Expression and genomic analyses of Camelus dromedarius T cell receptor delta (TRD) genes reveal a variable domain repertoire enlargement due to CDR3 diversification and somatic mutation.

By a combination of rapid amplification of cDNA ends (RACE) and reverse transcription-polymerase chain reaction (RT-PCR) we identified three T cell receptor delta variable (TRDV) subgroups and five joining (TRDJ) genes expressed in spleen, tonsils and blood of Camelus dromedarius. We provide evidence that the high diversity in sequence and length of the third complementarity determining region (CDR3) is a major component of the TR delta chain variability. Moreover, the identification of the corresponding germline genes allowed us to find out for the first time in a mammalian organism that productively rearranged TRDV genes undergo somatic mutation: the mutation rate of the analysed TRDV4 region is 0.013 per base pair in spleen and 0.009 in blood. The point mutations are scattered throughout the length of the variable domain from framework region FR1 to FR4. This random distribution of the amino acid changes, instead of its CDR clustering observed in immunoglobulins (IG), indicates that somatic mutation in dromedary, while contributing to the development of the TRDV repertoire, is not under antigen selection.

Identification of a new pathotype of Bean yellow mosaic virus (BYMV) infecting blue passion flower and some evolutionary characteristics of BYMV.

The modes of molecular evolution of the coat protein (CP) and 3' non-coding region (NCR) were investigated in Bean yellow mosaic virus (BYMV) isolates, including a new pathotype from blue passion fruit. In phylogenetic analysis, the new pathotype did not cluster with pathotypes or host groups described previously. Intraspecific recombinations involving the entire 3'-NCR and a variable portion of the 3'-terminal region of the CP gene were detected between a broad bean isolate and several isolates from monocots. Since the predicted secondary structure of the 3'-NCR correlated mostly with the botanical origin of isolates, a possible role of the 3'-NCR in BYMV host adaptation is proposed and discussed.

Genome duplication and gene-family evolution: the case of three OXPHOS gene families.

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.

Variability of the mitochondrial genome in mammals at the inter-species/intra-species boundary.

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.

Molecular evolution of the neurotrophin family members and their Trk receptors.

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.

Metazoan OXPHOS gene families: evolutionary forces at the level of mitochondrial and nuclear genomes.

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.

Evolution of ATP synthase subunit c and cytochrome c gene families in selected Metazoan classes.

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.

Evolution of nuclearly encoded mitochondrial genes in Metazoa.

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 evolution of the adenine nucleotide translocase family.

Homologous genes are grouped into families whose evolution may be different in the various organisms. For the variety of the processes and the well-known mechanism of gene gain and gene loss, which takes place in genome evolution, we deal in comparative analyses with a "one-to-many" or a "many-to-many" relationship between homologous genes going from invertebrates to vertebrates. In this scenario, it is important to understand how gene function has been preserved and in addition the innovations originated in a given lineage or species. The phylogenetic relations between gene family members and their molecular clock behavior may be very helpful to elucidate their functional fates in various organisms. This in turn can direct laboratory experiments and practical applications. In order to track the evolutionary history of the ANT gene family, we have collected and analyzed 46 sequences from fungi to mammals. Phylogenetic analyses have been performed on nucleotide and amino acidic sequences which have produced basically the same results. We observe the presence of multiple isoforms both in lower and higher eukaryotic species, thus a "many-to-many" correspondence between genes. The molecular phylogeny of ANT genes, reported in the present study, allows to date the time of divergence of ANT isoforms in various lineages. Furthermore, the logo analysis has been carried out to characterize the conservation features of ANT proteins particularly in their three similar domains originated by duplication.

Comparative genomics: the evolutionary history of the Bcl-2 family.

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.

A comparative study of the porin genes encoding VDAC, a voltage-dependent anion channel protein, in Anopheles gambiae and Drosophila melanogaster.

The protein called voltage-dependent anion-selective channel (VDAC), or mitochondrial porin, forms channels that provide the major pathway for small metabolites across the mitochondrial outer membrane. We have identified and sequenced agporin, a gene of the malaria vector mosquito Anopheles gambiae that conceptually encodes a protein with 73% identity to the VDAC protein encoded by the porin gene in Drosophila melanogaster. By in situ hybridization, we have localized agporin at region 35D on the right arm of A. gambiae chromosome 3, which is homologous to the 2L chromosomal arm of D. melanogaster where the porin gene resides. The comparison of agporin with its putative Drosophila counterpart revealed that both the nucleotide sequence and the structural organization of the two genes are strikingly conserved even though the ancestral lines of A. gambiae and D. melanogaster are thought to have diverged about 250 million years ago. Our results suggest that, while in yeast, plants, and mammals, VDAC isoforms are encoded by small multigene families and are able to compensate for each other at least partially, in A. gambiae a single gene encodes the VDAC protein.

Genetic analysis of HAV strains recovered from patients with acute hepatitis from Southern Italy.

Southern Italy is an endemic area for HAV infection contributing to the majority of Italian hepatitis A cases. Using molecular analysis, HAV strains have been classified in distinct genotypes and subgenotypes. To characterize HAV wild-type strains circulating in Southern Italy, sequence analysis of VP3-VP1 and VP1/2A junction regions of HAV isolates recovered from 25 patients with acute hepatitis during 2000 and 2001 was carried out. HAV isolates showed a degree of identity, after pairwise comparison with one another, ranging from 91.9-100% in the VP3-VP1 junction region and 89.9-100% in the VP1/2A junction region. All strains belonged to genotype I, with 84% (21/25) of samples clustering in subgenotype IA and 16% (4/25) in subgenotype IB. Cocirculation of subgenotypes IA and IB was observed among isolates from 2000, whereas all strains from 2001 were subgenotype IA. In addition, the subgenotype IA strains formed different clusters, one of which was related closely to some Cuban strains, showing a percent similarity of 98.8% in the 168-base pair segment encompassing the VP1/2A junction and the same amino acid substitution. The latter finding suggests that this subgenotype variant circulates also in the Mediterranean area. The results of the phylogenetic analysis confirm the genetic heterogeneity among HAV strains in Western Europe.

Molecular clock and gene function.

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.

Update of AMmtDB: a database of multi-aligned Metazoa mitochondrial DNA sequences.

The AMmtDB database (http://bighost.area.ba.cnr.it/mitochondriome) has been updated by collecting the multi-aligned sequences of Chordata and Invertebrata mitochondrial genes coding for proteins and tRNAs. Links to the multi-aligned mtDNA intraspecies variants, collected in VarMmtDB at the Mitochondriome web site, have been introduced. The genes coding for proteins are multi-aligned based on the translated sequences and both the nucleotide and amino acid multi-alignments are provided. AMmtDB data selected through SRS can be viewed and managed using GeneDoc or other programs for the management of multi-aligned data depending on the user's operative system. The multiple alignments have been produced with CLUSTALW and PILEUP programs and then carefully optimized manually.