Carla De Giorgi

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.

Comparison of the sequences of the D3 expansion of the 26S ribosomal genes reveals different degrees of heterogeneity in different populations and species of Pratylenchus from the Mediterranean region

Plant parasitic nematodes belonging to the genus Pratylenchus, also known as root lesion nematodes, cause serious economic damage to different crop plants. In order to explore genetic structures in different isolates, we investigated several P. thornei,P. neglectus and P. penetrans populations of different geographic origins. The analysis at the species level was also extended to P. penetrans, P. pinguicaudatus, P. vulnus and P. mediterraneus. Sequence analysis of a specific portion of DNA was carried out. In particular, the sequences of the D3 region of the 26S gene were obtained and compared with similar sequences available in databases. The results support the hypothesis that P. penetrans may represent a species complex, while in P. neglectus the intra-species heterogeneity observed is due to intra-individual variability. Furthermore, the specific conservation of some nucleotides in different P. thornei populations indicates their fixation in the rDNA repeats in this species. The presence of these nucleotides, the molecular signature of P. thornei, may assist in determining the nature of nematode infections.

Horizontal transfer of a bacterial gene involved in polyglutamate biosynthesis to the plant-parasitic nematode Meloidogyne artiellia

Analysis of a genomic fragment from the plant parasitic nematode Meloidogyne artiellia revealed the presence of a gene which, in bacteria, is involved in the formation of polyglutamate capsule. Searching of various databases, including the Caenorhabditis elegans genome sequence and the large EST datasets from a variety of parasitic nematodes, showed that no similar genes have been identified in other nematodes or in any other eukaryotic organisms. The M. artiellia gene has a typical eukaryotic structure and its mRNA is present in the intestine. The gene is expressed in all life cycle stages tested. These findings demonstrate horizontal gene transfer may be important in catalyzing the diversification of nematode lineages.

Characterization of the (GAAA) microsatellite region in the plant parasitic nematode Meloidogyne artiellia.

Microsatellites have become one of the most powerful genetic markers in biology. We have used DNA sequencing to characterize a highly variable microsatellite (GAAA) locus in the root-knot nematode Meloidogyne artiellia. The use of microsatellite flanking primers produced four amplification products that are defined as electromorphs, based on conventional length criteria. The sequencing of these four amplification products revealed the presence of new variants in the population due to sequence variability. The sum of electromorphs and sequence polymorphisms resulted in a total of six variants. The high degree of variability in the microsatellite containing region is due not only to variation in the number of tetranucleotide repeats but also to variation (length and site variation) in the flanking regions of the microsatellite. These investigations show that, in spite of the size homoplasy, the variability of the microsatellite flanking sequences of M. artiellia could be used as informative markers for phylogenetic reconstructions.

Different effects of polycyclic aromatic hydrocarbons in artificial and in environmental mixtures on the free living nematode C. elegans

Polycyclic aromatic hydrocarbons (PAHs) are known to exert mutagenic and carcinogenic effects. Research on extracted organic matter (EOM) from environmental mixtures has indicated several mechanisms of intracellular damage in living organisms. The toxic effect of environmental pollutants is usually assessed on cell systems or in single species. We used the model organism Caenorhabditis elegans to compare the effect of synthetic PAHs with that of the EOM from environmental mixtures. The biological effect was measured by monitoring the expression level of some crucial genes, sensitive parameters of the organisms response. The results indicate the ability of C. elegans to counteract damage by mounting a stress‐response only in the presence of EOM. On the other hand the exposure of C. elegans to a mixture of synthetic PAHs determines the silencing of the transcriptional machinery, thus preventing the synthesis of proteins that are important for both the damage repair mechanism and survival itself. The results strongly indicate that the study of environmental toxicant effects at the molecular level may provide information on their mechanism of action. Copyright

Silencing of FAD synthase gene in Caenorhabditis elegans upsets protein homeostasis and impacts on complex behavioral patterns

BACKGROUND FAD synthase is a ubiquitous enzyme that catalyses the last step of FAD biosynthesis, allowing for the biogenesis of several flavoproteins. In humans different isoforms are generated by alternative splicing, isoform 1 being localized in mitochondria. Homology searching in Caenorabditis elegans leads to the identification of two human FAD synthase homologues, coded by the single copy gene R53.1. METHODS The C. elegans R53.1 gene was silenced by feeding. The expression level of transcripts was established by semi-quantitative RT-PCR. Overall protein composition was evaluated by two-dimensional electrophoresis. Enzymatic activities were measured by spectrophotometry and oxygen consumption by polarography on isolated mitochondria. RESULTS From R53.1 two transcripts are generated by trans-splicing. Reducing by 50% the transcription efficiency of R53.1 by RNAi results in a 50% reduction in total flavin with decrease in ATP content and increase in ROS level. Significant phenotypical changes are noticed in knock-down nematodes. Among them, a significant impairment in locomotion behaviour possibly due to altered cholinergic transmission. At biochemical level, impairment of flavoenzyme activities and of some KCN-insensitive oxygen-consuming enzymes is detected. At proteomic level, at least 15 abundant proteins are affected by R53.1 gene silencing, among which superoxide dismutases. CONCLUSION AND GENERAL SIGNIFICANCE For the first time we addressed the existence of different isoforms of FAD-metabolizing enzymes in nematodes. A correlation between FAD synthase silencing and flavoenzyme derangement, energy shortage and redox balance impairment is apparent. In this aspect R53.1-interfered nematodes could provide an animal model system for studying human pathologies with alteration in flavin homeostasis/flavoenzyme biogenesis.

Characterization of nematode resistance gene analogs in tetraploid wheat

Abstract Using a PCR-based approach we have isolated and characterized a putative plant resistance gene from Triticum turgidum whose corresponding resistance gene has been identified in Triticum aestivum . Furthermore, the partial sequences of four new resistance gene analogs (RGA), were used to study their expression profiles in leaves and in roots of two different varieties of T. turgidum . The analysis of the conserved motifs clearly indicates that the sequences we have isolated belong to the NBS-LRR class of plant resistance genes. Expression studies in healthy plants, using the varieties Creso and Latino of T. turgidum , indicate different localizations of RGA transcripts and suggest that they function in the general mechanism of plant surveillance against pathogens.

Cuticlin Gene Organization in Meloidogyne Artiellia

The cuticlins are insoluble, non-collagenous components of the nematode cuticle. They were first described in Ascaris lumbricoides by Fujimoto and Kanaya in 1973. Their insolubility, even in the presence of strong detergents and reducing agents, indicates that they are highly cross-linked by non reducible bonds. Recently, Sebastiano et al. (1991) have identified and cloned two distinct cuticlin genes from Caenorhabditis elegans, namely cut-1 and cut-2.

Increased liver apoptosis and tumor necrosis factor expression in Atlantic bluefin tuna (Thunnus thynnus) reared in the northern Adriatic Sea

The Atlantic bluefin tuna Thunnus thynnus (ABFT) is intensely fished in the Mediterranean Sea to supply a prosperous capture-based mariculture industry. Liver apoptotic structures and tumor necrosis factor (TNF) gene expression were determined in: wild ABFT caught in the eastern Atlantic; juvenile ABFT reared in the central Adriatic Sea; juvenile ABFT reared in the northern Adriatic Sea; adult ABFT reared in the western Mediterranean. The highest density of liver apoptotic structures was found in the juveniles from the northern Adriatic. Two partial TNF cDNAs (TNF1 and TNF2) were cloned and sequenced. TNF1 gene expression was higher in juveniles than in adults. The highest expression of TNF2 was found in the juveniles from the northern Adriatic. These findings might be related to the juvenile exposure to environmental pollutants.

Evolutionary analysis of sea urchin mitochondrial tRNAs: folding of the molecules as suggested by the non-random occurrence of nucleotides.

Abstract 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.