Diana Ferro

Transcription of genes involved in glutathione biosynthesis in the solitary tunicate Ciona intestinalis exposed to metals.

Exposure to metals is known to generate oxidative stress risk in living organisms, which are able to respond with the induction of antioxidant defenses, both enzymatic and non-enzymatic. Glutathione (GSH) is considered to be an important cellular component involved in protecting cells, both as metal chelating agent and oxygen radical scavenger. In this work we used molecular techniques to analyze the nucleotide and predicted amino acid sequences of genes involved in GSH biosynthesis, γ-glutamyl-cysteine ligase catalytic subunit (ci-gclc), γ-glutamyl-cysteine ligase modifier subunit (ci-gclm) and GSH synthase (ci-gs) in the solitary tunicate Ciona intestinalis. We also studied the transcription of the above genes after in vivo exposure to Cd, Cu and Zn by semiquantitativ RT-PCR to improve our knowledge about the relationship between metal-induced oxidative stress and GSH production and locate mRNA expression by in situ hybridization (ISH). These genes exhibit a good level of sequence conservation with metazoan homologs generally, especially for residues important for the activity of the enzymes. Phylogenetic analyses indicate that the three enzymes evolved in different ways, Ci-GCLC and Ci-GS being mostly correlated with invertebrate proteins, Ci-GCLM being as sister group of vertebrate GCLMs. Our in silico analyses of the ci-gs and ci-gclc promoter regions revealed putative consensus sequences similar to mammalian metal-responsive elements (MRE) and antioxidant response elements (ARE), indicating that the transcription of these genes may directly depend on metals and/or reactive oxygen species. Results highlight a statistically significant increase in gene transcription, demonstrating that metal treatments have inducible effects on these genes. They can modulate gene transcription not only through MREs but also through AREs, as a consequence of metal-dependent ROS formation. The ISH location of Ci-GS and Ci-GCLC mRNAs shows that the cells most involved in glutathione biosynthesis are circulating hemocytes. The data presented here emphasize the importance of complex metal regulation of ci-gclc, ci-gclm and ci-gs transcription, which can create an efficient detoxification pathway allowing C. intestinalis to survive in continued elevated presence of metals in the environment.

Characterization and metal-induced gene transcription of two new copper zinc superoxide dismutases in the solitary ascidian Ciona intestinalis

Antioxidant enzymes are known to protect living organisms against the oxidative stress risk, also induced by metals. In the present study, we describe the purification and molecular characterization of two Cu,Zn superoxide dismutases (SODs), referred to as Ci-SODa and Ci-SODb, from Ciona intestinalis, a basal chordate widely distributed in temperate shallow seawater. The putative amino acid sequences were compared with Cu,Zn SODs from other metazoans and phylogenetic analyses indicate that the two putative Ci-SODs are more related to invertebrate SODs than vertebrate ones. Both phylogenetic and preliminary homology modeling analyses suggest that Ci-SODa and Ci-SODb are extracellular and intracellular isoform, respectively. The mRNA of the two Cu,Zn SODs was localized in hemocytes and in ovarian follicular cells, as revealed by in situ hybridization. The time course of SOD mRNA levels in the presence of three different metals showed upregulation of ci-soda and inhibition of ci-sodb. Spectrophotometric analysis confirms the presence of SOD activity in Ciona tissues. Our in silico analyses of the ci-soda promoter region revealed putative consensus sequences similar to mammalian metal-responsive elements (MRE), suggesting that the transcription of these genes directly depends on metals. These data emphasize the importance of complex metal regulation of ci-soda and ci-sodb transcription, as components of an efficient detoxification pathway allowing the survival of C. intestinalis in continued, elevated presence of metals in the environment.

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.

Peroxiredoxin 6 from the Antarctic emerald rockcod: molecular characterization of its response to warming.

In the present study, we describe the purification and molecular characterization of two peroxiredoxins (Prdxs), referred to as Prdx6A and Prdx6B, from Trematomus bernacchii, a teleost widely distributed in many areas of Antarctica, that plays a pivotal role in the Antarctic food chain. The two putative amino acid sequences were compared with Prdx6 orthologs from other fish, highlighting a high percentage of identity and similarity with the respective variant, in particular for the residues that are essential for the characteristic peroxidase and phospholipase activities of these enzymes. Phylogenetic analyses suggest the appearance of the two prdx6 genes through a duplication event before the speciation that led to the differentiation of fish families and that the evolution of the two gene variants seems to proceed together with the evolution of fish orders and families. The temporal expression of Prdx6 mRNA in response to short-term thermal stress showed a general upregulation of prdx6b and inhibition of prdx6a, suggesting that the latter is the variant most affected by temperature increase. The variations of mRNA accumulation are more conspicuous in heart than the liver, probably related to behavioral changes of the specimens in response to elevated temperature. These data, together with the peculiar differences between the molecular structures of the two Prdx6s in T. bernacchii as well as in the tropical species Stegastes partitus, suggest an adaptation that allowed these poikilothermic aquatic vertebrates to colonize very different environments, characterized by different temperature ranges.

Cu,Zn Superoxide Dismutases from Tetrahymena thermophila: Molecular Evolution and Gene Expression of the First Line of Antioxidant Defenses

In the present study, we describe the molecular and functional characterization of two Cu,Zn superoxide dismutase (SOD) genes, named tt-sod1a and tt-sod1b from Tetrahymena thermophila, a free-living ciliated protozoan widely used as model organism in biological research. The cDNAs and the putative amino acid sequences were compared with Cu,Zn SODs from other Alveolata. The primary sequences of T. thermophila Cu,Zn SODs are unusually long if compared to orthologous proteins, but the catalytically important residues are almost fully conserved. Both phylogenetic and preliminary homology modeling analyses provide some indications about the evolutionary relationships between the Cu,Zn SODs of Tetrahymena and the Alveolata orthologous enzymes. Copper-dependent regulation of Cu,Zn SODs expression was investigated by measuring mRNA accumulation and enzyme activity in response to chronic exposure to non-toxic doses of the metal. Our in silico analyses of the tt-sod1a and tt-sod1b promoter regions revealed putative consensus sequences similar to half Antioxidant Responsive Elements (hARE), suggesting that the transcription of these genes directly depends on ROS formation. These data emphasize the importance of complex metal regulation of tt-sod1a and tt-sod1b activation, as components of an efficient detoxification pathway allowing the survival of T. thermophila in continued, elevated presence of metals in the environment.

Molecular characterization and metal induced gene expression of the novel glutathione peroxidase 7 from the chordate invertebrate Ciona robusta

In the present study, we describe the identified and characterized the gene and the transcript of a novel glutathione peroxidase-7 (GPx7) from the solitary ascidian Ciona robusta, an invertebrate chordate widely distributed in temperate shallow seawater. The putative nucleotide and amino acid sequences were compared with those of GPx7 from other metazoans and phylogenetic analysis suggests the presence of a high evolutionary pressure in the contest of neutral evolution. The mRNA of CrGPx7 is located in hemocytes and ovarian follicular cells, as revealed by in situ hybridization. The time course of CrGPx7 mRNA levels in the presence of Cd, Cu and Zn, showed upregulation in the final stages of the experiments, suggesting a role of GPx7 in late protection from oxidative stress. Our in silico analyses of the crgpx7 promoter region revealed putative consensus sequences similar to mammalian metal-responsive elements (MRE) and xenobiotic-responsive elements (XRE), suggesting that the transcription of these genes directly depends on metals. Cell-free extract from C. robusta tissues show the presence of selenium-independent GPx activity that is inhibited by the presence of metals.