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Nowadays DNA meta-barcoding is a powerful instrument capable of quickly discovering the biodiversity of an environmental sample by integrating the DNA barcoding approach with High Throughput Sequencing technologies. It mainly consists of the parallel reading of informative genomic fragment/s able to discriminate living entities. Although this approach has been widely studied, it still needs optimization in some necessary steps requested in its advanced accomplishment. A fundamental element concerns the standardization of bioinformatic analyses pipelines. The aim of the present study was to underline a number of critical parameters of laboratory material preparation and taxonomic assignment pipelines in DNA meta-barcoding experiments using the cytochrome oxidase subunit-I (

Background Phylogenetic relationships between Lagomorpha, Rodentia and Primates and their allies (Euarchontoglires) have long been debated. While it is now generally agreed that Rodentia constitutes a monophyletic sister-group of Lagomorpha and that this clade (Glires) is sister to Primates and Dermoptera, higher-level relationships within Rodentia remain contentious. Results We have sequenced and performed extensive evolutionary analyses on the mitochondrial genome of the scaly-tailed flying squirrel Anomalurus sp., an enigmatic rodent whose phylogenetic affinities have been obscure and extensively debated. Our phylogenetic analyses of the coding regions of available complete mitochondrial genome sequences from Euarchontoglires suggest that Anomalurus is a sister taxon to the Hystricognathi, and that this clade represents the most basal divergence among sampled Rodentia. Bayesian dating methods incorporating a relaxed molecular clock provide divergence-time estimates which are consistently in agreement with the fossil record and which indicate a rapid radiation within Glires around 60 million years ago. Conclusion Taken together, the data presented provide a working hypothesis as to the phylogenetic placement of Anomalurus, underline the utility of mitochondrial sequences in the resolution of even relatively deep divergences and go some way to explaining the difficulty of conclusively resolving higher-level relationships within Glires with available data and methodologies.

Nowadays DNA meta-barcoding is a powerful instrument capable of quickly discovering the biodiversity of an environmental sample by integrating the DNA barcoding approach with High Throughput Sequencing technologies. It mainly consists of the parallel reading of informative genomic fragment/s able to discriminate living entities. Although this approach has been widely studied, it still needs optimization in some necessary steps requested in its advanced accomplishment. A fundamental element concerns the standardization of bioinformatic analyses pipelines. The aim of the present study was to underline a number of critical parameters of laboratory material preparation and taxonomic assignment pipelines in DNA meta-barcoding experiments using the cytochrome oxidase subunit-I (coxI) barcode region, known as a suitable molecular marker for animal species identification. We compared nine taxonomic assignment pipelines, including a custom in-house method, based on Hidden Markov Models. Moreover, we evaluated the potential influence of universal primers amplification bias in qPCR, as well as the correlation between GC content with taxonomic assignment results. The pipelines were tested on a community of known terrestrial invertebrates collected by pitfall traps from a chestnut forest in Italy. Although the present analysis was not exhaustive and needs additional investigation, our results suggest some potential improvements in laboratory material preparation and the introduction of additional parameters in taxonomic assignment pipelines. These include the correct setup of OTU clustering threshold, the calibration of GC content affecting sequencing quality and taxonomic classification, as well as the evaluation of PCR primers amplification bias on the final biodiversity pattern. Thus, careful attention and further validation/optimization of the above-mentioned variables would be required in a DNA meta-barcoding experimental routine.

DNA duplication is one of the main forces acting on the evolution of organisms because it creates the raw genetic material that natural selection can subsequently modify. Duplicated regions are mainly due to "errors" in different phases of meiosis, but DNA transposable elements and reverse transcription also contribute to amplify and move the genomic material to different genomic locations. As a result, redundancy affects genomes to variable degrees: from the single gene to the whole genome (WGD). Gene families are clusters of genes created by duplication and their size reflects the number of duplicated genes, called paralogs, in each species. The aim of this review is to describe the state of the art in the identification and analysis of gene families in eukaryotes, with specific attention to those generated by ancient large scale events in vertebrates (WGD or large segmental duplications). As a case study, we report our work on the evolution of gene families encoding subunits of the five OXPHOS (oxidative phosphorylation) complexes, fundamental and highly conserved in all respiring cells. Although OXPHOS gene families are smaller than the general trend in nuclear gene families, some exceptions are observed, such as three gene families with at least two paralogs in vertebrates. These gene families encode cytochrome c (Cyt c, the electron shuttle protein between complex III and IV), Lipid Binding Protein (LBP, the channel protein of complex V which transfers protons through the inner mitochondrial membrane) and the MLRQ subunit (MLRQ, a supernumerary subunit of the large complex I, with unknown function). We provide a two-step approach, based on structural genomic data, to demonstrate that these gene families should have arisen through WGD (or large segmental duplication) events at the origin of vertebrates and, only afterwards, underwent species-specific events of further gene duplications and loss. In summary, this review reflects the need to apply genome comparative approaches, deriving from both "classical" molecular phylogenetic analysis and "new" genome map analysis, to successfully define the complex evolutionary relations between gene family members which, in turn, are essential to obtain any other comparative phylogenetic or functional results.

Mitochondrial and nuclear DNAs contribute to encode the whole mitochondrial protein complement. The two genomes possess highly divergent features and properties, but the forces influencing their evolution, even if different, require strong coordination. The gene content of mitochondrial genome in all Metazoa is in a frozen state with only few exceptions and thus mitochondrial genome plasticity especially concerns some molecular features, i.e. base composition, codon usage, evolutionary rates. In contrast the high plasticity of nuclear genomes is particularly evident at the macroscopic level, since its redundancy represents the main feature able to introduce genetic material for evolutionary innovations. In this context, genes involved in oxidative phosphorylation (OXPHOS) represent a classical example of the different evolutionary behaviour of mitochondrial and nuclear genomes. The simple DNA sequence of Cytochrome c oxidase I (encoded by the mitochondrial genome) seems to be able to distinguish intra- and inter-species relations between organisms (DNA Barcode). Some OXPHOS subunits (cytochrome c, subunit c of ATP synthase and MLRQ) are encoded by several nuclear duplicated genes which still represent the trace of an ancient segmental/genome duplication event at the origin of vertebrates.

The 5' and 3' untranslated regions of eukaryotic mRNAs play crucial roles in the post-transcriptional regulation of gene expression through the modulation of nucleo-cytoplasmic mRNA transport, translation efficiency, subcellular localization and message stability. UTRdb is a curated database of 5' and 3' untranslated sequences of eukaryotic mRNAs, derived from several sources of primary data. Experimentally validated functional motifs are annotated (and also collated as the UTRsite database) and cross-links to genomic and protein data are provided. The integration of UTRdb with genomic and protein data has allowed the implementation of a powerful retrieval resource for the selection and extraction of UTR subsets based on their genomic coordinates and/or features of the protein encoded by the relevant mRNA (e.g. GO term, PFAM domain, etc.). All internet resources implemented for retrieval and functional analysis of 5' and 3' untranslated regions of eukaryotic mRNAs are accessible at http://www.ba.itb.cnr.it/UTR/.

The mitochondrial genome (mtDNA), due to its peculiar features such as exclusive presence of orthologous genes, uniparental inheritance, lack of recombination, small size and constant gene content, certainly represents a major model system in studies on evolutionary genomics in metazoan. In 800 million years of evolution the gene content of metazoan mitochondrial genomes has remained practically frozen but several evolutionary processes have taken place. These processes, reviewed here, include rearrangements of gene order, changes in base composition and arising of compositional asymmetry between the two strands, variations in the genetic code and evolution of codon usage, lineage-specific nucleotide substitution rates and evolutionary patterns of mtDNA control regions.

This paper reports an intraorder study on the D-loop-containing region of the mitochondrial DNA in rodents. A complete multialignment of this region is not feasible with the exception of some conserved regions. The comparative analysis of 25 complete rodent sequences from 23 species plus one lagomorph has revealed that only the central domain (CD), a conserved region of about 80 bp in the extended termination-associated sequences (ETAS) domain, adjacent to the CD, the ETAS1, and conserved sequence block (CSB) 1 blocks are present in all rodent species, whereas the presence of CSB2 and CSB3 is erratic within the order. We have also found a conserved region of 90 bp located between tRNAPro and ETAS1 present in fat dormouse, squirrel, guinea pig, and rabbit. Repeated sequences are present in both the ETAS and the CSB domain, but the repeats differ in length, copy number, and base composition in different species. The potential use of the D-loop for evolutionary studies has been investigated; the presence/absence of conserved blocks and/or repeated sequences cannot be used as a reliable phylogenetic marker, since in some cases they may be shared by distantly related organisms but not by close ones, while in other ones a relationship between tree topology and presence/absence of such motifs is observed. Better results can be obtained by the use of the CD, which, however, due to its reduced size, when used for tracing a phylogenetic tree, shows some nodes with low statistical support.

We present here a novel method to estimate the site-specific relative variability in large sets of homologous sequences. It is based on the simple idea that the more closely related are the compared sequences, the higher the probability of observing nucleotide changes at rapidly evolving sites. A simulation study has been carried out to support the reliability of the method, which has been applied also to analyzing the site variability of all available human sequences corresponding to the two hypervariable regions of the mitochondrial D-loop.

The phylogenetic relationships among major lineages of rodents is one of the issues most debated by both paleontologists and molecular biologists. In the present study, we have analyzed all complete mammalian mitochondrial genomes available in the databases, including five rodent species (rat, mouse, dormouse, squirrel and guinea-pig). Phylogenetic analyses were performed on H-strand amino acid sequences by means of maximum-likelihood and on H-strand protein-coding and ribosomal genes by means of distance methods. Also, log-likelihood ratio tests were applied to different tree topologies under the assumption of rodent monophyly, paraphyly or polyphyly. The analyses significantly rejected rodent monophyly and showed the existence of two differentiated clades, one containing non-murids (dormouse, squirrel and guinea-pig) and the other containing murids (rat and mouse). Long-branch attraction between murids and the outgroups could not be responsible for the existence of two different rodent clades, as no significant differences in evolutionary rate have been observed, except in the case of the squirrel, which shows a lower rate. The impact of among-site rate variation models on the phylogeny of rodents has been evaluated using the gamma distribution model. Results have shown that relationships among rodents remained unchanged, and the general topology of the tree was not affected, even though some branches were not properly resolved, most likely due to a lack of fit between estimated and real rate heterogeneity parameters.

The existence of a lineage-specific nucleotide substitution rate in mammalian mtDNA has been investigated by analyzing the mtDNA of all available species, that is, 35 complete mitochondrial genomes from 14 mammalian orders. A detailed study of their evolutionary dynamics has been carried out on both ribosomal RNA and first and second codon positions (P12) of H-strand protein-coding genes by using two different types of relative-rate tests. Results are quite congruent between ribosomal and P12 sites. Significant rate variations have been observed among orders and among species of the same order. However, rate variation does not exceed 1.8-fold between the fastest (Proboscidea and Primates) and the slowest (Perissodactyla) evolving orders. Thus, the observed mitochondrial rate variations among taxa do not invalidate the suitability of mtDNA for drawing mammalian phylogeny. Dependence of evolutionary rate differences on variations in mutation and/or fixation rates was examined. Body size, generation time, and metabolic rate were tested, and no significant correlation was observed between them and the taxon-specific evolutionary rates, most likely because the latter might be influenced by multiple overlapping variable constraints.

We report the sequence of the guinea pig p53 cDNA. The comparative analysis of the coding and noncoding regions of p53 cDNAs of all available complete vertebrate sequences has allowed us to single out new conserved signals possibly involved in p53 functional activity. We have focused our attention on the most variable region of the protein, the proline (P)-rich domain, suggested to play a fundamental role in antiproliferative pathways. In this domain we have identified the PXXXXP repeated motif and singled out a common consensus sequence that can be considered a signature for mammalian p53: PXXXXPX{0,4}PX{0,9}PA(T,P,I,)(S,P)WPL. We have demonstrated the significance of the PXXXXP motif in SH3-binding protein and suggested its structure to be a loop. Also, the 5' and 3' untranslated regions (UTRs) of the guinea pig were sequenced, and this study represents the first detailed structural analysis of the UTRs of the p53 mRNAs available in literature. The 5' UTR of guinea pig (233 nt) can be folded into a stable secondary structure resembling that predicted in mouse. The 3' UTR of guinea pig is 771 nt long and shows higher similarity with human than with rodent sequences, having a region of about 350 nt that is deleted in rat and mouse. In the 3' UTR we have identified the presence of a mammalian-wide interspersed repeat sequence and of a cytoplasmic polyadenylation element, which could be involved in translational activation by promoting polyadenylation of mRNA, providing information about a possible mechanism of regulation of p53 expression mediated by the 3' UTR of the mRNA. The observations presented here could open new avenues to targeted mutations and experimental approaches useful in investigating new regulation mechanisms of p53 translation, activity, and stability.

Forty-two patients with hepatocellular carcinoma (HCC) were resected and their tumours were analysed for p53 mutations by GC-clamped denaturing gradient gel electrophoresis (DGGE), single-strand conformation polymorphism (SSCP) and gene sequencing. All the exons have been analysed in this study. Eight of 12 HCCs with cirrhosis due to viral hepatitis and the two patients with sarcomatoid changes displayed p53 mutations. In contrast, no mutation was observed in the fibrolamellar variant (n = 9), non-cirrhotics (n = 13) and alcoholic cirrhosis (n = 6). The mutations observed were in exons 5-8. Two mutations were observed in codons 136 and 213 as well as a T insertion between residues 156 and 157 (exon 5) and these are reported for the first time in HCC. Likewise, the silent mutation polymorphism in codon 213 was noticed in 3 of the 42 patients. Survival analysis of these patients after surgery showed the mean and median survival in patients with wild-type p53 to be 60 and 43 months respectively. In the group with p53 mutations, the mean and median survival was 15 and 12 months. The difference was statistically significant (P= 0.003). Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5

This paper reports the first comprehensive analysis of Displacement loop (D-loop) region sequences from ten different mammalian orders. It represents a systematic evolutionary study at the molecular level on regulatory homologous regions in organisms belonging to a well defined class, mammalia, which radiated about 150 million years ago (Mya). We have aligned and analyzed 26 complete D-loop region sequences available in the literature and the fat dormouse sequence, recently determined in our laboratory. The novelty of our alignment consists of the extensive manual revision of the preliminary output obtained by computer program to optimize sequence similarity, particularly for the two peripheral domains displaying heterogeneity in length and the presence of repeated sequences. The multialignment is available at the WWW site: http://www.ba.cnr.it/dloop.html. Our comparative study has allowed us to identify new conserved sequence blocks present in all the species under consideration and events of insertion/deletion which have important implications in both functional and evolutionary aspects. In particular we have detected two blocks, about 60 bp long, extended termination associated sequences (ETAS1 and ETAS2) conserved in all the organisms considered. Evaluation against experimental work suggests a possible functional role of ETAS1 and ETAS2 in the regulation of replication and transcription and targeted experimental approaches. The analyses on conserved sequence blocks (CSBs) clearly indicate that CSB1 is the only very essential element, common to all mammalian mt genomes, while CSB2 and CSB3 could be involved in different though related functions, probably species specific, and thus more linked to nuclear mitochondrial coevolutionary processes. Our hypothesis on the different functional implications of the conserved elements, CSBs and TASs, reported so far as main regulatory signals, would explain the different conservation of these elements in evolution. Moreover the intra-order comparison of the D-loop regions highlights peculiar features useful to define the evolutionary dynamics of this region in closely related species.

Peculiar evolutionary properties of the subunit 8 of mitochondrial ATP synthase (ATPase8) are revealed by comparative analyses carried out between both closely and distantly related species of echinoderms. The analysis of nucleotide substitution in the three echinoids demonstrated a relaxation of amino acid functional constraints. The deduced protein sequences display a well conserved domain at the N-terminus, while the central part is very variable. At the C-terminus, the broad distribution of positively charged amino acids, which is typical of other organisms, is not conserved in the two different echinoderm classes of the sea urchins and of the sea stars. Instead, a motif of three amino acids, so far not described elsewhere, is conserved in sea urchins and is found to be very similar to the motif present in the sea stars. Our results indicate that the N-terminal region seems to follow the same evolutionary pattern in different organisms, while the maintenance of the C-terminal part in a phylum-specific manner may reflect the co-evolution of mitochondrial and nuclear genes.

The present paper describes the structure of MmtDB—a specialized database designed to collect Metazoa mitochondrial DNA variants. Priority in the data collection is given to the Metazoa species for which a large amount of variants is available, as it is the case for human variants. Starting from the sequences available in the Nucleotide Sequence Databases, the redundant sequences are removed and new sequences from other sources are added. Value-added information are associated to each variant sequence, e.g. analysed region, experimental method, tissue and cell lines, population data, sex, age, family code and information about the variation events (nucleotide position, involved gene, restriction site’s gain or loss). Cross-references are introduced to the EMBL Data Library, as well as an internal cross-referencing among MmtDB entries according to their tissual, heteroplasmic, familiar and aplotypical correlation. MmtDB can be accessed through the World Wide Web at URL http://WWW.ba.cnr.it/~areamt08/MmtDBWWW.htm.

We have analyzed the nad3-rps12 locus for eight angiosperms in order to compare the utility of mitochondrial DNA and edited mRNA sequences in phylogenetic reconstruction. The two coding regions, containing from 25 to 35 editing sites in the various plants, have been concatenated in order to increase the significance of the analysis. Differing from the corresponding chloroplast sequences, unedited mitochondrial DNA sequences seem to evolve under a quasi-neutral substitution process which undifferentiates the nucleotide substitution rates for the three codon positions. By using complete gene sequences (all codon positions) we found that genomic sequences provide a classical angiosperm phylogenetic tree with a clear-cut grouping of monocotyledons and dicotyledons with Magnoliidae at the basal branch of the tree. Conversely, owing to their low nucleotide substitution rates, edited mRNA sequences were found not to be suitable for studying phylogenetic relationships among angiosperms.

Comparative analyses of the mitochondrial tRNA sequences of the sea urchins Arbacia lixula, Paracentrotus lividus and Strongylocentrotus purpuratus revealed that conserved nucleotides may be involved in determining the typical L-shaped spatial conformation of tRNAs. These results shed light on the specific tertiary interactions that allow the folding of the atypical mitochondrial tRNAs into a functional form. A consensus mitochondrial tRNA secondary structure was derived. It shows the presence of nucleotides virtually conserved only in these organisms that represent a sort of molecular signature in sea urchins and suggests a possible physiological role. Finally, we speculate that the non-canonical structure of animal tRNAs, as well as the deviations from the universality of the genetic code, may be due to the reduction in size of the metazoan mitochondrial genome, with the concomitant acquisition of new functions by the mitochondrial tRNAs.

We report a detailed evolutionary study of the RNase P- and RNase MRP- associated RNAs. The analyses were performed on all the available complete sequences of RNase MRP (vertebrates, yeast, plant), nuclear RNase P (vertebrates, yeast), and mitochondrial RNase P (yeast) RNAs. For the first time the phylogenetic distance between these sequences and the nucleotide substitution rates have been quantitatively measured.The analyses were performed by considering the optimal multiple alignments obtained mostly by maximizing similarity between primary sequences. RNase P RNA and MRP RNA display evolutionary dynamics following the molecular clock. Both have similar rates and evolve about one order of magnitude faster than the corresponding small rRNA sequences which have been, so far, the most common gene markers used for phylogeny. However, small rRNAs evolve too slowly to solve close phylogenetic relationships such as those between mammals. The quicker rate of RNase P and MRP RNA allowed us to assess phylogenetic relationships between mammals and other vertebrate species and yeast strains. The phylogenetic data obtained with yeasts perfectly agree with those obtained by functional assays, thus demonstrating the potential offered by this approach for laboratory experiments.

Ribonuclease mitochondrial RNA processing cleaves RNAs from the mammalian mitochondrial main non-coding regulatory region, called the displacement loop. Our data demonstrate that rat cells contain a site-specific ribonuclease mitochondrial RNA processing activity. We found that this enzyme processes the rat mitochondrial displacement-loop RNA substrate at the level of the conserved sequence block 1, a result which is different from that for mouse. This finding correlates with the in-vivo transcriptional analysis of the rat displacement-loop region. Processing by homologous and heterologous ribonuclease mitochondrial RNA enzymes occurs in the same manner, suggesting a conserved mode of substrate recognition.

Mitochondria harbour some of the most critical functions of life [[1],[2],[3]]. As the site of ATP synthesis by oxidative phosphorylation, they are the primary energy‐generating system in almost all eukaryotic cells and are central to programmed cell death. Mitochondria also play major roles in a broad range of other key processes such as the synthesis of amino acids, haem, nucleotides and lipids, ion homeostasis, cell proliferation and motility. It is of major evolutionary and functional significance that they carry their own small DNA genome—a legacy of the endosymbiosis that created mitochondria, and possibly the eukaryotic cell, some 1.5–2 billion years ago [[4]]. In humans, mitochondrial DNA (mtDNA) is a double‐stranded circular molecule of 16.5 kilobase pairs that carries only 37 genes, 13 of …

Nucleic acids, the molecules responsible for the storage and transmission of genetic information, can disclose the beguiling history of the evolution of life on the Earth. In this paper, the molecular mechanisms underlying the evolutionary processes are illustrated together with the methods for the recollection of their history. The impact of molecular data on the evolutionary studies is stressed together with the importance of suitable mathematical models for the treatment of sequence data. Possible future scenarios are conceived for the evolutionary process in relation to environmental phenomena, on account of the new technologies and the development of genetic engineering.

We have analyzed the tRNALys, ATPase8, ATPase6, COIII region of mitochondrial DNA in several human tissues. Beside the mature tRNALys, ATPase8 and ATPase6 common mRNA, and COIII mRNA, we have characterized two new transcripts, called RNA 20 and RNA 21. The RNA 20 is a precursor species which contains the tRNALys plus the ATPase8 and ATPase6 common mRNA; the RNA 21 is an RNA species shorter than the ATPase8 and ATPase6 common mRNA. The relative concentration of the mature with respect to that of the new species proved different in the various tissues. These findings provide new insights into the mitochondrial transcription mechanism opening the question of a possibly regulatory role of the processing on the expression of the mitochondrial genome.