Mitochondrial DNA sequence variations in some Italian wild boar populations.

In order to investigate the relationships between Italian wild boar and major pig breeds, we studied the genetic variability of four wild boar populations in Italy (Arezzo, Pisa, Parma, Bergamo) using a 533-bp fragment of the mitochondrial control region. Sixty-nine wild boar samples were analysed, allowing the identification of 10 distinct haplotypes, which involve a total of 15 single nucleotide polymorphisms. Phylogenetic and network analyses were performed also considering several sequences of wild and domesticated forms available in the databases. The Bayesian phylogenetic tree and the Median-Joining network analyses show three main groups: the Italian (IT), European (EU) and Asian (AS) clades. The IT clade corresponds to the Maremma endemic wild boar population and also includes Sardinian individuals, while the EU and AS groups include wild boars as well as domestic pig breeds. Only two individuals from Pisa cluster in the IT group, whereas two haplotypes from Bergamo cluster in the AS group and all other samples cluster in the EU clade. These findings suggest that in Italy wild boar populations have a mixed origin, both EU and AS, and that an interbreeding between wild and domesticated strains has probably occurred. Eight of the 10 wild boars coming from the Migliarino-San Rossore-Massaciuccoli Regional Park (Pisa) belong to H2 and H3 haplotypes, and cluster into the EU clade, suggesting that this regional park is not anymore exclusive of the endemic Maremma wild boar.

Evolution of the mitochondrial genome of Metazoa as exemplified by comparison of congeneric species.

The mitochondrial genome (mtDNA) of Metazoa is a good model system for evolutionary genomic studies and the availability of more than 1000 sequences provides an almost unique opportunity to decode the mechanisms of genome evolution over a large phylogenetic range. In this paper, we review several structural features of the metazoan mtDNA, such as gene content, genome size, genome architecture and the new parameter of gene strand asymmetry in a phylogenetic framework. The data reviewed here show that: (1) the plasticity of Metazoa mtDNA is higher than previously thought and mainly due to variation in number and location of tRNA genes; (2) an exceptional trend towards stabilization of genomic features occurred in deuterostomes and was exacerbated in vertebrates, where gene content, genome architecture and gene strand asymmetry are almost invariant. Only tunicates exhibit a very high degree of genome variability comparable to that found outside deuterostomes. In order to analyse the genomic evolutionary process at short evolutionary distances, we have also compared mtDNAs of species belonging to the same genus: the variability observed in congeneric species significantly recapitulates the evolutionary dynamics observed at higher taxonomic ranks, especially for taxa showing high levels of genome plasticity and/or fast nucleotide substitution rates. Thus, the analysis of congeneric species promises to be a valuable approach for the assessment of the mtDNA evolutionary trend in poorly or not yet sampled metazoan groups.

Evolutionary genomics in Metazoa: the mitochondrial DNA as a model system.

One of the most important aspects of mitochondrial (mt) genome evolution in Metazoa is constancy of size and gene content of mtDNA, whose plasticity is maintained through a great variety of gene rearrangements probably mediated by tRNA genes. The trend of mtDNA to maintain the same genetic structure within a phylum (e.g., Chordata) is generally accepted, although more recent reports show that a considerable number of transpositions are observed also between closely related organisms. Base composition of mtDNA is extremely variable. Genome GC content is often low and, when it increases, the two complementary bases distribute asymmetrically, creating, particularly in vertebrates, a negative GC-skew. In mammals, we have found coding strand base composition and average degree of gene conservation to be related to the asymmetric replication mechanism of mtDNA. A quantitative measurement of mtDNA evolutionary rate has revealed that each of the various components has a different evolutionary rate. Non-synonymous rates are gene specific and fall in a range comparable to that of nuclear genes, whereas synonymous rates are about 22-fold higher in mt than in nuclear genes. tRNA genes are among the most conserved but, when compared to their nuclear counterparts, they evolve 100 times faster. Finally, we describe some molecular phylogenetic reconstructions which have produced unexpected outcomes, and might change our vision of the classification of living organisms.

Analysis of oligonucleotide AUG start codon context in eukariotic mRNAs.

The AUG start codon context features have been investigated by analyzing eukaryotic mRNAs belonging to various taxonomic groups. The functional relevance of each specific position surrounding the AUG start codon has been established as a function of the measured shift between base composition observed at that particular position, and base composition averaged over all the 5'untranslated regions. A more detailed analysis carried out on human genes belonging to different isochores showed significant isochore-specific fea-tures that cannot be explained only by a mutational bias effect. The most represented heptamers spanning from position -3 to +4 with respect to the initiator AUG have been determined for mRNAs belonging to different taxonomic groups and a web page utility has been set up (http://bigarea.area.ba.cnr.it:8000/BioWWW/ATG.html) to determine the relative abundance of a user submitted oligonucleotide context in a given species or taxon.

Evolution of the mitochondrial genetic system: an overview.

Mitochondria, semi-autonomous organelles possessing their own genetic system, are commonly accepted to descend from free-living eubacteria, namely hydrogen-producing alpha-proteobacteria. The progressive loss of genes from the primitive eubacterium to the nucleus of the eukaryotic cell is strongly justified by the Muller rachet principle, which postulates that asexual genomes, like mitochondrial ones, accumulate deleterious and sublethal mutations faster than sexual genomes, like the nucleus. According to this principle, the mitochondrial genome would be doomed to death; instead, we observe that the mitochondrial genome has a variable size and structure in the different organisms, though it contains more or less the same set of genes. This is an example of genetic conservation versus structural diversity. From an evolutionary point of view the genetic system of organelles is clearly under strong selective pressure and for its survival it needs to utilize strategies to slow down or halt the ratchet. Anyway, the mitochondrial genome changes with time, and the rate of evolution is different for both diverse regions of the mtDNA and between lineages, as demonstrated in the case of mammalian mt genomes. We report here our data on the evolution of the mitochondrial DNA in mammals which demonstrate the suitability of mtDNA as a molecular tool for evolutionary analyses.

Structural and functional features of eukaryotic mRNA untranslated regions.

The crucial role of the non-coding portion of genomes is now widely acknowledged. In particular, mRNA untranslated regions are involved in many post-transcriptional regulatory pathways that control mRNA localization, stability and translation efficiency. We review in this paper the major structural and compositional features of eukaryotic mRNA untranslated regions and provide some examples of bioinformatic analyses for their functional characterization.

The complete mitochondrial DNA sequence of the rabbit, Oryctolagus cuniculus.

The nucleotide sequence of the complete mitochondrial DNA (mtDNA) molecule of the rabbit (Oryctolagus cuniculus, order Lagomorpha) was determined. The length of the molecule is 17,245 nt, but the length is not absolute due to the presence of different numbers of repeated motifs in the control region. The organization and gene contents of the mtDNA of the rabbit conform to those of other eutherian species. The putative secondary structures of the tRNAs of the rabbit have been described. These structures as well as the structure of the L-strand origin of replication comply with those characteristic for eutherians in general. The compositional differences between the two mtDNA strands have also been detailed.

Lineage-specific evolutionary rate in mammalian mtDNA.

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.

Glutamine synthetase gene evolution in bacteria.

The evolution of the prokaryotic glutamine synthase (GS) genes, namely the GSI and GSII isoforms, has been investigated using the second codon positions, which have previously proven to behave as a good molecular clock. Our data confirm the early divergence between prokaryotic and eukaryotic GSII before the splitting between plants and animals. The phylogenetic tree of the GSI isoforms shows Archaebacteria to be more closely related to Eubacteria than to Eukaryotes. This finding is confirmed by the phylogenetic analysis carried out on both large and small subunits of rRNA. However, differently from the rRNA analyses, Crenarchaeota and Euryarchaeota Archaebacteria, as well as high- and low-GC gram-positive bacteria, appear to be polyphyletic. We provide evidence that the observed polyphyly of Archaebacteria might be only apparent, resulting from a gene duplication event preceding the split between Archaebacteria and Eubacteria and followed by the retention of only one isoform in the extant lineages. Both gram-negative bacteria and high-GC gram-positive bacteria, which appear closely related, have GS activity regulated by an adenylylation/deadenylylation mechanism. A lateral gene transfer from Archaebacteria to low-GC eubacteria is invoked to explain the observed polyphyly of gram-positive bacteria.

Molecular classification of living organisms.

Recent studies in molecular evolution have generated strong conflicts in opinion as to how world living organisms should be classified. The traditional classification of life into five kingdom has been challenged by the molecular analysis carried out mostly on rRNA sequences, which supported the division of the extant living organisms into three major groups: Archaebacteria, Eubacteria, and Eukaryota. As to the problem of placing the root of the tree of life, the analysis carried out on a few genes has provided discrepant results. In order to measure the genetic distances between species, we have carried out an evolutionary analysis of the glutamine synthetase genes, which previously have been revealed to be good molecular clocks, and of the small and large rRNA genes. All data demonstrate that archaebacteria are more closely related to eubacteria than to eukaryota, thus supporting the classical division of living organisms into two main superkingdoms, Prokaryota and Eukaryota.

Nucleotide substitution rate of mammalian mitochondrial genomes.

We present here for the first time a comprehensive study based on the analysis of closely related organisms to provide an accurate determination of the nucleotide substitution rate in mammalian mitochondrial genomes. This study examines the evolutionary pattern of the different functional mtDNA regions as accurately as possible on the grounds of available data, revealing some important "genomic laws." The main conclusions can be summarized as follows. (1) High intragenomic variability in the evolutionary dynamic of mtDNA was found. The substitution rate is strongly dependent on the region considered, and slow- and fast-evolving regions can be identified. Nonsynonymous sites, the D-loop central domain, and tRNA and rRNA genes evolve much more slowly than synonymous sites and the two peripheral D-loop region domains. The synonymous rate is fairly uniform over the genome, whereas the rate of nonsynonymous sites depends on functional constraints and therefore differs considerably between genes. (2) The commonly accepted statement that mtDNA evolves more rapidly than nuclear DNA is valid only for some regions, thus it should be referred to specific mitochondrial components. In particular, nonsynonymous sites show comparable rates in mitochondrial and nuclear genes; synonymous sites and small rRNA evolve about 20 times more rapidly and tRNAs about 100 times more rapidly in mitochondria than in their nuclear counterpart. (3) A species-specific evolution is particularly evident in the D-loop region. As the divergence times of the organism pairs under consideration are known with sufficient accuracy, absolute nucleotide substitution rates are also provided.

Asymmetrical directional mutation pressure in the mitochondrial genome of mammals.

The base composition of 25 complete mammalian mitochondrial (mt) genomes has been analyzed taking into account all three codon positions (P1230 and fourfold degenerate sites (P4FD) of H-strand genes. In the nontranscribed L strand, G is the less represented base and A is the most represented one in all cases, while C and T differ among species. H-strand protein-coding genes show an asymmetric distribution of the four bases between the two strands. The asymmetry indexes AT and GC skews on P4FD are much higher than those on P123, suggesting the existence of asymmetrical directional mutation pressure. Relationships between the compositional features and transcription of replication processes have been investigated in order to find a possible mechanism that could explain the origin of this asymmetry. AT and GC skews, the base composition in fourfold degenerate sites, and the number of variable sites for each gene are significantly correlated with the duration of single-stranded state of the H-stranded genes during replication. We tested different replication-related hypotheses, such as the existence of biased dNTP pools, gamma DNA polymerase mispairing, and the asymmetric replication itself. Most of them failed to explain the observed results, hydrolytic deaminations being the only one in agreement with our data. Thus, we hypothesize that one of the crucial processes for the origin of asymmetric and biased base composition of mammalian mitochondrial genomes is the spontaneous deamination of C and A in the H strand during replication.

GeneSyn: a tool for detecting conserved gene order across genomes.

UNLABELLED: GeneSyn is a software tool that allows automatic detection of conserved gene order from annotated genomes. AVAILABILITY: Available free of charge for Unix/Linux/Cygwin platforms at ftp://159.149.110.11/pub/GeneSyn_1.0/ SUPPLEMENTARY INFORMATION: ftp://159.149.110.11/pub/GeneSyn_1.0/

Evolution of the nad3-rps12 gene cluster in angiosperm mitochondria: comparison of edited and unedited sequences.

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.

The guinea-pig is not a rodent.

In 1991 Graur et al. raised the question of whether the guinea-pig, Cavia porcellus, is a rodent. They suggested that the guinea-pig and myomorph rodents diverged before the separation between myomorph rodents and a lineage leading to primates and artiodactyls. Several findings have since been reported, both for and against this phylogeny, thereby highlighting the issue of the validity of molecular analysis in mammalian phylogeny. Here we present findings based on the sequence of the complete mitochondrial genome of the guinea-pig, which strongly contradict rodent monophyly. The conclusions are based on cumulative evidence provided by orthologically inherited genes and the use of three different analytical methods, none of which joins the guinea-pig with myomorph rodents. In addition to the phylogenetic conclusions, we also draw attention to several factors that are important for the validity of phylogenetic analysis based on molecular data.

MitoNuc and MitoAln: two related databases of nuclear genes coding for mitochondrial proteins.

Mitochondria, besides their central role in energy metabolism, have recently been found to be involved in a number of basic processes of cell life and to contribute to the pathogenesis of many degenerative diseases. All functions of mitochondria depend on the interaction of nuclear and organellar genomes. Mitochondrial genomes have been extensively sequenced and analysed and the data collected in several specialised databases. In order to collect information on nuclear coded mitochondrial proteins we developed MitoNuc and MitoAln, two related databases containing, respectively, detailed information on sequenced nuclear genes coding for mitochondrial proteins in Metazoa and yeast, and the multiple alignments of the relevant homologous protein coding regions. MitoNuc and MitoAln retrieval through SRS at http://bio-www.ba.cnr.it:8000/srs6/ can easily allow the extraction of sequence data, subsequences defined by specific features and nucleotide or amino acid multiple alignments.

Functional annotation of a full-length mouse cDNA collection.

The RIKEN Mouse Gene Encyclopaedia Project, a systematic approach to determining the full coding potential of the mouse genome, involves collection and sequencing of full-length complementary DNAs and physical mapping of the corresponding genes to the mouse genome. We organized an international functional annotation meeting (FANTOM) to annotate the first 21,076 cDNAs to be analysed in this project. Here we describe the first RIKEN clone collection, which is one of the largest described for any organism. Analysis of these cDNAs extends known gene families and identifies new ones.

Analysis of the mouse transcriptome based on functional annotation of 60,770 full-length cDNAs.

Only a small proportion of the mouse genome is transcribed into mature messenger RNA transcripts. There is an international collaborative effort to identify all full-length mRNA transcripts from the mouse, and to ensure that each is represented in a physical collection of clones. Here we report the manual annotation of 60,770 full-length mouse complementary DNA sequences. These are clustered into 33,409 'transcriptional units', contributing 90.1% of a newly established mouse transcriptome database. Of these transcriptional units, 4,258 are new protein-coding and 11,665 are new non-coding messages, indicating that non-coding RNA is a major component of the transcriptome. 41% of all transcriptional units showed evidence of alternative splicing. In protein-coding transcripts, 79% of splice variations altered the protein product. Whole-transcriptome analyses resulted in the identification of 2,431 sense-antisense pairs. The present work, completely supported by physical clones, provides the most comprehensive survey of a mammalian transcriptome so far, and is a valuable resource for functional genomics.