In silico characterization of the neural alpha tubulin gene promoter of the sea urchin embryo Paracentrotus lividus by phylogenetic footprinting

During Paracentrotus lividus sea urchin embryo development one alpha and one beta tubulin genes are expressed specifically in the neural cells and they are early end output of the gene regulatory network that specifies the neural commitment. In this paper we have used a comparative genomics approach to identify conserved regulatory elements in the P. lividus neural alpha tubulin gene. To this purpose, we have first isolated a genomic clone containing the entire gene plus 4.5 Kb of 50 upstream sequences. Then, we have shown by gene transfer experiments that its non-coding region drives the spatiotemporal gene expression corresponding substantially to that of the endogenous gene. In addition, we have identified by genome and EST sequence analysis the S. purpuratus alpha tubulin orthologous gene and we propose a revised annotation of some tubulin family members. Moreover, by computational techniques we delineate at least three putative regulatory regions located both in the upstream region and in the first intron containing putative binding sites for Forkhead and Nkx transcription factor families

Hsp56 protein and mRNA distribution in normal and stressed P.lividus embryos

It was previously demonstrated that Paracentrotus lividus Hsp56 mitochondrial chaperonin is con- stitutively expressed during development, that it increases after heat-shock and cadmium treatment, and that it has a speci\ufb01c territorial distribution, both in normal and heat-shocked embryos, as shown by immunolocalization experiments. In this work, we analyzed by Western blot the territorial distribution of the protein in plutei exposed to heat-shock or sublethal cadmium concentrations, and we found that Hsp56 increases in both ectodermal and en- dodermal cells. Moreover, by \u201cin situ\u201d hybridization, we looked at Hsp56 mRNA during normal development and under stress conditions. We found that the territorial distribution of the messenger changes during development and that its amount is steadily increased in stressed embryos. Finally, by T1 RNase assay, we identi\ufb01ed a cytoplasmic factor that binds to the region of Hsp56 messenger containing the 5\u2019UT

Metallothionein gene family in the sea urchin Paracentrotus lividus: Gene structure, differential expression and phylogenetic analysis

Metallothioneins (MT) are small and cysteine-rich proteins that bind metal ions such as zinc, copper, cadmium, and nickel. In order to shed some light on MT gene structure and evolution, we cloned seven Paracentrotus lividus MT genes, comparing them to Echinodermata and Chordata genes. Moreover, we performed a phylogenetic analysis of 32 MTs from different classes of echinoderms and 13 MTs from the most ancient chordates, highlighting the relationships between them. Since MTs have multiple roles in the cells, we performed RT-qPCR and in situ hybridization experiments to understand better MT functions in sea urchin embryos. Results showed that the expression of MTs is regulated throughout development in a cell type-specific manner and in response to various metals. The MT7 transcript is expressed in all tissues, especially in the stomach and in the intestine of the larva, but it is less metal-responsive. In contrast, MT8 is ectodermic and rises only at relatively high metal doses. MT5 and MT6 expression is highly stimulated by metals in the mesenchyme cells. Our results suggest that the P. lividus MT family originated after the speciation events by gene duplications, evolving developmental and environmental sub-functionalization

An intronic cis-regulatory element is crucial for the alpha tubulin Pl-Tuba1a gene activation in the ciliary band and animal pole neurogenic domains during sea urchin development

In sea urchin development, structures derived from neurogenic territory control the swimming and feeding responses of the pluteus as well as the process of metamorphosis. We have previously isolated an alpha tubulin family member of Paracentrotus lividus (Pl-Tuba1a, formerly known as Pl-Talpha2) that is specifically expressed in the ciliary band and animal pole neurogenic domains of the sea urchin embryo. In order to identify cis-regulatory elements controlling its spatio-temporal expression, we conducted gene transfer experiments, transgene deletions and site specific mutagenesis. Thus, a genomic region of about 2.6 Kb of Pl-Tuba1a, containing four Interspecifically Conserved Regions (ICRs), was identified as responsible for proper gene expression. An enhancer role was ascribed to ICR1 and ICR2, while ICR3 exerted a pivotal role in basal expression, restricting Tuba1a expression to the proper territories of the embryo. Additionally, the mutation of the forkhead box consensus sequence binding site in ICR3 prevented Pl-Tuba1a expression

Maintenance of a Protein Structure in the Dynamic Evolution of TIMPs over 600 Million Years

Deciphering the events leading to protein evolution represents a challenge, especially for protein families showing complex evolutionary history. Among them, TIMPs represent an ancient eukaryotic protein family widely distributed in the animal kingdom. They are known to control the turnover of the extracellular matrix and are considered to arise early during metazoan evolution, arguably tuning essential features of tissue and epithelial organization. To probe the structure and molecular evolution of TIMPs within metazoans, we report the mining and structural characterization of a large data set of TIMPs over approximately 600 Myr. The TIMPs repertoire was explored starting from the Cnidaria phylum, coeval with the origins of connective tissue, to great apes and humans. Despite dramatic sequence differences compared with highest metazoans, the ancestral proteins displayed the canonical TIMP fold. Only small structural changes, represented by an α-helix located in the N-domain, have occurred over the evolution. Both the occurrence of such secondary structure elements and the relative solvent accessibility of the corresponding residues in the three-dimensional structures raises the possibility that these sites represent unconserved element prone to accept variations

RT-PCR and in situ hybridization analysis of apolipoprotein H expression in rat normal tissues

In this study, by using different techniques (i.e. Northern blot hybridization, RT-PCR and Southern blot hybridization) on various normal rat tissues, we were able to identify liver, kidney, heart, small intestine, brain, spleen, stomach and prostate as tissues in which the ApoH gene is transcribed. Moreover, for some of these tissues, by in situ hybridization, we found a specific localization of apoH transcripts. For instance epithelial cells of the bile ducts in liver and of the proximal tubules in kidney are the major sites of apoH synthesis. Our data suggest that some of the different physiological roles proposed for apoH could correlate with its direct expression, while others could correlate with its absorption from bloodstream or adjacent cells

New Mediterranean Marine biodiversity records

Based on recent biodiversity studies carried out in different parts of the Mediterranean, the following 19 species are included as new records on the floral or faunal lists of the relevant ecosystems: the green algae Penicillus capitatus (Maltese waters); the nemertean Amphiporus allucens (Iberian Peninsula, Spain); the salp Salpa maxima (Syria); the opistobranchs Felimida britoi and Berghia coerulescens (Aegean Sea, Greece); the dusky shark Carcharhinus obscurus (central-west Mediterranean and Ionian Sea, Italy); Randall’s threadfin bream Nemipterus randalli, the broadbanded cardinalfish Apogon fasciatus and the goby Gobius kolombatovici (Aegean Sea, Turkey); the reticulated leatherjack Stephanolepis diaspros and the halacarid Agaue chevreuxi (Sea of Marmara, Turkey); the slimy liagora Ganonema farinosum, the yellowstripe barracuda Sphyraena chrysotaenia, the rayed pearl oyster Pinctada imbricata radiata and the Persian conch Conomurex persicus (south-eastern Kriti, Greece); the blenny Microlipophrys dalmatinus and the bastard grunt Pomadasys incisus (Ionian Sea, Italy); the brown shrimp Farfantepenaeus aztecus (north-eastern Levant, Turkey); the blue-crab Callinectes sapidus (Corfu, Ionian Sea, Greece). In addition, the findings of the following rare species improve currently available biogeographical knowledge: the oceanic pufferfish Lagocephalus lagocephalus (Malta); the yellow sea chub Kyphosus incisor (Almuñécar coast of Spain); the basking shark Cetorhinus maximus and the shortfin mako Isurus oxyrinchus (north-eastern Levant, Turkey)

The first confirmed record of the Atlantic blue crab Callinectes sapidus Rathbun, 1896 (Decapoda, Brachyura) from Maltese waters

The invasive portunid species Callinectes sapidus is hereby recorded for the first time from Maltese waters, thus updating the known distribution of this decapod of Atlantic origin within the Mediterranean. Potential introduction pathways of the species to Maltese waters are discussed

Multiple Processes Regulate Long-Term Population Dynamics of Sea Urchins on Mediterranean Rocky Reefs

We annually monitored the abundance and size structure of herbivorous sea urchin populations (Paracentrotus lividus and Arbacia lixula) inside and outside a marine reserve in the Northwestern Mediterranean on two distinct habitats (boulders and vertical walls) over a period of 20 years, with the aim of analyzing changes at different temporal scales in relation to biotic and abiotic drivers. P. lividus exhibited significant variability in density over time on boulder bottoms but not on vertical walls, and temporal trends were not significantly different between the protection levels. Differences in densities were caused primarily by variance in recruitment, which was less pronounced inside the MPA and was correlated with adult density, indicating density-dependent recruitment under high predation pressure, as well as some positive feedback mechanisms that may facilitate higher urchin abundances despite higher predator abundance. Populations within the reserve were less variable in abundance and did not exhibit the hyper-abundances observed outside the reserve, suggesting that predation effects maybe more subtle than simply lowering the numbers of urchins in reserves. A. lixula densities were an order of magnitude lower than P. lividus densities and varied within sites and over time on boulder bottoms but did not differ between protection levels. In December 2008, an exceptionally violent storm reduced sea urchin densities drastically (by 50% to 80%) on boulder substrates, resulting in the lowest values observed over the entire study period, which remained at that level for at least two years (up to the present). Our results also showed great variability in the biological and physical processes acting at different temporal scales. This study highlights the need for appropriate temporal scales for studies to fully understand ecosystem functioning, the concepts of which are fundamental to successful conservation and management