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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 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.

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.

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.

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.

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 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.

The effect of the in vivo administration of some inhibitors of transcription and translation processes on the capacity of isolated rat liver mitochondria to synthesize RNA and protein has been studied. The in vivo treatment with cycloheximide, anisomycin and emetine inhibits the vitro synthesis of RNA and proteins, demonstrating that both mitochondrial processes are dependent on cytoplasmic protein synthesis. Of the three inhibitors used only emetine at higher doses acts directly on the mitochondrial proteinsynthesizing machinery because of the inhibition exerted in vitro at the level of mitochondrial ribsomes. The in vitro synthesis of RNA and protein by isolated mitochondria is sensitive to rifamycin SV that, however, has no effect when given in vivo, probably because the antibiotic is unable to reach the mitochondrial RNA polymerase molecule. The adminstration of α-amanitin produces an inhibition only at the level of mitochondrial protein synthesis, suggesting that mitochondrial transcription and translation can be independently controlled by extramitochondrial activities. Comparison of effects on mitochondrial protein synthesis during the course of adminstration of cycloheximide and α-amanitin suggests that the effects of α-amanitin is accounted for through its action on cytoplasmic protein synthesis.

High molecular weight mitochondrial (mt) RNAs were isolated from rat liver mitochondria and hybridized in the presence of excess competitor mt rRNA and/or mt tRNA to restriction fragments of mtDNA. The data reveals that there are a few areas of the mt-genome on which the complementary of these presumptive messenger RNAs is most pronounced. These areas are away from the parts of the genome which are coding for the mt rRNA or containing the D-loop.

We have estimated phylogenetic patterns and rates of nucleotide substitution in the hominoid primates using two different probabilistic models of molecular evolution as applied to three different data sets of nucleic acid sequences. The orang-utan was found to be the out-group of the other hominoids examined. Within the African apes and human clade the sister-group relationship of chimpanzee and human was found to be statistically the best, although the magnitude of the error estimates (a reflection of random statistical fluctuations) makes this conclusion tentative. The ψν-globin data sets were found to be statistically the most consistent and gave estimates of the times of divergence of chimpanzee and human from gorilla and of chimpanzee from human as 7·7 ± 1·5 Ma (Millions of years ago) and 7·4 ± 1·5 Ma respectively, although the speculative nature of these estimates is emphasized. In all cases the calibration point was the assumed divergence of the orang-utan from the remaining hominoids at 14·5 Ma. There was no statistically significant evidence of a slowdown in nucleotide substitution rate for the human lineage, or among the hominoids as a whole with respect to the Old and New World monkeys. We advocate the continued use and development of stochastic models of molecular evolution as a basis for phylogenetic estimation. On this basis one can choose between competing hypotheses of relationship in a statistical manner and can provide estimates of the errors involved in such estimations. The assumptions of all stochastic models are open to test and future refinement.

The 15,697-nucleotide sequence of Paracentrotus lividus mitochondrial DNA is reported. This genome codes for 2 rRNAs, 22 tRNAs, and 12 mRNAs which specify 13 subunits of the mitochondrial inner membrane respiratory complexes. The gene arrangement differs from that of other animal species. The two ribosomal genes 16 S and 12 S are separated by a stretch of about 3.3 kilobase pairs which contains the ND1 and ND2 genes and a cluster of 15 tRNA genes. The ND4L coding sequence is not contained in the ND4 mRNA but has its own mRNA which maps between the tRNA(Arg) and the Co II genes. The main noncoding region, located in the tRNA gene cluster, is only 132 nucleotides long, but contains sequences homologous to the mammalian displacement loop. Other short noncoding sequences are interspersed in the genome: they contain a conserved AT consensus which probably has a role in transcription or RNA processing. As regards the mitochondrial genetic code, the codons AGA and AGG specify serine and are recognized by a tRNA with a GCU anticodon, whereas AUA and AAA code for isoleucine and asparagine rather than for methionine and lysine. Except for ND4L which starts with AUC and ATPase 8 which starts with GUG, AUG is used as the initiation codon. In 11 out of 13 cases the genes terminate with the canonical stop codons UAA or UAG. These observations suggest that during invertebrate evolution each lineage developed its own mechanism of mitochondrial DNA replication and transcription and of RNA processing and translation.

In vitro mitochondrial DNA synthesis by sea urchin Paracentrotus lividus was characterized using either unfertilized eggs or embryos isolated at the mesenchyme blastula stage. The enzyme activity of isolated sea urchin mitochondria showed properties of true mitochondrial DNA dependent DNA polymerase either when unfertilized eggs or mesenchyme blastulae mitochondria were used. The reaction product was extracted and analyzed in neutral sucrose gradient. DNA polymerase activity was also measured at various stages of development. Until early blastula the activity was the same as in unfertilized eggs, then it rose, reaching a maximum at hatching. Thereafter the activity decreased to 50% of the maximum remaining constant up to gastrula stage.

1. The sequence of the small Hind III fragment F of rat-liver mitochondrial DNA (mtDNA) type A and type B was determined in order to investigate the nature of the differences between the two types of mtDNA and to determine its position in the Hind III fragment map. 2. The three differences found were point mutations. No deletion or insertion and no modification was observed. Two of the three differences affect the sequences which are recognition sites for Eco RI, Alu I and Taq I in type A, but not in type B, mtDNA. 3. The presence of an Eco RI restriction site only within the Hind III fragment F of type A shows that the fragment is situated in between the Hind III fragments A and E. 4. In one of the six reading frames, the Hind III fragment F contains the code for a carboxyl-terminal end of a polypeptide in which the three mutations do not lead to alterations in the possible aminoacid sequence. 5. The restriction sites for Taq I and a number of the sites for Alu I and Hae III were mapped. 6. The positions of the Hap II fragment J, and of a Hind III fragment G on the mtDNA were determined.

In order to assay the functionnal significance of the D-loop containing region of mitochondrial DNA, the authors characterized the transcripts from the replication origin of rat mt DNA