Characterization and transcription studies of a phytochelatin synthase gene from the solitary tunicate Ciona intestinalis exposed to cadmium.

The major thiol-containing molecules involved in controlling the level of intracellular ROS in eukaryotes, acting as a nonenzymatic detoxification system, are metallothioneins (MTs), glutathione (GSH) and phytochelatins (PCs). Both MTs and GSH are well-known in the animal kingdom. PC was considered a prerogative of the plant kingdom but, in 2001, a phytochelatin synthase (PCS) gene was described in the nematode Caenorhabditis elegans; additional genes encoding this enzyme were later described in the earthworm Eisenia fetida and in the parasitic nematode Schistosoma mansoni but scanty data are available, up to now, for Deuterostomes. Here, we describe the molecular characteristics and transcription pattern, in the presence of Cd, of a PCS gene from the invertebrate chordate Ciona intestinalis, a ubiquitous solitary tunicate and demonstrate the presence of PCs in tissue extracts. We also studied mRNA localization by in situ hybridization. In addition, we analyzed the behavior of hemocytes and tunic cells consequent to Cd exposure as well as the transcription pattern of the Ciona orthologous for proliferating cell nuclear antigen (PCNA), usually considered a proliferation marker, and observed that cell proliferation occurs after 96h of Cd treatment. This matches the hypothesis of Cd-induced cell proliferation, as already suggested by previous data on the expression of a metallothionein gene in the same animal.

Comparative study on metal homeostasis and detoxification in two Antarctic teleosts.

The main characteristic of Antarctic seawater is its low constant temperature and its high concentration of O(2), which can increase the formation rate of reactive oxygen species (ROS), together with a natural occurrence of elevated cadmium and copper levels. In the present paper, we studied the presence of cadmium, copper and zinc, metallothioneins (MTs) and glutathione (GSH), and antioxidant enzyme activities in the Antarctic teleosts Trematomus bernacchii and Trematomus newnesi, in order to determine the influence of the peculiar physico-chemical features of the Antarctic marine environment on these physiological defence systems in two species of teleosts. In both of them, cadmium and copper accumulation occurs mostly in the liver. T. bernacchii accumulates zinc mostly in the hepatic tissue, whereas T. newnesi does not show a preferential accumulation site. In addition to the intra-specific analysis, we decided to compare the two species of the Trematomus genus in order to verify if the different feeding habits and motility of these fish affects metal accumulation. Our results show that the liver of T. bernacchii accumulates cadmium and zinc at a higher extent with respect to T. newnesi. Glutathione (GSH) and metallothioneins (MTs) are present in great quantity in the liver of both species. Moreover liver is the tissue which generally showed the highest antioxidant enzyme levels. The results provide further insights in the physiological mechanisms evolved by animals living in this extreme environment.

Functional characterization of the 5'-upstream region of MTT5 metallothionein gene from Tetrahymena thermophila.

Metallothioneins are ubiquitous small, cysteine-rich, metal-binding proteins that play important roles in intracellular metal homeostasis and detoxification. Very few data are available on the promoter region and the mechanism of metallothionein transcription in Protozoa. In this study, we focused on Tetrahymena thermophila MTT5 5'-flanking region. To define the sequence elements underlying the metal-responsiveness of this promoter, we constructed a series of deletions and mutations starting with a 1777 bp fragment immediately upstream of the start codon of MTT5. As a reporter gene we used the previously tested IAG52B surface antigen from the protozoan fish parasite Ichthyophthirius multifiliis. The results suggest that a region spanning between -300 bp and -274 bp, dubbed Tetrahymena thermophila Cadmium-Response-Element (TtCdRE), is necessary to elicit high-level expression of the transgene following induction with cadmium. This is the first demonstration by in vivo analyses of a regulatory element essential for Cd-mediated control of protozoan metallothionein gene expression, where the sequence GATA appears to be involved.

MTT2, a copper-inducible metallothionein gene from Tetrahymena thermophila.

Metallothioneins (MTs) are ubiquitous, cysteine-rich, metal-binding proteins whose transcriptional activation is induced by a variety of stimuli, in particular heavy metals such as cadmium, copper and zinc. Here we describe the sequence and organization of a novel copper-inducible metallothionein gene (MTT2) from Tetrahymena thermophila. Based on its deduced sequence, the gene encodes a protein 108 amino acids, containing 29 cysteine residues (30%) arranged in motifs characteristic of vertebrate and invertebrate MTs. We demonstrate that the 5'-region of the MTT2 gene can act as an efficient promoter to drive the expression of heterologous genes in the Tetrahymena system. In the latter case, a gene for a candidate vaccine antigen against Ichthyophthirius multifiliis, a ubiquitous parasite of freshwater fish, was expressed at high levels in transformed T. thermophila cell lines. Moreover, the protein was properly folded and targeted to the plasma membrane in its correct three-dimensional conformation. This new copper-inducible MT promoter may be an attractive alternative to the cadmium-inducible MTT1 promoter for driving ectopic gene expression in Tetrahymena and could have a great impact on biotechnological perspectives.

Antioxidant responses to variations in dissolved oxygen of Scapharca inaequivalvis and Tapes philippinarum, two bivalve species from the lagoon of Venice.

The aim of the present work was to determine the activities of selected antioxidant enzymes (SOD, Se-GPX, CAT) in two species of bivalves, Scapharca inaequivalvis and Tapes philippinarum, from two sites of the lagoon of Venice that are characterized by different pO(2) (Marghera and Chioggia). The specimens were collected at four times during a 1-year period. In the two species studied, enzyme activities were found to be present in both digestive glands and gills, but with some species-specific differences that may also represent a different adaptation to seasonal variations. The presence of high SOD activities in the gills of both species may be related to their physiological role in respiration. Scapharca inaequivalvis is less sensitive than T. philippinarum to environmental changes, perhaps due to the presence of hemoglobins in this species. Moreover, in the digestive gland of T. philippinarum we found a significant negative correlation between the activities of SOD and GPX that may indicate the presence of oxidative stress. Some correlations between temperature/dissolved oxygen and antioxidant enzyme activity were present in specimens sampled in Marghera. Only GPX adequately responded to changes in dissolved oxygen and temperature, while the decrease in the activity of SOD and CAT in winter may be directly responsible for an enhanced susceptibility of mussels to oxidative stress during this period. We can conclude that the observed differences between Chioggia and Marghera are due to different concentrations of dissolved oxygen. Marghera is an appropriate location to study seasonal variations in water temperature. In fact, in this site, the differences between hot and cold months are quite evident.