Emmanuel D. Ladoukakis

Animal mitochondrial DNA recombination revisited

Exchange of homologous sequences between mitochondrial DNA (mtDNA) molecules is thought to be absent in animals, primarily because of a failure to observe clear cases of recombinant haplotypes in natural populations. However, whether mtDNA recombination occurs is a different issue from whether it produces new haplotypes. A requirement for the latter is heteroplasmy – the presence of more than one type of mtDNA in an individual, which is rare in animals. In male mussels, in which heteroplasmy is the rule, recombination is common, arguing against an innate impediment to mtDNA recombination in animals. In addition, recent biochemical studies suggest that recombination is an indispensable part of the mtDNA replication and repair machinery and that most animal genomes have the necessary enzymes for mtDNA recombination. When strict maternal mtDNA transmission is compromised, recombinant haplotypes can be generated and eventually become fixed. Although the pervasiveness of mtDNA recombination in animals is unknown, its presence could have important consequences for phylogenetic studies of closely related taxa (e.g. leading to incorrect phylogenetic inferences and incorrect rejection of the molecular clock) and human mtDNA-associated diseases.

Cryptic Variation in the Human Mutation Rate

The mutation rate is known to vary between adjacent sites within the human genome as a consequence of context, the most well-studied example being the influence of CpG dinucelotides. We investigated whether there is additional variation by testing whether there is an excess of sites at which both humans and chimpanzees have a single-nucleotide polymorphism (SNP). We found a highly significant excess of such sites, and we demonstrated that this excess is not due to neighbouring nucleotide effects, ancestral polymorphism, or natural selection. We therefore infer that there is cryptic variation in the mutation rate. However, although this variation in the mutation rate is not associated with the adjacent nucleotides, we show that there are highly nonrandom patterns of nucleotides that extend ∼80 base pairs on either side of sites with coincident SNPs, suggesting that there are extensive and complex context effects. Finally, we estimate the level of variation needed to produce the excess of coincident SNPs and show that there is a similar, or higher, level of variation in the mutation rate associated with this cryptic process than there is associated with adjacent nucleotides, including the CpG effect. We conclude that there is substantial variation in the mutation that has, until now, been hidden from view.

Mitonuclear interactions: evolutionary consequences over multiple biological scales

Fundamental biological processes hinge on coordinated interactions between genes spanning two obligate genomes—mitochondrial and nuclear. These interactions are key to complex life, and allelic variation that accumulates and persists at the loci embroiled in such intergenomic interactions should therefore be subjected to intense selection to maintain integrity of the mitochondrial electron transport system. Here, we compile evidence that suggests that mitochondrial–nuclear (mitonuclear) allelic interactions are evolutionarily significant modulators of the expression of key health-related and life-history phenotypes, across several biological scales—within species (intra- and interpopulational) and between species. We then introduce a new frontier for the study of mitonuclear interactions—those that occur within individuals, and are fuelled by the mtDNA heteroplasmy and the existence of nuclear-encoded mitochondrial gene duplicates and isoforms. Empirical evidence supports the idea of high-resolution tissue- and environment-specific modulation of intraindividual mitonuclear interactions. Predicting the penetrance, severity and expression patterns of mtDNA-induced mitochondrial diseases remains a conundrum. We contend that a deeper understanding of the dynamics and ramifications of mitonuclear interactions, across all biological levels, will provide key insights that tangibly advance our understanding, not only of core evolutionary processes, but also of the complex genetics underlying human mitochondrial disease.

Mitochondrial DNA variation in a species with two mitochondrial genomes: the case of Mytilus galloprovincialis from the Atlantic, the Mediterranean and the Black Sea.

We have examined mitochondrial DNA (mtDNA) variation in samples of the mussel Mytilus galloprovincialis from the Black Sea, the Mediterranean and the Spanish Atlantic coast by scoring for presence or absence of cleavage at 20 restriction sites of a fragment of the COIII gene and at four restriction sites of the 16S RNA gene. This species contains two types of mtDNA genomes, one that is transmitted maternally (the F type) and one that is transmitted paternally (the M type). The M genome evolves at a higher rate than the F genome. Normally, females are homoplasmic for an F type and males are heteroplasmic for an F and an M type. Occasionally molecules from the F lineage invade the paternal transmission route, resulting in males that carry two F‐type mtDNA genomes. These features of the mussel mtDNA system give rise to a new set of questions when using mtDNA variation in population studies and phylogeny. We show here that the two mtDNA types provide different information with regard to amounts of variation and genetic distances among populations. The F genome exhibits higher degrees of diversity within populations, while the M genome produces higher degrees of differentiation among populations. There is a strong differentiation between the Atlantic and the Black Sea. The Mediterranean samples have intermediate haplotype frequencies, yet are much closer to the Black Sea than to the Atlantic. We conclude that in this species gene flow among the three Seas is restricted and not enough to erase the combined effect of mutation and random drift. In one sample, that from the Black Sea, the majority of males did not contain an M mtDNA type. This suggests that a molecule of the maternal lineage has recently invaded the paternal route and has increased its frequency in the population to the point that the present pool of paternally transmitted mtDNA molecules is highly heterogeneous and cannot be used to read the population’s history. This liability of the paternal route means that in species with doubly uniparental inheritance, the maternal lineage provides more reliable information for population and phylogenetic studies.

Hundreds of putatively functional small open reading frames in Drosophila

BackgroundThe relationship between DNA sequence and encoded information is still an unsolved puzzle. The number of protein-coding genes in higher eukaryotes identified by genome projects is lower than was expected, while a considerable amount of putatively non-coding transcription has been detected. Functional small open reading frames (smORFs) are known to exist in several organisms. However, coding sequence detection methods are biased against detecting such very short open reading frames. Thus, a substantial number of non-canonical coding regions encoding short peptides might await characterization.ResultsUsing bio-informatics methods, we have searched for smORFs of less than 100 amino acids in the putatively non-coding euchromatic DNA of Drosophila melanogaster, and initially identified nearly 600,000 of them. We have studied the pattern of conservation of these smORFs as coding entities between D. melanogaster and Drosophila pseudoobscura, their presence in syntenic and in transcribed regions of the genome, and their ratio of conservative versus non-conservative nucleotide changes. For negative controls, we compared the results with those obtained using random short sequences, while a positive control was provided by smORFs validated by proteomics data.ConclusionsThe combination of these analyses led us to postulate the existence of at least 401 functional smORFs in Drosophila, with the possibility that as many as 4,561 such functional smORFs may exist.

A Phage Tail-Derived Element with Wide Distribution among Both Prokaryotic Domains: A Comparative Genomic and Phylogenetic Study

Prophage sequences became an integral part of bacterial genomes as a consequence of coevolution, encoding fitness or virulence factors. Such roles have been attributed to phage-derived elements identified in several Gram-negative species: The type VI secretion system (T6SS), the R- and F-type pyocins, and the newly discovered Serratia entomophila antifeeding prophage (Afp), and the Photorhabdus luminescens virulence cassette (PVC). In this study, we provide evidence that remarkably conserved gene clusters, homologous to Afp/PVC, are not restricted to Gram-negative bacteria but are widespread throughout all prokaryotes including the Archaea. Even though they are phylogenetically closer to pyocins, they share key characteristics in common with the T6SS, such as the use of a chaperon-type AAA+ ATPase and the lack of a host cell lysis mechanism. We thus suggest that Afp/PVC-like elements could be classified as phage-like-protein-translocation structures (PLTSs) rather than as pyocins. The reconstruction of phylogeny and the conserved gene content suggest that the diversification of prophage sequences to PLTS occurred in bacteria early in evolution and only once, but PLTS clusters have been horizontally transferred to some of the bacterial lineages and to the Archaea. The adaptation of this element in such a wide host range is suggestive of its versatile use in prokaryotes.

Homologous Recombination between Highly Diverged Mitochondrial Sequences: Examples from Maternally and Paternally Transmitted Genomes

Homologous recombination is restricted to sequences of low divergence. This is attributed to the mismatch repairing system (MMR), which does not allow recombination between sequences that are highly divergent. This acts as a safeguard against recombination between nonhomologous sequences that could result in genome imbalance. Here, we report recombination between maternal and paternal mitochondrial genomes of the sea mussel, whose sequences differ by >20%. We propose that the strict maternal inheritance of the animal mitochondrial DNA and the ensuing homoplasmy has relieved the MMR system of the animal mitochondrion from the pressure to tolerate recombination only among sequences with a high degree of similarity.

The Pattern of Change in the Abundances of Specific Bacterioplankton Groups Is Consistent across Different Nutrient-Enriched Habitats in Crete

ABSTRACT A common source of disturbance for coastal aquatic habitats is nutrient enrichment through anthropogenic activities. Although the water column bacterioplankton communities in these environments have been characterized in some cases, changes in α-diversity and/or the abundances of specific taxonomic groups across enriched habitats remain unclear. Here, we investigated the bacterial community changes at three different nutrient-enriched and adjacent undisturbed habitats along the north coast of Crete, Greece: a fish farm, a closed bay within a town with low water renewal rates, and a city port where the level of nutrient enrichment and the trophic status of the habitat were different. Even though changes in α-diversity were different at each site, we observed across the sites a common change pattern accounting for most of the community variation for five of the most abundant bacterial groups: a decrease in the abundance of the Pelagibacteraceae and SAR86 and an increase in the abundance of the Alteromonadaceae, Rhodobacteraceae, and Cryomorphaceae in the impacted sites. The abundances of the groups that increased and decreased in the impacted sites were significantly correlated (positively and negatively, respectively) with the total heterotrophic bacterial counts and the concentrations of dissolved organic carbon and/or dissolved nitrogen and chlorophyll α, indicating that the common change pattern was associated with nutrient enrichment. Our results provide an in situ indication concerning the association of specific bacterioplankton groups with nutrient enrichment. These groups could potentially be used as indicators for nutrient enrichment if the pattern is confirmed over a broader spatial and temporal scale by future studies.

Different degree of paternal mtDNA leakage between male and female progeny in interspecific Drosophila crosses

Maternal transmission of mitochondrial DNA (mtDNA) in animals is thought to prevent the spread of selfish deleterious mtDNA mutations in the population. Various mechanisms have been evolved independently to prevent the entry of sperm mitochondria in the embryo. However, the increasing number of instances of paternal mtDNA leakage suggests that these mechanisms are not very effective. The destruction of sperm mitochondria in mammalian embryos is mediated by nuclear factors. Also, the destruction of paternal mitochondria in intraspecific crosses is more effective than in interspecific ones. These observations have led to the hypothesis that leakage of paternal mtDNA (and consequently mtDNA recombination owing to ensuing heteroplasmy) might be more common in inter- than in intraspecific crosses and that it should increase with phylogenetic distance of hybridizing species. We checked paternal leakage in inter- and intraspecific crosses in Drosophila and found little evidence for this hypothesis. In addition, we have observed a higher level of leakage among male than among female progeny from the same cross. This is the first report of sex-specific leakage of paternal mtDNA. It suggests that paternal mtDNA leakage might not be a stochastic result of an error-prone mechanism, but rather, it may be under complex genetic control.

Evolutionary genetics: Direct evidence of recombination in human mitochondrial DNA

O ver the last 5 years, there has been considerable debate as to whether there is recombination in human mitochondrial DNA (mtDNA) (for references, see Piganeau and Eyre-Walker, 2004). That debate appears to have finally come to an end with the publication of some direct evidence of recombination. Schwartz and Vissing (2002), 2 years ago, presented the case of a 28year-old man who had both maternal and paternally derived mtDNA in his muscle tissue – in all his other tissues he had only maternally derived mtDNA. It was the first time that paternal leakage and, consequently, heteroplasmy was observed in human mtDNA. In a recent paper, Kraytsberg et al (2004) take this observation one step further, and claim to show that there has been recombination between the maternal and paternal mtDNA in this individual. There is a major possibility, in experiments of this nature, that the recombinants have been produced in laboratory, either by PCR, or by some other mistake. However, the authors have gone to great lengths to ensure that the recombinants are genuine, including repeating the experiment with a mix of maternal and paternal mtDNAs. They did not observe any recombination in this latter experiment, so we can be very confident that recombinants that they detected in the muscle tissue are genuine. The direct demonstration of recombination in human mtDNA has a number of important implications. First, the results show that recombination between maternal and paternal mtDNA is possible. It has been known for sometime that paternal mtDNA enters the egg in humans (see Cummins, 2000), and that mammalian mitochondria contain the enzymes necessary to promote homologous recombination (Thyagarajan et al, 1996). However, there are efficient mechanisms that target paternal mtDNA for destruction once it is in the egg (Sutovsky et al, 2000), and there is no evidence that different mtDNAs would ever have the chance to recombine. If different mtDNAs are introduced into the same cell in different mitochondria, can be maintained in that state for many generations, without ever appearing in the same mitochondria (Enriquez et al, 2000). Second, the results suggest that human mitochondria have an active recombination pathway. Human mtDNA has a high rate of evolution and this has been attributed to a lack of repair enzymes (Brown, 2001). The possibility of a recombination pathway suggested by these current results may also provide a further reason. Human mitochondrial DNA has been used extensively to study the evolution of our species. However, most of the conclusions from these studies are likely to remain unaffected, either because they do not rely on the assumption of clonality, or because the level of recombination is such that its effects will be small. For example, mtDNA has been used to study the spread of humans across the globe (Cann, 2001). The genetic differences were probably established as human populations spread to new localities and have persisted because of reduced gene flow between distant populations. If there is little gene flow, there will be little opportunity for recombination. The one area in which recombination may have implications is dating events in human evolution. This is for two reasons – first, the phylogenetic tree may not represent the particular evolutionary events of interest; for example, a mtDNA tree may not represent the phylogeny solely of females if there is paternal leakage and/or recombination; it could not, therefore, be used to estimate the date of our most recent female common ancestor. Second, recombination will tend to generate homoplasies and therefore variation in the rate of nucleotide substitution between sites. This in turn will lead to an overestimate of the rate of nucleotide substitution if clonal inheritance is wrongly assumed. Although Kraytsberg et al (2004) show very clear evidence of crossing-over in human mtDNA, there is little or no population genetic evidence of recombination. How can this be? There are two possibilities. One possibility is that, although Kraytsberg et al (2004) have detected recombination in a somatic tissue, it never occurs in the germ line. This seems unlikely, however. Alternatively, the explanation may be that recombination is difficult to detect in population genetic data, even if it is occurring at appreciable frequencies. This is because all methods for detecting recombination have low statistical power. The results of Kraytsberg et al (2004) show that recombination has been fairly frequent in the individual they study. Even if we assume that all identical sequences are the product of the same recombination event, there must still have been 16 events. Unfortunately, this observation does not give us a handle on how frequent recombination is likely to be in the population, since this depends on the rate of paternal leakage; yet, we have no precise estimate of this parameter. At most, one presumes it must be less than 1 in 1000, since there are 100 000 mitochondria in the human egg and only 100 in the sperm (Satoh and Kuroiwa, 1991). The challenge for the future will be to determine how frequent paternal leakage and recombination are in humans, and how frequent these processes are in other species.