Structure and chromosomal distribution of human mitochondrial pseudogenes

Nuclear mitochondrial pseudogenes (Numts) have been found in the genome of many eukaryote species, including humans. Using a BLAST approach, we found 1105 DNA sequences homologous to mitochondrial DNA (mtDNA) in the August 2001 Goldenpath human genome database. We assembled these sequences manually into 286 pseudogenes on the basis of single insertion events and constructed a chromosomal map of these Numts. Some pseudogenes appeared highly modified, containing inversions, deletions, duplications, and displaced sequences. In the case of four randomly selected Numts, we used PCR tests on cells lacking mtDNA to ensure that our technique was free from genome-sequencing artifacts. Furthermore, phylogenetic investigation suggested that one Numt, apparently inserted into the nuclear genome 25-30 million years ago, had been duplicated at least 10 times in various chromosomes during the course of evolution. Thus, these pseudogenes should be very useful in the study of ancient mtDNA and nuclear genome evolution.     

Incorporation of mitochondrial fragments in the nuclear genome (Numts) of the longhorned beetle Monochamus galloprovincialis (Coleoptera, Cerambycidae)

A mitochondrial DNA (mtDNA) study, based on 43 European populations (33 of them sampled in France) of Monochamus galloprovincialis , vector of the pinewood nematode, and 14 populations of its sister species Monochamus sutor was realized. Sequencing of 792 bp of the cytochrome oxidase I (COI) and 521 bp of the COII genes revealed numerous ambiguities on multiple nucleotide sites for half of M. galloprovincialis specimens studied (44.8%). Hypotheses of heteroplasmy and pseudogenes ( Numts ) were examined. The mtDNA isolation by alkaline lysis and cloning (for three successfully used individuals) both support the hypothesis that the ambiguous sequences amplified were not of mtDNA nature and validate the presence of Numts in the nuclear genome of M. galloprovincialis . Multiple copies of mtDNA-like sequences were found paralogous to COI, tRNA leucin and COII regions. Phenetic analysis placed different recently diverged mtDNA-like sequences as a close relative of mtDNA sequences, and grouped 10 closely related mtDNA-like sequences as a more basal clade, closer to ancestral states and to M. sutor . This result supports that this nuclear family of pseudogenes arose independently of the other events and may represent mitochondrial haplotypes sampled from M. galloprovincialis ancestral populations. This is the first time that Numts are proved for a longhorned beetle, whereas no Numts were found within its sister species M. sutor. The incorporation mechanism of Numts in unknown for M. galloprovincialis , however, excess of ambiguous sites corresponding to synonymous mutations placed on third codon position as well as the absence of Numts in M. sutor , conducted to the hypothesis of a recent transfer of these Numts in the nuclear genome of M. galloprovincialis .     

Frequent assimilation of mitochondrial DNA by grasshopper nuclear genomes

Multiple copies of mitochondrial-like DNA were found in the brown mountain grasshopper, Podisma pedestris (Orthoptera: Acrididae), paralogous to COI and ND5 regions. The same was discovered using the ND5 regions of nine other grasshopper species from four separate subfamilies (Podisminae, Calliptaminae, Cyrtacanthacridinae, and Gomphocerinae). The extra ND5-like sequences were shown to be nuclear in the desert locust, Schistocerca gregaria (Cyrtacanthacridinae), and probably so in P. pedestris and an Italopodisma sp. (Podisminae). Eighty-seven different ND5-like nuclear mitochondrial pseudogenes (Numts) were sequenced from 12 grasshopper individuals. Different nuclear mitochondrial pseudogenes, if descended from the same mitochondrial immigrant, will have diverged from each other under no selective constraints because of their loss of functionality. Evidence of selective constraints in the differences between any two Numt sequences (e.g., if most differences are at third positions of codons) implies that they have separate mitochondrial origins. Through pairwise comparisons of pseudogene sequences, it was established that there have been at least 12 separate mtDNA integrations into P. pedestris nuclear genomes. This is the highest reported rate of horizontal transfer between organellar and nuclear genomes within a single animal species. The occurrence of numerous mitochondrial pseudogenes in nuclear genomes derived from separate integration events appears to be a common phenomenon among grasshoppers. More than one type of mechanism appears to have been involved in generating the observed grasshopper Numts.     

家马核基因组中线粒体核内插入序列分析

在各种真核生物核基因组中,存在一些由线粒体基因组转移进入核基因组中的DNA片段,这些被认为是分子化石的片段叫做线粒体核内插入序列(Numt)。由于Numt与真实的线粒体序列高度相似,因此它的存在必然会成为PCR扩增线粒体DNA的不利因素。利用已经公布的家马(Equus caballus)基因组序列(2007年9月公布,GenBank登录号为NC_009144-NC_009175)对家马Numt进行了深入分析,共发现200个可能的Numt,长度范围为29到3727bp,其中有10个的长度大于800bp。分析结果显示由于不存在线粒体控制区域的疑似Numt,因此对基于此区域的群体遗传学研究不会产生影响。本研究还发现在家马进化过程中,第1号和27号染色体更倾向于接受线粒体序列的转移。以上结果将为今后马科动物的研究提供重要的参考信息,有助于避免在线粒体DNA研究中由于Numt污染的存在而得出错误的实验结果。     

青蟹线粒体COI假基因的分离和特征分析

线粒体DNA标记在遗传结构和系统进化研究中得到广泛应用,然而核假基因的存在对此有很大威胁。本文以中国东南沿海的青蟹(Scylla paramamosain)为研究对象,利用线粒体COI基因的通用引物和特异性引物进行扩增,分别得到34个假基因（nuclear mitochondrial pseudogenes, Numts）和5个线粒体COI基因序列。在所获得的34个假基因中共定义了29种单倍型,根据序列的相似度,这些假基因可以分为2类,每类假基因都有各自保守的核苷酸序列。第Ⅰ类假基因存在2处插入序列和1处8 bp的缺失序列,这些位点导致了整个阅读框的移位;在第Ⅱ类假基因和5个线粒体COI序列中只有碱基替换,未发现插入和缺失序列。实验结果分析表明,这两类假基因分别代表了2次核整合事件,即核转移事件的最低值。研究结果提示了     

Fugu genome does not contain mitochondrial pseudogenes

Contrary to previous observations that fish genomes are devoid of nuclear mitochondrial pseudogenes, a genome-wide survey identified a large number of "recent" and "ancient" nuclear mitochondrial DNA fragments (Numts) in the whole-genome sequences of the fugu (Takifugu rubripes), Tetraodon nigroviridis, and zebrafish (Danio rerio). We have analyzed the latest assembly (v4.0) of the fugu genome and show that, like the Anopheles genome, the fugu nuclear genome does not contain mitochondrial pseudogenes. Fugu assembly v4.0 contains a single scaffold representing the near complete sequence of the fugu mitochondria. The "recent" Numts identified by the previous study in fugu assembly v2.0 are in fact shotgun sequences of mitochondrial DNA that were misassembled with the nuclear sequences, whereas the "ancient" Numts appear to be the result of spurious matches. It is likely that the Numts identified in the genomes of Tetraodon and zebrafish are also similar artifacts. Shotgun sequences of whole genomes often include some mitochondrial sequences. Therefore, any Numts identified in shotgun-sequence assemblies should be verified by Southern hybridization or PCR amplification.     

Mitochondrial COII Introgression into the Nuclear Genome of Gorilla gorilla

Numts are nonfunctional mitochondrial sequences that have translocated into nuclear DNA, where they evolve independently from the original mitochondrial DNA (mtDNA) sequence. Numts can be unintentionally amplified in addition to authentic mtDNA, complicating both the analysis and interpretation of mtDNA-based studies. Amplification of numts creates particular issues for studies on the noncoding, hypervariable 1 mtDNA region of gorillas. We provide data on putative numt sequences of the coding mitochondrial gene cytochrome oxidase subunit II (COII). Via polymerase chain reaction (PCR) and cloning, we obtained COII sequences for gorilla, orangutan, and human high-quality DNA and also from a gorilla fecal DNA sample. Both gorilla and orangutan samples yielded putative numt sequences. Phylogenetically more anciently transferred numts were amplified with a greater incidence from the gorilla fecal DNA sample than from the high-quality gorilla sample. Data on phylogenetically more recently transferred numts are equivocal. We further demonstrate the need for additional investigations into the use of mtDNA markers for noninvasively collected samples from gorillas and other primates.     

Nuclear mitochondrial pseudogenes in Austinograea alayseae hydrothermal vent crabs (Crustacea: Bythograeidae): effects on DNA barcoding

Members of the brachyuran crab family, Bythograeidae, are among the most abundant and common crabs in vent fields. However, their identification based on morphological characteristics often leads to incorrect species recognition due to a lack of taxonomic factors and the existence of sibling (or cryptic) species. For these reasons, we used DNA barcoding for vent crabs using mitochondrial cytochrome c oxidase subunit 1 (CO1). However, several nuclear mitochondrial pseudogenes (Numts) were amplified from Austinograea alayseae Guinot, 1990, using universal primers (Folmer primers). The Numts were characterized in six haplotypes, with 13.58–14.11% sequence divergence from A. alayseae, a higher nonsynonymous substitution ratio than true CO1, and the formation of an independent clade in bythograeids. In a neighbour‐joining tree, the origin of the Numts would be expected to incorporate into the nucleus at an ancestral node of Austinograea, and they mutated more slowly in the nucleus than CO1 in the mitochondria. This evolutionary process may have resulted in the higher binding affinity of Numts for the Folmer primers than CO1. In the present study, we performed long PCR for the amplification of CO1 in A. alayseae. We also present evidence that Numts can introduce serious ambiguity into DNA barcoding, including overestimating the number of species in bythograeids. These results may help in conducting taxonomic studies using mitochondrial genes from organisms living in hydrothermal vent fields.     

动物线粒体假基因的识别及其在进化生物学中的应用

在真菌、昆虫、无脊椎动物和脊椎动物等许多分类单元中,都已发现mtDNA序列的核转座现象。在PCR扩增时,往往同时扩增出mtDNA和细胞核中线粒体假基因(Numts),Numts混淆系统发育和群体遗传研究,得出错误的结果。本文综述了Numts的检查和避免的方法,以及在进化生物学研究中的应用进展。     

Mitochondrial pseudogenes are pervasive and often insidious in the snapping shrimp genus Alpheus

Here we show that multiple DNA sequences, similar to the mitochondrial cytochrome oxidase I (COI) gene, occur within single individuals in at least 10 species of the snapping shrimp genus Alpheus. Cloning of amplified products revealed the presence of copies that differed in length and (more frequently) in base substitutions. Although multiple copies were amplified in individual shrimp from total genomic DNA (gDNA), only one sequence was amplified from cDNA. These results are best explained by the presence of nonfunctional duplications of a portion of the mtDNA, probably located in the nuclear genome, since transfer into the nuclear gene would render the COI gene nonfunctional due to differences in the nuclear and mitochondrial genetic codes. Analysis of codon variation suggests that there have been 21 independent transfer events in the 10 species examined. Within a single animal, differences between the sequences of these pseudogenes ranged from 0.2% to 20.6%, and those between the real mtDNA and pseudogene sequences ranged from 0.2% to 18.8% (uncorrected). The large number of integration events and the large range of divergences between pseudogenes and mtDNA sequences suggest that genetic material has been repeatedly transferred from the mtDNA to the nuclear genome of snapping shrimp. Unrecognized pseudogenes in phylogenetic or population studies may result in spurious results, although previous estimates of rates of molecular evolution based on Alpheus sister taxa separated by the Isthmus of Panama appear to remain valid. Especially worrisome for researchers are those pseudogenes that are not obviously recognizable as such. An effective solution may be to amplify transcribed copies of protein-coding mitochondrial genes from cDNA rather than using genomic DNA.     

Few mitochondrial DNA sequences are inserted into the turkey (Meleagris gallopavo) nuclear genome: evolutionary analyses and informativity in the domestic lineage

Mitochondrial DNA (mtDNA) insertions have been detected in the nuclear genome of many eukaryotes. These sequences are pseudogenes originated by horizontal transfer of mtDNA fragments into the nuclear genome, producing nuclear DNA sequences of mitochondrial origin (numt). In this study we determined the frequency and distribution of mtDNA‐originated pseudogenes in the turkey (Meleagris gallopavo) nuclear genome. The turkey reference genome (Turkey_2.01) was aligned with the reference linearized mtDNA sequence using last . A total of 32 numt sequences (corresponding to 18 numt regions derived by unique insertional events) were identified in the turkey nuclear genome (size ranging from 66 to 1415 bp; identity against the modern turkey mtDNA corresponding region ranging from 62% to 100%). Numts were distributed in nine chromosomes and in one scaffold. They derived from parts of 10 mtDNA protein‐coding genes, ribosomal genes, the control region and 10 tRNA genes. Seven numt regions reported in the turkey genome were identified in orthologues positions in the Gallus gallus genome and therefore were present in the ancestral genome that in the Cretaceous originated the lineages of the modern crown Galliformes. Five recently integrated turkey numts were validated by PCR in 168 turkeys of six different domestic populations. None of the analysed numts were polymorphic (i.e. absence of the inserted sequence, as reported in numts of recent integration in other species), suggesting that the reticulate speciation model is not useful for explaining the origin of the domesticated turkey lineage.     

单个拟穴青蟹核内多拷贝线粒体COI假基因序列的研究(简报)

线粒体DNA(mtDNA)因它的高拷贝数、易扩增、高突变率、中性、低重组和母系遗传等特征,已广泛应用在系统进化和群体遗传研究方面[1,2].但是,由于mtDNA本身的特征,如异质性.     

A cryptic, intergeneric cytochrome c oxidase I pseudogene in tyrant flycatchers (family: Tyrannidae)

Nuclear mitochondrial pseudogenes, or "numts", are nonfunctional copies of mitochondrial genes that have been translocated to the nuclear genome. Numts have been used to study differences in mutation rates between the nuclear and mitochondrial genomes, but have also been implicated as troublesome for phylogenetic studies and DNA-based species identification (i.e., DNA barcoding). In this study, a suspected numt discovered during a study of mitochondrial cytochrome c oxidase I (COI) diversity in North American birds was targeted and sequenced from tyrant flycatchers (family: Tyrannidae). In total, the numt was found in five taxa representing two genera. Substitution rates were compared between COI and numt sequences. None of the numt sequences harboured stop codons nor frameshift mutations, but phylogenetic analysis revealed they had accumulated more amino acid substitutions than the mitochondrial COI sequences. Mitochondrial COI appeared to be preferentially amplified in most cases, but methods for numt detection are discussed for cases like this where sequences lack obvious features for identification. Because of its persistence across a broad taxonomic lineage, this numt could form a valuable model system for studying evolution in numts. The full size of the numt and its location within the nuclear genome are yet to be determined.     

Estimates of linkage disequilibrium and effective population size in rainbow trout

Background

Mitochondrial DNA (mtDNA) is widely used in population genetic and phylogenetic studies in animals. However, such studies can generate misleading results if the species concerned contain nuclear copies of mtDNA (Numts) as these may amplify in addition to, or even instead of, the authentic target mtDNA. The aim of this study was to determine if Numts are present in Aedes aegypti mosquitoes, to characterise any Numts detected, and to assess the utility of using mtDNA for population genetics studies in this species.

Results

BLAST searches revealed large numbers of Numts in the Ae. aegypti nuclear genome on 146 supercontigs. Although the majority are short (80% < 300 bp), some Numts are almost full length mtDNA copies. These long Numts are not due to misassembly of the nuclear genome sequence as the Numt-nuclear genome junctions could be recovered by amplification and sequencing. Numt evolution appears to be a complex process in Ae. aegypti with ongoing genomic integration, fragmentation and mutation and the secondary movement of Numts within the nuclear genome. The PCR amplification of the putative mtDNA nicotinamide adenine dinucleotide dehydrogenase subunit 4 (ND4) gene from 166 Southeast Asian Ae. aegypti mosquitoes generated a network with two highly divergent lineages (clade 1 and clade 2). Approximately 15% of the ND4 sequences were a composite of those from each clade indicating Numt amplification in addition to, or instead of, mtDNA. Clade 1 was shown to be composed at least partially of Numts by the removal of clade 1-specific bases from composite sequences following enrichment of the mtDNA. It is possible that all the clade 1 sequences in the network were Numts since the clade 2 sequences correspond to the known mitochondrial genome sequence and since all the individuals that produced clade 1 sequences were also found to contain clade 2 mtDNA-like sequences using clade 2-specific primers. However, either or both sets of clade sequences could have Numts since the BLAST searches revealed two long Numts that match clade 2 and one long Numt that matches clade 1. The substantial numbers of mutations in cloned ND4 PCR products also suggest there are both recently-derived clade 1 and clade 2 Numt sequences.

Conclusion

We conclude that Numts are prevalent in Ae. aegypti and that it is difficult to distinguish mtDNA sequences due to the presence of recently formed Numts. Given this, future population genetic or phylogenetic studies in Ae. aegypti should use nuclear, rather than mtDNA, markers.     

Nuclear insertions help and hinder inference of the evolutionary history of gorilla mtDNA

Numts are fragments of mitochondrial DNA (mtDNA) that have been translocated to the nucleus, where they can persist while their mitochondrial counterparts continue to rapidly evolve. Thus, numts represent 'molecular fossils' useful for comparison with mitochondrial variation, and are particularly suited for studies of the fast-evolving hypervariable segment of the mitochondrial control region (HV1). Here we used information from numts found in western gorillas (Gorilla gorilla) and eastern gorillas (Gorilla beringei) to estimate that these two species diverged about 1.3 million years ago (Ma), an estimate similar to recent calculations for the divergence of chimpanzee and bonobo. We also describe the sequence of a gorilla numt still possessing a segment lost from all contemporary gorilla mtDNAs. In contrast to that sequence, many numts of the HV1 are highly similar to authentic mitochondrial organellar sequences, making it difficult to determine whether purported mitochondrial sequences truly derive from that genome. We used all available organellar HV1 and corresponding numt sequences from gorillas in a phylogenetic analysis aimed at distinguishing these two types of sequences. Numts were found in several clades in the tree. This, in combination with the fact that only a limited amount of the extant variation in gorillas has been sampled, suggests that categorization of new sequences by the indirect means of phylogenetic comparison would be prone to uncertainty. We conclude that for taxa such as gorillas that contain numerous numts, direct approaches to the authentication of HV1 sequences, such as amplification strategies relying upon the circularity of the mtDNA molecule, remain necessary.     

Distinguishing gorilla mitochondrial sequences from nuclear integrations and PCR recombinants: guidelines for their diagnosis in complex sequence databases

Nuclear integrations of mitochondrial DNA (Numts) are widespread in many taxa and if left undetected can confound phylogeny interpretation and bias estimates of mitochondrial DNA (mtDNA) diversity. This is particularly true in gorillas, where recent studies suggest multiple integrations of the first hypervariable (HV1) domain of the mitochondrial control region. Problems can also arise through the inadvertent incorporation of artifacts produced by in vitro recombination between sequence types during polymerase chain reaction amplification. This issue has attracted little attention yet could potentially exacerbate errors in databases already contaminated by Numts. Using a set of existing diagnostic tools, this study set out to systematically inventory Numts and PCR recombinants in a gorilla HV1 sequence database and address the degree to which existing public databases are contaminated. Phylogenetic analysis revealed three distinct gorilla HV1 Numt groups (I, II, and III) that could be readily differentiated from mtDNA sequences by Numt-specific diagnostic sites and sequence-based motifs. Several instances of genuine recombination were also identified by a suite of detection methods. The location of putative breakpoints was identified by eye and by likelihood analysis. Findings from this study reveal widespread nuclear contamination of gorilla HV1 GenBank databases and underline the importance of recognizing not only Numts but also PCR recombinant artifacts as potential sources of data contamination. Guidelines for the routine identification of Numts and in vitro recombinants are presented and should prove useful in the detection of similar artifacts in other species mtDNA databases.     

Rampant Nuclear Insertion of mtDNA across Diverse Lineages within Orthoptera (Insecta)

Nuclear mitochondrial pseudogenes (numts) are non-functional fragments of mtDNA inserted into the nuclear genome. Numts are prevalent across eukaryotes and a positive correlation is known to exist between the number of numts and the genome size. Most numt surveys have relied on model organisms with fully sequenced nuclear genomes, but such analyses have limited utilities for making a generalization about the patterns of numt accumulation for any given clade. Among insects, the order Orthoptera is known to have the largest nuclear genome and it is also reported to include several species with a large number of numts. In this study, we use Orthoptera as a case study to document the diversity and abundance of numts by generating numts of three mitochondrial loci across 28 orthopteran families, representing the phylogenetic diversity of the order. We discover that numts are rampant in all lineages, but there is no discernable and consistent pattern of numt accumulation among different lineages. Likewise, we do not find any evidence that a certain mitochondrial gene is more prone to nuclear insertion than others. We also find that numt insertion must have occurred continuously and frequently throughout the diversification of Orthoptera. Although most numts are the result of recent nuclear insertion, we find evidence of very ancient numt insertion shared by highly divergent families dating back to the Jurassic period. Finally, we discuss several factors contributing to the extreme prevalence of numts in Orthoptera and highlight the importance of exploring the utility of numts in evolutionary studies.     

Nuclear mitochondrial pseudogenes

As has been demonstrated recently, the transfer of genetic material from mitochondria to the nucleus and its integration into the nuclear genome is a continuous and dynamic process. Fragments of mitochondrial DNA (mtDNA) are incorporated in the nuclear genome as noncoding sequences, which are called nuclear mitochondrial pseudogenes (NUMT pseudogenes or NUMT inserts). In various eukaryotes, NUMT pseudogenes are distributed through different chromosomes to form a “library” of mtDNA fragments, providing important information on genome evolution. The escape of mtDNA from mitochondria is mostly associated with mitochondrial damage and mitophagy. Fragments of mtDNA may be integrated into nuclear DNA (nDNA) during repair of double-strand breaks (DSBs), which are caused by endogenous or exogenous agents. DSB repair of nDNA with a capture of mtDNA fragments may occur via nonhomologous end joining or a similar mechanism that involves microhomologous terminal sequences. An analysis of the available data makes it possible to suppose that the NUMT pseudogene formation rate depends on the DSB rate in nDNA, the activity of the repair systems, and the number of mtDNA fragments leaving organelles and migrating into the nucleus. Such situations are likely after exposure to damaging agents, first and foremost, ionizing radiation. Not only do new NUMT pseudogenes change the genome structure in the regions of their integration, but they may also have a significant impact on the actualization of genetic information. The de novo integration of NUMT pseudogenes in the nuclear genome may play a role in various pathologies and aging. NUMT pseudogenes may cause errors in PCR-based analyses of free mtDNA as a component of total cell DNA because of their coamplification.     

Reliability of mitochondrial DNA in an acanthocephalan: the problem of pseudogenes

The utility of mitochondrial DNA as a molecular marker for evolutionary studies is well recognized. However, several problems can arise when using mitochondrial DNA, one of which is the presence of nuclear mitochondrial pseudogenes, or Numts. Pseudogenes of cytochrome oxidase I were preferentially amplified from Acanthocephalus lucii (Acanthocephala) using a universal PCR approach. To verify the presence and abundance of pseudogenes, length heterogeneity analysis of the PCR fragments was performed. PCR products obtained with universal primers often contained fragments of different sizes. Cloned sequences from universal PCR products nearly always contained sequence abnormalities such as indels and/or stop codons. Based on these sequences, new primers were developed to specifically target mitochondrial DNA. Sequences obtained with these specific primers lacked abnormalities. Phylogenetic analysis produced a single most parsimonious tree in which pseudogenes obtained with universal primers grouped together as did putative mitochondrial DNA sequences obtained with specific primers. The pattern of codon bias observed in the pseudogenes suggests a single nuclear integration event from the mitochondria. This is the first reported occurrence of pseudogenes in an acanthocephalan, and it demonstrates the potential dangers associated with the use of universal primers.     

Primers for a PCR-based approach to mitochondrial genome sequencing in birds and other vertebrates.

A PCR-based approach to sequencing complete mitochondrial genomes is described along with a set of 86 primers designed primarily for avian mitochondrial DNA (mtDNA). This PCR-based approach allows an accurate determination of complete mtDNA sequences that is faster than sequencing cloned mtDNA. The primers are spaced at about 500-base intervals along both DNA strands. Many of the primers incorporate degenerate positions to accommodate variation in mtDNA sequence among avian taxa and to reduce the potential for preferential amplification of nuclear pseudogenes. Comparison with published vertebrate mtDNA sequences suggests that many of the primers will have broad taxonomic utility. In addition, these primers should make available a wider variety of mitochondrial genes for studies based on smaller data sets.