Department of Biology, University of Crete, Crete, Greece
Institute of Marine Biology of Crete, Crete, Greece
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Abstract |
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Introduction |
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Other observations are, however, suggestive of the possibility of recombination in animal mtDNA. Thyagarajan, Padua, and Campbell (1996)
have demonstrated that human mitochondria contain the enzymes necessary for homologous recombination. Fusion of mitochondria is well demonstrated in Drosophila (Yaffe 1999
). Nonhomologous recombination was proposed as the mechanism responsible for variation in the number of tandemly repeated motifs (variant number of tandem repeats [VNTR] polymorphism) observed in noncoding regions of mtDNA in a number of animal species (such as Drosophila [Solignac, Monnerot, and Mounolou 1986
], scallops [Snyder et al. 1987
], crickets [Rand and Harrison 1989
], and sturgeons [Buroker et al. 1990
]). Lunt and Hyman (1997)
have indeed provided direct evidence of this type of recombination in the nematode Meloidogyne javanica. Two population studies have also raised the possibility of recombination in primate mtDNA. One of these was based on a high frequency of homoplasies (Eyre-Walker, Smith, and Maynard Smith 1999
), and the other was based on a negative correlation between linkage disequilibrium and physical distance along the molecule of mtDNA variants (Awadalla, Eyre-Walker, and Maynard Smith 1999
). These studies have, however, been criticized on several grounds (Arctander 1999
; Merriweather and Kaestle 1999
; Kivisild et al. 2000
). Moreover, two more recent studies failed to produce evidence for recombination in human mtDNA (Ingman et al. 2000
; Elson et al. 2001)
. Here, we present direct evidence of homologous recombination in the mussel Mytilus galloprovincialis.
Mussels of the families Mytilidae (sea mussels) and Unionidae (freshwater mussels) possess two mtDNA lineages, one transmitted through the egg (the F lineage) and one transmitted through the sperm (the M lineage) (Skibinski, Gallagher, and Beynon 1994a, 1994b
; Zouros et al. 1994a, 1994b
; Liu, Mitton, and Wu 1996
). Sperm mtDNA is delivered into the oocyte and eliminated within the first 24 h after fertilization in females (Sutherland et al. 1998
) but is retained in males which develop into "homoplasmic mosaics," with their gonads dominated by the M molecule and their somatic tissues dominated by the F molecule (Garido-Ramos et al. 1998
). The phenomenon has become known as doubly uniparental inheritance (DUI; Zouros et al. 1994a
). M and F DNA sequences normally differ by >20% (Hoeh et al. 1997
). This difference is considered prohibitive for homologous recombination because it necessitates a high activity of mismatch repair, which in turn suppresses homologous recombination (Rayssiguier, Thayler, and Radman 1989
; Selva et al. 1995
; de Wind et al. 1995
). However, occasionally F genomes invade the M transmission route (Hoeh et al. 1997
). This reversal of roles results in newly established M molecules (for which we here use the symbol Mf) whose sequences are initially similar to F but diverge rapidly owing to the high rate of mutation accumulation that characterizes male-transmitted lineages (Stewart et al. 1996
). Males from natural populations that inherit an Mf from the father and an F from the mother are heteroplasmic for molecules that differ by about 4% (Hoeh et al. 1997
), an amount not large enough to prevent homologous recombination and yet large enough to allow its detection if it has occurred. Our demonstration of homologous recombination was performed using this type of male.
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Materials and Methods |
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In contrast to gonadal tissue, which produced complex restriction profiles, somatic tissues always produced simple patterns, compatible with the presence of a single F molecule. As a result, the PCR product from the DNA of somatic tissues was directly sequenced. The resulting chromatograms were indeed consistent with a single molecule. An additional check of somatic tissue homoplasmy was made by examining a large number of clones from somatic DNA of individual 68, which contained the largest number of different sequences in the gonadal tissue (table 1
). It is known that somatic tissues of males contain predominantly the maternal genome, whereas the gonad contains both the paternal and the maternal genomes (Garido-Ramos et al. 1998
). This allows the identification of the maternal genome as the one found in somatic tissues. No such identification is possible for the paternal genome (or genomes) when more than two molecules are recovered from the gonad and when the possibility of recombination exists between maternal and paternal genomes.
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The PCR assay for verification of the presence of recombinant molecules in extracts from gonadal tissues (see Results) was performed using primers RECF (5'-GCT TTA AGG CCC TCA-3') and RECR (5'-GCC TCA AAC CCA AAG-3'), an annealing temperature of 48°C, and a MgCl2 concentration of 1.5 mM.
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Results |
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In phylad 5, sequences 12 and 3 can be seen as results of simple recombination exchange between sequences 5 and 2. For sequence 12, the beginning of the segment that is derived from phylad 2 is between the diagnostic sites 519 and 639, and the end is between sites 663 and 672; thus, the recombinant fragment has a length between 24 and 153 nt. The substitution at site 171 of this sequence can be either a PCR error or a homoplasy (given that it also occurs in phylad 2). For sequence 3, the beginning of recombination is between sites 507 and 516, and the recombinant fragment apparently extends beyond the cloned region.
In phylad 2 we may recognize three subgroups of sequences. One consists of sequence 2 and sequences 4, 13, and 8, all of which can be derived from simple recombination exchanges between sequences 2 and 5. The second subgroup consists of sequences 9 and 10. Sequence 9 differs from sequence 2 at seven sites (sites 42, 54, 324, 375, 418, 467, and 672) and must represent a mutational derivative of sequence 2. Sequence 10 shares the same differences as sequence 9 from sequence 2 but, in addition, contains a stretch of sequence 5 and therefore can be seen as a recombination result between sequences 5 and 9. The third subgroup consists of sequences 6 and 1, which share six nucleotide differences from sequence 2 (at sites 111, 342, 366, 467, 672, and 681). In total, after ignoring single-nucleotide differences, we are left with six unambiguous recombinant sequences, of which five (sequences 3, 12, 4, 13, and 8) are products of recombination between sequences 2 and 5 and one (sequence 10) is a product of recombination between sequences 9 and 5. For sequences for which both ends of the recombinant fragment lie within the sequenced region, the length of the fragment varies from 24 to 255 bp. In sequences 4 and 3, the length may be longer (fig. 3 ).
Amino acid substitutions have been observed in 3 of the 41 variable sites (sites 25, 142, and 467; fig. 3 ). The singleton substitution at site 142 in sequence 11 is, as noted, most likely the result of PCR error. At site 25, all sequences of phylad 2 have leucine and all sequences of phylad 5 have phenylalanine. At site 467, all sequences of phylad 5 and sequences 9, 10, 6, and 1 of phylad 2 have methionine, while all other sequences have threonine. The important observation is that both of these sites lie in regions that have not recombined. As a result, none of the recombination exchanges we observed resulted in any amino acid replacement. At present, we cannot say whether this is simply an accidental observation or that products of recombination with amino acid changes do not persist.
Elimination of the Possibility of Artifactual PCR Recombination
A previous study (Paabo, Irwin, and Wilson 1990
) has shown that when the template DNA of the PCR reaction contains a mixture of DNA molecules, Taq DNA polymerase may "jump" from one molecule to another, thus producing hybrid products. In our protocol, the first PCR reaction was carried out on DNA extracts from gonad tissue, and the products were subsequently cloned and sequenced. Theoretically, there is a possibility that in the presence of more than one mtDNA molecule in the original DNA extract, PCR jumping could produce recombinant molecules. The original study by Paabo, Irwin, and Wilson (1990)
was designed to facilitate PCR jumping. The source DNA contained highly homologous sequences digested with restriction enzymes to produce fragments with single-strand protruding ends. These conditions did not apply to our DNA source. Nevertheless, we performed two experiments to eliminate the possibility that the recombinant sequences we observed were caused by PCR jumping.
The first experiment consisted of mixing DNA from two clones of known sequence, using the mixture as the PCR template, cloning the PCR products, and assaying the cloned sequences by restriction analysis following steps 1, 3, and 4 from Materials and Methods. In such an experiment, any restriction pattern other than that of the two source sequences would be due to PCR error. This experiment was performed twice. In the first experiment, we used a pair of clones, one containing sequence 2 and one containing sequence 5. These were the sequences which upon recombination would have produced sequences 4, 13, 8, 3, and 12 in our experimental animals. Forty clones from the PCR product were digested with RsaI. Fifteen clones produced the restriction pattern of sequence 2, and 25 produced the pattern of sequence 5. In the second experiment, we used a pair of clones, one containing sequence 9 and one containing sequence 5, the sequences which upon recombination would have produced sequence 10. Thirty-seven clones were assayed as above, of which 27 had the restriction pattern of sequence 9, 9 had the pattern of sequence 5, and 1 had a novel restriction pattern. The clone with the novel restriction pattern was sequenced and found to differ from sequence 5 by a single substitution: it had C in position 443 instead of T. Thus, the experiment failed to produce recombinant sequences by PCR jumping but demonstrated that PCR errors of single-nucleotide substitution occur with low frequency.
The purpose of the second experiment was to recover the recombinant sequences we observed from the original DNA extracts. The rationale was that if we designed a PCR reaction which would specifically amplify a recombinant sequence, the sequence would be recovered if it was present in the original extract (and thus in the animal itself), but it would not be recovered if in the original experiment it was produced by PCR jumping. We used the primers RECF (5'-GCT TTA AGG CCC TCA-3') and RECR (5'-GCC TCA AAC CCA AAG-3'), which corresponded to sites 130144 and 516530 of figure 2 , respectively. The first primer anneals on sequence 5, and the second anneals on sequence 2. Before they were used on the source DNA, the primers were used on a mixture of two clones, one containing sequence 2 and one containing sequence 5. The same was done with another pair of clones, one containing sequence 5 and one containing sequence 9. In both cases, we failed to produce a PCR product. The primers were also tried against a clone containing sequence 8 and against a clone containing sequence 13 (both of which consisted of recombinant sequences) and produced the expected PCR product. After these pilot trials, the primers were used directly on the DNA that was extracted from the gonads of individuals 59 and 35. The PCR product from animal 59 was sequenced and found to be identical to the recombinant sequence 13. The product from animal 35 was identical to the recombinant sequence 8 except at position 411, where the new product had T instead C, which must be a PCR point error.
How Recent Is the Recombination?
How recent might the recombination events that generated the observed sequences be? This question is important because different sequences found in an individual may be the results of recombination that happened in the individual itself, or they can be parts of a long-standing polymorphism generated by past recombination events. Four individuals (individuals 20, 59, 35, and 68) carry recombinant sequences and can be used to examine this question. The other individuals are either homoplasmic (individuals 1, 12, and 13) or heteroplasmic (individuals 23, 61, and 81) for the two common sequences (sequences 2 and 5) and thus are assumed to carry no recombinant sequences (table 1
).
In individual 68, sequence 10 can be seen as a recombination product between sequences 9 and 5 (fig. 4
), both of which were found in this individual (as mentioned earlier, the difference at position 483 is either a PCR error or a postrecombination mutation). Given the rarity of sequence 9 in the population as a whole (Ladoukakis 1998
), its presence in the same individual with its recombination product implies a recent recombination event. In individual 20, sequences 3 and 4 are products of recombination between sequences 2 and 5 (fig. 4
). Sequence 2 was recovered in individual 20, but sequence 5 was not. Unlike nuclear recombination, there is no cellular mechanism that would prevent loss upon cytokinesis of one or more mtDNA sequences. Indeed, loss of either a parental or a recombinant sequence is more probable than retention of the full complement of sequences (Koehler et al. 1991
). Thus, sequences 3 and 4 are most likely products of a recombination event between sequences 2 and 5 that happened either in individual 20 itself or in a recent ancestral individual from which they were cotransmitted to individual 20. This transmission is unlikely to have occurred through the egg because these sequences were not found in the somatic tissues. Transmission of more than one type of mtDNA genome through the sperm is a possibility for which we have no evidence one way or the other. Whatever the case, there is evidence from two of the four informative animals that the events of recombination are recent and may in fact have occurred in the individuals in which they were observed. No such information can be extracted from animals 35 and 59, but this is compatible with stochastic loss of mitotypes.
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Discussion |
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We performed two experiments in order to eliminate the possibility that the recombination we observed was an artifact of the PCR reaction. Paabo, Irwin, and Wilson (1990)
have demonstrated that under certain conditions, Taq DNA polymerase may move from one template molecule to another, with the result that the PCR product may contain stretches of DNA from two different molecules. In the first experiment, the PCR polymerase was offered two types of DNA molecules, but we failed to detect any hybrid molecule among the products. The second experiment was designed to recover the observed recombinant sequences from the original DNA extract. The specific primers that were used for this experiment failed to produce a product when used against a mixture of sequences that could produce the recombinants by PCR jumping. When the same primers were used on the original DNA source, they produced the expected recombinant sequences. We conclude that the recombinant sequences we observed were present in the gonads of the individuals and cannot be attributed to PCR error.
The demonstration of recombination in mussel mtDNA raises several questions about the mussel mtDNA system itself and about animal mtDNA in general. One question is how common homologous recombination might be in mussel mtDNA. Our results suggest that it may be very common. Of the 24 sequences we obtained from the gonads (table 1 ), 13 were different, and of these, 6 were recombinant. Unless this region happened to be a recombination hot spot, we have to conclude that recombinant fragments would possibly be found in all molecules if longer stretches of DNA were examined. Thus, we have to assume that in the male gonad of the mussel, homologous mtDNA recombination is common, and it may be limited only by the degree of sequence divergence among the molecules that are contributed by the egg and the sperm.
Another question is to what extent do the results from the mussel mtDNA apply to the animal mtDNA as a whole? MtDNA transmission in mussels, now known as DUI, differs from that of standard maternal inheritance in several important ways (see Saavedra, Reyero, and Zouros 1997
). For the question at hand, the issue is whether the homologous recombination we observed was an integral part of the DUI mechanism or a consequence. Whereas we have at present no evidence for the former, we can cite two arguments in favor of the latter. One is the claim for homologous recombination in primate mtDNA from population data (Awadalla, Eyre-Walker, and Maynard Smith 1999
; Eyre-Walker, Smith, and Maynard Smith 1999
). Even though these studies have been criticized (Arctander 1999
; Merriweather and Kaestle 1999
; Kivisild et al. 2000
) and were not corroborated by two more recent studies (Ingman et al. 2000
; Elson et al. 2001)
, they remain suggestive of the possibility of rare recombination in primate mtDNA. The second argument arises from the demonstration that mammalian mitochondria house the enzyme apparatus that is necessary for recombination (Thyagarajan, Padua, and Campbell 1996
). Rather than assume that this apparatus is somehow inoperative in the animal mitochondria, it would be more inductive to further research on the issue of animal mtDNA recombination if one assumes that it is active and that the success in detecting recombination might simply be proportional to the degree of differentiation of the recombining genomes. In the face of rare heteroplasmy or even relatively common heteroplasmy for molecules with low degrees of molecular divergence, recombination will be difficult to detect either in present or in historical time. Transmission of paternal mtDNA has been reported in a number of species that cover a wide spectrum of the animal kingdom (Drosophila [Kondo et al.1990
], mice [Gyllensten et al. 1991
; Kaneda et al. 1995
; Shitara et al. 1998
], and anchovies [Magoulas and Zouros 1993
]). For mice, the "leakage" of paternal mtDNA was estimated at about 10-4 of an individual's mtDNA pool. These observations suggest that incidental paternal mtDNA transmission may be the rule in animals, despite the presence of mechanisms for sperm mtDNA elimination in the fertilized ovum (Shitara et al. 1998
; Sutovsky et al. 1999
). In this study, the degree of differentiation between a genuine F genome inherited from the mother and an F-like genome inherited from the father has made the detection of recombination relatively easy.
If homologous recombination occurs in animal mtDNA, it will have an important effect on our understanding of mtDNA mutation and repair mechanisms and rates of mutation accumulation. Homologous recombination is essential for DNA repair in yeast (Ling et al. 1995
) and is expected to play a similar role in animal mtDNA (Thyagarajan, Padua, and Campbell 1996
; Howell 1997
). Recombination errors may lead to unequal crossing over and deletions between short direct repeats of the type associated with mitochondrial diseases (Holt, Harding, and Morgan-Hughes 1988
). It must be emphasized that the implications of homologous recombination of mtDNA for mitochondrial disease, of which there is a growing list (Chinnery and Turnbull 1999
), aging (Ozawa 1999
), and certain methods of cloning (Evans et al. 1999
; Steinborn et al. 2000
) do not depend on initial heteroplasmy or on whether or not it generates new haplotypes in the population.
The implications are equally serious for the use of mtDNA in studies of phylogeny, phylogeography, and dating where the genome is assumed to be clonally transmitted. Recent simulation studies by Schierup and Hein (2000a, 2000b)
suggest that assuming no recombination when recombination does occur leads to longer basal branches and shorter terminal branches than the real situation might and also to apparent violation of the molecular clock. To this we can suggest two other complications that could be of special importance in the reconstruction of phylogenies of closely related but reproductively isolated taxa. One is the possibility of concerted evolution in mtDNA. Concerted evolution was suggested in the A/T-rich region of Drosophila (Solignac, Monnerot, and Mounolou 1986
) but is now a possibility for the whole mitochondrial genome. The second complication may result from variation (either among individuals or among populations) in the recombinational activity of the mitochondrion, which must be controlled by nuclear genes. All of these will have the effect of adding a heavy layer of noise around the phylogenetic signal. At the limit, mtDNA would be reduced to just another molecule with anastomosing lineages (Hey 2000)
.
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Conclusions |
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Acknowledgements |
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Footnotes |
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1 Keywords: mtDNA recombination
doubly uniparental inheritance
Mytilus
COIII
2 Address for correspondence and reprints: Eleftherios Zouros, Institute of Marine Biology of Crete, P.O. Box 2214, 71003 Iraklio Crete, Greece. E-mail: zouros{at}imbc.gr
.
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