View source for Nuclear mitochondrial DNA segment

{{Short description|Sporadic biological phenomenon at the molecular scale}}
{{Lead too long|date=January 2023}}

'''Nuclear mitochondrial DNA''' (NUMT) segments or genetic loci describe a transposition of any type of cytoplasmic mitochondrial DNA into the nuclear genome of [[eukaryotic]] organisms.<ref name="Gaziev">{{cite journal|last1=Gaziev|first1=A. I.|last2=Shaikhaev|first2=G. O|title=Nuclear Mitochondrial Pseudogenes|journal=Molecular Biology|date=2010|volume=44|issue=3|pages=358–368|doi=10.1134/s0026893310030027|pmid=20608164|s2cid=22819449}}</ref><ref name=":0">{{Cite journal|last=Lopez|first=J.V., Yuhki, N., Modi, W., Masuda, R. and O'Brien, S.J.|date=1994|title=Numt, a recent transfer and tandem amplification of mitochondrial DNA in the nuclear genome of the domestic cat|journal=J Mol Evol|volume=39|issue=2|pages=174–190|doi=10.1007/BF00163806|pmid=7932781|bibcode=1994JMolE..39..174L |s2cid=22165470 }}</ref><ref>{{cite journal | last1 = Lopez | first1 = J.V. | last2 = Stephens | first2 = J.C | last3 = O'Brien | first3 = S.J. | year = 1997 | title = The long and short of nuclear mitochondrial lineages | journal = Trends Ecol. Evol. | volume = 12 | issue = 3| page = 114 | doi=10.1016/s0169-5347(97)84925-7| pmid = 21238001 }}</ref>

More NUMT sequences of different sizes and lengths in the diverse number of eukaryotes have been detected as [[whole genome sequencing]] of different [[organisms]] accumulates.<ref name="Nomiyama">{{cite journal|last1=Nomiyama|first1=Hisayuki|title=Molecular Structures of Mitochondrial-DNA-Like Sequences in Human Nuclear DNA|journal= Nucleic Acids Research|date=1985|volume=13|issue=5|pages=1649–658|display-authors=etal|doi=10.1093/nar/13.5.1649|pmid=2987834|pmc=341102}}</ref> They have often been unintentionally discovered by researchers who were looking for [[mitochondrial DNA]] (mtDNA).<ref name="Hazkami-Covo" /> NUMTs have been reported in all studied eukaryotes, and nearly all mitochondrial genome regions can be integrated into the nuclear genome.<ref name=":1">{{Cite journal|last=Mishmar|first=D., Ruiz-Pesini, E., Brandon, M. and Wallace, D.C.|date=2004|title=Mitochondrial DNA-like sequences in the nucleus (NUMTs): Insights into our African origins and the mechanism of foreign DNA integration|journal=Hum Mutat|volume=23|issue=2|pages=125–133|doi=10.1002/humu.10304|pmid=14722916|s2cid=25109836|doi-access=free}}</ref><ref>{{Cite journal|last=Qu|first=H., Ma, F. and Li, Q.|date=2008|title=Comparative analysis of mitochondrial fragments transferred to the nucleus in vertebrate|journal=J Genet Genomics|volume=35|issue=8|pages=485–490|doi=10.1016/S1673-8527(08)60066-1|pmid=18721785}}</ref> However, NUMTs differ in number and size across different species.<ref name=":1" /><ref>{{Cite journal|last=Sacerdot|first=C., Casaregola, S., Lafontaine, I., Tekaia, F., Dujon, B. and Ozier-Kalogeropoulos, O.|date=2008|title=Promiscuous DNA in the nuclear genomes of hemiascomycetous yeasts|journal=FEMS Yeast Res|volume=8|issue=6|pages=846–857|doi=10.1111/j.1567-1364.2008.00409.x|pmid=18673395|doi-access=free}}</ref><ref>{{Cite journal|last=Schizas|first=N.V.|date=2012|title=Misconceptions regarding nuclear mitochondrial pseudogenes (Numts) may obscure detection of mitochondrial evolutionary novelties|journal=Aquat Biol|volume=17|pages=91–96|doi=10.3354/ab00478|doi-access=free}}</ref> Such differences may be accounted for by interspecific variation in such factors as [[germline]] stability and mitochondria number.<ref name="Richly" /> After the release of the mtDNA into the [[cytoplasm]], due to the mitochondrial alteration and [[morphology (biology)|morphological]] changes, it is transferred into the nucleus<ref name="Gaziev" /><ref name="Hazkami-Covo">{{cite journal|last1=Hazkani-Covo|first1=Einat|title=Molecular Poltergeists: Mitochondrial DNA Copies (NUMT) in Sequenced Nuclear Genomes|journal=PLOS Genetics|date=2010|volume=6|issue=2|display-authors=etal|doi=10.1371/journal.pgen.1000834|pmid=20168995|page=e1000834|pmc=2820518 |doi-access=free }}</ref> and inserted by double-stranded break repair processes into the [[nuclear DNA]] (nDNA).<ref name="Gaziev" /> A correlation has been found between the fraction of noncoding DNA and NUMT abundance in the genome,<ref name="Richly">{{cite journal|last1=Richly|first1=E.|title=NUMTs in Sequenced Eukaryotic Genomes|journal=Molecular Biology and Evolution|date=2004|volume=21|issue=6|pages=1081–084|doi=10.1093/molbev/msh110|pmid=15014143|url=http://edoc.mpg.de/222288|hdl=11858/00-001M-0000-0012-3BE0-F|hdl-access=free}}</ref><ref name="Rogers">{{cite journal|last1=Rogers|first1=Hubert H|last2=Griffiths-Jones|first2=Sam|title=Mitochondrial pseudogenes in the nuclear genomes of Drosophila|journal=PLOS ONE|date=2012|volume=7|issue=3|pages=e32593|doi=10.1371/journal.pone.0032593 |pmid=22412894|pmc=3296715|bibcode=2012PLoSO...732593R|doi-access=free}}</ref><ref name="Tsuji">{{cite journal |last1=Tsuji |first1=J. |display-authors=etal |date=2012 |title=Mammalian NUMT Insertion Is Non-random |journal=Nucleic Acids Research |volume=40 |issue=18 |pages=9073–088 |doi=10.1093/nar/gks424 |pmc=3467031 |pmid=22761406}}</ref> and NUMTs are observed to have non-random distribution and a higher likelihood of being inserted in certain genomic regions.<ref name="Tsuji" /> Depending on the location of the insertion, NUMTs might disrupt gene function.<ref name="Gaziev" /> In addition, de novo integration of NUMT pseudogenes into the nuclear genome can have adverse effects.<ref name="Dayama">{{cite journal|last1=Dayama|first1=G|title=The Genomic Landscape of Polymorphic Human Nuclear Mitochondrial Insertions|journal=Nucleic Acids Res|date=2014|volume=42|issue=20|pages=12640–12649|display-authors=etal|pmc=4227756|pmid=25348406|doi=10.1093/nar/gku1038}}</ref><ref name="HAzkani-Covo">{{cite journal|last1=Hazkani-Covo|first1=E|last2=Graur|first2=D|title=A Comparative Analysis of NUMT Evolution in Human and Chimpanzee|journal=Molecular Biology and Evolution|date=2006|volume=24|issue=1|pages=13–18|doi=10.1093/molbev/msl149|pmid=17056643|doi-access=free}}</ref><ref name="Chatre">{{cite journal |last1=Chatre |first1=Lauren |last2=Ricchetti |first2=Miria |date=2011 |title=Nuclear Mitochondrial DNA Activates Replication in Saccharomyces Cerevisiae |journal=PLOS ONE |volume=6 |issue=3 |page=e17235 |bibcode=2011PLoSO...617235C |doi=10.1371/journal.pone.0017235 |pmc=3050842 |pmid=21408151 |doi-access=free}}</ref><ref name="bensasson2001">{{cite journal|last1=Bensasson|first1=D|title=Mitochondrial Pseudogenes: Evolution's Misplaced Witnesses|journal=Trends in Ecology & Evolution|date=2001|volume=16|issue=6|pages=314–321|doi=10.1016/s0169-5347(01)02151-6|pmid=11369110|doi-access=free}}</ref>

In the [[Cat|domestic cat]], mitochondrial gene number and content were amplified 38 to 76 times in the cat's nuclear genome besides being transposed from the cytoplasm.<ref name="Lopez">{{cite journal |last1=Lopez |first1=Jose V |display-authors=etal |date=1996 |title=Complete Nucleotide Sequences of the Domestic Cat (Felis Catus) Mitochondrial Genome and a Transposed MtDNA Tandem Repeat (Numt) in the Nuclear Genome |url=https://zenodo.org/record/1229675 |journal=Genomics |volume=33 |issue=2 |pages=229–46 |doi=10.1006/geno.1996.0188 |pmid=8660972}}</ref> Cat NUMT sequences did not appear to be functional due to the discovery of multiple mutations, differences in mitochondrial and nuclear genetic codes, and the apparent insertion within typically inert [[centromere]] regions. The presence of NUMT fragments in the genome is not problematic in all species; for instance, it is shown that sequences of mitochondrial origin promote nuclear DNA replication in ''[[Saccharomyces cerevisiae]]''.<ref name="Chatre" /> Although the extended translocation of mtDNA fragments and their co-amplification with free mitochondrial DNA has been problematic in the diagnosis of mitochondrial disorders, in the study of population genetics and [[phylogenetic analyses]],<ref name="Gaziev" /> scientists have used NUMTs as genetic markers to determine the relative rate of nuclear and mitochondrial mutation and recreating the evolutionary tree.<ref name="bensasson2001" />

In 2022, scientists reported the discovery of ongoing [[Mitochondrial DNA#mtDNA in nuclear DNA|transfer of mitochondrial DNA into DNA in the cell nucleus]]. Previously, NUMTs were thought to have arisen before the existence of humans. 66,000 [[Whole genome sequencing|whole-genome sequences]] indicate this occurs as frequently as approximately once every 4,000 human births.<ref>{{cite news |title=Mitochondrial DNA Is Working Its Way Into the Human Genome |url=https://www.technologynetworks.com/genomics/news/mitochondrial-dna-is-working-its-way-into-the-human-genome-366341 |access-date=17 November 2022 |work=Genomics Research from Technology Networks |language=en}}</ref><ref>{{cite journal |last1=Wei |first1=Wei |last2=Schon |first2=Katherine R. |last3=Elgar |first3=Greg |last4=Orioli |first4=Andrea |last5=Tanguy |first5=Melanie |last6=Giess |first6=Adam |last7=Tischkowitz |first7=Marc |last8=Caulfield |first8=Mark J. |last9=Chinnery |first9=Patrick F. |title=Nuclear-embedded mitochondrial DNA sequences in 66,083 human genomes |journal=Nature |date=November 2022 |volume=611 |issue=7934 |pages=105–114 |doi=10.1038/s41586-022-05288-7 |pmid=36198798 |pmc=9630118 |bibcode=2022Natur.611..105W |language=en |issn=1476-4687|doi-access=free}}
* University press release: {{cite news |title=A new route to evolution: How DNA from our mitochondria works its way into our genomes |url=https://phys.org/news/2022-10-route-evolution-dna-mitochondria-genomes.html |access-date=17 November 2022 |work=[[University of Cambridge]] via phys.org |language=en}}</ref>

==History==
According to the [[endosymbiosis theory]],<ref name="Hazkami-Covo" /> which gained acceptance around the 1970s,<ref name="Ju">{{cite journal|last1=Ju|first1=Young Seok|title=Abstract LB-161: Frequent Somatic Transfer of Mitochondrial DNA into the Nuclear Genome of Human Cancer Cells|journal=Cancer Research Cancer Res|date=2015|volume=75|issue=15}}</ref> the [[mitochondrion]], as a major energy producer in the cell, was previously a free-living prokaryote that invaded a eukaryotic cell. Under this theory, symbiotic organelles gradually transferred their genes to the eukaryotic genome, implying that mitochondrial DNA (mtDNA) was gradually integrated into the nuclear genome.<ref name=":0" /> Despite the metabolic alterations and functional adaptations in the host eukaryotes, circular mitochondrial DNA is contained within the organelles. mtDNA has an essential role in the production of necessary compounds, such as required [[enzymes]] for the proper function of mitochondria.<ref>{{cite web|title=Help Me Understand Genetics|url=https://ghr.nlm.nih.gov/primer|website=Genetics Home Reference|publisher=U.S. National Library of Medicine|access-date=4 May 2016}}</ref> Specifically, it has been suggested that certain genes (such as the genes for [[cytochrome c oxidase subunit I|cytochrome oxidase subunits I]] and II) within the organelle are necessary to regulate [[redox]] balance throughout membrane-associated [[electron transport chain]]s.<ref name="Hazkami-Covo" /><ref name=":2">{{Cite journal|last=Zhang|first=D-X. and Hewitt, G.M.|date=1996|title=Nuclear integrations: challenges for mitochondrial DNA markers|journal=Trends Ecol Evol|volume=11|issue=6|pages=247–251|doi=10.1016/0169-5347(96)10031-8|pmid=21237827}}</ref> These parts of the mitochondrial genome have been reported to be the most frequently employed.<ref name=":2" /> Mitochondria are not the only locations within which mtDNA can be found; sometimes mtDNA can be transferred from organelles to the nucleus; the evidence of such [[Chromosomal translocation|translocation]] has been seen by comparing mtDNA sequences with the genome sequence in the nucleus.<ref name="Gaziev" /><ref name="Nomiyama" /><ref name="Richly" /> The integration and recombination of cytoplasmic mtDNA into the nuclear DNA is called nuclear mitochondrial DNA (NUMT).<ref name="Gaziev" />

The possible presence of organelle DNA inside the nuclear genome was suggested after discovering [[homologous structure]]s to the mitochondrial DNA in the nucleus, which was shortly after the discovery of independent DNA within the organelles in 1967.<ref name="bensasson2001" /> This topic stayed untouched until the 1980s. Initial evidence that DNA could move among cell compartments came when fragments of chloroplast DNA were found in the maize mitochondrial genome with the help of cross-hybridization, chloroplast and mitochondrial DNA, and physical mapping of homologous regions.<ref name="Gaziev" /><ref name="Stern">{{cite journal|last1=Stern|first1=David B|last2=Lonsdale|first2=David M.|title=Mitochondrial and Chloroplast Genomes of Maize Have a 12-kilobase DNA Sequence in Common|journal=Nature|date=1982|volume=299|issue=5885|pages=698–702|doi=10.1038/299698a0|pmid=6889685|bibcode=1982Natur.299..698S|s2cid=4252809}}</ref><ref name="Ellis">{{cite journal|last1=Ellis|first1=John|title=Promiscuous DNA—chloroplast Genes inside Plant Mitochondria|journal=Nature|date=1982|volume=299|issue=5885|pages=678–679|doi=10.1038/299678a0|pmid=7121600|bibcode=1982Natur.299..678E|s2cid=31189305}}</ref> After this initial observation, John Ellis coined the term ''promiscuous DNA'' to signify the transfer of DNA intracellularly from one organelle to the other and denote the presence of organelle DNA in multiple cellular compartments.<ref name="Ellis" /> The search for mtDNA in nuclear DNA continued until 1994, when the transposition of 7.9 kb of a typically 17.0-kb mitochondrial genome to a specific nuclear chromosomal position in the domestic cat was reported by evolutionary geneticist,  Jose V. Lopez, who coined the term ''NUMT'' to designate the large stretches of mitochondrial DNA in the nuclear genome.<ref name="bensasson2001" /><ref name="Lopez" />

Currently, the whole genomes of many eukaryotes, both [[vertebrate]] and [[invertebrate]], have been sequenced and NUMTs have been observed in the nuclear genome of various organisms, including yeast, ''[[Podospora]]'', [[sea urchin]], [[locust]], honey bee, ''[[Tribolium (beetle)|Tribolium]]'', rat, maize, rice, and [[primates]].<ref name="Nomiyama" /><ref name="Song">{{cite journal|last1=Song|first1=Shen|title=Exceptionally High Cumulative Percentage of NUMTs Originating from Linear Mitochondrial DNA Molecules in the Hydra Magnipapillata Genome|journal=BMC Genomics|date=2013|volume=14|issue=1|page=447|display-authors=etal|doi=10.1186/1471-2164-14-447|pmid=23826818|pmc=3716686 |doi-access=free }}</ref> In ''[[Plasmodium]]'', ''[[Anopheles gambiae]]'' and ''[[Aedes aegypti]]'', NUMTs can barely be detected.<ref name="Hoy">{{cite book|last1=Hoy|first1=Marjorie A.|title=Insect Molecular Genetics: An Introduction to Principles and Applications|date=2013|publisher=Academic Press|location=San Diego|isbn=978-0-12-415874-0|pages=613–620|edition=3}}</ref><ref name="Hlaing">{{cite journal|last1=Hlaing|first1=Thaung|title=Mitochondrial Pseudogenes in the Nuclear Genome of Aedes Aegypti Mosquitoes: Implications for the past and Future Population Genetic Studies|journal=BMC Genetics|date=2009|volume=10|issue=1|page=11|display-authors=etal|doi=10.1186/1471-2156-10-11|pmid=19267896|pmc=2660364 |doi-access=free }}</ref> In contrast, conserved fragments of NUMT were identified in genome data for ''[[Ciona intestinalis]]'', ''[[Neurospora crassa]]'', ''[[Schizosaccharomyces pombe]]'', ''[[Caenorhabditis elegans]]'', ''[[Drosophila melanogaster]]'', and ''[[Rattus norvegicus]]''.<ref name="Gaziev" /><ref name="Richly" /><ref name="Rogers" /><ref name="Song" /> Agostinho Antunes and Maria João Ramos discovered the presence of NUMTs in the fish genome in 2005 by using [[BLAST (biotechnology)|BLAST]], [[MAFFT]], genome mapping, and phylogenic analysis.<ref name="Rogers" /><ref name="antunes1">{{cite journal|last1=Antunes|first1=Agostinho|last2=Ramos|first2=Maria João|title=Discovery of a Large Number of Previously Unrecognized Mitochondrial Pseudogenes in Fish Genomes.|journal=Genomics|date=2005|volume=86|issue=6|pages=708–717|doi=10.1016/j.ygeno.2005.08.002|pmid=16176867|doi-access=free}}</ref> The [[Apis mellifera|western honey bee]] and ''[[Hydra magnipapillata]]'' are, respectively, the first and second animals with the highest ratio of NUMTs to the total size of the nuclear genome while the [[gray short-tailed opossum]] is the record holder for NUMT frequency among vertebrates.<ref name="Hazkami-Covo" /><ref name="Song" /> Like animals, NUMTs are abundant in plants, and the longest NUMT fragment known so far is a 620 kb partially-duplicated insertion of the 367 kb mtDNA of ''[[Arabidopsis thaliana]]''.<ref name="Hazkami-Covo" />

==Mechanism of NUMT insertion==
NUMT insertion into the nuclear genome and its persistence in the nuclear genome is initiated by the physical delivery of mitochondrial DNA to the nucleus.<ref name="Hazkami-Covo" /> This step follows by the mtDNA integration into the genome through a [[non-homologous end joining]] mechanism during the [[double-strand break]] (DSB) repair process as envisioned by studying ''[[Saccharomyces cerevisiae]]'',<ref name="Dayama" /><ref name="Blanchard">{{cite journal|last1=Blanchard|first1=J.L|last2=Schmidt|first2=G.W|title=Mitochondrial DNA migration events in yeast and humans: integration by a common end-joining mechanism and alternative perspectives on nucleotide substitution patterns|journal=Mol. Biol. Evol.|date=1996|volume=13|issue=3|pages=537–548|doi=10.1093/oxfordjournals.molbev.a025614|pmid=8742642|doi-access=free}}</ref> and terminates by intragenomic dynamics of amplification, mutation, or deletion, collectively known as post-insertion modifications.<ref name="Hazkami-Covo" /> The mechanism of mtDNA transfer into nucleus is not yet fully understood.{{Citation needed|date=March 2024}}
[[File:Different ways mtDNA moves into the nucleus.PNG|thumb|Different ways mtDNA moves into the nucleus]]

===Transfer of the released mtDNA into the nucleus===
The first step in the transfer process is the release of mtDNA into the cytoplasm.<ref name="Gaziev" /> Peter Thorsness and Thomas Fox demonstrated the rate of relocation of mtDNA from mitochondria into the nucleus using ''ura3-'' yeast strain with an engineered ''URA3 ''[[plasmid]], a required gene for [[uracil]] biosynthesis, in the mitochondria. During the propagation of such yeast strains carrying a nuclear ''ura3'' mutation, plasmid DNA that escapes from the mitochondrion to the nucleus complements the uracil [[biosynthetic]] defect, restoring growth in the absence of uracil, and easily scored phenotype.<ref name="Thorsness1990">{{cite journal|last1=Thorsness|first1=Peter E|last2=Fox|first2=Thomas D.|title=Escape of DNA from Mitochondria to the Nucleus in Saccharomyces Cerevisiae|journal=Nature|date=1990|volume=346|issue=6282|pages=376–79|doi=10.1038/346376a0|pmid=2165219|bibcode=1990Natur.346..376T|s2cid=4303713}}</ref> The rate of DNA transfer from the mitochondria to the nucleus was estimated as 2 x 10<sup>−5</sup> {{Clarify|reason=Missing unit|date=March 2024}} per cell per generation, while in the case of the ''cox2'' mutant the rate of transfer of the plasmid from the nucleus to the mitochondria is approximately at least 100,000 times less.<ref name="Thorsness1990" /> Many factors control the rate of mtDNA escapes from mitochondria to the nucleus. The higher rate of mutation in mtDNA in comparison with nDNA in the cells of many organisms is an important factor promoting the transfer of mitochondrial genes into the nuclear genome.<ref name="Gaziev" /><ref name="Thorsness 1993">{{cite journal|last1=Thorsness|first1=P. E.|last2=Fox|first2=T. D.|title=Nuclear Mutations in Saccharomyces Cerevisiae That Affect the Escape of DNA from Mitochondria to the Nucleus|journal= Genetics|volume=134|issue=1|pages=21–28|date=1993|doi=10.1093/genetics/134.1.21|pmc=1205423|pmid=8514129}}</ref> One of the [[intergenic]] factors that results in more frequent destruction of mitochondrial macromolecules, including mtDNA, is the presence of high level of reactive oxygen species generated in mitochondria as the by-products in ATP synthesis.<ref name="Gaziev" /> Some other factors influencing the escape of mtDNA from mitochondria include the action of mutagenic agents and other forms of cellular stress that can damage mitochondria or their membranes,<ref name="bensasson2001" /> which makes assuming that [[exogenous]] damaging agents (for example, ionizing radiation and chemical genotoxic agents) increase the rate of mtDNA escape into the cytoplasm possible.<ref name="Wallace2005">{{cite journal|last1=Wallace|first1=D. C.|title=A mitochondrial paradigm of metabolic and degenerative diseases, aging, and cancer: A dawn for evolutionary medicine|journal=Annu. Rev. Genet.|date=2005|volume=39|pages=359⎯407|doi=10.1146/annurev.genet.39.110304.095751|pmid=16285865|pmc=2821041}}</ref>  Thorsness and Fox continued their research to find the endogenous factors effecting mtDNA escape into the nucleus. They isolated and studied 21 nuclear mutants with different combinations of mutations in at least 12 nuclear loci called the ''yme'' (yeast mitochondrial escape) mutations, in different environmental conditions since some of these mutations cause temperature sensitivity. They discover that these mutations which perturb mitochondrial functions affect mitochondrial integrity and led to mtDNA escaping into the cytoplasm.<ref name="Thorsness 1993" /> Additionally, defects in the proteins change the rate of mtDNA transfer into the nucleus; for instance, in the case of the ''yme1 ''mutant, abnormal mitochondria are targeted for degradation by the vacuole with the help of ''pep4'', a major [[Protease|proteinase]], and degradation increases mtDNA escape into the nucleus through [[mitophagy]].<ref name="Gaziev" /><ref name="Campbell">{{cite journal|last1=Campbell|first1=Corey L|last2=Thorsness|first2=Peter E|title=Escape of Mitochondrial DNA to the Nucleus in ''Yme1'' Yeast Is Mediated by Vacuolar-dependent Turnover of Abnormal Mitochondrial Compartments|journal=Journal of Cell Science|date=30 July 1998|pmid=9683639|volume=111|pages=2455–64|issue=16|doi=10.1242/jcs.111.16.2455}}</ref> Thorsness and Corey Campbell found that by disrupting ''pep4'', the frequency of mtDNA escape in ''yme1'' strains decreases. Similarly, the disruption of ''PRC1'', which encodes [[carboxypeptidase]] Y, lowers the rate of mtDNA escape in ''yme1'' yeast.<ref name="Campbell" />

Evidence shows that mitophagy is one of the possible ways for mtDNA transfer into the nucleus and determined to be the most supported pathway up to now. The first pathway is a ''yme1'' mutant that results in inactivation of ''YMe1p'' protein, a mitochondrial-localized ATP-dependent [[metalloproteinase]], leading to high escape rate of mtDNA to the nucleus.<ref name="Campbell" /> Mitochondria of the ''yme1'' strain are taken up for degradation by the vacuole more frequently than the wild-type strain.<ref name="Campbell" /> Moreover, cytological investigations have suggested several other possible pathways in the diverse number of [[species]], including a [[lysis]] of the mitochondrial compartment, direct physical connection and membrane fusion between mitochondria and nucleus, and the encapsulation of mitochondrial compartments inside the nucleus.<ref name="Hazkami-Covo" />

===Pre-insertion preparation===
[[File:Different possible ways of mtDNA processing before insertion into the nDNA.PNG|thumb|Different possible ways of mtDNA processing before insertion into the nDNA]]
After reaching the nucleus, mtDNA has to enter the nuclear genome. The rate of mtDNA integration into the nuclear genome relies on the DSB number in nDNA, the activity of DSB repair systems, and the rate of mtDNA escape from organelles.<ref name="Gaziev" /> The insertion of mtDNA comprises three main processes: first, the mtDNA must have the proper form and sequence; in other words, the mtDNA has to be edited, which creates the new edited site in the polynucleotide structure. Mitochondrial DNA is not universal and, in animals similar to plants, mitochondrial editing shows very erratic patterns of taxon-specific occurrence.<ref name="Henz">{{cite journal |last1=Henz |first1=K |last2=Martin |first2=William |date=2001 |title=How Do Mitochondrial Genes Get into the Nucleus |journal=Trends in Genetics |volume=17 |issue=7 |pages=383–387 |doi=10.1016/s0168-9525(01)02312-5 |pmid=11418217}}</ref>

There are three possible ways that mtDNA can become prepared to be inserted into the nuclear DNA. The process mainly depends on the time mtDNA transfers into the nucleus.<ref name="Henz" />  Direct integration of unedited mtDNA fragments into the nuclear genomes is the most plausible, and is observed in plants, the ''Arabidopsis'' genome, and animals with the help of different methods, including BLAST-based analysis.<ref name="Gaziev" /><ref name="Henz" /> In this case, mtDNA is transferred into the nucleus while editing and the creation of [[intron]]s occur later in the mitochondrion. If a gene was transferred to the nucleus in one lineage before mitochondrial editing evolved, but remained in the organelle in other lineages where editing arose, the nuclear copy would appear more similar to an edited transcript than to the remaining mitochondrial copies at the edited sites.<ref name="Henz" /> Another represented and less supported model is the [[cDNA]]-mediated model, in which intron-contained mtDNA enters the nucleus, and by reverse transcription of spliced and edited mitochondrial transcript, integrates into the nDNA.<ref name="Gaziev" /><ref name="Henz" /> The third proposed mechanism is the direct transfer and integration of intronless mtDNA into the nucleus, where editing and introns in the mitochondrion come and go during evolution. In this case, the introduction and removal of the intron, as well as [[reverse transcription]], occur within mitochondria and the final product, the edited intronless mtDNA, integrates into nDNA after being transferred into the nucleus.<ref name="Henz" />

===Insertion into the nuclear genome===
[[File:Mechanism of NUMT insertion into the nuclear DNA.PNG|thumb|Mechanism of NUMT insertion into the nuclear DNA]]

After the preparatory step is over, mtDNA is ready to be inserted into the nuclear genome. Based on NUMT integration site and the analyzed obtained results from the baker's yeast experiment, Blanchard and Schmidt hypothesized that mtDNA are inserted into the DSB via non-homologous end joining machinery; the hypothesis has been widely accepted.<ref name="Blanchard" />  Later analyses were consistent with the involvement of NHEJ{{Clarify|reason=What does this stand for?|date=March 2024}} in NUMT integration in humans.<ref name="Hazkami-Covo" /> These processes occur in both somatic and [[germline]] cells. In animals and humans, however, the capability of DSB repair in germline cells depends on the oogenetic and spermatogenetic stage, nonetheless, due to the low repair activity, mature spermatozoa are incapable of DSB repair.<ref name="Gaziev" /><ref name="Ju" /> DSB can also be repaired by [[homologous recombination]], which is more accurate and introduces fewer errors in the process of repair.<ref name="Gaziev" /><ref name="Ju" /> Apart from canonical NHEJ, DSBs are repaired via [[microhomology-mediated end joining]] (MMEJ), which involves sequences containing a few homologous nucleotides at the ends of a DSB to be [[Ligation (molecular biology)|ligated]].<ref name="Gaziev" /> MMEJ is the most [[mutagenic]] DSB repair mechanism due to generating deletions, insertions of various sizes, and other genome rearrangements in mammals.<ref name="Gaziev" />

The processes of mtDNA insertion and DSB repair include DNA segment alignment, DNA end-processing, DNA synthesis, and ligation.<ref name="Gaziev" /> In each step, certain protein complexes are required to facilitate the occurrence of the indicated events. In NHEJ, the ''Ku70/Ku80'' [[heterodimer]] and DNA-dependent protein kinase (DNA-PK) for bringing DNA fragments end together, the [[Artemis]] nuclease and [[polynucleotide]] [[kinase]] 3' phosphatase(PNKP) for end processing, X family [[DNA polymerases]] ''(Pol μ ''and'' Pol λ) '' and [[terminal deoxynucleotidyl transferase]] (TdT) for DNA synthesis, and the ''XLF/XRCC4/LigIV '' complex for completing the repair and joining the ends via a [[phosphodiester bond]], are the protein complexes involved in DSB repair process in many higher organisms.<ref name="Gaziev" /> DNA polymerases Pol μ and Pol λ and the ''XLF/XRCC4/LigIV ''complex are shared between two NHEJ and MMEJ repair machinery and have the same function in both repair processes.<ref name="Gaziev" /> The first step of MMEJ is performed by WRN{{Clarify|reason=No mention of it earlier|date=March 2024}}, Artemis, DNA-PK, and [[XRCC4]] protein complexes, which process the ends of DSB and mtDNA fragments in addition to aligning them in order for polymerases and ligases to be able to complete NUMT insertion.

===Post-insertion modification===
The complex pattern of NUMT in comparison with the single mitochondrial piece, the appearance of non-continuous mitochondrial DNA in the nuclear genome, and different orientations of these fragments demonstrate post-insertion processes of NUMT within the nuclear genome.<ref name="Hazkami-Covo" /> The cause of these complex patterns might be the result of multiple NUMT insertions at insertional hotspots.<ref name="Hazkami-Covo" /> In addition, duplication after insertion contributes to NUMT diversity.<ref name="Gaziev" /> NUMTs do not have self-replicating mechanisms or transposition mechanisms, so NUMT duplication is expected to occur in tandem or to involve larger segmental duplication at rates representative of the rest of the genome.  Evidence for NUMT duplications that are not in proximity to other NUMTs is present in many genomes and probably happens as part of segmental duplication.<ref name="bensasson 2003">{{cite journal |last1=Bensasson |first1=D |last2=Feldman |first2=MW |last3=Petrov |first3=DA |date=2003 |title=Rates of DNA duplication and mitochondrial DNA insertion in the human genome |journal=J Mol Evol |volume=57 |issue=3 |pages=343–354 |bibcode=2003JMolE..57..343B |doi=10.1007/s00239-003-2485-7 |pmid=14629044 |s2cid=42754186}}</ref> However, duplicates of recent human-specific NUMTs as part of segmental duplication seem to be rare; in humans, only a few NUMTs are found to overlap with segmental duplication, and those NUMTs were found in only one of the copies while missing from the others, suggesting that the NUMTs were inserted after duplication.<ref name="bensasson 2003" /> Deletion is another NUMT post-insertional modification method that has not yet been studied in the same amount of detail as insertion.<ref name="Hazkami-Covo" /> Constant erosion of phylogenic signals and high mutation rate in animal mtDNA make recognition of such modification, especially deletion, difficult. Bensasson and his team members studied cases in which NUMT patterns of appearance do not agree with the [[phylogenetic tree]] to estimate the oldest inserted NUMT in humans, which are dated around 58 million years ago.<ref name="bensasson 2003" />

==General characteristics==

As the number of mitochondria and their functional level differ across eukaryotic organisms, the length, structure, and sequence of NUMTs vary significantly.<ref name="antunes1" /> Researchers have found that the recent NUMT insertions are derived from different segments of the mitochondrial genome, including the [[D-loop]] and, in some cases, NUMTs almost encompassing the entire mitochondrial genome.<ref name="Richly" /><ref name="Dayama" />  The sequence, frequency, size distribution,<ref name="Richly" /> and even the difficulties of finding these sequences in the genome vary substantially among species.<ref name="Gaziev" /><ref name="Hazkami-Covo" /> The majority of DNA fragments transferred from mitochondria and plastids into the nuclear genome are less than 1 kb in size,<ref name="Gaziev" /><ref name="Dayama" /> though large fragments of organelle DNA are found in some the plant genomes.<ref name="Hazkami-Covo" />

As the genome changes over time, the number of NUMTs in it differs over the course of evolution.<ref name="Hazkami-Covo" /> NUMTs enter the nucleus and insert into the nDNA at different points of time. Due to constant mutations and the instability of NUMTs, the resemblance of this genome stretch to the mtDNA varies widely.<ref name="Gaziev" /><ref name="Hazkami-Covo" /> For instance, the latest number of NUMTs recorded in the human genome is 755 fragments ranging from 39 [[Base pair|bp]] to almost the entire mitochondrial sequence in size.<ref name="Dayama" /> There are 33 paralogous sequences with over 80% sequence similarity and of a greater length than 500 bp.<ref name="Ramos">{{cite journal|last1=Ramos|first1=Amanda|title=Nuclear Insertions of Mitochondrial Origin: Database Updating and Usefulness in Cancer Studies|journal=Mitochondrion|date=2011|volume=11|issue=6|pages=946–53|display-authors=etal|doi=10.1016/j.mito.2011.08.009|pmid=21907832}}</ref> Not all of the NUMT fragments in the genome are the result of mtDNA migration; some are the outcome of amplification after insertion.<ref name="Dayama" /> Old NUMTs are found to be more abundant in the human genome than recent integrants, indicating that mtDNA can be amplified once inserted.<ref name="Dayama" /> Dayama ''et al.'' developed a high-yield new technique for the exact detection of the number of NUMTs in the human genome called the discovery of nuclear mitochondrial insertions (dinumt).<ref name="Dayama" /> This method enabled her and her team to identify NUMT insertions of all sizes in the whole genomes sequenced using paired-end sequencing technology. They applied dinumt to 999 individuals from the [[1000 Genomes Project]] and [[Human Genome Diversity Project]], and conducted an updated enrichment analysis in humans using these [[Genetic polymorphism|polymorphic]] insertions.<ref name="Dayama" /> Further investigation and genotyping of the discovered NUMTs also analyses age of insertion, origin, and sequence characteristics. Finally, they assessed their potential impact on ongoing studies of mitochondrial heteroplasmy.<ref name="Dayama" />

Although mtDNA is inserted into the nuclear genome only when a DSB is produced by endogenous or exogenous damaging factors,<ref name="Gaziev" /> it is not randomly inserted into the genome.<ref name="Tsuji" /> Moreover, there is no correlation between the fraction of noncoding DNA and NUMT abundance.<ref name="Richly" /><ref name="Rogers" /><ref name="Tsuji" /> Antunes and Ramos found that old NUMTs are inserted preferentially into the known and predicted loci, as inferred for recent NUMTs in the human genome, during their work on NUMT sequence in fishes using BLASTN analysis method.<ref name="antunes1" /> One of the best studies supporting non-random distribution and insertion of NUMTs in the nuclear genome is done by Tsuji ''et al''.<ref name="Tsuji" /> Using the LAST method instead of BLAST, which makes computing [[E-values]] with higher accuracy possible and does not underrepresent the repetitive elements in NUMT flanks, they were able to precisely characterize the location of NUMT insertion, and found that NUMT fragments tend to be inserted in the regions with high local DNA curvature or bendability and high A+T rich oligomers, especially TAT,<ref name="Dayama" /> in primarily open chromatin regions.<ref name="Tsuji" /> Using the same method, Tsuji showed that NUMTs are not usually clustered together, and the NUMTs produced by D-loop are usually underrepresented, which is more evident in monkey and human genomes when compared to their rat and mouse counterparts due to the total length of their NUMTs.<ref name="Tsuji" /> However, Tsuji also found that although  [[retrotransposon]] structures are highly enriched in NUMT flanks and most NUMTs are inserted in close proximity to a retrotransposon, while 10 out of 557 NUMTs were inserted within a retrotransposon, there was no clear relation between the size of [[non-coding DNA]] and the number of NUMT.<ref name="Tsuji" />

==Consequences of de novo integration of NUMT inserts==

NUMTs are not utterly functionless and certain functions are associated with them.<ref name="Gaziev" /> Although NUMTs were previously considered functionless pseudogenes, recent human NUMTs have been shown to be a potentially mutagenic process that could damage the functional integrity of the human genome.<ref name="antunes1" /> The processes of NUMT migration into the nucleus can cause mutations and dramatic alterations of the genome structure at the integration site, interfere with the function of the genome, and exert substantial effects on the expression of genetic information.<ref name="Gaziev" /> The integration of mtDNA sequences substantially affects the spatial organization of nDNA and may have an important role in the evolution of eukaryotic genomes.<ref name="Gaziev" /> In addition to the negative effects of mtDNA, conserved old NUMTs in the genome are likely to represent evolutionary successes and they should be considered as a potential evolutionary mechanism for the enhancement of genomic coding regions.<ref name="antunes1" /> Lauren Chatre and Miria Ricchetti found that migratory mitochondrial DNAs can impact the replication of the nuclear region in which they are inserted.<ref name="Chatre" /> They observed sequences of mitochondrial origin promoting nDNA replication in ''Saccharomyces cerevisiae''. The NUMTs are 11 bp [[Autonomously replicating sequence|autonomously replicating]] (ARS) core-A consensus sequences (ACS), which are necessary but not sufficient for the function of replication origin and any mutation that consensus causes the reduction or loss of DNA replication activity.<ref name="Chatre" /> Given the high density of ACS motifs, some NUMTs appear essentially as ACS carriers.<ref name="Chatre" /> In contrast, replication efficiency is higher in yeast strains that have plasmids containing both NUMTs and ARS.<ref name="Chatre" /> They also found that some NUMTs can work as an independent replication fork and late chromosomal origins and NUMTs located close to or within ARS provide key sequence elements for replication. Thus, NUMTs can act as the independent origins, when inserted in an appropriate genomic context or affect the efficiency of pre-existing origins.{{Original research inline|date=March 2024}}<ref name="Chatre" />

=== Disease and disorders ===
NUMT insertion into the genome can be problematic. The transposition of NUMTs into the genome has been associated with human diseases.<ref name="Dayama" /><ref name="HAzkani-Covo" /><ref name="Chatre" /> De novo integration of NUMT pseudogenes into the nuclear genome has an adverse effect, in some cases promoting various disorders and aging.<ref name="Gaziev" /> Mitochondrial DNA integration into coding genes in the germline cells has dramatic consequences for embryo development and is lethal in many cases.<ref name="Gaziev" /> Few NUMT pseudogenes associated with diseases are found within [[exon]]s or at the exon–intron boundaries of human genes.<ref name="Gaziev" /> For example, patients with [[mucolipidosis]] inherit a mutation caused by the insertion of a 93 bp fragment of mitochondrial ND5 into exon 2 of the R403C mucolipin gene. This is the first case of a heritable disorder due to the NUMT insert.<ref name="Gaziev" /> Despite the small treatment group, [[stem cell transplant]] has been found to be effective and [[Lysosomal enzymes|lysosomal enzyme]] levels seemed to normalize after transplant in at least one case.<ref>{{cite web|last1=Orchard|first1=Paul MD.|title=Stem Cell Transplant for Inborn Errors of Metabolism|url=https://clinicaltrials.gov/ct2/show/NCT00176904|website=Clinicaltrials.gov|publisher=U.S. National Institutes of Health|access-date=4 May 2016}}</ref> [[Pallister–Hall syndrome]], a developmental disorder, results from a ''[[Mutation#By inheritance|de novo]]'' [[insertion (genetics)|insertion]] of a 72 bp mtDNA fragment into ''GLI3'' [[exon]] 14 in [[chromosome 7]],<ref name="Gaziev" /> causing central and postaxial [[polydactyly]], bifid epiglottis, imperforate anus, renal abnormalities including cystic malformations, [[renal hypoplasia]], ectopic ureteral implantation, and pulmonary segmentation [[Genetic anomaly|anomalies]] such as bilateral bilobed lungs.<ref name="biesecker">{{cite journal|last1=Biesecker|first1=LG|last2=Johnston|first2=JJ|title=Pallister-Hall syndrome (PHS)|journal=Atlas Genet Cytogenet Oncol Haematol|date=2007|volume=11|issue=2|pages=145–147}}</ref> A splice site mutation in the human gene for plasma factor VII that causes severe plasma factor VII deficiency, bleeding disease, results from a 251 bp NUMT insertion.<ref name="Hazkami-Covo" /> Finally, a 36 bp insertion in exon 9 of the USH1C gene associated with [[Usher syndrome]] type IC is the NUMT.<ref name="Hazkami-Covo" /> No certain cause has been found for Usher syndrome yet, but a clinical study on 18 volunteers is taking place to determine the influence both in a short- and a long-term period.<ref>{{cite web|last1=Weleber|first1=Richard MD.|title=A Study to Determine the Long-Term Safety, Tolerability and Biological Activity of UshStat® in Patients With Usher Syndrome Type 1B|url=https://clinicaltrials.gov/ct2/show/NCT02065011|website=ClinicalTrials.gov|publisher=U.S. National Institutes of Health|access-date=4 May 2016}}</ref>

=== Aging ===
Several studies indicated that de novo appearance of NUMT pseudogenes in the genome of somatic cells may be of [[etiological]] importance for carcinogenesis and aging.<ref name="Gaziev" /><ref name="Dayama" /> To show the relation between aging and NUMT in the nuclear genome, Xin Cheng and Andreas Ivessa used ''yme1-1'' mutant strains of ''Saccharomyces cerevisiae'' that have a higher rate of mtDNA migration,<ref name="Ivessa">{{cite journal |last1=Cheng |first1=Xin |last2=Ivessa |first2=Andreas S |date=2010 |title=The Migration of Mitochondrial DNA Fragments to the Nucleus Affects the Chronological Aging Process of Saccharomyces Cerevisiae |journal=Aging Cell |volume=9 |issue=5 |pages=919–923 |doi=10.1111/j.1474-9726.2010.00607.x |pmc=3394387 |pmid=20626726}}</ref> using the same method Thorsness and Fox used to determine the important mechanisms and factors for mtDNA migration into the nucleus.<ref name="Thorsness 1993" /><ref name="Ivessa" /> They discovered that the yeast strains with elevated migration rates of mtDNA fragments to the nucleus showed accelerated chronological aging, whereas strains with decreased mtDNA transfer rates to the nucleus exhibited an extended chronological life span,<ref name="Ivessa" /> which could possibly be due to the effect of NUMT on nuclear processes including DNA replication, recombination, and repair as well as gene transcription.<ref name="Chatre" /><ref name="Ivessa" /> The effect of NUMT on the higher eukaryotic organisms was investigated by Caro ''et al''., using rats as a model organism. Using real-time [[polymerase chain reaction]] (PCR) quantification, ''in situ'' hybridization of mtDNA to [[nDNA]], and comparison of young and old rats, they not only could determine the high concentration of [[cytochrome oxidase]] III and 16S [[rRNA]] from mtDNA in both young and old rats, but also the increase in the number of mitochondrial sequences in nDNA as rats age.<ref name="Caro">{{cite journal |last1=Caro |first1=Pilar |display-authors=etal |date=2010 |title=Mitochondrial DNA sequences are present inside nuclear DNA in rat tissues and increase with age |journal=Mitochondrion |volume=10 |issue=5 |pages=479–486 |doi=10.1016/j.mito.2010.05.004 |pmid=20546951}}</ref> Based on these findings, mitochondria can be a major trigger of aging, but the final target could also be the nucleus.<ref name="Ivessa" /><ref name="Caro" />

=== Cancer ===
The worst cases of NUMT insertion happen when mtDNA is inserted into the regulatory region or nuclear structural genes and disrupts or alters vital cell processes.<ref name="Gaziev" /><ref name="Campbell" /> For instance, in primary low-grade brain neoplasms, fluorescent ''in situ'' hybridization analysis helped with recognizing mtDNA localized in the nucleus in correlation with an overall increase in mtDNA in the cell.<ref name="Liang">{{cite journal|last1=Liang|first1=B. C|title=Evidence for the association of mitochondrial DNA sequence amplification and nuclear localization in human low-grade gliomas|journal=Mutat. Res.|date=1996|volume=354|issue=1|pages=27–33|doi=10.1016/0027-5107(96)00004-8|pmid=8692203|url=https://zenodo.org/record/1258242}}</ref> In [[hepatoma]] cells mtDNA sequences are present in the nuclear genome at a higher copy number than normal tissue.<ref name="Ju" /><ref name="Campbell" /> Another example would be [[HeLa]] nDNA that contains sequences which hybridize with mtDNA fragments of approximately 5 kb. An analysis showed that nDNA of malignant cells contains sequences of the mitochondrial ''cytochrome oxidase I'', ''ND4 '', ''ND4L '', and ''12S'' rRNA genes.<ref name="Ju" /> Based on these findings, mtDNA fragments were assumed to act as a mobile genetic element in the initiation of [[carcinogenesis]].<ref name="Gaziev" /> [[Southern blotting]] is used to determine the frequency of mitochondrial insertion in nDNA of normal and tumor cells of mice and rats, which supported mtDNA sequences being more numerous and abundant in nDNA of rodent tumor cells in comparison with normal cells.<ref name="Gaziev" /> Using FISH probes; PCR; and data sequencing, mapping, and comparison, Ju found that the 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. He also investigated the timing of somatic mtDNA integration into the nuclear genome by assessing cases in which a [[Metastasis|metastatic]] sample had been sequenced in addition to the primary tumor. In some cases, mtDNA transference into the nucleus in somatic cells is very frequent and can occur after neoplastic formation and during the course of subclonal evolution of cancer, suggesting the event occurs in the common ancestral cancer clones or in normal somatic cells prior to the neoplastic change.<ref name="Ju" /> These findings demonstrated that the presence of direct correlation between NUMTs and cancer in different organs.<ref name="bensasson2001" />

==Experimental uses and errors==
{{Very long section|date=November 2022}}
Although understanding non-random NUMT insertions that causes different effects helps with revealing the structure and determining the complete function of genomes, NUMTs have been used as experimental tools and have been beneficial in different biological fields before being recognized as such. For instance, NUMTs have been used as genetic markers, and also as a tool for understanding the relative mutation rate in the nucleus and the mitochondria, as well as recreating evolutionary trees.<ref name="bensasson2001" /> The continuing process of NUMT integration into the nuclear genome is evidenced by the finding of NUMTs that have been inserted into the human genome after the human–chimpanzee divergence.<ref name="HAzkani-Covo" /> Some of these NUMTs indicate that they have only arisen recently in the human population, making them useful as genetic markers of lineage.<ref name="HAzkani-Covo" /> Using a protocol based on genome alignment to estimate the number of NUMT in closely related species, Hazkani-Covo and Graur could identify evolutionary events that may have affected NUMT composition in each genome and reconstruct the NUMT makeup in the human and chimpanzee's common ancestor.<ref name="HAzkani-Covo" /> NUMTs can be also used to compare the rate of nonfunctional nuclear sequence evolution to that of functional mtDNA and determine the rate of evolution by the rate of mutation accumulation along NUMT sequences over time. The least selectively constrained regions are the segments with the most divergence from the mitochondrial sequence.<ref name="HAzkani-Covo" /><ref name="bensasson2001" /> One of the most promising applications of NUMT study is its use in the study of nuclear mutation.<ref name="bensasson2001" /> In [[metazoans]], NUMTs are considered non-functional. Therefore, nuclear mutations can be distinguished from mitochondrial changes and the study of nucleotide substitution, insertion, and deletion would be possible.{{Original research inline|date=March 2024}} The homology of paralogous NUMT sequences with mtDNA allows testing for local sequence effects on mutation.<ref name="bensasson2001" />

NUMTs offer an opportunity to study ancient diversity of mitochondrial lineages and to discover prehistoric interspecies hybridization. Ancient hybridization was first detected with NUMTs in bristletails,<ref name="BaldoL">{{cite journal|last1=Baldo|first1=L|last2=de Queiroz|first2=A.|last3=Hayashi|first3=C.Y.|last4 =Gatesy |first4=J.|title=Nuclear-mitochondrial sequences as witnesses of past interbreeding and population diversity in the jumping bristletail Mesomachilis.|journal=Mol Biol Evol|date=2011|volume=28|issue=1|pages=195–210|doi=10.1093/molbev/msq193|pmid=20667982|doi-access=free}}</ref> colobine monkeys,<ref name="WangB">{{cite journal|last1=Wang|first1=B|last2=Zhou|first2=X.|last3=Shi|first3=F.|title=Full-length Numt analysis provides evidence for hybridization between the Asian colobine genera Trachypithecus and Semnopithecus.|journal=Am J Primatol|date=2015|volume=77|issue=8|pages=901–910|doi=10.1002/ajp.22419|pmid=25903086|s2cid=205330921}}</ref> and most recently in a direct human ancestor. The hominid hybridization happened about the time of human/chimpanzee/gorilla separation.<ref name="PopadinK">{{cite bioRxiv|last1=Popadin|first1=K.|last2=Gunbin|first2=K.|last3=Peshkin|first3=L.|title=Mitochondrial pseudogenes suggest repeated inter-species hybridization in hominid evolution|date=2017|display-authors=etal|biorxiv=10.1101/134502}}</ref>

Another problem arose from the presence of NUMT in the genome associated with the hardship of concluding the exact number of mitochondrial insertions into the nDNA. Determining the exact number of NUMT pseudogenes for a species is difficult task for several reasons.<ref name="Gaziev" /> One reason that makes detection of NUMT sequences more difficult is the alteration of these sequences by mutation and deletion.<ref name="Hazkami-Covo" />  Two further substantial obstacles make recognition of NUMT very difficult: first, there is a lack of correlation between the proportion of noncoding nDNA and the number of NUMT inserts in the nuclear genome;<ref name="Gaziev" /> NUMT insertion could occur in the known or predicted coding region in introns and exons, rather than only in intergenic and intronic regions.<ref name="Tsuji" /><ref name="antunes1" />  Second, mitochondrial DNA integrated into animal nuclear genomes is primarily limited to animals with circular mitochondrial genomes without introns.<ref name="Song" />

[[File:Use of Sanger sequencing to locate NUMT insertion in the nuclear genome.PNG|thumb|Use of Sanger sequencing to locate NUMT insertion in the nuclear genome]]

These difficulties in detecting the presence of NUMT can be problematic. Translocated mitochondrial sequences in the nuclear genome have the potential to be amplified in addition to, or even instead of, the authentic target mtDNA sequence that can confound population genetic and phylogenetic analyses since mtDNA has been widely used for population mapping, evolutionary and phylogenic studies, species identification by DNA barcode, diagnosis of various pathologies, and forensic medicine.<ref name="Gaziev" /><ref name="Hlaing" /> This simultaneous amplification of NUMT with free extrachromosomal mtDNA prevents the exact number of NUMT fragments in the genome of different organisms from being determined,<ref name="Hlaing" /> especially those in which extended translocation of mtDNA fragments occur.<ref name="Gaziev" /> For instance, a large NUMT pseudogene was found on chromosome 1, while more recent analysis of the same sequence concluded that sperm mtDNA has mutations that cause low sperm mobility.<ref name="Gaziev" /><ref>{{cite journal|last1=Thangaraj|first1=K.|title=Sperm mitochondrial mutations as a cause of low sperm motility|journal=J. Androl.|date=2003|volume=24|issue=3|pages=388⎯392|doi=10.1002/j.1939-4640.2003.tb02687.x|pmid=12721215|doi-access=free}}</ref> Another example would be the recent report describing a heteroplasmic mtDNA molecule containing five linked [[missense mutation]]s dispersed over the contiguous mtDNA [[Cytochrome Oxidase Subunit 1|''{{not a typo|CO1}}'']] and [[Cytochrome c oxidase subunit II|''{{not a typo|CO2}}'']] genes in [[Alzheimer's disease]] patients;<ref name="Wallace1997">{{cite journal|last1=Wallace|first1=D. C.|title=Ancient MtDNA Sequences in the Human Nuclear Genome: A Potential Source of Errors in Identifying Pathogenic Mutations|journal=Proceedings of the National Academy of Sciences|date=1997|volume=94|issue=26|pages=14900–4905|display-authors=etal|doi=10.1073/pnas.94.26.14900|pmid=9405711|pmc=25135|bibcode=1997PNAS...9414900W|doi-access=free}}</ref> however, studies using PCR, restriction endonuclease site variant assays, and phylogenic analysis proposed that the nuclear ''{{not a typo|CO1}}'' and ''{{not a typo|CO2}}'' sequences revealed that they diverged from modern human mtDNA early in hominid evolution about 770,000 years before and these preserved NUMTs could cause Alzheimer's disease.<ref name="Gaziev" /><ref name="Wallace1997" />  One of the possible ways of preventing from such erroneous result is an amplification and comparison of heterogeneous sequence, comprises both mtDNA and nDNA, with the obtained results from Sanger sequencing of purified and enriched mtDNA.<ref name="Hlaing" /><ref name="Ramos" />

==Detection==
Signs that a mitochondrial DNA sequence may be contaminated with one or more NUMT sequences include double peaks (heterozygotes), [[indel]]s, and premature [[stop codon]]s.<ref name="Cristiano2014">{{cite journal |last1=Cristiano |first1=Maykon Passos |last2=Cardoso |first2=Danon Clemes |last3=Fernandes-Salomão |first3=Tânia Maria |title=Could pseudogenes be widespread in ants? Evidence of numts in the leafcutter ant Acromyrmex striatus (Roger, 1863) (Formicidae: Attini) |journal=Comptes Rendus Biologies |date=February 2014 |volume=337 |issue=2 |pages=78–85 |doi=10.1016/j.crvi.2013.11.007|pmid=24581801 |url=http://www.locus.ufv.br/handle/123456789/18520 |url-access=subscription }}</ref>

==See also==
*[[Human mitochondrial genetics]]
*[[Mitochondrial DNA]]
*[[CoRR hypothesis]]

==References==
{{reflist}}
		 	
{{Use dmy dates|date=April 2017}}

[[Category:Mitochondrial genetics]]
[[Category:DNA]]