that somatic mtDNA mutations may play a critical role in the progression of breast cancer.     

Physical mapping and genome organization of mitochondrial DNA from Candida maltosa

Summary Mitochondrial (mt) DNA of the ascomycetous yeast Candida maltosa was isolated and characterized. The mtDNA is circular and the size estimated from restriction analysis performed with 7 endonucleases was 52 kb pairs. A restriction map was constructed, using the cleavage data of four endonucleases. Using mt genes from Saccharomyces cerevisiae, six structural genes (large rRNA, apocytochrome b, cytochrome c oxidase subunit I and subunit 11, ATPase subunit 6 and subunit 9) were located on the C. maltosa chondriome by cross hybridization experiments. The comparison between the mt genomes of C. maltosa and six other yeasts showed differences in the overall genome organization.     

Molecular fossils in the human genome: identification and analysis of the pseudogenes in chromosomes 21 and 22

We have developed an initial approach for annotating and surveying pseudogenes in the human genome. We search human genomic DNA for regions that are similar to known protein sequences and contain obvious disablements (i.e., mid-sequence stop codons or frameshifts), while ensuring minimal overlap with annotations of known genes. Pseudogenes can be divided into "processed" and "nonprocessed"; the former are reverse transcribed from mRNA (and therefore have no intron structure), whereas the latter presumably arise from genomic duplications. We annotate putative processed pseudogenes based on whether there is a continuous span of homology that is >70% of the length of the closest matching human protein (i.e., with introns removed), or whether there is evidence of polyadenylation. We have applied our approach to chromosomes 21 and 22, the first parts of the human genome completely sequenced, finding 190 new pseudogene annotations beyond the 264 reported by the sequencing centers. In total, on chromosomes 21 and 22, there are 189 processed pseudogenes, 195 nonprocessed pseudogenes, and, additionally, 70 pseudogenic immunoglobulin gene segments. (Detailed assignments are available at http://bioinfo.mbb.yale.edu/genome/pseudogene or http://genecensus.org/pseudogene.) By extrapolation, we predict that there could be up to approximately 20,000 pseudogenes in the whole human genome, with a little more than half of them processed. We have determined the main populations and clusters of pseudogenes on chromosomes 21 and 22. There are notable excesses of pseudogenes relative to genes near the centromeres of both chromosomes, indicating the existence of pseudogenic "hot-spots" in the genome. We have looked at the distribution of InterPro families and Gene Ontology (GO) functional categories in our pseudogenes. Overall, the families in both processed and nonprocessed pseudogene populations occur according to a similar power-law distribution as that found for the occurrence of gene families, with a few big families and many small ones. The processed population is, in particular, enriched in highly expressed ribosomal-protein sequences (approximately 20%), which appear fairly evenly distributed across the chromosomes. We compared processed pseudogenes of different evolutionary ages, observing a high degree of similarity between "ancient" and "modern" subpopulations. This may be attributable to the consistently high expression of ribosomal proteins over evolutionary time. Finally, we find that chromosome 22 pseudogene population is dominated by immunoglobulin segments, which have a greater rate of disablement per amino acid than the other pseudogene populations and are also substantially more diverged.     

Evolution of mitochondrial DNA in yeast: gene order and structural organization of the mitochondrial genome of Saccharomyces uvarum

We have determined the size, the restriction map and the gene order of the mitochondrial genome of the yeast Saccharomyces uvarum. Sequence analysis of the mitochondrial COXII gene confirmed the position of this yeast in the Saccharomyces cerevisiae-like group, near Saccharomyces cerevisiae and Saccharomyces douglasii. Most mitochondrial genes have been positioned on this approximately 57-kb long genome and three regions containing putative replication origins have been identified. The gene order of S. uvarum suggests that the mitochondrial genome of the S.cerevisiae-like yeasts could have evolved from an ancestral molecule, similar to that of S. uvarum, through specific genome rearrangements. Received: 22 April / 2 September 1997     

The mitochondrial DNA of the yeast Hansenula petersonii: genome organization and mosaic genes

Summary The mitochondrial genomes of yeasts are circular DNA molecules that vary greatly in size in different species. The mitochondrial DNA of the yeast H. petersonii is about 42 kbp in length, about one half the size of the corresponding genome in S. cerevisiae. Sequences homologous to protein-encoding genes from S. cerevisiae have been identified and localized on this genome by hybridization with DNA from petite mutants. The comparison between the mitochondrial genomes of H. petersonii and S. cerevisiae showed differences in the overall genome organization, but both include genes with mosaic organization. In fact, sequences homologous to the first intron of the S. cerevisiae cob short gene are found in (or adjacent to) the cob and cox1 genes present in the genome of H. petersonii. Moreover, an intron homologous to that present in the 21S rRNA gene of S. cerevisiae seems to have been conserved in the large ribosomal RNA gene of H. petersonii, in a similar position.     

Complete sequence and organization of the human adenovirus serotype 46 genome

Out of 51 human adenoviral serotypes recognized to date, 32 of them belong to species D. Members of species D adenoviruses are commonly isolated from immune suppressed patients (organ transplant) and patients suffering from AIDS. The role of species D adenoviruses in pathogenesis is currently unclear. To derive new insights into the genetic content and evolution of species D adenoviruses and as a first step towards development of human adenovirus serotype 46 (Ad46) as vector, the complete nucleotide sequence of the virus was determined. The size of the genome is 35,178 bp in length with a G+C content of 56.9%. All the early and late region genes are present in the expected locations of the genome. The deduced amino acid sequences of all late region genes, with the exception of fiber, exhibited high degree of homology with the corresponding proteins of other adenoviruses. The deduced amino acid sequences of early regions E1, E3 and E4 showed a high degree of homology with the corresponding proteins of adenoviruses belonging to species D and less homology with the corresponding proteins of adenoviruses of other species. The homologues of Ad5 E3 region genes encoding 12.5K, gp19K, 10.4K, 14.5K and 14.7K are conserved in the genome of Ad46. However, the E3 region of Ad46 lacks genes encoding 6.7K and adenovirus death protein (ADP) but contains two additional open reading frames with a coding capacity of 433 and 281 amino acids. The fiber protein of Ad46 is 200 amino acids smaller than the fiber protein of Ad5 and contains only 10 pseudo-repeats in the shaft region. To facilitate the manipulation of the genome, the complete genome of Ad46 was cloned into a single bacterial plasmid. Following transfection into E1 complementing cell lines, the virus was recovered demonstrating the feasibility of viral genome manipulation for generation of recombinant viruses.     

Two pathogenic point mutations exist in the authentic mitochondrial genome, not in the nuclear pseudogene

Technical advancements in molecular genetics have shown various mitochondrial DNA (mtDNA) abnormalities in patients with mitochondrial myopathies. Recently, it has been revealed that, in these patients, the nuclear DNA carries sequences similar to those of the mtDNA (nuclear pseudogene) and it has several point mutations previously reported to be pathogenic. We verified the existence of the T3250C and T3291C mutations, which we have found in patients with mitochondrial myopathy, in the authentic mitochondrial genome. A long polymerase chain reaction provides a powerful tool for avoiding nuclear pseudogene amplification and for ruling out ambiguity in the detection of the mutation for diagnosis. Received: August 2, 2000 / Accepted: August 30, 2000     

线粒体基因组特征性变异与人类进化

自然选择究竟在人类基因组留下怎样的印记,是我们今天一直想解开的秘密.由于线粒体基因组缺少重组和母系遗传等区别于核基因组的诸多特性,使在基因组水平上一些重要的生物学特征如自然选择等较易被检测.因此,为我们探讨自然选择与人类进化和适应的关系提供了具有巨大优势和潜力的研究工具.在本文中,依据线粒体基因组的系统发生和编码基因的变异分析,系统地阐述了基因组特征性变异与人类进化的关系及研究进展.