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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.

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.

A detailed computer analysis of the untranslated regions, 5′-UTR and 3′- UTR, of human mRNA sequences is reported. The compositional properties of these regions, compared with those of the corresponding coding regions, indicate that 5′-UTR and 3′-UTR are less affected by the isochore compartmentalization than the corresponding third codon positions of mRNAs. The presence of higher functional constraints in 5′-UTR is also reported. Dinucleotide analysis shows a depletion of CpG and TpA in both sequences. A search for significant sequence motifs using the WORDUP algorithm reveals the patterns already known to have a functional role in the mRNA UTR, and several other motifs whose functional roles remain to be demonstrated. This type of analysis may be particularly useful for guiding site-directed mutagenesis experiments. In addition, it can be used for assessing the nature of anonymous sequences now produced in large amounts in megabase sequencing projects.

The mitochondrial genome is a fundamental component of the eukaryotic domain of life, encoding for several important subunits of the respiratory chain, the main energy production system in cells. The processes by means of which mitochondrial DNA (mtDNA) replicates, expresses itself and evolves have been explored over the years, although various aspects are still debated. In this review, we present several key points in modern research on the role of evolutionary forces in affecting mitochondrial genomes in Metazoa. In particular, we assemble the main data on their evolution, describing the contributions of mutational pressure, purifying, and adaptive selection, and how they are related. We also provide data on the evolutionary fate of the mitochondrial synonymous variation, related to the nonsynonymous variation, in comparison with the pattern detected in the nucleus. Elevated mutational pressure characterizes the evolution of the mitochondrial synonymous variation, whereas purging selection, physiologically due to phenomena such as cell atresia and intracellular mtDNA selection, guarantees coding sequence functionality. This enables mitochondrial adaptive mutations to emerge and fix in the population, promoting mitonuclear coevolution. © The Author(s) 2010.

In this paper we report the comparison of the sequences of the cytochrome oxidase subunit III from three different sea urchin species. Both nucleotide and amino acid sequences have been analyzed. The nucleotide sequence analysis reveals that the sea urchin sequences obey some rules already found in mammals. The base substitution analysis carried out on the sequences of the three species pairs, shows that the evolutionary dynamics of the first and the second codon positions are so slow that do not allow a quantitative measurement of their genetic distances, thus demonstrating that also in these species the COIII gene is strongly conserved during evolution. Changes occurring at the third codon positions indicate that the three species evolved from a common ancestor under different directional mutational pressure. The multi-alignment of the sea urchin proteins indicates the existence of the amino acid sequence motif N R T that represents a possible glycosylation site. Another glycosylation site has been detected in the mammalian cytochrome oxidase subunit III, in a position slightly different. Such an analysis revealed, for the first time, a new functional aspect of this sequence.

The origin of modern man is still a highly debated problem. The new contributions of molecular biologists to paleontological data are numerous but discordant as these data obtained with biosequences are based on different methodologies. In order to obtain a more accurate measure of the genetic distance between extant humans and thus the deepest root of the human tree, we have reanalyzed the sequences of the regulatory region of mitochondrial DNA (D-loop) by using an improved version of the Markov Clock model devised in our laboratory. Our analysis, as well as other studies, supports the African origin of modern man. However the quantitative estimate of mtDNA evolution carried out with the corrected Markov Clock model pushes back irradiation time from Africa to 400±100 Kya. Thus according to our model there would be a genetic continuity betweenHomo erectus andHomo sapiens, who would therefore be the result of regional evolution differentiating himself under appropriate environmental pressures.

The Polymerase Chain reaction technique has been used in order to detect and amplify a specific region of mtDNA, in a total DNA preparation extracted from the sperm of the sea urchin Arbacia lixula. The amplified fragment is the D-loop region which hybridizes with the homologous region extracted from the egg mtDNA. The results demonstrate that mtDNA is present in sperm cell, and, since the replication origin is present it is potentially able to replicate in the zygote. Furthermore, the technique used allowed us to estimate mtDNA copy number in sea urchin sperm, which has never been done before. Our results are that sea urchin sperm cell contains between 4 and 28 mtDNA molecules.

Background A standardized and cost-effective molecular identification system is now an urgent need for Fungi owing to their wide involvement in human life quality. In particular the potential use of mitochondrial DNA species markers has been taken in account. Unfortunately, a serious difficulty in the PCR and bioinformatic surveys is due to the presence of mobile introns in almost all the fungal mitochondrial genes. The aim of this work is to verify the incidence of this phenomenon in Ascomycota, testing, at the same time, a new bioinformatic tool for extracting and managing sequence databases annotations, in order to identify the mitochondrial gene regions where introns are missing so as to propose them as species markers. Methods The general trend towards a large occurrence of introns in the mitochondrial genome of Fungi has been confirmed in Ascomycota by an extensive bioinformatic analysis, performed on all the entries concerning 11 mitochondrial protein coding genes and 2 mitochondrial rRNA (ribosomal RNA) specifying genes, belonging to this phylum, available in public nucleotide sequence databases. A new query approach has been developed to retrieve effectively introns information included in these entries. Results After comparing the new query-based approach with a blast-based procedure, with the aim of designing a faithful Ascomycota mitochondrial intron map, the first method appeared clearly the most accurate. Within this map, despite the large pervasiveness of introns, it is possible to distinguish specific regions comprised in several genes, including the full NADH dehydrogenase subunit 6 (ND6) gene, which could be considered as barcode candidates for Ascomycota due to their paucity of introns and to their length, above 400 bp, comparable to the lower end size of the length range of barcodes successfully used in animals. Conclusion The development of the new query system described here would answer the pressing requirement to improve drastically the bioinformatics support to the DNA Barcode Initiative. The large scale investigation of Ascomycota mitochondrial introns performed through this tool, allowing to exclude the introns-rich sequences from the barcode candidates exploration, could be the first step towards a mitochondrial barcoding strategy for these organisms, similar to the standard approach employed in metazoans.

In order to clarify some controverisal phylogenies such as those regarding the triplet of human, rodent, and cow and the evolutionary position of Lagompopha with respect to other mammals, we have analyzed both nuclear and mitochondrial genes using the stationary Markov model developed in our laboratory. We found that the two sets of genes give different results. In particular the mitochondrial tree showed rabbit linked first to rodents and the the rabbit-rodents branch linked to artiodactyls with human as the outgroup. The most favorite nuclear tree showed human linked first to artiocactlys and the human-artiocactyls branch linked to rabbit with rodents as the outgroup. The obvious questions, (1) which tree is the correct one, or (2) both trees can be incorrect, and (3) how can we explain such an evolutionary pattern, are discussed on the basis of our limited knowledge of factors that influence the clocklike behavior of biological macromolecules.

Glutamine synthetase (EC 6.3.1.2) gene evolution in various animals, plants, and bacteria was evaluated by a general stationary Markov model. The evolutionary process proved to be unexpectedly regular even for a time span as long as that between the divergence of prokaryotes from eukaryotes. This enabled us to draw phylogenetic trees for species whose phylogeny cannot be easily reconstructed from the fossil record. Our calculation of the times of divergence of the various organelle-specific enzymes led us to hypothesize that the pea and bean chloroplast genes for these enzymes originated from the duplication of nuclear genes as a result of the different metabolic needs of the various species. Our data indicate that the duplication of plastid glutamine synthetase genes occurred long after the endosymbiotic events that produced the organelles themselves.

The cytochrome c oxidase enzyme complex of eukaryotes is made up of three mitochondrial-coded subunits and a variable number of nuclear-coded subunits. Some nuclear-coded subunits are present in multiple forms and probably perform a tissue- or development-specific function. A detailed evolutionary analysis of the cytochrome c oxidase subunits that have been sequenced to date is reported here. We have found that gene duplication events from which the liver and heart isoforms of rat subunits VIa and subunit VIII originated can both be dated at about 240 +/- 90 million years ago, long before the radiation of mammalian lineages. Sequence divergence between the processed-type pseudogenes for the subunits IV, VIc and VIII have been estimated. Our results indicate that they arose fairly recently, thus suggesting that retroposition is a continuing process. We show that the rate of silent substitution in mitochondrial-coded subunits is 5-10 times higher than in nuclear-coded subunits; on the other hand replacement rates, although differing from gene to gene, are roughly of the same order of magnitude in both nuclear and mitochondrial genes. In the case of most of the nuclear-coded proteins we observed a slightly greater similarity between rats and cow, which agrees with the data obtained for mitochondrial-coded subunits.

The efficiency of AUG start codon recognition in translation initiation is modulated by its sequence context. Here we investigated a non-redundant set of 5914 human genes and show that this context is different in genes located in different isochores. In particular, of the two main consensus start sequences, RCCaugR is five-fold more represented than AARaugR in genes from the GC-rich H3 isochores compared to genes from the GC-poor L isochores. Furthermore, genes located in GC-rich isochores have shorter 5′ UTRs and stronger avoidance of upstream AUG than genes located in GC-poor isochores. This suggests that genes requiring highly efficient translation are located in GC-rich isochores and genes requiring fine modulation of expression are located in GC-poor isochores. This is in agreement with independent data from the literature concerning the location of housekeeping and tissue-specific genes, respectively.

BackgroundTo a greater or lesser extent, eukaryotic nuclear genomes contain fragments of their mitochondrial genome counterpart, deriving from the random insertion of damaged mtDNA fragments. NumtS (Nuclear mt Sequences) are not equally abundant in all species, and are redundant and polymorphic in terms of copy number. In population and clinical genetics, it is important to have a complete overview of NumtS quantity and location. Searching PubMed for NumtS or Mitochondrial pseudo-genes yields hundreds of papers reporting Human NumtS compilations produced byin silicoor wet-lab approaches. A comparison of published compilations clearly shows significant discrepancies among data, due both to unwise application of Bioinformatics methods and to a not yet correctly assembled nuclear genome. To optimize quantification and location of NumtS, we produced a consensus compilation of Human NumtS by applying various bioinformatics approaches.ResultsLocation and quantification of NumtS may be achieved by applying database similarity searching methods: we have applied various methods such as Blastn, MegaBlast and BLAT, changing both parameters and database; the results were compared, further analysed and checked against the already published compilations, thus producing the Reference Human Numt Sequences (RHNumtS) compilation. The resulting NumtS total 190.ConclusionThe RHNumtS compilation represents a highly reliable reference basis, which may allow designing a lab protocol to test the actual existence of each NumtS. Here we report preliminary results based on PCR amplification and sequencing on 41 NumtS selected from RHNumtS among those with lower score. In parallel, we are currently designing the RHNumtS database structure for implementation in the HmtDB resource. In the future, the same database will host NumtS compilations from other organisms, but these will be generated only when the nuclear genome of a specific organism has reached a high-quality level of assembly.

The experiments here reported demonstrate that the main non-coding region of rat mitochondrial DNA is symmetrically transcribed. We have identified stable heavy and light transcripts, whose pattern is rather complex, in the D-loop region of rat mitochondrial DNA. Their relative concentrations have been determined. We detected heavy transcripts which encompass the whole D-loop and more abundant heavy RNA species which we interpreted as transcripts terminating downstream of the 3' end of the last coded gene (Thr-tRNA). The processed heavy RNA species contain polyA, suggesting a strict association between cleavage and polyadenylation. The pattern of light transcripts shows a long RNA, which, starting from the light strand promoter, covers the whole segment, and shorter RNA species which seems to be actively processed at the level of the conserved sequence boxes, probably acting as primers. The symmetric transcription of the D-loop containing region of rat mitochondrial DNA, and in particular the presence of stable transcripts complementary to the putative RNA primers, suggest that mechanisms mediated by interaction between complementary transcripts (antisense RNAs) might play a role in the regulation of mitochondrial DNA replication and expression.

Neurotrophins are structurally related proteins regulating brain development and function. Molecular evolution studies of neurotrophins and their receptors are essential for understanding the mechanisms underlying the coevolution processes of these gene families and how they correlate with the increased complexity of the vertebrate nervous system. In order to improve our current knowledge of the molecular evolution of neurotrophins and receptors, we have collected all information available in the literature and analyzed the genome database for each of them. Statistical analysis of aminoacid and nucleotide sequences of the neurotrophin and Trk family genes was applied to both complete genes and mature sequences, and different phylogenetic methods were used to compare aminoacid and nucleotide sequences variability among the different species. All collected data favor a model in which several rounds of genome duplications might have facilitated the generation of the many different neurotrophins and the acquisition of specific different functions correlated with the increased complexity of the vertebrate nervous system during evolution. We report findings that refine the structure of the evolutionary trees for neurotrophins and Trk receptors families, indicate different rates of evolution for each member of the two families, and newly demonstrate that the NGF-like genes found in Fowlpox and Canarypox viruses are closely related to reptile NGF.

Genomic variations represent the molecular basis of the biodiversity of living organisms on which selection operates to generate evolution. In eukaryotes, genomic variability can be experienced in both nuclear and organellar, i.e. mitochondrial and plastid (where present), genomes, which can follow completely different evolution pathways, as revealed by comparative genomics analyses. In Metazoa, for which a substantial number of complete genome sequences are available (nuclear, but mainly mitochondrial), we are just starting to grasp the selective pressures operating on some basic features of the genome as a whole. In this brief review, we discuss the variability of the mitochondrial metazoan genome, with particular reference to mitochondrial DNA in mammals. In light of the recent assumption that a small segment of mitochondrial DNA may be used, particularly in Metazoa, as a species marker, some data on mitochondrial gene variability at the inter-species/intra-species boundary are reported. Intra-species variability has been evaluated in four mammalian species, Homo sapiens, Bos taurus, Sus scrofa and Canis familiaris, whereas the relationship between intra- and inter-species variability has been investigated in Bos taurus and Bos indicus.

Background The p53 gene family consists of the three genes p53, p63 and p73, which have polyhedral non-overlapping functions in pivotal cellular processes such as DNA synthesis and repair, growth arrest, apoptosis, genome stability, angiogenesis, development and differentiation. These genes encode sequence-specific nuclear transcription factors that recognise the same responsive element (RE) in their target genes. Their inactivation or aberrant expression may determine tumour progression or developmental disease. The discovery of several protein isoforms with antagonistic roles, which are produced by the expression of different promoters and alternative splicing, widened the complexity of the scenario of the transcriptional network of the p53 family members. Therefore, the identification of the genes transactivated by p53 family members is crucial to understand the specific role for each gene in cell cycle regulation. We have combined a genome-wide computational search of p53 family REs and microarray analysis to identify new direct target genes. The huge amount of biological data produced has generated a critical need for bioinformatic tools able to manage and integrate such data and facilitate their retrieval and analysis. Description We have developed the p53FamTaG database (p53 FAMily TArget Genes), a modular relational database, which contains p53 family direct target genes selected in the human genome searching for the presence of the REs and the expression profile of these target genes obtained by microarray experiments. p53FamTaG database also contains annotations of publicly available databases and links to other experimental data. The genome-wide computational search of the REs was performed using PatSearch, a pattern-matching program implemented in the DNAfan tool. These data were integrated with the microarray results we produced from the overexpression of different isoforms of p53, p63 and p73 stably transfected in isogenic cell lines, allowing the comparative study of the transcriptional activity of all the proteins in the same cellular background. p53FamTaG database is available free at http://www2.ba.itb.cnr.it/p53FamTaG/ Conclusion p53FamTaG represents a unique integrated resource of human direct p53 family target genes that is extensively annotated and provides the users with an efficient query/retrieval system which displays the results of our microarray experiments and allows the export of RE sequences. The database was developed for supporting and integrating high-throughput in silico and experimental analyses and represents an important reference source of knowledge for research groups involved in the field of oncogenesis, apoptosis and cell cycle regulation.

The oxidative phosphorylation (OXPHOS) is the primary energy-producing process of all aerobic organisms and the only cellular function under the dual control of both the mitochondrial and the nuclear genomes. Functional characterization and evolutionary study of the OXPHOS system is of great importance for the understanding of many as yet unclear aspects of nucleus-mitochondrion genomic co-evolution and co-regulation gene networks. The MitoDrome database is a web-based database which provides genomic annotations about nuclear genes of Drosophila melanogaster encoding for mitochondrial proteins. Recently, MitoDrome has included a new section annotating genomic information about OXPHOS genes in Drosophila pseudoobscura and Anopheles gambiae and their comparative analysis with their Drosophila melanogaster and human counterparts. The introduction of this new comparative annotation section into MitoDrome is expected to be a useful resource for both functional and structural genomics related to the OXPHOS system.

To investigate the integrated evolution of mitochondrial and nuclear genomes in the eukaryotic cell, we have focused our attention on OXPHOS (oxidative phosphorylation) gene families which encode proteins involved in the main mitochondrial function. The present study reports the phylogenetic analysis of two OXPHOS gene families: ATP synthase subunit c (or lipid binding protein, LBP) and Cytochrome c (Cytc). Both gene families possess a higher expansion trend than the typically low duplication rate of OXPHOS genes in Metazoa, but follow a completely different evolutionary history, especially in mammals. LBP is represented by three well conserved isoforms in all mammals (P1, P2, P3): only P3 possesses a clearly conserved isoform in all Vertebrates, P1 and P2 were already present before the bird-mammal divergence and there are preliminary evidence from the in silico analysis that P1, the most evolutionary divergent isoform, is poorly expressed and not regulated by NRF1. In contrast, Cytc family presents at least two duplicated genes in all the analysed Vertebrates, is subject to a high expansion trend, especially of processed pseudogenes in mammals, and some events of gain and loss of function can be supposed.

Background Mitochondria are sub-cellular organelles that have a central role in energy production and in other metabolic pathways of all eukaryotic respiring cells. In the last few years, with more and more genomes being sequenced, a huge amount of data has been generated providing an unprecedented opportunity to use the comparative analysis approach in studies of evolution and functional genomics with the aim of shedding light on molecular mechanisms regulating mitochondrial biogenesis and metabolism. In this context, the problem of the optimal extraction of representative datasets of genomic and proteomic data assumes a crucial importance. Specialised resources for nuclear-encoded mitochondria-related proteins already exist; however, no mitochondrial database is currently available with the same features of MitoRes, which is an update of the MitoNuc database extensively modified in its structure, data sources and graphical interface. It contains data on nuclear-encoded mitochondria-related products for any metazoan species for which this type of data is available and also provides comprehensive sequence datasets (gene, transcript and protein) as well as useful tools for their extraction and export. Description MitoRes http://www2.ba.itb.cnr.it/MitoRes/ consolidates information from publicly external sources and automatically annotates them into a relational database. Additionally, it also clusters proteins on the basis of their sequence similarity and interconnects them with genomic data. The search engine and sequence management tools allow the query/retrieval of the database content and the extraction and export of sequences (gene, transcript, protein) and related sub-sequences (intron, exon, UTR, CDS, signal peptide and gene flanking regions) ready to be used for in silico analysis. Conclusion The tool we describe here has been developed to support lab scientists and bioinformaticians alike in the characterization of molecular features and evolution of mitochondrial targeting sequences. The way it provides for the retrieval and extraction of sequences allows the user to overcome the obstacles encountered in the integrative use of different bioinformatic resources and the completeness of the sequence collection allows intra- and interspecies comparison at different biological levels (gene, transcript and protein).

All Metazoan nuclear genomes underwent a continuous process of both complete and partial genetic material gain and loss. The forces modulating these events are also subject to the strict interaction between nuclear and mitochondrial (mt) genome. In this context we investigate the evolution of nuclear genes encoding proteins which target the mitochondrion, with a particular attention to genes involved in oxidative phosphorylation (OXPHOS), one of the most ancient and conserved functions. To examine thoroughly the evolutionary strategies that preserve OXPHOS and coordinate the two cellular genomes, a comparative analysis has been carried out for 78 OXPHOS gene families in several Metazoa (insects, tunicates, fishes and mammals). We demonstrate that the duplication rate of OXPHOS genes increases passing from invertebrates to vertebrates consistently with the total increase in genome size, but all species are prone to negatively select OXPHOS duplicates compared to the general trend of nuclear gene families. These results are consistent with the ‘balance hypothesis’ and, at least in insects, the expression of duplicate genes is low and strongly testis-biased.

The complete genome sequences with their annotations are a considerable resource in biology, particularly in understanding the global structure of the genetic material at the molecular level. The reason why some eukaryotic genomes contain large quantities of apparently unnecessary DNA, namely pseudogenes, while others seem to invest in more efficient thinning processes or are equipped with protection systems against parasitic elements still remains a mystery. Several genome-wide surveys have been undertaken to identify pseudogenes in the completely sequenced genome, bringing to light some differences both in their amount and distribution. Since pseudogenes are important resources in evolutionary and comparative genomics - as 'molecular fossils' - in this paper, a survey on the origins, features, abundance and localisation of the different pseudogenes is reported. As an example of genes producing processed pseudogenes, some experimental data obtained in the authors' laboratories from the study of a nuclear gene coding for the mitochondrial transcription factor A (mtTFA), a key regulator of mitochondrial biogenesis, are also reported.

This chapter focuses on the most popular methods used in the study of the evolution of the mitochondrial genome. This theoretically implies the knowledge of the structure and function of this organellar genome, which has several peculiar features. Therefore, its study often requires the adaptation and/or modification of already existing methodologies, and sometimes even the implementation of new tools.

Molecular phylogenies based on the molecular clock require the comparison of orthologous genes. Orthologous and paralogous genes usually have very different evolutionary fates. In general, orthologs keep the same functions in species, whereas, particularly over a long time span, paralogs diverge functionally and may become pseudogenes or get lost. In eukaryotic genomes, because of the degree of redundancy of genetic information, homologous genes are grouped in gene families, the evolution of which may differ greatly between the various organisms. This implies that each gene in a species does not always have an ortholog in another species and thus, due to multiple duplication events following a speciation, many orthologous clades of paralogs are generated. We are often dealing with a one-to-many or many-to-many relationship between genes. In this paper, we analyze the evolution of two gene families, the p53 gene family and the porin gene family. The evolution of the p53 family shows a one-to-many gene relationship going from invertebrates to vertebrates. In invertebrates only a single gene has been found, while in vertebrates three members of the family, namely p53, p63, and p73, are present. The evolution of porin (VDAC) genes (VDAC1, VDAC2, and VDAC3) is an example of a many-to-many gene relationship going from yeast to mammals. However, the porin gene redundancy found in invertebrates and possibly in some fishes may indicate a tendency to duplicate the genetic material, rather than a real need for function innovation.

Summary: DNAfan (DNA Feature ANalyzer) is a tool combining sequence-filtering and pattern searching. DNAfan automatically extracts user-defined sets of sequence fragments from large sequence sets. Fragments are defined by annotated gene feature keys and co- or non-occurring patterns within the feature or close to it. A gene feature parser and a pattern-based filter tool localizes and extracts the specific subset of sequences. The selected sequence data can subsequently be retrieved for analyses or further processed with DNAfan to find the occurrence of specific patterns or structural motifs. DNAfan is a powerful tool for pattern analysis. Its filter features restricts the pattern search to a well-defined set of sequences, allowing drastic reduction in false positive hits. Availability: http://bighost.ba.itb.cnr.it:8080/Framework

Analyses of mitochondrial and nuclear gene sequences have often produced different mammalian tree topologies, undermining confidence in the merit of molecular approaches with respect to "traditional" morphological classification. The recent sequencing of the complete mitochondrial genomes of two additional rodents (Spalax judaei and Jaculus jaculus) and one lagomorph (Ochotona princeps) has prompted us to reinvestigate the issue. Using Bayesian phylogenetics, we found phylogenetic relationships between mammalian species highly congruent with previous results based on nuclear genes. Our results show the existence of four primary lineages of placental mammals: Xenarthra, Afrotheria, Laurasiatheria, and Euarchontoglires. Relationships between and within these lineages strongly suggest that the gene trees may also be congruent with the underlying species phylogeny.

The discovery of the p73 and p63 genes, homologous to p53 tumor suppressor has uncovered a family of transcription factors and widened the scenario of cell cycle control and apoptosis. We have identified a putative p53-responsive element in the human adenosine deaminase (ADA) gene, an important enzyme involved in nucleotide metabolism, the deficit of which causes the inhibition of DNA synthesis and repair. Here, we demonstrate that the ectopic expression of p73 isoforms leads to the ADA gene upregulation, showing for the first time a correlation between p73 and ADA. We found that p73 promotes ADA gene expression following a dNTP unbalance generated by ADA enzyme deficiency and 2′deoxyadenosine accumulation. The abrogation of p73 transcriptional activity by the specific dominant-negative p73DD abolishes ADA induction. By contrast, the ADA gene does not appear to be a functional p53 target in the physiological conditions we tested. On the whole, our results contribute to the emerging picture that p73 could play a different role from p53 in normal growth and development by inducing alternative target genes, which are not shared by p53.

The Bcl-2 family is a huge family composed of various members, occurring in all animals, which are key regulators of apoptosis, the cell death program critical for cell survival and development, tissue homeostasis, and protection against pathogens. The members of the Bcl-2 family can be divided into pro-apoptotic and anti-apoptotic proteins. A delicate balance between these members exists in each cell and the regulations of these two groups of proteins determines whether the cell survives or undergoes apoptosis. Bcl-2 family proteins are characterized by distinct domains. All members possess at least one of the four motifs known as Bcl-2 homology domains (BH1 to BH4). Most pro-survival members which can inhibit apoptosis facing a wide variety of cytotoxic insults, contain at least BH1 and BH2 domains; those most similar to Bcl-2 have all four BH domains. All the pro-apoptosis family members possess BH3 domain which is the central domain. For the first time, a global phylogenetic analysis of all Bcl-2 family members is presented here. We have analyzed the genes known so far that have a different composition of the functional domains BH1, BH2, BH3 and BH4. The analyses were performed both on complete sequences (124 sites analyzed) and on single domains. We present the results obtained using both approaches. We have also analyzed the amino acid profile and the degree of conservation of the BH3 domains of pro- and anti-apoptotic proteins. The results of our phylogenetic analyses show that a clear-cut clustering into pro- and anti-apoptotic products, reproducible with different evolutionary methods, could also be obtained by analyzing restricted areas such as the BH1 and BH2 domains. It is noteworthy that even when the analysis is performed only on the BH3 domain, we have two clear-cut clusters. The evolutionary analysis of gene family members is a valuable tool to predict their functions and guide experimental assays to validate predictions. Once the functions of all the components are known, it will be possible to study the process in a holistic way.