p63 in Mytilus galloprovincialis and p53 family members in the phylum Mollusca.

Genes of the p53 family are known to be critical regulators of the cell cycle. They have already been established as possible biomarkers. Elaborate regulation mechanisms result in numerous cDNA and protein isoforms being expressed from each gene of the p53 family. Their similarity caused an often misleading nomenclature in non-vertebrate species. The aim of the present work is a clarification of the nomenclature of molluscan p53 family sequences, an essential prerequisite for reliable interpretation of gene expression and protein function studies. Here, we report five partial cDNA and one partial genomic p63 sequences, all originating from two Mytilus galloprovincialis individuals. DNA, deduced protein sequences, and the exon/intron architecture were analyzed and compared to p53, p63 and p73 sequences from other organisms. Along with our sequences, we analyzed all similar molluscan sequences found in the GenBank database. The analysis showed our cDNA sequences code for the TAp63gamma isoform of the p63 protein, and identified all other molluscan p53 family sequences as p63 genes or their expression isoforms. Our results also indicate p63 as the ancestral gene of the p53 family as well as the only gene of the family present in non-chordate metazoan species.

Flow cytometric detection of DNA cell cycle alterations in hemocytes of mussels (Mytilus galloprovincialis) off the Adriatic coast, Croatia.

Studies were carried out to determine the alteration in DNA cell cycle characteristics of hemocytes of the mussel Mytilus galloprovincialis collected at 17 different locations (146 individuals) along the Adriatic coast, Croatia. In order to connect possible genomic manifestation to urban and/or industrial waste flow cytometry was used. We studied incidence of altered DNA profile reflective of chromosomal fragmentation phenomena or aneuploid mosaicism, coefficient of variation (CV) in DNA fluorescence as a measure of intraindividual genome size variability and DNA index (DI) as a measure of ploidy. The different classes of DNA cell cycle alterations found in this study mirror either acute or cumulative genotoxic effects of the surrounding environment on mussel hemocyte DNA. These are intraindividual genome size variability (CV>8, seven individuals from four sites), aneuploidy (altered DNA profile and DI<0.9, 45 individuals from 14 sites) and accidental apoptotic processes (altered DNA profile and presence of apoptotic cells, two individuals from two sites). Normal cell cycle DNA profiles were obtained for 89 (60.9%) individuals from all 17 sites and for 146 examined samples polyploids were absent. Flow cytometry proved to be a powerful technique for the determination of alterations in cell cycle characteristics in mussel hemocyte DNA. Therefore, it may be used in pollution control measurements to distinguish affected or vulnerable populations from healthy populations living in the presence of a wide variety of marine environmental contaminants.

Species-specific aggregation factor in sponges : VIII. Nature and alteration of cell surface charge.

Isolated cells from the siliceous spongeGeodia cydonium have been studied with respect to their partition behaviour in a two-phase aqueous polymer system. With this method it is possible to determine subtle changes in the cell surface charge. Addition of a homologous aggregation factor to the isolated cells lowers the partition rate, a finding which indicates that after binding of the aggregation factor to the cells their surface charge is reduced. The partition rate of the cells is strongly correlated with their content of membranebound sialic acid.Sixty-nine percent of the total, membrane-bound hexuronic acid is associated with the aggregation receptor; 1.8×107 aggregation receptor molecules are present on the surface of one cell which means that the average surface density amounts to 2.8×105 molecules per µm2.Removal of the aggregation receptor molecules from the cell surface results in a decrease of the partition rate in the two-phase system. After charging the receptor-depleted cells with soluble aggregation receptor, the partition behaviour of these cells can be reconstituted.

[Morphological and biochemical characterization of the developmental stages of fertilized eggs inSphaerechinus granularis Lam : II. DNA content, DNA polymerase activity and DNase activity]. / Morphologische und biochemische Charakterisierung der Entwicklung befruchteter Eier des SeeigelsSphaerechinus granularis Lam : II. Teil: DNA, DNA-Polymerase und DNase.

The investigations were performed with the eggs of the sea urchin speciesSphaerechinus granularis Lam. They were kept at 22° C under continuous aeration for up to 45 hours with stirring to compensate for sedimentation. 1. The change in DNA content, 2. the change in DNA dependent DNA polymerase activity, and 3. the change in DNase activity with time have been evaluated. 1. DNA Content of Embryos. The DNA content of the embryo development was determined by two different methods. Before and immediately after fertilization DNA content has been found to be 1.7±0.5·10-10 g per egg. This amount is about 100 times higher than in diploid nuclei. Three periods with different rates of DNA synthesis may be distinguished: a) the first one, lasting from fertilization to about the time of the volume maximum just before the onset of gastrulation with an average rate of synthesis of 1.2·10-10g DNA per minute per embryo; b) a second one, lasting from then on to the gastrula stage with a lower average rate of synthesis of about 0.7·10-12 g DNA per minute per embryo; c) a third one, starting from the gastrula stage up to the experimental end point in the pluteus stage. The rate of synthesis in this case is 2.3·10-12 g DNA per minute per embryo. On a relative base the rates of synthesis are 100∶58∶192. The cytoplasmic, extramitochondrial DNA persists through the stage of the first period of the embryogenesis, up to the blastula stage. The amount of extranuclear DNA increases in the first 6 hours of embryo development; then the cytoplasmic DNA disappears. 2. DNA Dependent DNA Polymerase Activity. The DNA polymerase has been isolated from embryos. Its activity has been determined in relation to the activity of the total embryo as well as per embryonic cell. The polymerase activity is much higher at the start of the development than in later stages, reaching a minimum in the blastula stage, the time at which cytoplasmic DNA has been exhausted. In the subsequent period the polymerase activity parallels the rate of DNA synthesis in vivo. The level of the DNA polymerase activity per cell remains constant. 3. DNase Activity. The DNase activity has been determined using the Lanthanum-Nitrate-Method. Three distinct maxima were found: A first maximum is reached immediately upon fertilization. The second one coincides with the onset of mesenchyme formation in the blastula, and the third one coincides with the end of gastrulation. The average specific activity is roughly equivalent to about 10-6 g DNase I per g of embryo. The possibility is discussed that rises in nucleolytic activities may trigger differentiation events in the developing egg. The influence of DNA polymerase activity and DNase activity on in vivo DNA synthesis is discussed.