Rosanna del Gaudio

Organization and nucleotide sequence of the cluster of five histone genes in the polichaete worm Chaetopterus variopedatus: first record of a H1 histone gene in the phylum Annelida.

Abstract. Histone genes were identified and their nucleotide sequences were determined in the polychaete marine worm Chaetopterus variopedatus. The genes are organized in about 390 clusters of 7.3 kbp. Each cluster contains one copy of the five histone genes. The H1 histone gene present in the clusters is the first ever isolated in the phylum Annelida. The cluster has the unique peculiarity that all genes contain both the replication-dependent and the replication-independent 3′ mRNA termination signals. Despite the differences in cluster organization and transcription polarity of the individual histone genes between C. variopedatus and Platynereis dumerilii, the other annelid in which histone genes have been studied, phylogenetic analysis of the encoded amino acid sequences clearly groups together those two organisms in a tree in which the other studied worms find closely related positions on the same evolutionary branch.

A network system for vitellogenin synthesis in the mussel Mytilus galloprovincialis (L.)

The aim of this study is to assess, by RT‐PCR, in situ hybridization, electron microscopy, and immunohistochemistry, the site/s of vitellogenin (VTG) synthesis in the mussel Mytilus galloprovincialis. Our investigations demonstrate that, among the analyzed tissues, the synthesis of VTG occurs only in the female gonad, that is, within the oocyte and follicle and connective cells. Such a synthesis is just evident in early vitellogenic oocytes, whose cytoplasm is characterized by numerous RER cisternae and an extended Golgi complex surrounded by nascent yolk platelets. The synthesis of VTG goes on in vitellogenic oocytes assuming a pear form, and progressively reduces once the oocyte shows the pear or polygonal form, typical of those oocytes that have concluded the growth. The expression of VTG occurs also within follicle (auxiliary) and connective cells. In particular, it is noteworthy that follicle cells are characterized by numerous RER cisternae and an active Golgi complex surrounded by numerous vesicles and vacuoles containing electron dense material. The same material is also present along their plasma membrane, within the intercellular space between oocyte and follicle cells, and finally within invaginations of the oocyte surface, thus suggesting a VTG transfer to the oocyte via endocytosis. Differently, no VTG synthesis was observed within digestive gland. All together the findings here reported strongly suggest that in M. galloprovincialis, inside the gonad, the VTG synthesis occurs in the oocyte (autosynthesis) and in the follicle and adipogranular cells (heterosynthesis). J. Cell. Physiol. 228: 547–555, 2013.

Genome methylation of the marine annelid worm Chaetopterus variopedatus: methylation of a CpG in an expressed H1 histone gene.

Hydrolysis by methylation‐dependent restriction enzymes shows that the genomic DNA of the polychaete annelid worm Chaetopterus variopedatus is methylated. Electrophoretic analyses of the digestion products indicate that the degree of methylation is lower in adult tissues than in sperm and embryonic DNA. 5‐Methylcytosine was identified by HPLC, absorption spectroscopy and mass spectrometry analyses of free bases obtained by acid hydrolysis of the DNA. An average value of 1.6% methylated cytosines was determined in sperm DNA. Partial methylation was also found in an actively expressed H1 histone gene. This is the first time that genomic DNA methylation is demonstrated to occur in a worm.

Differential Expression of Duplicated Genes for Prothymosin Alpha During Zebrafish Development

We show that ptma, a single copy gene found in all organisms investigated so far, is duplicated in zebrafish. The two genes, ptmaa and ptmab, are individually controlled as indicated by their different expression patterns during embryonic development. Only the ptmab transcript is observed at 4 and 8 hpf of development in all embryonic cells, whereas both genes are expressed at later stages as revealed by in situ hybridization studies. In most cases, the two genes are expressed in the same territories, but only the ptmaa transcript was found in the trigeminal ganglion and in endodermal pouches. In the eye, at 72 hpf, the ptmaa and ptmab transcripts were found in amacrine cells, whereas only the ptmab transcript appeared in horizontal cells. The existence of two prothymosin genes indicates that their function in cell proliferation and differentiation is more complex in fishes than in mammals. Developmental Dynamics 237:1112–1118, 2008.

Cloning and Molecular Characterization of the First Innexin of the Phylum Annelida—Expression of the Gene During Development

A novel member of the innexin family (cv-inx) has been isolated from the annelid polychaete worm Chaetopterus variopedatus using a PCR approach on genomic DNA and sequence analysis on genomic DNA clones. The gene is present in a HindIII-HindIII segment of 2250 bp containing an uninterrupted open reading frame of 1196 bp encoding a protein of 399 amino acids. The predicted protein shows the typical structural features of innexins and consensus sites for phosphorylation. Analyses on genomic DNA demonstrate that cv-inx is a single copy gene with no introns in the coding region, exactly corresponding to the cDNA sequence. The gene expression is regulated during development as shown by Northern blots analyses of the RNA and by immunoreaction with antibodies against the protein at several embryonic stages. The finding of an innexin in the phylum Annelida, outside of the Ecdysozoa clade, and its peculiar gene structure suggest the necessity to reconsider the current hypothesis on the origin and evolution of gap junctional proteins.

Characterization of a new variant DNA (cytosine-5)-methyltransferase unable to methylate double stranded DNA isolated from the marine annelid worm Chaetopterus variopedatus.

The enzyme S‐adenosylmethionine‐DNA (cytosine‐5)‐methyltransferase has been identified, first time for invertebrates, in embryos of the marine polychaete annelid worm Chaetopterus variopedatus. The molecule has been isolated from embryos at 15 h of development. It is a single peptide of about 200 kDa molecular weight, cross‐reacting with antibodies against sea urchin DNA methyltransferase. The enzymatic properties of the molecule are similar to those of Dnmt1 methyltransferases isolated from other organisms, but with the peculiarity to be unable to make ‘de novo’ methylation on double stranded DNA.

Characterization and developmental expression pattern of the relaxin receptor rxfp1 gene in zebrafish

We report the gene characterization, the cDNA cloning and the temporal and spatial expression pattern of the relaxin receptor rxfp1 gene in the zebrafish Danio rerio. The zebrafish rxfp1 gene has the same syntenic genomic organization, and a similar exon‐intron structure to the homologue human gene. Furthermore, the deduced Rxfp1 protein sequence shows a high degree of amino acid similarity when compared with the human protein and the conservation of all amino acid identity necessary for the binding with relaxin. Our results show that rxfp1 gene is active either during embryogenesis or in the adult organism, showing a wide expression pattern. Moreover, we provide the first description of rxfp1 spatial expression pattern during embryo development, showing that the transcript is already present at the early developmental stage and is distributed in all of the embryonic cells until somitogenesis. Starting at the pharyngula stage the gene expression becomes mainly restricted in the brain territories. In fact, at the larval stage, the transcript is detectable in the epiphysis, postoptic region, posterior tuberculum, hypothalamus, optic tectum, tegmentum/pons, medulla and also in the structure of a peripheral nervous system, the terminal nerve. The rxfp1 expression pattern in Danio rerio embryos is very similar to that reported in the adult mammalian brain, suggesting a pivotal role of this receptor in the neurophysiology processes already at very early developmental stages.

Microbes in rocks and meteorites: a new form of life unaffected by time, temperature, pressure

Crystals, rocks and mineral ores of different origins contain viable microbial life that appears actively swimming under the microscope when the sample is properly fragmented and suspended in a nutrient medium. This form of life in rocks is unaffected by time, since microbes have been found in samples of all geological ages, from about 2.8 Ga to recent rocks, and by pressure and temperature, since it is present in metamorphic and in igneous rocks. From the tests performed, among which those to secure from sample pollution, it emerges that this form of life is not destroyed, as indeed expected, when the rock is heated above 500 °C in a kiln. However, all cloned microbes are sensitive to growth inhibition by specific antibiotics. A similar search, for the presence of microbes in meteorites, shows that also these materials are rich in microorganisms, indicating that these already existed in early Earth formation stages. Some different microbial species, derived from different samples of rocks and meteorites, have been cultured, cloned and classified by 16S rDNA typing and found to be not essentially different from present day organisms. An interesting consequence of these findings, among others, is the support to the hypothesis that life came from outside Earth with the additional indication that it was already present in those materials that accreted to form the solar planetary system.RiassuntoCristalli, rocce e minerali di diversa origine contengono microrganismi vitali che si osservano nuotare attivamente al microscopio quando il campione solido è frammentato in modo appropriato, raccolto su un vetrino portaoggetti e sospeso in un mezzo nutriente. Questa forma di vita, quando è all’interno della roccia, non è influenzata dal tempo, perché sono stati trovati microrganismi vitali e coltivabili in campioni di diverse età, a partire da circa 2.8 Ga a rocce recenti, e dalla temperatura e pressione, perché è presente in rocce metamorfiche e in rocce ignee. In alcune prove, fra le molte fatte per assicurarsi da possibili contaminazioni, è risultato che questa forma di vita non è distrutta, come ci si sarebbe effettivamente aspettato, quando la roccia è riscaldata al di sopra di 500 °C in un forno per ceramica, mentre tutte le specie clonate non crescono in presenza di antibiotici specifici. La ricerca con lo stesso approccio di forme microbiche in meteoriti ha mostrato che esse sono ricche in microrganismi, indicando che questi già esistevano durante i primi stadi di formazione della Terra. Alcune specie microbiche, derivate da campioni di rocce e di meteoriti, sono state ottenute in coltura, clonate e classificate con il metodo della tipizzazione del 16S rDNA e sono risultate non dissimili dai microrganismi attuali. Questi risultati avvalorano l’ipotesi che la vita sia venuta dall’esterno della Terra e suggeriscono che fosse già presente nei materiali che, condensandosi, hanno generato i pianeti del sistema solare.

Specificity of cellular expression of C. variopedatus polychaete innexin in the developing embryo: Evolutionary aspects of innexins' heterogeneous gene structures

Innexins are a family of membrane proteins involved in the formation of gap junctions in invertebrates. They have been found to participate in several aspects of cell differentiation and in embryonic patterning through the formation of specific intercellular communication channels. We present here data showing that the recently identified innexin of the marine worm Chaetopterus variopedatus is expressed only in particular cells of the early stage, demonstrating cell specificity of innexin expression also in polychaete annelids. Phylogenetic analysis of all known innexins results in a phylogenetic tree clearly distinguishing insect, nematode, and other invertebrate innexins. Comparative analysis of proteins and known related genes shows that the apparent similarity of protein composition, overall structural organization, and specificity of cellular expression, typical of innexins of all studied organisms, correspond to highly heterogeneous gene structures even for genes that are in close contiguity on the same chromosome. A possible evolutionary motive producing this situation is discussed.

Developmental expression pattern of two zebrafish rxfp3 paralogue genes

In mammals, the RXFP3 is the cognate receptor of the relaxin‐3 peptide (RLN3). In teleosts, many different orthologue genes for RXFP3 are present. In particular, two paralogue genes, rxfp3‐2a and rxfp3‐2b, likely encode the receptors for the Rln3a peptide. The transcription of these two rxfp3 genes is differentially regulated early during zebrafish embryogenesis. Indeed, reverse transcription–polymerase chain reaction analyses show that the rxfp3‐2b transcript is always present during embryo development, while the rxfp3‐2a transcript is detectable only at larval stage. By in situ hybridization experiments on embryos and larvae, the rxfp3‐2b transcript was revealed in the brain and in the retinal ganglion cell layer and thymus. Particularly in the brain, many territories are involved in the rxfp3‐2b expression, among them the optic tectum, thalamus, preoptic area, different nerve nuclei, habenula and pineal gland. The RXFP3 spatiotemporal expression pattern appears to be conserved between Danio rerio and mammals, as also previously showed for the corresponding ligand, the RLN3. Interestingly, the brain areas expressing the rxfp3‐2b receptor gene are involved in the visual system, emotional behaviors and circadian rhythm and could be functionally related to the neurotransmitter Rln3a‐expressing territories.