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Riboflavin (Rf), or vitamin B2, is the precursor of FMN and FAD, redox cofactors of several dehydrogenases involved in energy metabolism, redox balance and other cell regulatory processes. FAD synthase, coded by FLAD1 gene in humans, is the last enzyme in the pathway converting Rf into FAD. Mutations in FLAD1 gene are responsible for neuromuscular disorders, in some cases treatable with Rf. In order to mimic these disorders, the Caenorhabditis elegans (C. elegans) gene orthologue of FLAD1 (flad-1) was silenced in a model strain hypersensitive to RNA interference in nervous system. Silencing flad-1 resulted in a significant decrease in total flavin content, paralleled by a decrease in the level of the FAD-dependent ETFDH protein and by a secondary transcriptional down-regulation of the Rf transporter 1 (rft-1) possibly responsible for the total flavin content decrease. Conversely an increased ETFDH mRNA content was found. These biochemical changes were accompanied by significant phenotypical changes, including impairments of fertility and locomotion due to altered cholinergic transmission, as indicated by the increased sensitivity to aldicarb. A proposal is made that neuronal acetylcholine production/release is affected by alteration of Rf homeostasis. Rf supplementation restored flavin content, increased rft-1 transcript levels and eliminated locomotion defects. In this aspect, C. elegans could provide a low-cost animal model to elucidate the molecular rationale for Rf therapy in human Rf responsive neuromuscular disorders and to screen other molecules with therapeutic potential.

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 organism's 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 © 2011 John Wiley & Sons, Ltd.

The sequence of a 13.423 nucleotide genomic fragment has been determined for the plant parasitic nematode Meloidogyne artiellia. It contains an entire rDNA cluster, the bordering intergenic regions and portions of the flanking coding regions. The sequence analysis of the rDNA repeats suggests homogeneity in M. artiellia, thus providing a further indication of the usefulness of these genes for the diagnostic identification of this species. The comparison of the secondary structures of the internal transcribed spacer 2 region in several Meloidogyne species indicates that RNA folding predictions can be used as a tool of potential diagnostic relevance. The other ribosomal gene, 5S rDNA, has been demonstrated to be functional and located near the trans-spliced leader sequences, in the same arrangement found in the distantly related nematode Caenorhabditis elegans but never in other Meloidogyne thus providing species-specific markers for the identification of several Thylenchida parasitic nematodes.

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.

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.

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.

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.

A study concerning the detection and characterization of a DNA fragment from plant-parasitic nematodes to be used as a molecular marker for the identification of different nematodes is described. A fragment of DNA, which is known to consist of a variable region flanked by two conserved regions, has been studied by using PCR amplification. A portion of about 600 nucleotides at the 5’end of the larger rRNA gene has been amplified in different nematodes, using heterologous primers which hybridize with the conserved regions. The results obtained clearly indicate that the same primers can be used for the amplification of this segment in nematodes of different species and of different genera: Meloidogyne artiellia, M. incognita, Xiphinema index, X. diversicaudatum and Globodera pallida. The amplified regions have been partially sequenced. The nucleotide sequences have been analysed by comparison with the published sequence of Caenorhabditis elegans.

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.

Le recenti politiche europee sono rivolte alla valutazione dei possibili effetti nocivi di miscele complesse di sostanze chimiche (REACH) utilizzando metodi che escludano l'impiego di vertebrati. In questa relazione vengono illustrati gli effetti di tossicità di particolato atmosferico su due organismi modello: Caenorhabditis elegans e Rhodobacter sphaeroides. I campioni di particolato sono stati raccolti in due siti della città di Bari e da questi sono stati estratti i composti organici solubili in acetone ed esano. I risultati ottenuti da tali test hanno mostrato un effetto tossico sul nematode C. elegans, mentre nel caso del batterio fotosintetico gli effetti sono limitati ad una leggera diminuzione della velocità di crescita. Gli sviluppi futuri di questo studio saranno orientati verso frazioni granulometriche quali PM1 e nano particolato e verso altri sistemi modello.

C. elegans provides a suitable model to study basic and conserved nematode biology. The short life-cycle, adult size, easy maintenance in large numbers and the tractability of C. elegans facilitate its use in translational biology. The C. elegans genome project has greatly assisted the mapping, sequencing and annotation of parasitic nematode genomes. Furthermore, the development of RNAi in C. elegans has not only aided functional analysis of conserved proteins in parasitic species but has also provided a platform for the development of RNAi in these target species. Application of RNAi in plant-parasitic nematodes has enhanced gene functional analysis as well as highlighted the potential for its development as a transgenic control.

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.

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 organism's 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.

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 full-length cDNA and the corresponding gene of the heat shock protein 90, Mt-Hsp90, were isolated and characterized in the plant parasitic nematode Meloidogyne artiellia. The full-length Mt-Hsp90 cDNA contained a 5' untranslated region (UTR) of 45 bp with the 22 bp trans-spliced leader SL1, an ORF of 2172 bp encoding a polypeptide of 723 amino acids and a 3' UTR of 191 bp. The deduced amino acid sequence of Mt-hsp90 showed high similarity with other known Hsp90s. Five conserved amino acid signatures indicated that Mt-hsp90 is a cytosolic member of the Hsp90 family. The gene consists of 10 exons and 9 introns, a more expanded gene structure compared to the corresponding Caenorhabditis elegans gene, daf-21. Mt-hsp90 gene was constitutively expressed at high levels in all developmental stages of M. artiellia. Egg masses and second stage juveniles (J2s) were exposed at 5 degrees and 30 degrees C for different periods of times in order to explore the impact of adverse temperature on Mt-hsp90 gene expression. Expression levels of Mt-hsp90 were examined by fluorescent real-time PCR. At 30 degrees C a burst of expression for Mt-hsp90 was observed in J2s after 2 h of heat shock treatment, then expression dropped with longer exposing times, although remaining still relatively high after 24 h. This temperature did not affect Mt-hsp90 gene expression in the egg masses. However, egg masses exposed at 5 degrees C showed a little but gradual increase in the mRNA level with time. By contrast, no significant changes in the Mt-hsp90 level were observed in J2s exposed to cold. These data show that egg masses and J2s exposed to cold and heat stresses have different expression profiles suggesting that Mt-Hsp90 may provide a link between environmental conditions and the life cycle of the nematode.

Plant-parasitic nematodes are important pests of crop plants worldwide, and also among the most difficult animals to identify. Their identification based on nuclear ribosomal DNA (rDNA) cistron (18S, 28S, and 5.8S RNA genes, and internal transcribed spacers, ITS1 and ITS2) is becoming a popular tool. Sequences from nuclear ribosomal RNA repeats have been used to demonstrate the identity of isolates from various hosts and to unravel the relationships of cryptic and complex species. In addition, the availability of RNA sequences allows study of phylogenetic relationships between nematodes, also for more complete understanding of their biology as agricultural pests. PPNEMA is a plant-parasitic nematode bioinformatic resource. It consists of a database of ribosomal cistron sequences from various species grouped according to nematode genera, and a search system allowing data to be extracted according to both text and pattern searching. PPNEMA offers to the scientific community a preprocessed archive of plant parasitic nematode sequences useful for nematologists. It is a tool to retrieve plant nematode multialigned sequences for phylogenetic studies or to recognize a nematode by comparing its rDNA sequence with the PPNEMA available genus specific multialignments.

Animals and plants both respond rapidly to pathogens by inducing the expression of defence-related genes. Within this context, a prominent role has been assigned to the lysozyme. In the present study we isolated and carried out detailed analysis of the lysozyme gene in the plant nematode Meloidogyne artiellia. The expression of lysozyme was up-regulated following exposure of M. artiellia juveniles to the Gram-negative bacterium Serratia marcescens. On the other hand, when isolated eggs containing embryos at various developmental stages were challenged with bacteria, no increase in lysozyme expression was detected. Evidence of lysozyme expression regulation was obtained in the case of adult male and females worms collected from soil. The lysozyme gene was expressed solely in the nematode intestine and, as it is predicted to be secreted, may protect the nematode from microbial infections originating in the intestinal lumen or in the pseudocoelom. This paper demonstrates, to our knowledge for the first time, the immune response to infection in a plant parasitic nematode.

The Ras-MAPK signal transduction pathway controls multiple developmental events and is involved in the processing of olfactory information in the free living nematode Caenorhabditis elegans. We have studied the Ras-MAPK pathway in the plant parasitic nematode Meloidogyne artiellia. The genes Mt-let-60, Mt-lin-45, Mt-mek-2 and Mt-mpk-1 have been isolated and sequenced. Each of them shows a high level of sequence similarity to its presumed ortholog in C. elegans and key functional domains are structurally conserved. Furthermore, we show that the M. artiellia recombinant MEK-2 protein can phosphorylate and activate the M. artiellia recombinant MPK-1 and the recombinant MEK-2 itself can be phosphorylated and activated by immunoprecipitated mammalian Raf. Surprisingly, the Mt-lin-45 message is not detectable in freshly emerged juveniles or in male specimens, suggesting that it may be quickly degraded in these life stages.

A single chitin synthase gene is responsible for chitin production in the eggshells of the plant parasitic nematode Meloidogyne artiellia. In this paper we describe a functional analysis of this gene using RNAi as well as further analysis of two similar genes from the free-living nematode Caenorhabditis elegans. The parasitic life-style of M. artiellia required the development of a novel method for delivery of dsRNA to nematode eggs that may be of utility in other experimental systems. C. elegans chitin synthase genes were silenced by feeding nematodes bacteria expressing appropriate chitin synthase dsRNA from a plasmid vector, while M. artiellia egg masses were soaked in dsRNA solution. The results obtained demonstrated that the synthesis of chitin continues to take place in nematode eggs within the egg sac in the parasitic nematode, and that the removal of this activity affects egg development in both C. elegans and M. artiellia. The method described here provides a new way of investigating gene function in plant parasitic nematodes allowing the validity of potential target genes for novel control methods to be assessed. Furthermore, since intact egg cells within the gelatinous matrix of M. artiellia are permeable to dsRNA, eggs of other nematodes may also be similarly permeable to dsRNA and therefore amenable to use with dsRNAi.

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.

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.

A gene (cut-1) coding for a cuticular protein, cuticlin-1, has been isolated and sequenced in the plant parasitic nematode, Meloidogyne artiellia. The nucleotide sequence revealed a typical eukaryotic-like organization, exons and introns bordered by canonical sequences. The 5' flanking region presents nematode-specific sequence motifis, including a trans-splicing signal. Studies on the expression of this gene demonstrated that, while in the adult females cut-1 is not expressed, the removal of the introns occurs in the eggs. These experiments also indicate that cis-splicing precedes the processing of the 5' untranslated region. In no case has a trans-spliced transcript been detected.

The complete nucleotide sequence (15,719 nucleotides) of the mitochondrial DNA (mtDNA) from the sea urchin Arbacia lixula is presented. The comparison of gene arrangement between different echinoderm orders of the same class provides evidence that the gene organization is conserved within the same echinoderm class. The peculiarities of sea urchin mtDNA features, already described, are confirmed by the A. lixula mtDNA sequence. The comparison of the entire sequences of mtDNA among A. lixula, Paracentrotus lividus, and Strongylocentrotus purpuratus allowed us to detect peculiar features, common to the three sea urchin species, that can represent the molecular signature of the mt genome in the sea urchin group. Analysis of the nucleotide composition indicates that A. lixula mtDNA, in contrast with the mtDNA of other sea urchins, shows a bias in the use of T and tends to avoid the use of C, most evident in the neutral part of the molecule, such as the third codon positions. This observation indicates that the three sea urchin mtDNAs evolve under different mutation pressure. Analysis of the sequence evolution allowed us to confirm the phylogenetic tree. However, the absolute divergence time, calculated on the basis of paleontological estimates, largely diverged from the expected one.

Mitochondria from all organisms studied to date have their own mitochondrial DNA (mtDNA), distinct from that of the nucleus. Animal mtDNA carries the genetic information for two rRNAs, a complete set of tRNAs and only a few essential components of the respiratory complexes: three subunits of the cytochrome oxidase (COI, COII and COIII), the cytochrome b (Cyt b), the subunits 6 and 8 of the Fo ATPase complex and several subunits of the NADH dehydrogenase (ND1-ND6 and ND4L). In addition, each molecule contains a control region in which signals of DNA duplication and transcription are included (for a review see Wolstenholme, 1993).

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.

Two polymorphic forms of mitochondrial DNA in the eggs of the sea urchin Arbacia lixula have been detected by BamHI endonuclease analysis. The two genomes are unequally distributed throughout the population. Few individuals are heteroplasmic, that is containing both types of mitochondrial DNA, while the majority contain only one type. Mapping of several other restriction enzymes is also reported. The results show a new example of mitochondrial DNA polymorphism in which restriction site variants instead of restriction size variants of mitochondrial DNA in single individuals are detected. The findings presented may be a useful tool for the study of mitochondrial genes segregation during development of the sea urchin.

Dialyzed rat liver cytosol possesses the ability to stimulate the incorporation of [35S]-methionine into mitochondrial proteins. The addition of dialyzed cytosol stimulates sevenfold the rate of protein synthesis by isolated mitochondria after 10 minutes incubation and the extent of this stimulation increases with the time. Various control experiments indicate that the effect is not artifactual nor does it appear to be brought about by GDP or GTP.

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.

Mitochondrial DNA, RNA and protein synthesis in normal and hypothyroid rat liver between the ages of -3 and 21 days were followed. In normal rats DNA polymerase activity and protein synthesis behaved similarly, showing two peaks of activity, one at -3 and the other at 21 days of age. RNA polymerase activity did not change between days -3 and 14, whereas it increased by 21 days of age. Hypothyroidism delayed the developmental pattern of DNA polymerase activity, affected RNA polymerase activity only at 21 days, whereas it inhibited protein synthesis at birth and in the third week of life. The cytochrome aa3 content appeared to be affected by hypothyroidism at birth and at 21 days of age.