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Enriching mitochondrial DNA (mtDNA) for sequencing entire mitochondrial genomes (mitogenomes) can be achieved by single long-range PCR. This avoids interference from the omnipresent nuclear mtDNA sequences (NUMTs). The approach is currently restricted to the use of samples collected from humans and ray-finned fishes. Here, we extended the use of single long-range PCR by introducing back-to-back oligonucleotides that target a sequence of extraordinary homology across vertebrates. The assay was applied to five hibernating rodents, namely alpine marmot, Arctic and European ground squirrels, and common and garden dormice, four of which have not been fully sequenced before. Analysis of the novel mitogenomes focussed on the prediction of mitochondrialderived peptides (MDPs) providing another level of information encoded by mtDNA. The comparison of MOTS-c, SHLP4 and SHLP6 sequences across vertebrate species identified segments of high homology that argue for future experimentation. In addition, we evaluated four candidate polymorphisms replacing an amino acid in mitochondrially encoded subunits of the oxidative phosphorylation (OXPHOS) system that were reported in relation to cold-adaptation. No obvious pattern was found for the diverse sets of mammalian species that either apply daily or multiday torpor or otherwise cope with cold. In summary, our single long-range PCR assay applying a pair of back-to-back primers that target a consensus sequence motif of Vertebrata has potential to amplify (intact) mitochondrial rings present in templates from a taxonomically diverse range of vertebrates. It could be promising for studying novel mitogenomes, mitotypes of a population and mitochondrial heteroplasmy in a sensitive, straightforward and flexible manner. [ABSTRACT FROM AUTHOR]

Abstract Background In mammals, males typically have shorter lives than females. This difference is thought to be due to behavioural traits which enhance competitive abilities, and hence male reproductive success, but impair survival. Furthermore, in many species males usually show higher parasite burden than females. Consequently, the intensity of selection for genetic factors which reduce susceptibility to pathogens may differ between sexes. High variability at the major histocompatibility complex (MHC) genes is believed to be advantageous for detecting and combating the range of infectious agents present in the environment. Increased heterozygosity at these immune genes is expected to be important for individual longevity. However, whether males in natural populations benefit more from MHC heterozygosity than females has rarely been investigated. We investigated this question in a long-term study of free-living Alpine chamois (Rupicapra rupicapra), a polygynous mountain ungulate. Results Here we show that male chamois survive significantly (P = 0.022) longer if heterozygous at the MHC class II DRB locus, whereas females do not. Improved survival of males was not a result of heterozygote advantage per se, as background heterozygosity (estimated across twelve microsatellite loci) did not change significantly with age. Furthermore, reproductively active males depleted their body fat reserves earlier than females leading to significantly impaired survival rates in this sex (P < 0.008). This sex-difference was even more pronounced in areas affected by scabies, a severe parasitosis, as reproductively active males were less likely to survive than females. However, we did not find evidence for a survival advantage associated with specific MHC alleles in areas affected by scabies. Conclusions Increased MHC class II DRB heterozygosity with age in males, suggests that MHC heterozygous males survive longer than homozygotes. Reproductively active males appear to be less likely to survive than females most likely because of the energetic challenge of the winter rut, accompanied by earlier depletion of their body fat stores, and a generally higher parasite burden. This scenario renders the MHC-mediated immune response more important for males than for females, which implies a relatively stronger selection pressure on MHC genes in males than in females.

Abstract Background Mitotic recombination is important for inactivating tumour suppressor genes by copy-neutral loss of heterozygosity (LOH). Although meiotic recombination maps are plentiful, little is known about mitotic recombination. The APC gene (chr5q21) is mutated in most colorectal tumours and its usual mode of LOH is mitotic recombination. Methods We mapped mitotic recombination boundaries ("breakpoints") between the centromere (~50 Mb) and APC (~112 Mb) in early colorectal tumours. Results Breakpoints were non-random, with the highest frequency between 65 Mb and 75 Mb, close to a low copy number repeat region (68–71 Mb). There were, surprisingly, few breakpoints close to APC, contrary to expectations were there constraints on tumorigenesis caused by uncovering recessive lethal alleles or if mitotic recombination were mechanistically favoured by a longer residual chromosome arm. The locations of mitotic and meiotic recombination breakpoints were correlated, suggesting that the two types of recombination are influenced by similar processes, whether mutational or selective in origin. Breakpoints were also associated with higher local G+C content. The recombination and gain/deletion breakpoint maps on 5q were not, however, associated, perhaps owing to selective constraints on APC dosage in early colorectal tumours. Since polymorphisms within the region of frequent mitotic recombination on 5q might influence the frequency of LOH, we tested the 68–71 Mb low copy number repeat and nearby tagSNPs, but no associations with colorectal cancer risk were found. Conclusion LOH on 5q is non-random, but local factors do not greatly influence the rate of LOH at APC or explain inter differential susceptibility to colorectal tumours.

Background: Recent studies of selection on mitochondrial (mt) OXPHOS genes suggest adaptation due mainly to environmental variation. In this context, Tunisian hares that display several external phenotypes with phylogenetically rather homogenous gene pool and shallow population structure provide a good precondition to detect positive selection on mt genes related to environmental/climatic variation, specifically ambient temperature and precipitation. Results: We used codon-based methods along with population genetic data to test for positive selection on ATPase synthase 6 (ATP6) and NADH dehydrogenase 2 (ND2) of cape hares (Lepus capensis) collected along a steep ecological gradient in Tunisia. We found significantly higher differentiation at the ATP6 locus across Tunisia, with sub-humid Mediterranean, semi-arid, and arid Sahara climate than for fourteen unlinked supposedly neutrally evolving nuclear microsatellites and mt control region sequences. This suggested positive selection on ATP6 sequences, which was confirmed by several codon-based tests for one sequence site that together with a second site translated into four different amino acids. Positive selection on ND2 sequences was also confirmed by several codon-based tests. The corresponding frequencies of the two most prevalent variants at each locus varied significantly across climate regions, and our logistic general linear models of occurrence of those proteins indicated significant effects of mean annual temperature for ATP6 and mean minimum temperature of the coldest month of the year for ND2, independent of geographical location, annual precipitation, and the respective co-occurring protein at the second locus. Moreover, presence of the ancestral ATP6 protein, as inferred from phylogenetic networks, was positively affected by the simultaneous presence of the derived ND2 protein and vice versa, independent of temperature, precipitation, or geographic location. Finally, we obtained a significant coevolution signal for the ancestral ATP6 and derived ND2 sequences and vice versa. Conclusions: positive selection was strongly suggested by the population genetic approach and the codon-based tests in both mtDNA genes. Moreover, the two most prevalent proteins at the ATP6 locus were distributed at significantly varying frequencies across the study area with a significant effect of mean annual temperature on the occurrence of the ATP6 proteins independent of geographical coordinates and the co-occuring ND2 protein variant. For ND2, occurrence of the two most frequent protein variants was significantly influenced by the mean minimum temperature of the coldest month, independent of the co-occurring ATP6 protein variant and geographical coordinates. This strongly suggests direct involvement of ambient temperature in the adaptation of the studied mtOXPHOS genes. [ABSTRACT FROM AUTHOR]

We surveyed the genetic diversity of the expressed major histocompatibility complex class II DQA locus in natural populations of European brown hares, Lepus europaeus, from Austria and Belgium (267 individuals in total). Based on cDNA sequences, we designed hare-specific primers to amplify the highly variable second exon of the DQA gene. Using cloning–sequencing methodology and capillary electrophoresis single-strand conformation polymorphism, we found ten alleles of the DQA exon 2 locus across these two European regions, of which eight are described for the first time. To search for signals of selection and recombination in the evolution of the DQA gene within the leporids, we augmented our sample with orthologous DQA alleles from the European rabbit, Oryctolagus cuniculus, in order to carry out a species level, species pairwise comparison. We found evidence of recombination in the history of the DQA sequences in leporids with some recombinant alleles bridging the species divide. In both species, selection on peptide binding site codons can be detected, though stronger for the rabbit. This result suggests that there may be a differential selection pressure in the deeper evolutionary history of these two species due to differences in several demographic and ecological traits likely subjecting them to differential selection by parasites. Finally, evolutionary relationships show a widespread and statistically significant intermingling of alleles from the two species. The many macroparasites shared between hares and rabbits may explain this pattern of trans-species polymorphism. [ABSTRACT FROM AUTHOR]

We examined the evolutionary processes contributing to genetic diversity at the major histocompatibility complex (MHC) class IIDRBlocus in chamois (Rupicapraspp., subfamily Caprinae). We characterised the pattern of intragenic recombination (or homologous gene conversion) and quantified the amount of recombination in the genealogical history of the two chamois species, Pyrenean chamois (Rupicapra pyrenaica) and Alpine chamois (Rupicapra rupicapra). We found evidence for intragenic recombination, and the estimated amount of population recombination suggests that recombination has been a significant process in generatingDRBallelic diversity in the genealogical history of the genusRupicapra. Moreover, positive selection appears to act on the same peptide-binding residues in both analysed chamois species, but not in identical intensity. Recombination coupled with positive selection drives the rapid evolution at the peptide-binding sites in the MHC class IIDRBgene. Many chamois MHC class IIDRBalleles are thus much younger than previously assumed. [ABSTRACT FROM AUTHOR]

In lagomorphs, lymphocyte subset distributions and the importance of CD4+ T cell levels has so far only been considered in the frame of rabbit disease models. In this study, the first assessment of CD4+ T lymphocytes in peripheral blood cells in brown hares (Lepus europaeus L., 1758), a further leporid species using a cross-reactive rabbit anti-CD4 antibody in flow cytometry, is presented. In addition, the entire coding region of the hare CD4 gene (1380 bp) coding for a polypeptide of 459 amino acids has been sequenced. Using generalized least squares fitting by maximum likelihood (GLS) test, significantly (p = 0.0095) higher CD4+ T cell frequencies in males than in females and significantly (p = 0.0001) higher frequencies for leverets (younger than 2 months of age) than for subadult and adult (older than 7 months of age) individuals were detected. No significant age influence, however, was found for subadult and adult hares. The study is particularly meant to provide a first step in establishing a toolbox for the assessment of the immune response in this leporid species. [ABSTRACT FROM AUTHOR]