School of Biological Sciences, University of East Anglia, Norwich, England
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Abstract |
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Introduction |
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Hundreds of mitochondrial pseudogenes appear to reside in the human nuclear genome (Fukuda et al. 1985
). A similar estimate has been made for the migratory locust, Locusta migratoria (Gellissen and Michaelis 1987
). In the course of studying postglacial colonization events for the brown mountain grasshopper, Podisma pedestris, using mtDNA as a genetic marker, high copy numbers of mitochondrial pseudogenes were encountered. This finding complements earlier observations of Numts in the desert locust Schistocerca gregaria (Zhang and Hewitt 1996b
). As knowledge about Numts in insects is generally poor (Gellissen et al. 1983
; Sunnucks and Hales 1996
; Zhang and Hewitt 1996a
), we have carried out genomic in situ hybridization (Vaughan, Heslop-Harrison, and Hewitt 1999
) and PCR cloning studies of various acridid grasshoppers to investigate the genomic abundance, organization, taxonomic distribution, and evolutionary dynamics of these nuclear pseudogenes.
Presented here are the results of a comparative study of 87 different ND5-like Numt sequences taken from P. pedestris, its Italian relative, Italopodisma sp., a more distant Japanese grasshopper, Parapodisma mikado, and other acridid species of different subfamilies. We address the following questions: How abundant are these mitochondrial-like sequences in the nuclear genome? How different are they from one another? Could most of them be identical in sequence? How were they produced: by a single major event or through separate integrations? Other aspects revealed by these data, such as rate and patterns of Numt sequence evolution, differences among grasshopper populations in which Numts are held, and ancient population processes, will be discussed in detail elsewhere.
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Materials and Methods |
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Enrichment of mtDNA
The grasshoppers used in this study are relatively small, so not enough tissue was available from single individuals for a complete purification of mtDNA. DNA was enriched for mtDNA relative to total DNA using the protocol described in Zhang and Hewitt (1996b)
, which was adapted from Lansman et al. (1981
). This protocol was modified for Podisma and Italopodisma in that DNA was not purified by CsCl/ethidium bromide gradient ultracentrifugation, as DNA yields were prohibitively low.
To check whether the protocol had yielded DNA which was mostly mitochondrial, the ND5 region was amplified from the enriched mtDNA and from total DNA (from the same individual) and digested with AluI, Sau3AI, and DraI. Digestion with these enzymes of the PCR products amplified from total genomic DNA had previously revealed more than one type of PCR product. If after completion of mitochondrial enrichment, only one type of PCR product is observed after digestion with these enzymes, this suggests that the enrichment has been successful and the extra types of PCR product digest were nuclear. Restriction analysis showed the successful enrichment for mtDNA (fig. 1 ). The PCR product was also cloned, and several clones were sequenced. Only one type of sequence was common among the clones after enrichment (table 4 ).
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PCR Amplification
ND5 PCR (688 bp)
The primer N5-J-6579, designed in R. Harrison's laboratory for use on the flea and described by Simon et al. (1994
), was extended and adapted for use in Orthoptera in our laboratory. Its sequence is now based on L. migratoria, but the position implied in its name refers to the 3' base position in Drosophila yakuba as in the convention of Simon et al. (1994
): N5-J-6578 (Imelda) (5'-ACTCACCTCAACCAGAATCAA-3'). A second primer was designed for the reverse direction: N5-N-7225 (Ferdinand) (5'-ACTCATGCTTTATTTAAGGCTTTA-3'). These primers always amplified well together and matched the template DNA of S. gregaria, Chorthippus parallelus (the meadow grasshopper), P. pedestris, and Italopodisma sp., except for one mismatch for Ferdinand in Podisma and Italopodisma (5'-...CATTA-3').
Conditions when using BIOTAQ polymerase (Bioline) were as follows: 1 U of enzyme (in 50-µl reactions), 0.3 µM of each primer, 200 µM of each dNTP, and 1 x KCl buffer (Bioline). Cycling parameters were 94°C for 4 min; 35 cycles of 94°C for 40 s, 52°C for 1 min, and 72°C for 1 min 20 s; and 72°C for 7 min.
Conditions when using a higher fidelity polymerase, Pfu DNA polymerase (Stratagene), were as follows: 3.5 U of enzyme (in 50-µl reactions), 0.6 µM of each primer, 144 µM of each dNTP, and 1 x Pfu reaction buffer (Stratagene). Cycling parameters were the same as for the Taq PCR except with the annealing temperature at 50°C and 25 cycles.
COI PCR (324 bp)
The primers C1-J-1763 (UEA3) and C1-N-2087 (UEA4) (Lunt et al. 1996
; Zhang and Hewitt 1997
) were used. Conditions were as follows: 2 U of Taq DNA polymerase (Promega) in 50-µl reactions, 0.3 µM of each primer, 200 µM of each dNTP, 1 x reaction buffer (Promega), and 1 mM MgCl2. Cycling parameters were same as for the ND5 PCR with Taq except with the annealing temperature at 43°C.
Universal PCR
The modified M13 universal primer (5'-CGACGTTGTAAAACGAGGCCAGT-3') and the M13 reverse primer (extended to 5'-ACAGGAAACAGCTATGACCATGAT-3') were used to amplify the cloned ND5 insert from colonies dissolved in DNA-free water. Conditions and cycling parameters used were same as for the ND5 Taq PCR except with the annealing temperature at 55°C.
Cloning and Sequencing of the ND5 PCR Products
Taq PCR products were cloned using the pMOS Blue T-Vector cloning kit from Amersham according to the manufacturer's instructions. Pfu PCR products were ligated into SmaI (Gibco BRL) cut pUC18 using T4 DNA ligase (Gibco BRL), and Epicurian Coli XL1-Blue competent cells (Stratagene) were transformed according to the manufacturer's instructions with this recombinant DNA before plating on standard color selection plates. Colonies were picked into 100 µl DNA-free water, vortexed, heated to 95°C for 2 min, vortexed, then spun for 5 min at about 10,000 x g; 3 µl was used for the subsequent PCR.
The universal PCR products (amplified from single colonies) were purified using the Wizard PCR Purification Kit from Promega and then sequenced using the M13 primers described above. The Thermo Sequenase dye terminator cycle sequencing pre-mix kit (Amersham Life Science) was used for sequencing, and samples were run on an Applied Biosystems (ABI) 377 automated DNA sequencer.
For the two individuals whose Taq-amplified PCR products were sequenced (see table 3 SpA and Parapodisma), plasmid DNA was prepared using the QIAGEN Plasmid Midi Kit, and the purified double-stranded plasmid templates were sequenced using the AutoRead sequencing kit (Pharmacia) and run on an Automated Laser Fluorescent A.L.F. DNA sequencer (Pharmacia).
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Pairwise distances were estimated using the F84 model (Felsenstein 1984
). This measure allows for considerable base frequency bias and for a transition : transversion ratio bias, which may be expected when handling insect mtDNA sequences. Our sequences are very AT-rich. A neighbor-joining approach was used to construct a tree from these distances in order to illustrate approximately how diverged different ND5-like regions are from each other. Gaps were treated as missing data and distributed proportionally to unambiguous changes. Bootstrap values calculated were based on 1,000 replicates. The DNA regions included in the analysis may be evolving at different rates, and some are apparently under selective constraints, while others are not; thus, many of the assumptions made when estimating genetic distances are violated. Therefore, this tree may not represent a true phylogeny of the nuclear and mitochondrial sequences it includes.
Deduction of Mitochondrial DNA Sequences
Mitochondrial DNA was successfully enriched for one P. pedestris individual, two Italopodisma individuals and one S. gregaria individual. Amplifications of the ND5 region from the (unenriched) DNA of these individuals yielded more than one type of ND5-like sequence. In contrast, after the enrichment for mitochondrial DNA, only one type of ND5 sequence was abundant in the PCR product (fig. 1
and table 4
). Under this circumstance, and because no unusual traits were observed in the sequence (e.g., frameshift mutations, stop codons), this sequence was assumed to be the mitochondrial sequence in each of these individuals.
PCR products from the total DNA of eight ethanol-preserved grasshoppers (one P. mikado, one C. parallelus, and six P. pedestris individuals) were also cloned and sequenced. Because they were stored in ethanol and their mitochondria were therefore not well preserved (Dowling et al. 1996
), their mtDNA sequences could not be identified directly by mtDNA enrichment. However, for P. pedestris and P. mikado, the most likely mtDNA sequence candidates could be deduced. In general, a copy was assumed to be mitochondrial (or at least very recently mitochondrial) if it contained no frameshifts or stop codons, if there were several identical copies of it present within a grasshopper, and if there were no other likely candidates.
Determination of Separate Nuclear Integration Events
A pair of nuclear mitochondrial pseudogenes could have descended from the same mitochondrial immigrant; for example, they could be the result of an intragenomic (nuclear) duplication or amplification event. If this is the case, and if mitochondrial sequences do not retain their function after their nuclear integration (a reasonable assumption for animal Numts discussed in Brennicke et al. [1993
] and Gellissen and Michaelis [1987]
), then they will have diverged from each other under no selective constraints. The lack of selective constraints during nuclear sequence divergence should ensure that there is no significant bias in favor of the accumulation of changes at otherwise selectively constrained sites (e.g., at third positions of codons). If significant bias among codon positions is observed in the changes accumulated during the evolutionary divergence of two pseudogenes, this implies that the pseudogenes are descended from different (selectively constrained) mitochondrial immigrants. Therefore, if significant codon position bias is observed in the differences between two Numts, this implies that they originated from separate transfers from mitochondria to nucleus.
Each of the 61 nuclear mitochondrial pseudogenes shown in figure 2 was compared with every other ND5-like pseudogene (a total of 1,769 pairwise comparisons). The number of nucleotide differences between each pair were counted separately using MEGA, version 1.0 (Kumar, Tamura, and Nei 1993
), for the first, second, and third codon positions. For each pairwise comparison, a
2 test was performed to test if the pair of Numts showed significant codon position bias (df = 2, P < 0.05) in the differences between them. These pairwise comparisons generate a list of Numts which could not have descended from the same mitochondrial immigrants. This list can be used to infer the minimum number of separate integration events required to explain the data.
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Results |
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The possibility of the cross-contamination of samples was thoroughly controlled for, as was the possibility of somatic differences (Hadler, Daniel, and Pratt 1971
; Nielsen et al. 1994
; Liang 1996
; Hadler, Devadas and Mahalingam 1998
) in ND5-like regions (data not shown). The results from sequencing and restriction enzyme approaches are in close agreement (data not shown). In particular, the majority of sequences obtained were from PCR products which had been amplified with Pfu DNA polymerase, a high-fidelity enzyme. Controls were carried out to check the error rate, which was shown not to be significant (data not shown). It is therefore unlikely that the multiple different mtDNA-like regions observed within single individuals are artifacts of the techniques employed.
Extra ND5 Types Are Not Heteroplasmic mtDNAs
Nuclear and mitochondrial DNA were purified for S. gregaria (Zhang and Hewitt 1996b
). After this separation, many different ND5-like regions (many of which contained frameshift mutations) had been amplified from the purified nuclear DNA, and only one sequence (with no frameshift mutations) appeared to be amplified from the purified mtDNA. This suggests that the extra ND5-like sequences are nuclear in S. gregaria.
After enriching P. pedestris and Italopodisma DNA for mtDNA, the DNA amplified was mostly of one type, whereas multiple ND5-like regions had been amplified before enrichment (fig. 1 and table 4 ). This separation of the extra ND5-like sequences (successful for P. pedestris and two of the Italopodisma) implies that the extra ND5 types observed are not mitochondrial. Although heteroplasmy may occur in grasshoppers, it has not been detected here and therefore cannot explain the many extra ND5-like sequences reported in this study.
Although it is possible that these extra ND5-like sequences exist as extrachromosomal, episomal DNA (Sunnucks and Hales 1996
), several lines of evidence suggest they are of nuclear origin. The extra ND5-like copies are nuclear in S. gregaria, as are the extra 12S rRNA, D-loop, and tRNAIle regions observed by Zhang and Hewitt (1996b
). Fluorescent in situ hybridization (FISH) data have shown COI and control region-like sequences to map to the telomeres in S. gregaria, COI-like sequences are centromeric in C. parallelus, and preliminary results for Italopodisma suggest a dispersed distribution of numerous COI-like sequences in the nuclear genome (Vaughan, Heslop-Harrison, and Hewitt 1999
). There are thought to be hundreds of tRNALeu, 12S, and 16S rRNA-like Numts in L. migratoria (Orthoptera, Acrididae) (Gellissen and Michaelis 1987
). All of these studies suggest that nuclear locations for mitochondrial-like sequences are common in grasshoppers.
In addition, most of the extra ND5-like copies were lost after amplification of a large fragment of the mtDNA molecule by long-PCR (data not shown). Studies of nonmitochondrial ND5-like sequence evolution in P. pedestris and Italopodisma sp. confirm that the extra ND5-like regions are evolving in a manner that would be expected in the nucleus (data not shown). Some of the extra ND5-like regions appear to have escaped the mitochondria before P. pedestris diverged from Italopodisma sp. (fig. 2 ). This suggests that the extra ND5-like regions can persist in grasshoppers over long periods of evolutionary time, which is most likely if they are stably integrated into the nuclear genome.
Numts Observed in Different Acridid Species Are the Result of Independent Migrations to the Nucleus
It is clear from the distance tree (fig. 2
), that the nuclear ND5-like pseudogenes observed in acridid species from different acridid subfamilies (the Podisminae, the Cyrtacanthacridinae, and the Gomphocerinae) did not result from a single mitochondrial invasion of the nuclear genome in ancient acridid history. The Numts of S. gregaria (Cyrtacanthacridinae) and C. parallelus (Gomphocerinae) are distinct from those of the Podisminae and appear to have arisen relatively recently in the evolution of these species.
Independent Mitochondrial Origins of the Numts Observed Within Species and in Single Individuals
Many of the pseudogenes observed within single individuals displayed significant codon position bias (more changes at third and first than at second codon positions) when compared with each other (1,450 of the 1,769 pairwise comparisons). This implies that they are descended from significantly diverged mtDNA sequences, and therefore from separate mitochondrial migrations to the nucleus. At least 12 independent mitochondrion to nucleus transfer events are required to explain the codon position biases revealed through the pairwise comparisons of P. pedestris pseudogenes, two for Italopodisma, two for the three sequenced pseudogenes of P. mikado, three for S. gregaria, and the five different ND5-like Numts sequenced for C. parallelus originate from at least three significantly diverged mtDNAs.
If two pseudogenes have separate mitochondrial origins but the two mtDNAs they were descended from were similar, there will not be enough differences between them for any resulting codon position bias to be significant. In addition, if many nuclear mutations have accumulated in a Numt, they will swamp selectively constrained changes between it and other Numts. Therefore, the number of independent integration events has probably been underestimated.
A Nuclear Family of Pseudogenes in Italopodisma
There is a large group of Italopodisma pseudogenes which are closely related (fig. 3
). Examination of their aligned sequences reveals that these copies differ from each other only by small insertions, deletions (indels), and unique substitutions. These are not the result of polymerase error; the clones that were sequenced from Italopodisma are from a Pfu-amplified PCR product, and the Pfu error rate was empirically found to be too low to be significant for the purpose of this study (data not shown). There is no significant codon position bias among the pseudogenes belonging to this family; the pooled numbers of changes among the 30 different pseudogene sequences of this family are 12 at first codon positions, 19 at second codon positions, and 12 at third codon positions, and this does not significantly differ from a 1:1:1 ratio (2 test: df = 2, P = 0.3). Therefore, this family of nuclear pseudogenes is likely to have descended from a single mitochondrial immigrant which has been amplified since it escaped from the mitochondria.
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Discussion |
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This study demonstrates the use of a novel approach for distinguishing Numts with separate mitochondrial origins. This is done by examining the codon position bias in the differences revealed through pairwise comparisons of the sequences (where they show a reasonable degree of divergence). Such an approach could be used to establish the independent origins of other types of DNA which lose their functions upon nuclear incorporation ("dead-on-arrival sequences" as described in Graur, Shuali, and Li 1989
), e.g., processed pseudogenes or non-LTR retrotransposable elements.
The many different Numts observed in different grasshopper subfamilies appear to be the result mainly of horizontal transfer events from mitochondria to nuclei which have occurred since these grasshopper species diverged from each other. Similar observations of independent mitochondrial origins of nuclear pseudogenes among related species have been made for aphids of the Sitobion genus (Sunnucks and Hales 1996
) and for diving ducks of the Aythyini tribe (Sorenson and Fleischer 1996
).
Independent horizontal transfer events also explain much of the Numt diversity observed within individuals. There have been at least 12 separate integrations of mtDNA into the nuclear genomes of P. pedestris. This is the highest reported frequency of mitochondrial integration in a single species. Podisma pedestris individuals are not unusual among grasshoppers. Every individual of the other grasshopper species studied in depth (Italopodisma sp., P. mikado, C. parallelus, and S. gregaria) harbors Numts apparently arising from significantly different mitochondrial ancestors.
The work presented here suggests that more than one mechanism exists in grasshoppers for the generation of Numt sequences. The Italopodisma individuals studied show evidence of multiple horizontal transfer events from mitochondrion to nucleus; however, a family of almost identical Numts also exists (figs. 2 and 3 ). Given the patterns of nucleotide substitutions observed in these sequences, it is likely that this family of Numts was generated by amplification of a single type of mtDNA-like sequence. Such amplification events have been described for cats (Lopez et al. 1994
) and humans (Hu and Thilly 1995
). In cats, the amplification event is thought to have occurred prior to the nuclear integration of the mitochondrial-like DNA in question (Lopez et al. 1994
), while in humans, amplifications are thought to have occurred since nuclear integration (Hu and Thilly 1995
). Further work is needed to explain how and where the family of Numts reported here arose. At least in grasshoppers, more than one process appears to be involved in the generation of numerous mitochondrial-like sequences in the nuclear genome: intergenomic horizontal transfer (see above), which involves the migration of DNA from mitochondrion to nucleus and is responsible for the independence of mitochondrial origins among Numts, and posttransfer amplification, which is probably a nuclear event (or a cytoplasmic episomal event), responsible for the repetitive presence of some Numt sequences.
The ND5-like Numts observed in S. gregaria are only up to 1.4% diverged from the current mtDNA sequence, while in P. pedestris this divergence can be as high as 12.5% (table 3
and fig. 2
). Such heterogeneity has been noticed in other animal species, but never before in species which are this closely related (Zhang and Hewitt 1996a
). This suggests heterogeneity among these species in the frequency of Numt generation, possibly reflecting differences in the ability of different genomes to gain or lose Numts.
Grasshoppers are not unique in the persistence of mtDNA-like sequences in their nuclear genomes. Numts are common in other species groups, for example, in hominoids and Old World monkeys (Collura and Stewart 1995
; van der Kuyl et al. 1995
), in birds (Quinn and White 1987
; Arctander 1995
; Sorenson and Fleischer 1996
; Kidd and Friesen 1998
; K. Nielsen, personal communication; N. Harvey, personal communication), and in three species of Sitobion aphid (Sunnucks and Hales 1996
). However, the stable nuclear absorption of mtDNA sequences does not appear to be universal; no Numts have been reported in Plasmodium falciparum, Caenorhabditis elegans, or Drosophila, although these organisms are well studied (Blanchard and Schmidt 1996
). The occurrence of Numts is not phylogenetically continuous in felines (Lopez et al. 1994
), and they also appear to be absent from the nuclear genomes of aphid species not belonging to the genus Sitobion (Sunnucks and Hales 1996
).
At present, it is not known which factors ultimately determine whether Numts are common in a species. Interestingly, some of the organisms which lack Numts (e.g., the aforementioned P. falciparum, C. elegans, and Drosophila) have small nuclear genomes, while species in which many Numts have been found (e.g., the grasshoppers studied here) possess much larger nuclear genomes. When more data on the taxonomic distribution of Numts become available, we will be able to explore the possibility of a correlation between these two phenomena.
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Acknowledgements |
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Footnotes |
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1 Abbreviations: digestype, restriction digest type; Numt, nuclear mitochondrial DNA.
2 Keywords: Acrididae
numtDNA
gene amplification
heteroplasmy
transposition
DNA migration
nuclear copies
horizontal gene transfer
dead-on-arrival sequences
3 Address for correspondence and reprints: Godfrey M. Hewitt, School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom. E-mail: g.hewitt{at}uea.ac.uk
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