Methylation profile of a satellite DNA constituting the intercalary G+C-rich heterochromatin of the cut trough shell Spisula subtruncata (Bivalvia, Mactridae).

Tandemly repeated DNAs usually constitute significant portions of eukaryotic genomes. In bivalves, however, repetitive DNAs are habitually not widespread. In our search for abundant repetitive DNAs in trough shells, we discovered a novel satellite DNA, SSUsat, which constitutes at least 1.3% of the genome of Spisula subtruncata. As foreseen by the satellite DNA library hypothesis, we confirmed that this satellite DNA is also present in two other Mactridae species, showing a highly conserved nucleotide sequence together with a dramatic diminution in the number of repeats. Predominantly located at the G + C-rich intercalary heterochromatin of S. subtruncata, SSUsat displays several DNA methylation peculiarities. The level of methylation of SSUsat is high (3.38%) in comparison with bivalve standards and triplicates the mean of the S. subtruncata genome (1.13%). Methylation affects not only the cytosines in CpG dinucleotides but also those in CHH and CHG trinucleotides, a feature common in plants but scarce and without any clear known relevance in animals. SSUsat segments enriched in methylated cytosines partly overlap those showing higher sequence conservation. The presence of a chromosome pair showing an accumulation of markedly under-methylated SSUsat monomers additionally indicates that the methylation processes that shape repetitive genome compartments are quite complex.

Localization of rRNA transcribed spacer domains in the nucleolinus and maternal procentrosomes of surf clam (Spisula) oocytes.

The nucleolinus is a nuclear subcompartment long ago posited to play a role in cell division. In a recent study using surf clam oocytes, cytoplasmic foci containing a nucleolinar protein were shown to later recruit γ-tubulin, identifying them as centrosomal precursors. (1) We now demonstrate the presence of structural RNAs from the nucleolinus in these procentrosomes. They include the well-known but poorly understood rRNA-transcribed spacer regions. In situ hybridization revealed a specific and dynamic association of these structural RNAs with the cell division apparatus that extends through the early stages of meiosis. In addition to their bearing on the debate over the nature of centrosome- and spindle-associated RNAs, the observations also suggest that rRNA spacer regions are not simply waste products to be discarded immediately, but may be functional byproducts that play a role in formation of the cell division apparatus.

Discovery of centrosomal RNA and centrosomal hypothesis of cellular ageing and differentiation.

In 2006, a group of scientists studying centrosomes of Spisula solidissima mollusc oocytes under the leadership of Alliegro (Alliegro, M.C.; Alliegro, M.A.; Palazzo, R.E. Centrosome-associated RNA in surf clam oocytes. Proc. Natl. Acad. Sci. USA 2006, 103(24), 9034-9038) reliably demonstrated the existence of specific RNA in centrosome, called centrosomal RNA (cnRNA). In their first article, five different RNAs (cnRNAs 11, 102, 113, 170, and 184) were described. During the process of full sequencing of the first transcript (cnRNA 11), it was discovered that the transcript contained a conserved structure-a reverse transcriptase domain located together with the most important centrosomal protein, γ-tubulin. In an article published in 2005, we made assumptions about several possible mechanisms for determining the most important functions of centrosomal structures and referred to one of them as a "RNA-dependent mechanism." This idea about participation of hypothetic centrosomal small interference RNA and/or microRNA in the process was made one year prior to the discovery of cnRNA by Alliegro's group. The discovery of specific RNA in a centrosome is indirect evidence of a centrosomal hypothesis of cellular ageing and differentiation. The presence of a reverse transcriptase domain in this type of RNA, together with its uniqueness and specificity, makes the centrosome a place of information storage and reproduction.

Centrosomal RNA correlates with intron-poor nuclear genes in Spisula oocytes.

The evolutionary origin of centriole/kinetosomes, centrosomes, and other microtubule organizing centers (MTOCs), whether by direct filiation or symbiogenesis, has been controversial for >50 years. Centrioles, like mitochondria and chloroplasts, duplicate independently of the nucleus and constitute a heritable system independent of chromosomal DNA. Nucleic acids endogenous to the MTOC would support evolutionary origin by symbiogenesis. To date, most reports of centrosome-associated nucleic acids have used generalized reagents such as RNases and nucleic acid dyes. Here, from a library of RNAs extracted from isolated surf clam (Spisula solidissima) centrosomes, we describe a group of centrosome-associated transcripts representing a structurally unique intron-poor collection of nuclear genes skewed toward nucleic acid metabolism. Thus, we resolve the debate over the existence of centrosome-associated RNA (cnRNA). A subset of cnRNAs contain functional domains that are highly conserved across distant taxa, such as nucleotide polymerase motifs. In situ localization of cnRNA65, a molecule with an RNA polymerase domain, showed it is present in the intact oocyte nucleus (germinal vesicle). Its expression, therefore, precedes the appearance of gamma-tubulin-containing centrosomes. At this stage, the in situ signal resembles the nucleolinus, a poorly understood organelle proposed to play a role in spindle formation. After oocyte activation and germinal vesicle breakdown, cnRNA65 persists as a cytoplasmic patch within which gamma-tubulin-stained centrosomes can be seen. These observations provoke the question of whether cnRNAs and the nucleolinus serve as cytological progenitors of the centrosome and may support a symbiogenetic model for its evolution.

Comparative evaluation of the alkaline comet assay with the micronucleus test for genotoxicity monitoring using aquatic organisms.

A comparative analysis between the in vivo comet assay and the in vivo micronucleus test (MNT) was carried out in three aquatic organisms suitable for genotoxicity monitoring, carp (Cyprinus carpio), rainbow trout (Oncorhynchus mykiss), and clam (Spisula sachalinensis), using a direct-acting mutagen, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), and an indirect mutagen, benzo[a]pyrene (B[a]P). By optimizing the conditions for cell isolation, gill and liver (or digestive glands) were selected as test tissues of the comet assay for MNNG and B[a]P. The MNT employed the erythrocytes (or hemocytes), the most universal cell type for the assay. The analysis of DNA strand breaks using the comet assay and the micronucleus frequencies using the MNT revealed dose- and time-dependent increases between animals exposed to several concentrations of mutagens. But the statistical significance (P<0.05) obtained was higher by the comet assay than by the MNT. When the time profiles of genotoxic signals resulting from B[a]P exposure to carp were plotted representatively, clear distinctions between all concentrations were made in the comet assay, but not in the MNT. The correlation index defined in this study also showed a higher correlation between concentration and signal in the comet assay than in the MNT. It is suggested that the standardization of the comet assay is necessary for its methodological evaluation and use as a genotoxicity biomarker. We conclude that the comet assay has an excellent suitability for aquatic genotoxicity monitoring because of its high and reliable sensitivity.

The nerve hemoglobin of the bivalve mollusc Spisula solidissima: molecular cloning, ligand binding studies, and phylogenetic analysis.

Members of the hemoglobin (Hb) superfamily are present in nerve tissue of several vertebrate and invertebrate species. In vertebrates they display hexacoordinate heme iron atoms and are typically expressed at low levels (microM). Their function is still a matter of debate. In invertebrates they have a hexa- or pentacoordinate heme iron, are mostly expressed at high levels (mM), and have been suggested to have a myoglobin-like function. The native Hb of the surf clam, Spisula solidissima, composed of 162 amino acids, does not show specific deviations from the globin templates. UV-visible and resonance Raman spectroscopy demonstrate a hexacoordinate heme iron. Based on the sequence analogy, the histidine E7 is proposed as a sixth ligand. Kinetic and equilibrium measurements show a moderate oxygen affinity (P(50) approximately 0.6 torr) and no cooperativity. The histidine binding affinity is 100-fold lower than in neuroglobin. Phylogenetic analysis demonstrates a clustering of the S. solidissima nerve Hb with mollusc Hbs and myoglobins, but not with the vertebrate neuroglobins. We conclude that invertebrate nerve Hbs expressed at high levels are, despite the hexacoordinate nature of their heme iron, not essentially different from other intracellular Hbs. They most likely fulfill a myoglobin-like function and enhance oxygen supply to the neurons.