Giuseppe Geraci

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.

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.

An H1 histone and a protamine molecule organize the sperm chromatin of the marine worm Chaetopterus variopedatus

Only two basic proteins are present in the chromatin of the sperm cells of the marine worm Chaetopterus variopedatus. They have distinctive electrophoretic mobilities on acetic acid-urea polyacrylamide. The two molecules have been purified by CM-cellulose column chromatography and their amino acid compositions determined. One component is a very lysine-rich H1 histone with Mr 22 000 as determined by SDS gel electrophoresis and from minimal amino acid composition. It shows a Lys/Arg ratio of about 2, typical of sea urchin sperm H1 histones. The other component is a protamine with high arginine and lysine content and minimal Mr 5600 as derived from amino acid composition. The H1 and the protamine are contained in the chromatin in a mass ratio of about 1:2.

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.

High sensitivity method for fluorofore detection in gradient polyacrylamide slab gels through excitation by laser light: Application to glycoproteins stained with concanavalin A-fluorescein isothiocyanate

An easy-to-assemble apparatus for the laser-light excitation of fluorofores in polyacrylamide gels is described. The assemblage is made up of a continuous-wave ion-argon laser with adjustable power output, a beam diffuser, appropriate filters to block excitation light, and a photographic camera. With this setup a minimum 20-fold increase of sensitivity was obtained for fluorofore detection in polyacrylamide gels as compared to the more conventional uv-light excitation using a commercial preparation of Con A-FITC (concanavalin A-fluorescein isothiocyanate) as reference molecule in the gel. The same apparatus, used to analyze the Con A-positive glycoproteins contained in serum Cohn fraction IV separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, showed a number of fluorescent components in a wide range of relative intensities while uv-light excitation showed none. Acrylamide concentration in the gel is critical, since a working limit of between 10 and 12% has been found, above which the diffusion of Con A-FITC in the gel, necessary to label glycoprotein bands, is hampered. The system described here also permits the optimization of detection of minor components not otherwise observable by conventional light excitation, because light power, angle of incidence, and beam divergence can be adapted to analyze specific areas of the sample gel.

Isolation of a new H3.3 histone variant cDNA of P. lividus sea urchin: Sequence and embryonic expression

A cDNA encoding a new H3 histone variant has been isolated from a Paracentrotus lividus sea urchin embryo cDNA library. The encoded protein is identical to the H3.3 histone subtype identified in other species, with the difference that E replaces D at position 81. The clone corresponds to a transcript of about 1.6 kb, not dependent on DNA replication, present in the unfertilized egg and at all stages of embryonic development. The coding part of the cDNA cross-reacts also with a 0.5 kb H3 late histone mRNA.

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.

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.

Multiple hemoglobins in the electric ray: Torpedo marmorata

Abstract The elasmobranch Torpedo marmorata has multiple hemoglobins. Some specimens have at least 6–8 components, others 10 or 12–13. We did not observe hemoglobins of molecular weight greater than tetramers. The analyses of hemoglobins polypeptide composition seem to indicate that each hemoglobin contains more than two types of globin chains. We show evidence for the existence of hybrid hemoglobins. Post-translational modifications of globin chains could occur. All these phenomena can contribute to the multiplicity of hemoglobins