Molecular dating of caprines using ancient DNA sequences of Myotragus balearicus, an extinct endemic Balearic mammal

Background: Myotragus balearicus was an endemic bovid from the Balearic Islands (Western Mediterranean) that became extinct around 6,000-4,000 years ago. The Myotragus evolutionary lineage became isolated in the islands most probably at the end of the Messinian crisis, when the desiccation of the Mediterranean ended, in a geological date established at 5.35 Mya. Thus, the sequences of Myotragus could be very valuable for calibrating the mammalian mitochondrial DNA clock and, in particular, the tree of the Caprinae subfamily, to which Myotragus belongs. Results: We have retrieved the complete mitochondrial cytochrome b gene (1,143 base pairs), plus fragments of the mitochondrial 12S gene and the nuclear 28S rDNA multi-copy gene from a well preserved Myotragus subfossil bone. The best resolved phylogenetic trees, obtained with the cytochrome b gene, placed Myotragus in a position basal to the Ovis group. Using the calibration provided by the isolation of Balearic Islands, we calculated that the initial radiation of caprines can be dated at 6.2 ± 0.4 Mya. In addition, alpine and southern chamois, considered until recently the same species, split around 1.6 ± 0.3 Mya, indicating that the two chamois species have been separated much longer than previously thought. Conclusion: Since there are almost no extant endemic mammals in Mediterranean islands, the sequence of the extinct Balearic endemic Myotragus has been crucial for allowing us to use the Messinian crisis calibration point for dating the caprines phylogenetic tree.

Molecular dating of caprines using ancient DNA sequences of Myotragus balearicus, an extinct endemic Balearic mammal

Background: Myotragus balearicus was an endemic bovid from the Balearic Islands (Western Mediterranean) that became extinct around 6,000-4,000 years ago. The Myotragus evolutionary lineage became isolated in the islands most probably at the end of the Messinian crisis, when the desiccation of the Mediterranean ended, in a geological date established at 5.35 Mya. Thus, the sequences of Myotragus could be very valuable for calibrating the mammalian mitochondrial DNA clock and, in particular, the tree of the Caprinae subfamily, to which Myotragus belongs. Results: We have retrieved the complete mitochondrial cytochrome b gene (1,143 base pairs), plus fragments of the mitochondrial 12S gene and the nuclear 28S rDNA multi-copy gene from a well preserved Myotragus subfossil bone. The best resolved phylogenetic trees, obtained with the cytochrome b gene, placed Myotragus in a position basal to the Ovis group. Using the calibration provided by the isolation of Balearic Islands, we calculated that the initial radiation of caprines can be dated at 6.2 ± 0.4 Mya. In addition, alpine and southern chamois, considered until recently the same species, split around 1.6 ± 0.3 Mya, indicating that the two chamois species have been separated much longer than previously thought. Conclusion: Since there are almost no extant endemic mammals in Mediterranean islands, the sequence of the extinct Balearic endemic Myotragus has been crucial for allowing us to use the Messinian crisis calibration point for dating the caprines phylogenetic tree.

Ancient DNA of the extinct Lava shearwater (Puffinus olsoni) from the Canary Islands reveals incipient differentiation within the P. puffinus complex

The loss of species during the Holocene was, dramatically more important on islands than on continents. Seabirds from islands are very vulnerable to human-induced alterations such as habitat destruction, hunting and exotic predators. For example, in the genus Puffinus (family Procellariidae) the extinction of at least five species has been recorded during the Holocene, two of them coming from the Canary Islands.

Ancient DNA analysis of 8000 B.C. near eastern farmers supports an early neolithic pioneer maritime colonization of Mainland Europe through Cyprus and the Aegean Islands

The genetic impact associated to the Neolithic spread in Europe has been widely debated over the last 20 years. Within this context, ancient DNA studies have provided a more reliable picture by directly analyzing the protagonist populations at different regions in Europe. However, the lack of available data from the original Near Eastern farmers has limited the achieved conclusions, preventing the formulation of continental models of Neolithic expansion. Here we address this issue by presenting mitochondrial DNA data of the original Near-Eastern Neolithic communities with the aim of providing the adequate background for the interpretation of Neolithic genetic data from European samples. Sixty-three skeletons from the Pre Pottery Neolithic B (PPNB) sites of Tell Halula, Tell Ramad and Dja'de El Mughara dating between 8,700-6,600 cal. B.C. were analyzed, and 15 validated mitochondrial DNA profiles were recovered. In order to estimate the demographic contribution of the first farmers to both Central European and Western Mediterranean Neolithic cultures, haplotype and haplogroup diversities in the PPNB sample were compared using phylogeographic and population genetic analyses to available ancient DNA data from human remains belonging to the Linearbandkeramik-Alföldi Vonaldiszes Kerámia and Cardial/Epicardial cultures. We also searched for possible signatures of the original Neolithic expansion over the modern Near Eastern and South European genetic pools, and tried to infer possible routes of expansion by comparing the obtained results to a database of 60 modern populations from both regions. Comparisons performed among the 3 ancient datasets allowed us to identify K and N-derived mitochondrial DNA haplogroups as potential markers of the Neolithic expansion, whose genetic signature would have reached both the Iberian coasts and the Central European plain. Moreover, the observed genetic affinities between the PPNB samples and the modern populations of Cyprus and Crete seem to suggest that the Neolithic was first introduced into Europe through pioneer seafaring colonization.

Fragmentation of contaminant and endogenous DNA in ancient samples determined by shotgun sequencing; prospects for human palaeogenomics

Despite the successful retrieval of genomes from past remains, the prospects for human palaeogenomics remain unclear because of the difficulty of distinguishing contaminant from endogenous DNA sequences. Previous sequence data generated on high-throughput sequencing platforms indicate that fragmentation of ancient DNA sequences is a characteristic trait primarily arising due to depurination processes that create abasic sites leading to DNA breaks.

Genetic origin, admixture, and asymmetry in maternal and paternal human lineages in Cuba

Background: Before the arrival of Europeans to Cuba, the island was inhabited by two Native American groups, the Tainos and the Ciboneys. Most of the present archaeological, linguistic and ancient DNA evidence indicates a South American origin for these populations. In colonial times, Cuban Native American people were replaced by European settlers and slaves from Africa. It is still unknown however, to what extent their genetic pool intermingled with and was 'diluted' by the arrival of newcomers. In order to investigate the demographic processes that gave rise to the current Cuban population, we analyzed the hypervariable region I (HVS-I) and five single nucleotide polymorphisms (SNPs) in the mitochondrial DNA (mtDNA) coding region in 245 individuals, and 40 Y-chromosome SNPs in 132 male individuals. Results: The Native American contribution to present-day Cubans accounted for 33% of the maternal lineages, whereas Africa and Eurasia contributed 45% and 22% of the lineages, respectively. This Native American substrate in Cuba cannot be traced back to a single origin within the American continent, as previously suggested by ancient DNA analyses. Strikingly, no Native American lineages were found for the Y-chromosome, for which the Eurasian and African contributions were around 80% and 20%, respectively. Conclusion: While the ancestral Native American substrate is still appreciable in the maternal lineages, the extensive process of population admixture in Cuba has left no trace of the paternal Native American lineages, mirroring the strong sexual bias in the admixture processes taking place during colonial times.

Population connectivity buffers genetic diversity loss in a seabird

Background Ancient DNA has revolutionized conservation genetic studies as it allows monitoring of the genetic variability of species through time and predicting the impact of ecosystems" threats on future population dynamics and viability. Meanwhile, the consequences of anthropogenic activities and climate change to island faunas, particularly seabirds, remain largely unknown. In this study, we examined temporal changes in the genetic diversity of a threatened seabird, the Cory"s shearwater (Calonectris borealis). Findings We analysed the mitochondrial DNA control region of ancient bone samples from the late-Holocene retrieved from the Canary archipelago (NE Atlantic) together with modern DNA sequences representative of the entire breeding range of the species. Our results show high levels of ancient genetic diversity in the Canaries comparable to that of the extant population. The temporal haplotype network further revealed rare but recurrent long-distance dispersal between ocean basins. The Bayesian demographic analyses reveal both regional and local population size expansion events, and this is in spite of the demographic decline experienced by the species over the last millennia. Conclusions Our findings suggest that population connectivity of the species has acted as a buffer of genetic losses and illustrate the use of ancient DNA to uncover such cryptic genetic events.

Array de DNA per identificar espècies de Fusarium: ampliació d'un array existent per incloure espècies del Sud d'Europa

Projecte de recerca elaborat a partir d’una estada al Department for Feed and Food Hygiene del National Veterinary Institute, Noruega, entre novembre i desembre del 2006. Els grans de cereal poden estar contaminats amb diferents espècies de Fusarium capaces de produir metabolits secundaris altament tòxics com trichotecenes, fumonisines o moniliformines. La correcta identificació d’aquestes espècies és de gran importància per l’assegurament del risc en l’àmbit de la salut humana i animal. La identificació de Fusarium en base a la seva morfologia requereix coneixements taxonòmics i temps; la majoria dels mètodes moleculars permeten la identificació d’una única espècie diana. Per contra, la tecnologia de microarray ofereix l’anàlisi paral•lel d’un alt nombre de DNA dianes. En aquest treball, s’ha desenvolupat un array per a la identificació de les principals espècies de Fusarium toxigèniques del Nord i Sud d’Europa. S’ha ampliat un array ja existent, per a la detecció de les espècies de Fusarium productores de trichothecene i moniliformina (predominants al Nord d’Europa), amb l’addició de 18 sondes de DNA que permeten identificar les espècies toxigèniques més abundants al Sud d’Europa, les qual produeixen majoritàriament fumonisines. Les sondes de captura han estat dissenyades en base al factor d’elongació translació- 1 alpha (TEF-1alpha). L’anàlisi de les mostres es realitza mitjançant una única PCR que permet amplificar part del TEF-1alpha seguida de la hibridació al xip de Fusarium. Els resultats es visualitzen mitjançant un mètode de detecció colorimètric. El xip de Fusarium desenvolupat pot esdevenir una eina útil i de gran interès per a l’anàlisi de cereals presents en la cadena alimentària.

Solid-phase synthesis and NMR studies of modified oligonucleotides forming triplex helix and of oligonucleopeptides mimicking the topoisomerase I-DNA covalent complex

Report for the scientific sojourn carried out at the Institut de Biologia Molecular de Barcelona of the CSIC –state agency – from april until september 2007. Topoisomerase I is an essential nuclear enzyme that modulates the topological status of DNA, facilitating DNA helix unwinding during replication and transcription. We have prepared the oligonucleotide-peptide conjugate Ac-NLeu-Asn-Tyr(p-3’TTCAGAAGC5’)-LeuC-CONH-(CH2)6-OH as model compound for NMR studies of the Topoisomerase I- DNA complex. Special attention was made on the synthetic aspects for the preparation of this challenging compound especially solid supports and protecting groups. The desired peptide was obtained although we did not achieve the amount of the conjugate needed for NMR studies. Most probably the low yield is due to the intrinsic sensitive to hydrolysis of the phosphate bond between oligonucleotide and tyrosine. We have started the synthesis and the structural characterization of oligonucleotides carrying intercalating compounds. At the present state we have obtained model duplex and quadruplex sequences modified with acridine and NMR studies are underway. In addition to this project we have successfully resolved the structure of a fusion peptide derived from hepatitis C virus envelope synthesized by the group of Dr. Haro and we have synthesized and started the characterization of a modified G-quadruplex.

Development of specific ITS markers for plant DNA identification within herbivorous insects

DNA based techniques have proved to be very useful methods to study trophic relationships 17 between pests and their natural enemies. However, most predators are best defined as omnivores, 18 and the identification of plant-specific DNA should also allow the identification of the plant 19 species the predators have been feeding on. In this study, a PCR approach based on the 20 development of specific primers was developed as a self-marking technique to detect plant DNA 21 within the gut of one heteropteran omnivorous predator (Macrolophus pygmaeus) and two 22 lepidopteran pest species (Helicoverpa armigera and Tuta absoluta). Specific tomato primers 23 were designed from the ITS 1-2 region, which allowed the amplification of a tomato DNA 24 fragment of 332 bp within the three insect species tested in all cases (100% of detection at t = 0) 25 and did not detect DNA of other plants nor of the starved insects. Plant DNA half-lives at 25ºC 26 ranged from 5.8h, to 27.7h and 28.7h within M. pygmaeus, H. armigera and T. absoluta, 27 respectively. Tomato DNA detection within field collected M. pygmaeus suggests dietary mixing 28 in this omnivorous predator and showed a higher detection of tomato DNA in females and 29 nymphs than males. This study provides a useful tool to detect and to identify plant food sources 30 of arthropods and to evaluate crop colonization from surrounding vegetation in conservation 31 biological control programs.

Análisis de la expresión de las moléculas relacionadas con el complejo principal de histocompatibilidad de clase I y de NKG2D como respuesta al daño oxidativo del DNA en el cáncer urotelial de vejiga. Relación con las características clínico patológicas y supervivencia

Es tracta d'un estudi retrospectiu de casos de 280 pacients diagnosticats de tumor vesical primari amb un seguiment mínim de 8 anys. S'ha construït un Tissue microarray i mitjançant mètodes semiquantitatius d’inmunohistoquímica es determinarà l'expressió de les molècules MICA (MHC class I chain-related gene A) i del seu receptor NKG2D (Natural-Killer group 2-member D) a nivell tissular, relacionant-lo amb variables anatomopatològiques segons els grups de risc, hàbit tabàquic i sexe. Finalment valorarem l'expressió de MICA/NKG2D com a factor independent de recidiva / progressió tumoral. En la literatura només existeixen 2 treballs que relacionin MICA amb el càncer vesical.

Electronic couplings and on-site energies for hole transfer in DNA: systematic quantum mechanical/molecular dynamic study

The electron hole transfer (HT) properties of DNA are substantially affected by thermal fluctuations of the π stack structure. Depending on the mutual position of neighboring nucleobases, electronic coupling V may change by several orders of magnitude. In the present paper, we report the results of systematic QM/molecular dynamic (MD) calculations of the electronic couplings and on-site energies for the hole transfer. Based on 15 ns MD trajectories for several DNA oligomers, we calculate the average coupling squares 〈 V2 〉 and the energies of basepair triplets X G+ Y and X A+ Y, where X, Y=G, A, T, and C. For each of the 32 systems, 15 000 conformations separated by 1 ps are considered. The three-state generalized Mulliken-Hush method is used to derive electronic couplings for HT between neighboring basepairs. The adiabatic energies and dipole moment matrix elements are computed within the INDO/S method. We compare the rms values of V with the couplings estimated for the idealized B -DNA structure and show that in several important cases the couplings calculated for the idealized B -DNA structure are considerably underestimated. The rms values for intrastrand couplings G-G, A-A, G-A, and A-G are found to be similar, ∼0.07 eV, while the interstrand couplings are quite different. The energies of hole states G+ and A+ in the stack depend on the nature of the neighboring pairs. The X G+ Y are by 0.5 eV more stable than X A+ Y. The thermal fluctuations of the DNA structure facilitate the HT process from guanine to adenine. The tabulated couplings and on-site energies can be used as reference parameters in theoretical and computational studies of HT processes in DNA

Estimates of electronic coupling for excess electron transfer in DNA

Electronic coupling Vda is one of the key parameters that determine the rate of charge transfer through DNA. While there have been several computational studies of Vda for hole transfer, estimates of electronic couplings for excess electron transfer (ET) in DNA remain unavailable. In the paper, an efficient strategy is established for calculating the ET matrix elements between base pairs in a π stack. Two approaches are considered. First, we employ the diabatic-state (DS) method in which donor and acceptor are represented with radical anions of the canonical base pairs adenine-thymine (AT) and guanine-cytosine (GC). In this approach, similar values of Vda are obtained with the standard 6-31 G* and extended 6-31++ G* basis sets. Second, the electronic couplings are derived from lowest unoccupied molecular orbitals (LUMOs) of neutral systems by using the generalized Mulliken-Hush or fragment charge methods. Because the radical-anion states of AT and GC are well reproduced by LUMOs of the neutral base pairs calculated without diffuse functions, the estimated values of Vda are in good agreement with the couplings obtained for radical-anion states using the DS method. However, when the calculation of a neutral stack is carried out with diffuse functions, LUMOs of the system exhibit the dipole-bound character and cannot be used for estimating electronic couplings. Our calculations suggest that the ET matrix elements Vda for models containing intrastrand thymine and cytosine bases are essentially larger than the couplings in complexes with interstrand pyrimidine bases. The matrix elements for excess electron transfer are found to be considerably smaller than the corresponding values for hole transfer and to be very responsive to structural changes in a DNA stack

Charge transfer in DNA: hole charge is confined to a single base pair due to solvation effects

We include solvation effects in tight-binding Hamiltonians for hole states in DNA. The corresponding linear-response parameters are derived from accurate estimates of solvation energy calculated for several hole charge distributions in DNA stacks. Two models are considered: (A) the correction to a diagonal Hamiltonian matrix element depends only on the charge localized on the corresponding site and (B) in addition to this term, the reaction field due to adjacent base pairs is accounted for. We show that both schemes give very similar results. The effects of the polar medium on the hole distribution in DNA are studied. We conclude that the effects of polar surroundings essentially suppress charge delocalization in DNA, and hole states in (GC)n sequences are localized on individual guanines

An assessment of gene prediction accuracy in large DNA sequences

One of the first useful products from the human genome will be a set of predicted genes. Besides its intrinsic scientific interest, the accuracy and completeness of this data set is of considerable importance for human health and medicine. Though progress has been made on computational gene identification in terms of both methods and accuracy evaluation measures, most of the sequence sets in which the programs are tested are short genomic sequences, and there is concern that these accuracy measures may not extrapolate well to larger, more challenging data sets. Given the absence of experimentally verified large genomic data sets, we constructed a semiartificial test set comprising a number of short single-gene genomic sequences with randomly generated intergenic regions. This test set, which should still present an easier problem than real human genomic sequence, mimics the approximately 200kb long BACs being sequenced. In our experiments with these longer genomic sequences, the accuracy of GENSCAN, one of the most accurate ab initio gene prediction programs, dropped significantly, although its sensitivity remained high. Conversely, the accuracy of similarity-based programs, such as GENEWISE, PROCRUSTES, and BLASTX was not affected significantly by the presence of random intergenic sequence, but depended on the strength of the similarity to the protein homolog. As expected, the accuracy dropped if the models were built using more distant homologs, and we were able to quantitatively estimate this decline. However, the specificities of these techniques are still rather good even when the similarity is weak, which is a desirable characteristic for driving expensive follow-up experiments. Our experiments suggest that though gene prediction will improve with every new protein that is discovered and through improvements in the current set of tools, we still have a long way to go before we can decipher the precise exonic structure of every gene in the human genome using purely computational methodology.