MBE Advance Access originally published online on October 20, 2006
Molecular Biology and Evolution 2007 24(1):13-18; doi:10.1093/molbev/msl149
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Letters |
A Comparative Analysis of numt Evolution in Human and Chimpanzee
* Department of Zoology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv 69978, Israel
Department of Biology and Biochemistry, University of Houston
E-mail: dgraur{at}uh.edu.
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Abstract |
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Mitochondrial DNA sequences are frequently transferred into the nuclear genome, giving rise to numts (nuclear DNA sequences of mitochondrial origin). So far, the evolutionary history of numts has largely been studied by using single genomes. Here, we present the first attempt to study numt evolution in a comparative manner by using a pairwise genomic alignment. The total number of numts was estimated to be 452 in human and 469 in chimpanzee. numts that were found in both genomes at identical loci were deemed to be orthologous; 391 numts (>80%) were classified as such. The preponderance of orthologous numts is due to the very short divergence time between the 2 hominoids. The rest of numts were deemed to be nonorthologous. Nonorthologous numts were subdivided into 1) ancestral numts that have lost an ortholog in one species through deletion (12 in human and 11 in chimpanzee), 2) new numts acquired by the insertion of a mitochondrial sequence after the divergence of the 2 species (34 in human and 46 in chimpanzee), and 3) paralogous numts created by the tandem duplication of a preexisting numt (2 in human). This approach also enabled us to reconstruct the numt repertoire in the common ancestor of humans and chimpanzees (409 numts). Our comparative approach is also useful in identifying the exact boundaries of numts.
Key Words: numts • comparative evolution • promiscuous DNA • human genome • chimpanzee genome • mitochondrial DNA • genome evolution • pseudogenes
Mitochondrial DNA sequences are frequently transferred into the nuclear genome, giving rise to numts (nuclear DNA sequences of mitochondrial origin, Lopez et al. 1994
). numts have been described in more than 80 species (Bensasson et al. 2001). For most species, the estimate of numt content and abundance is still incomplete. However, with fully sequenced genomes, it is possible to obtain an accurate estimate of numt abundance (Richly and Leister 2004). There is no correlation between the fraction of noncoding DNA and numt abundance (Richly and Leister 2004). The reason for the variation in numt abundance among genomes is not known. Conceptually, the differences might be due to 1) different rates of numt insertion, 2) different rates of numt deletion, and 3) different rates of numt postinsertional duplication.
All mammalian numt studied to date were found to be functionless, and it is thought that they became pseudogenized on arrival into the nucleus because of the differences between the nuclear and mitochondrial genetic codes (Gellissen and Michaelis 1987; Perna and Kocher 1996). In yeast, numts are transferred under natural conditions during the repair of double-strand breaks (Ricchetti et al. 1999), and it was suggested that this is the cause for the ongoing colonization of different genomes by numts. 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 (Ricchetti et al. 2004). Some of these numts are variable with respect to genomic presence or absence, indicating that they have only arisen recently in the human population. Transposition of numts into genes has also been associated with human diseases (Willett-Brozick et al. 2001; Turner et al. 2003; Goldin et al. 2004).
From human genome data, different estimates of the number of numts have been put forward in the literature (Mourier et al. 2001; Tourmen et al. 2002; Woischnik and Moraes 2002; Bensasson et al. 2003; Richly and Leister 2004). Additionally, phylogenetic methods have been suggested for dating the insertion of numts into the nuclear genome (Mourier et al. 2001; Woischnik and Moraes 2002). Initial results indicated a fairly rapid process of numt insertion, however, some studies ignored the possibility of postinsertional nuclear duplication (e.g., Bensasson et al. 2000) resulting in overestimation of numt insertion rates. Hazkani-Covo et al. (2003) suggested a methodology for dating the insertion of numts into the nuclear genome by using a single nuclear genome sequence and a mitochondrial phylogenetic tree. This methodology had the advantage of being able to detect numt duplication events. We discovered that the rate of numt insertion on the branch leading to humans was much lower than previously reported (Mourier et al. 2001; Woischnik and Moraes 2002). Most numts turned out to be paralogs of preexisting numts, rather than new insertions.
Two numts are defined as orthologous if they are derived from a speciation event, but as paralogous if they are derived from a duplication event. So far, the evolutionary history of numts has largely been studied by means of paralogous comparisons within single genomes (Mourier et al. 2001; Woischnik and Moraes 2002; Hazkani-Covo et al. 2003). The availability of closely related completely sequenced genomes has enabled us to use comparative methods to study directly orthologous numt evolution. We note that by using the methodology of Hazkani-Covo et al. (2003), the existence of orthologous numt in species other than humans was inferred indirectly. That inference, however, yielded a testable prediction. Thus, for example, a numt that was inferred to have been inserted in the common ancestor of human and chimpanzee should possess orthologs in both species. However, this prediction could be wrong if the mitochondrial phylogenetic tree is not the true tree. In addition, this methodology is only applicable to long numts that have sufficient phylogenetic signal. With 2 or more genomes, the presence of orthologous numts can be inferred directly, even when the numts are short.
In the following, we suggest a protocol based on genome alignment to estimate the number of numts in closely related species. We apply this approach to the genomes of human (Lander et al. 2001) and chimpanzee (Pan troglodytes; Mikkelsen et al. 2005), and use the alignment to identify evolutionary events that may have affected numt composition in each genome, as well as to reconstruct the numt makeup in the common ancestor of human and chimpanzee.
Because there are no hot spots for numt insertion (Zischler 2000), the presence of a numt at a particular locus in both genomes was taken to imply orthology (fig. 1). Nonorthologous numts that are present in only one genome are further classified into insertions, partial or total deletions, or tandem duplications (fig. 1). Each such event can take place in either lineage. Nonorthologous numts are identified by a gap in the alignment. The distinction between insertions and deletions is based on the fact that there exists no known mechanism for the precise excision of numts. Thus, if the gap coincides precisely with the boundaries of the numt, an insertion is inferred. If the gap is smaller or larger than the numt in the other genome, we infer the occurrence of a partial or total deletion, respectively. Tandem numt duplications are characterized by adjacent homologous numts and a gap coinciding perfectly with the boundaries of the homolog from the other species. The assumptions used for numt classifications here were also used in PCR-based numt recognition (e.g., Lopez et al. 1994; Zischler et al. 1998; Herrnstadt et al. 1999).
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Our analyses were based on genomic sequences and annotations from the University of California at Santa Cruz (Karolchik et al. 2004
) Genome Center. First, Blast was used to search each of the human and chimpanzee genomes for regions of similarity with conspecific mitochondrial sequences (fig. 2, frame 1). Closely spaced mitochondrial hits were concatenated (fig. 2, frame 2). The distinction between orthologous and nonorthologous numts (as described in fig. 1) was accomplished through a comparison of human and chimpanzee numt preliminary datasets. The comparison was based on the University of California-Santa Cruz genome alignment between human and chimpanzee. The analysis was performed in a reciprocal manner: comparing the human genome to the chimpanzee genome and comparing the chimpanzee genome to the human genome. For a detailed description of the methodology, see Supplementary Material online.
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We found a similar number of numts in both genomes: 452 numts in human and 469 numts in chimpanzee (table 1). The total number of numts in the 2 genomes was found to be similar to previous estimates in the literature. Unsurprisingly, because of the short time that has passed since the divergence of the 2 hominoids, 391 numts (87% in human and 84% in chimpanzee) were classified as orthologous, that is, were inserted into the nuclear genome before the divergence between the 2 lineages (table S1 in Supplementary Material online).
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We identified 46 previously undescribed postspeciation numts in the chimpanzee. These ranged in size between 37 and 3,076 bp. In addition, we identified 34 numts in human. Our study, thus, increases the number of known human-specific numts (Ricchetti et al. 2004
) by 26%, and identifies the shortest (29 vs. 47 bp) and the longest (5,219 vs. 1,323 bp) new numts. Human and chimpanzee postspeciation numts that were found in this study are listed in table 2. The common ancestor of human and chimpanzee is estimated to have lived about 6 Myr ago (Goodman et al. 1998). Thus, the average rates of numt insertion are 5.7 insertions per 1 Myr in human and 7.7 numt insertions per 1 Myr in chimpanzee. The difference is not statistically significant (P < 0.179).
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From among the postspeciation numts, only 2 cases of tandem duplication were found (both in the human genome). In the first case, an internal segment of 30 bp within a numt located on chromosome 10 was duplicated once. The second case, in chromosome 12, includes 18 tandem duplications of a 47-bp sequence (fig. 3).
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The number of events in which numts were deleted from the genome is fairly similar between the 2 species. There are 12 deletion events in human, of which 11 are total deletions and 1 is a partial one. In chimpanzee, there are 11 deletion events, of which 7 are total deletions and 4 are partial. As far as the total deletions are concerned, one can distinguish between 2 separate groups: most of the numts seem to have been deleted from the genome as part of a much larger segment. However, in a few cases, the numt deletion included only a limited flanking region.
The number of nonorthologous numts is not large enough to be able to detect differences in numt evolutionary dynamics (insertion, deletion, or tandem duplication) between the 2 lineages. Still, we are now able to reconstruct the numts constitution in the common ancestor of the 2 hominoids. The number of numts in the common ancestor of human and chimpanzee is estimated at 409. This number includes 391 numts that are still found in the 2 genomes, and a total of 18 numts that were lost from 1 of the 2 genomes. Given the very low rate of numt deletion, the possibility that a numt has been lost in both genomes seems negligible.
We suggest that in comparison to single genome analyses, our methodology resulted not only in a more accurate estimate of the number of numts but also in a more precise identification of their boundaries. First, this protocol distinguishes between orthologous and nonorthologous numts. Second, by using genome alignment, we identified orthologous numts that escaped detection by the usual Blasting of mitochondrial sequences against the nuclear genome. In 145 out of 391 cases, numts were identified in only one of the genomes when the Blast analysis was used. However, in the majority of cases, alignment of those numts to the corresponding fragment in the second genome revealed a cryptic or quasi-cryptic ortholog. In 15 cases, the existence of orthologous numts in chimpanzee was inferred on the basis of a small stretch of Ns similar in size to the human numt in the homologous position. Finally, our protocol enables a more precise identification of the genomic coordinates of numts. The comparative method allows concatenation of fragments that may otherwise be identified as independent numts.
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Supplementary Material |
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TOPAbstractSupplementary MaterialAcknowledgementsReferences |
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Supplementary data and tables are available at Molecular Biology and Evolution online (http://www.mbe.oxfordjournals.org/).
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Acknowledgements |
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TOPAbstractSupplementary MaterialAcknowledgementsReferences |
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We thank Shay Covo and Tal Dagan for their help. This work was supported in part by a grant (DBI-0543342) from the National Science Foundation.
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Footnotes |
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1 Present address: National Evolutionary Synthesis Center, Durham, North Carolina, USA
William Martin, Associate Editor
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References |
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TOPAbstractSupplementary MaterialAcknowledgementsReferences |
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