C. De Giorgi

Nematode chitin synthases: gene structure, expression and function in Caenorhabditis elegans and the plant parasitic nematode Meloidogyne artiellia

Abstract. Although the presence of chitin in nematodes is well documented little is known about its synthesis in this phyletic group. The recently completed genome sequence of Caenorhabditis elegans predicts two sequences with homology to chitin synthases (chitin-UDP acetyl-glucosaminyl transferase; EC 2.4.1.16). We show that these genes are differentially expressed in a pattern that may reflect different functional roles. One gene is expressed predominantly in the adult hermaphrodite (the main egg-producing stage in the nematode) and later larval stages, which is consistent with a role in production of chitin for the eggshell. The other gene, however, is expressed in the cells that form the pharynx, and only in the period directly preceding a moult. These data suggest that the product of this gene is involved in synthesis of the feeding apparatus, which is replaced during each moult. We have also isolated a full-length genomic sequence of a chitin synthase orthologue from the plant parasitic nematode Meloidogyne artiellia. The single gene present in M. artiellia shows an expression pattern that is consistent with a role for the protein in production of the eggshell.

Comparative study of liver vitellogenin gene expression and oocyte yolk accumulation in wild and captive Atlantic bluefin tuna (Thunnus thynnus L.).

The sequence of vitellogenin A (VgA) and vitellogenin B (VgB) cDNAs in Atlantic bluefin tuna (Thunnus thynnus L.) were determined, and vitellogenin expression levels in the liver and oocyte yolk accumulation were compared in wild and captive-reared individuals. Liver and ovary samples were taken from 31 individuals reared experimentally in three commercial Atlantic bluefin tuna fattening sites in the Mediterranean Sea and from 33 wild individuals caught by commercial traps during the fishs migration towards their Mediterranean spawning grounds. The total length of VgA cDNA was 5585 nucleotides and that of VgB was 5267 nucleotides. The identity and similarity between deduced amino acid sequences of VgA and VgB were 60% and 78%, respectively. The Atlantic bluefin tuna VgA and VgB amino acid sequences have high similarities with those of other teleost fishes. Relative levels of VgA and VgB mRNAs were low in April, increased significantly during the reproductive period in May and June, and declined in July. There was a trend towards higher relative levels of VgA and VgB mRNAs in captive fish compared to wild individuals during the reproductive period. The surface occupied by eosinophilic yolk granules in fully vitellogenic oocytes, as well as the frequency of oocytes in late vitellogenesis, was significantly higher in captive compared to wild individuals. The study suggests that the experimental conditions under which Atlantic bluefin tuna individuals were reared allowed the occurrence of normal vitellogenesis, based on gene expression of VgA and VgB in the liver and yolk accumulation in the oocytes. The higher yolk accumulation and frequency of vitellogenic oocytes observed in the ovaries of captive fish suggest that improvements in feeding practices may result in an improved vitellogenic process.

Modulation of expression at the level of splicing of cut-1 RNA in the infective second-stage juvenile of the plant parasitic nematode Meloidogyne artiellia

Abstract The cut-1 gene coding for cuticlin-1 has been isolated from the plant parasitic nematode Meloidogyne artiellia. The sequence of the cut-1 gene was compared with the corresponding sequence from the free-living nematode Caenorhabditis elegans. The high degree of similarity between the amino acid sequences, together with the occurrence of characteristic sequence motifs, indicates that the cuticlin-1 is a non-collagenous component of the cuticle also in plant parasitic nematodes. Studies on the expression pattern during the development of M. artiellia indicate that there is a burst of expression of this gene during moulting. Then, the expression rate is reduced in the infective juveniles, which migrate in the soil. In the sedentary females, in contrast, no expression is detected, while in the males which move freely through the soil, the gene is expressed and the transcript fully processed. These data strongly suggest that the gene is developmentally regulated. It is proposed that the production of cuticlin plays an important role in determining the mechanical properties of the cuticle. Furthermore, evidence is provided to indicate that the modulation of cut-1 expression is achieved by regulation of the cis-splicing mechanism in the infective second-stage juvenile.

Mitochondrial genome in animal cells: structure, organization, and evolution

In the past decade, the development of new DNA, RNA, and protein technologies has greatly incremented the knowledge about the organization and expression of mitochondrial DNA. The complete base sequence of mitochondrial DNA of several animals is known and many data are rapidly accumulating on the mitochondrial genomes of other systems. Here we discuss the results so far obtained that disclosed unexpected features of mitochondrial genetics. Furthermore, mitochondrial DNA has become established as a powerful tool for evolutionary studies in animals. Evidences are preented demonstrating that the evolution of mitochondrial DNA has proceeded in different ways in the various taxonomic groups. Data on heteroplasmic animals, which demonstrate the rapid evolution of mitochondrial DNA, are also presented.

Expression of vitellogenin receptor gene in the ovary of wild and captive Atlantic bluefin tuna (Thunnus thynnus)

The cDNA sequences of vitellogenin receptor proteins (VgR(+) and VgR(-)), containing or lacking the O-linked sugar domain, were determined in Atlantic bluefin tuna (Thunnus thynnus L.). VgR(-) gene expression in the ovary was compared in captive-reared and wild Atlantic bluefin tuna during the reproductive cycle. Gonad samples from adult fish were sampled from 2008 to 2010 from stocks reared in captivity at different commercial fattening operations in the Mediterranean Sea and from wild individuals caught either by traditional tuna traps during their migration towards the spawning grounds in the Mediterranean Sea or by the long-line artisanal fishery. In addition, juvenile male and female Atlantic bluefin tuna were sampled from a farming facility, to obtain baseline information and pre-adulthood amounts of VgR(-). The total length of VgR(+) cDNA was 4006 nucleotides (nt) and that of VgR(-) was 3946 nt. Relative amounts of VgR(-) were greater in juvenile females and in those adults having only previtellogenic oocytes (119 ± 55 and 146 ± 26 folds more than juvenile males, respectively). Amounts of VgR(-) were less in individuals with yolked oocytes (ripening stage, May-June) and increased after spawning in July (92 ± 20 and 113 ± 13 folds more than juvenile males in ripening and post-spawning fish, respectively). These data suggest that regulation of VgR(-) is not under oestrogen control. During the ripening period, greater VgR(-) gene expression was observed in wild fish than in fish reared in captivity, possibly because of (a) differences in water temperature exposure and/or energy storage, and/or (b) an inadequate diet in reared Atlantic bluefin tuna.

LINEAGE-SPECIFIC EVOLUTION OF ECHINODERM MITOCHONDRIAL ATP SYNTHASE SUBUNIT 8

Peculiar evolutionary properties of the subunit 8 of mitochondrial ATP synthase (ATPase8) are revealed by comparative analyses carried out between both closely and distantly related species of echinoderms. The analysis of nucleotide substitution in the three echinoids demonstrated a relaxation of amino acid functional constraints. The deduced protein sequences display a well conserved domain at the N-terminus, while the central part is very variable. At the C-terminus, the broad distribution of positively charged amino acids, which is typical of other organisms, is not conserved in the two different echinoderm classes of the sea urchins and of the sea stars. Instead, a motif of three amino acids, so far not described elsewhere, is conserved in sea urchins and is found to be very similar to the motif present in the sea stars. Our results indicate that the N-terminal region seems to follow the same evolutionary pattern in different organisms, while the maintenance of the C-terminal part in a phylum-specific manner may reflect the co-evolution of mitochondrial and nuclear genes.

Synthesis of poly(A) containing RNA in isolated mitochondria from rat liver

Rat liver mitochondria are able to incorporate labelled nucleotides in RNA species which are retained by Millipore filters or oligo(dT)-cellulose column. The radioactivity retained was completely RNAse sensitive when [3H] UTP was used as radioactive precursor whereas by using [3H] ATP this fraction was only 30% RNAse sensitive. These results demonstrate that rat liver mitochondria are able to synthesize poly(A) containing RNA species and furthermore that the polyadenylation reaction occurs within the organelle.

Direct evidence that restriction endonucleases may under estimate the degree of divergence between molecules

SummaryWe studied two polymorphic forms of mtDNA extracted from A. lixula eggs. In order to compare and to quantitate the variability, we sequenced specific regions of the two molecules. In this way, we obtained a precise measurement of the variability within two haplotypes. We also obtained a direct demonstration that some differences in nucleotide sequence can escape detection when restriction endonuclease analysis is used. Our results underline the unreliability of the use of restriction mapping to estimate divergence between relatively short and closely related DNA sequences.

The mitochondrial genome in sea urchin

mtDNA has been completely sequenced in three different sea urchin species. The comparison of the nucleotide sequences demonstrates that the sizes, the structure and the gene organization are highly conserved in the three sea urchin genomes. The nucleotide composition indicates that the mtDNA of Arbacia lixula, in contrast with that of Paracentrotus lividus and Strongylocentrotus purpuratus, shows a bias toward the use of T and against the use of C. Therefore, the G+T distribution between the two strands, which gives rise to the base composition skewing in Paracentrotus lividus and in Strongylocentrotus purpuratus, is absent in Arbacia lixula. Analysis of molecular evolution suggests that the separation between the two different orders of the three sea urchin species might be a more recent event than the previous estimates, derived from fossil records.

The Physical Map of the Various Transcripts of Rat Liver Mitochondrial DNA

It is well known that mitochondria and chloroplasts possess their own DNA. Eukaryotic cell contains therefore at least two or, in the case of plants and plastid-containing micro-organisms, even three different genetic systems (1–4). The organelle genomes show a pattern of cytoplasmic and maternal inheritance. For a number of features in lower organisms this has been known already for a long time. Recently it has been shown that the same holds for the mitochondrial genome of animal cells (5–8). Although the genetic function of the organelle genomes is not yet known in all details, it is evident they are indispensable and vital for eukaryotic organisms. For the expression of their genomes, the organelles depend on the nucleus. They constitute heteronomous genetic entities, their expression and continuity being ensured by the existence within the organelles of elements coded for and synthesized by the main genetic system of the cell, the nuclear-cytoplasmic genetic system. This implies that a much higher level of genetic complexity is reached in the eukaryotic cell as compared to the prokaryotic cell. Although also in prokaryotes extrachromosomal genetic elements such as plasmids may exist, the obvious difference is that the products of transcription and translation of these elements are not obligatory for the cells to survive as is the case for the mitochondrial and chloroplast genomes.