Gene duplication is considered an important evolutionary mechanism leading to new gene functions. According to the classical model, one gene copy arising from gene duplication retains the ancestral function, whilst the other becomes subject to directional selection for some novel functions. Hence, according to this model, long-term persistence of two paralogous genes is possible only with the acquisition of functional innovation. In the absence of neofunctionalization, one of the duplicate genes may be lost following accumulation of deleterious mutations, ultimately leading to the loss of function. Recently, new mechanisms have been proposed according to which both paralogs are maintained without apparent neofunctionalization. In this paper we describe the molecular evolution of the aspartic proteinase gene family, with particular regard for the nothepsin gene, a sex- and tissue-specific form of aspartic proteinase active in fish. The finding of nothepsin in a reptile is indicative of the presence of this gene in organisms other than fish. However, the failure to find any nothepsin-like gene in avian, murine and human genome suggests that the gene has been lost in certain lineages during evolution. At variance with piscine nothepsin expressed exclusively in female liver under the estrogens action, the reptilian counterpart lacks both tissue and sex specificity, as it is constitutively expressed in different tissues of male and female specimens. The expression of the nothepsin gene in fish and lizard is accompanied by the expression of a paralogous gene encoding for cathepsin D. Functional divergence analysis indicates that cathepsin D accumulated amino acid substitutions, whereas nothepsin retained most of the ancestral functions. Phylogenetic analysis shows a preponderance of replacement substitutions compared to silent substitutions in the branch leading to the cathepsin D clade, whilst nothepsin evolves under negative selection. To explain the loss of the nothepsin gene in certain lineages, we propose a model that takes into account the complementary degenerative mutations occurring in regulatory elements of the promoter regions of the two genes. According to this model, gene loss occurs whenever the two genes acquire the same expression pattern. The coexistence of cathepsin D and nothepsin is explained in terms of metabolic cooperation of the two enzymes.

Structural studies show that fish and mammalian metallothioneins are endowed of distinctive features. In particular, the ninth cysteine residue present in the alpha domain of fish metallothionein is shifted of two positions with respect to the mammalian metallothionein, introducing a conformational modification in the protein structure. In addition, the fish metallothionein is less hydrophobic and more flexible than its mammalian counterpart. Our previous studies showed that the hydropathy of piscine and mammalian metallothioneins is significantly correlated with organismal temperature. In the present paper we have performed phylogenetic comparative analysis on metallothioneins of 24 species of fish and mammals. The results of such analysis failed to indicate that metallothionein hydropathy is an adaptive response to the thermal regime of the species. We concluded that metallothionein hydropathy is a trait that did not evolve in association with environmental changes.

In previous studies, we showed that the chemical and dynamic properties of fish and mouse MTs (metallothioneins) present a number of distinctive differences linked to their primary structures, and that phylogenetic relationships of mammal and fish MTs correlate with their three-dimensional structures. The different behaviours of MTs may also be linked to the interaction between their two domains. In the present study, we have compared the physicochemical properties of the isolated recombinant domains constituting Notothenia coriiceps and mouse MTs, and compared them with those of the corresponding whole MTs. NMR spectra of the separated domains of N. coriiceps are almost superimposable on those of the parent MT, suggesting an apparent lack of interaction between the two domains in the protein. However, certain dynamic and physicochemical features of the isolated domains are unlike those of the whole protein. In particular, the temperature-induced changes in the chiroptical properties, thiol reactivity of the Zn-MT domains and the Zn2+/Cd2+ rate of exchange are different for the two domains and with respect to the whole protein. Taken together, these results provide a strong argument in favour of the interaction of the two domains in the MT molecule, in spite of the elusive evidence provided by the structural analyses.

In vertebrates, a large proportion of genes is organized in gene families. Paralogous gene groups generated by gene duplication are related by homology, high degree of sequence identity and similar structural architecture of their products. Aspartic proteinases form a widely distributed protein superfamily including cathepsins, pepsins, renin and napsin. In the present study, the nucleotide sequences coding for various pepsins in 30 vertebrate species have been used to derive a gene phylogeny. Gene duplication and losses have been inferred from a reconciled tree, reconstructed by combining information from gene tree and species tree. Our findings based on the results of the relative rate ratio test and maximum likelihood analysis suggest that each round of gene duplication is characterized by adaptive evolution, although instances of evolution under positive selection have been found also long after divergence of gene families. The results of functional divergence analysis provided statistical evidence for shifted evolutionary rate after gene duplication.

The climatic features of Antarctic waters are more extreme and constant than in the Arctic. The Antarctic has been isolated and cold longer than the Arctic. The polar ichthyofaunas differ in age, endemism, taxonomy, zoogeographic distinctiveness and physiological tolerance to environmental parameters. The Arctic is the connection between the Antarctic and the temperate-tropical systems. Paradigmatic comparisons of the pathways of adaptive evolution of fish from both poles address the oxygen-transport system and the antifreezes of northern and southern species, (i) Haemoglobin evolution has included adaptations at the biochemical, physiological and molecular levels. Within the study of the molecular bases offish cold adaptation, and taking advantage of the information on haemoglobin amino acid sequence, we analysed the evolutionary history of the ? and ? globins of Antarctic, Arctic and temperate haemoglobins as a basis for reconstructing phylogenetic relationships. In the trees, the constant physico-chemical conditions of the Antarctic waters are matched by clear grouping of globin sequences, whereas the variability typical of the Arctic ecosystem corresponds to high sequence variation, reflected by scattered intermediate positions between the Antarctic and non-Antarctic clades. (ii) Antifreeze (glyco)proteins and peptides allow polar fish to survive at sub-zero temperatures. In Antarctic Notothenioidei the antifreeze gene evolved from a trypsinogen-like serine protease gene. In the Arctic polar cod the genome contains genes which encode nearly identical proteins, but have evolved from a different genomic locus–a case of convergent evolution.

Cold-adapted organisms have developed a number of adjustments at the molecular level to maintain metabolic functions at low temperatures. Among other features, they can produce enzymes characterized by a high turnover number or a high catalytic efficiency. The present work is aimed at investigating the process of food digestion at low temperature through the study of pepsins in Antarctic notothenioids. For such a purpose, we have cloned and sequenced three forms of pepsin A and a single form of gastricsin from the gastric mucosa of Trematomus bernacchii (rock cod). Phylogenetic analysis has suggested that the three pepsin A isotypes arose from two gene duplication events leading to the most ancestral pepsin A3 and to the most recent forms represented by pepsin A1 and pepsin A2. Molecular modeling has unraveled significant structural differences in these enzymes with respect to their mesophilic counterparts. Hydropathy and flexibility determined on the substrate-binding subsites of Antarctic and mesophilic pepsins have shown for pepsin A2 reduced hydropathy and increased flexibility at the level of the substrate cleft, features typical of cold-adapted enzymes. Northern blot analysis of RNA from rock cod gastric mucosa hybridized with molecular probes designed on specific regions of different pepsin forms has shown that rock cod pepsin genes are expressed at comparable levels. The present results suggest that the Antarctic rock cod adopted two different strategies to accomplish efficient protein digestion at low temperature. One mechanism is the gene duplication that increases enzyme production to compensate for the reduced kinetic efficiency, the other is the expression of a new enzyme provided with features typical of cold-adapted enzymes.

The suborder Notothenioidei dominates the Antarctic ichthyofauna. The non‐Antarctic monotypic family Pseudaphritidae is one of the most primitive families. The characterization of the oxygen‐transport system of euryhaline Pseudaphritis urvillii is herewith reported. Similar to most Antarctic notothenioids, this temperate species has a single major hemoglobin (Hb 1, over 95% of the total). Hb 1 has strong Bohr and Root effects. It shows two very uncommon features in oxygen binding: At high pH values, the oxygen affinity is exceptionally high compared to other notothenioids, and subunit cooperativity is modulated by pH in an unusual way, namely the curve of the Hill coefficient is bell‐shaped, with values approaching 1 at both extremes of pH. Molecular modeling, electronic absorption and resonance Raman spectra have been used to characterize the heme environment of Hb 1 in an attempt to explain these features, particularly in view of some potentially important nonconservative replacements found in the primary structure. Compared to human HbA, no major changes were found in the structure of the proximal cavity of the α‐chain of Hb 1, although an altered distal histidyl and heme position was identified in the models of the β‐chain, possibly facilitated by a more open heme pocket due to reduced steric constraints on the vinyl substituent groups. This conformation may lead to the hemichrome form identified by spectroscopy in the Met state, which likely fulfils a potentially important physiological role.

Organisms living in the Arctic and Antarctic are exposed to strong environmental constraints, especially temperature. Consequently, haemoglobin evolution has included adaptations with implications at the biochemical, physiological and molecular levels. The northern and southern polar oceans have very different oceanographic characteristics. Within the study of the molecular bases of cold adaptation in fish inhabiting polar habitats, and taking advantage of the information available on haemoglobin structure and function, we analysed the evolutionary history of the α and β globins of Antarctic and Arctic haemoglobins, under the assumption of the molecular-clock hypothesis, as a basis for reconstructing the phylogenetic relationships between species. Temperate fish, including two non-Antarctic notothenioids of special evolutionary interest, were also considered. Phylogenetic analysis was performed on the multiple sequence alignments constructed with the programme Clustal X. Tree topologies indicate that the chains of Antarctic major and minor haemoglobins cluster in two well separated groups and diverged prior to cold adaptation, forming a monophyletic group. In Arctic haemoglobins, the structure/function relationship reveals important differences in comparison with Antarctic ones, indicating a distinct evolutionary pathway. The Arctic ichthyofauna (unlike the Antarctic, dominated by one taxonomically uniform group) is characterized by high diversity, reflected in the phylogeny of a given trait. The constant physico-chemical conditions of the Antarctic waters are matched by a clear grouping of fish globin sequences, whereas the variability typical of the Arctic Ocean corresponds to high sequence variation, reflected in the trees by scattered intermediate positions between the Antarctic and non-Antarctic clades. The evolutionary history of the Root effect, an important physiological feature of fish haemoglobin, was investigated. Analysis of the fate of the residues of the β chains suggested to be correlated with the Root effect indicate that they should rather be regarded as ancestral characters, inherited by some species but not by others.

The structure of [113Cd7]-metallothionein (MT_nc) of the Antarctic fish Notothenia coriiceps, the first three-dimensional structure of a fish metallothionein, was determined by homonuclear 1H NMR experiments and heteronuclear [1H, 113Cd]-correlation spectroscopy. MT_nc is composed of an N-terminal β domain with 9 cysteines and 3 metal ions and a carboxy-terminal α-domain with 11 cysteines and 4 metal ions. The position of the ninth Cys of the α domain of MT_nc is different from the corresponding Cys of mammalian MTs. As a result, the last CXCC motif in the mammalian MT sequence becomes CXXXCC in the fish MT. This difference leads to a structural change of the α domain and, in turn, to a different charge distribution with respect to that observed in mammalian metallothioneins.

The possible role of metallothionein (MT) in metal homeostasis has been investigated in growing oocytes and eggs of the lizard Podarcis sicula . Chromatographic analysis does not reveal the presence of MT in both ovary and eggs, the only metal‐binding proteins detected being represented by high molecular mass components. De novo synthesis of MT could be observed in the ovary of cadmium‐treated lizards. A cDNA encoding MT was obtained from the liver of P. sicula by RT‐PCR followed by a RACE strategy, using primers designed on consensus motifs of vertebrate MT. In spite of the lack of MT in the ovary of untreated animals, Northern blot analysis demonstrates that the maternal untranslated MT transcript is expressed constitutively in the ovary in all the periods of the ovarian cycle. MT mRNA content increases during the oocyte growth, reaching the highest level in ovulated eggs, concomitantly with the accumulation of zinc and copper. Our findings suggest that maternal MT mRNA accumulates in the egg and is translated sometime during development to cope with the future needs of the growing embryo. The appearance of MT after cadmium treatment suggests that the block that makes the oocytarian MT mRNA untranslatable is removed by the metal. Mol. Reprod. Dev. 66: 374–382, 2003. © 2003 Wiley‐Liss, Inc.

The hematological features of the Arctic spotted wolffish Anarhichas minor, a benthic sedentary species, differ markedly from those of Antarctic species suborder Notothenioidei. Most notothenioids have low hemoglobin multiplicity and possess a single major component, sometimes accompanied by a second, functionally indistinguishable minor hemoglobin. In contrast, the blood of A. minor has three major hemoglobins (Hb 1, Hb 2, Hb 3), which were structurally and functionally characterised. They showed important functional differences in PH and organophosphate regulation, subunit cooperativity and oxygen binding responses to temperature. Similar to Antarctic fish hemoglobins known so far, each A. minor hemoglobin has one of the two chains in common with another one. Homology modelling revealed an unusual structure of the organophosphate binding site in the beta cleft of Hb 3. Molecular phylogeny indicates that the alpha and beta globins are divergent from those of notothenioids and display mutually different paralogous relationships with Antarctic species. These findings at the molecular level suggest that the two polar environments have followed markedly distinct pathways in the evolution of regulatory mechanisms of the respiratory system.

In the present report, we investigated zinc, copper and metallothionein (MT) contents in zebrafish oocytes and embryos. Our results demonstrate that the metal content increases during oocytes maturation. Zinc increases from 30 ng/oocyte (stage-1 oocytes) to 100 ng/oocyte (stage-3 oocytes); copper varied from 1 ng/oocyte (stage-1 oocytes) to 3.5 ng/oocyte (stage-3 oocytes). During embryogenesis, zinc and copper contents dramatically increase after fertilisation around the 512-cells stage, then slowly decrease until the mid-gastrula stage. During oocyte growth, the changes in the MT level are proportional to metal content, whereas during embryogenesis the pattern of MT accumulation does not parallel that of the two metals. Indeed, the maternal pool of MT decreases steadily during the early stages of the development until the gastrula stage. We have examined the effect of cadmium on the expression of MT during zebrafish development. After cadmium exposure, MT content increases in embryos at the blastula stage, whereas no induction occurs in embryos at the gastrula stage. However, pre-treatment of embryos at the gastrula stage with 5-aza-2'-deoxycytidine induces MT synthesis following exposure to cadmium. These observations show that changes in metal levels are not correlated to MT content in the embryo, whereas DNA methylation is one of the factors regulating MT expression.

Copper (Cu) is an essential element required in many biological processes including cellular growth and development. The molecular mechanisms involved in copper homeostasis include proteins that play a role in Cu uptake. Genes encoding high affinity copper transporters (Ctr) have been identified in yeast, plant and mammalian cells. Analysis of copper and zinc content in growing ovarian follicles and ovulated eggs of the reptilian Podarcis sicula demonstrated that the levels of both metals rise during oocyte growth, reaching the maximum in ovulated eggs. By exploiting the remarkable evolutionary conservation of the primary structure of Ctr proteins, cDNA encoding a Ctr was isolated from the liver of the lizard P. sicula by reverse transcriptase PCR and RACE strategy by using primers designed based on consensus motifs present in mammalian Ctr. The predicted protein sequence contains three transmembrane domains and a putative hydrophilic extracellular amino-terminal domain. Besides complementing the respiratory deficiency of yeast cells defective in high affinity Cu transport, expression of lizard Ctr11 in Hek293 cells stimulates Cu uptake. Gene expression assessed by Northern blot hybridization of RNA from different tissues of P. sicula shows the highest levels of transcript in both intestine and liver. The profile of Ctr1 mRNA in growing ovarian follicles and eggs demonstrates that the transcript accumulates during the oocyte growth and reaches the highest levels in ovulated eggs. These results suggest that lizard Ctr1 protein may function in Cu acquisition in growing oocytes and eggs.

Aspartic proteinases are a group of endoproteolytic proteinases active at acidic pH and characterized by the presence of two aspartyl residues in the active site. They include related paralogous proteins such as cathepsin D, cathepsin E and pepsin. Although extensively investigated in mammals, aspartic proteinases have been less studied in other vertebrates. In a previous work, we cloned and sequenced a DNA complementary to RNA encoding an enzyme present in zebrafish liver. The sequence resulted to be homologous to a novel form of aspartic proteinase firstly described by us in Antarctic fish. In zebrafish, the gene encoding this enzyme is expressed only in the female liver, in contrast with cathepsin D that is expressed in all the tissues examined independently of the sex. For this reason we have termed the new enzyme liver-specific aspartic proteinase (LAP). Northern blot analyses indicate that LAP gene expression is under hormonal control. Indeed, in oestrogen-treated male fish, cathepsin D expression was not enhanced in the various tissues examined, but the LAP gene product appeared exclusively in the liver. Our results provide evidence for an oestrogen-induced expression of LAP gene in liver. We postulate that the sexual dimorphic expression of the LAP gene may be related to the reproductive process.

The structural properties and the conformational dynamics of antarctic fish Notothenia coriiceps and mouse metallothioneins were studied by Fourier-transform infrared and fluorescence spectroscopy. Infrared data revealed that the secondary structure of the two metallothioneins is similar to that of other metallothioneins, most of which lack periodical secondary structure elements such as alpha-helices and beta-sheets. However, the infrared spectra of the N. coriiceps metallothionein indicated the presence of a band, which for its typical position in the spectrum and for its sensitivity to temperature was assigned to alpha-helices whose content resulted in 5% of the total secondary structure of the protein. The short alpha-helix found in N. coriiceps metallothionein showed an onset of denaturation at 30 degrees C and a T(m) at 48 degrees C. The data suggest that in N. coriiceps metallothionein a particular cysteine is involved in the alpha-helix and in the metal-thiolate complex. Moreover, infrared spectra revealed that both proteins investigated possess a structure largely accessible to the solvent. The time-resolved fluorescence data show that N. coriiceps metallothionein possesses a more flexible structure than mouse metallothionein. The spectroscopic data are discussed in terms of the biological function of the metallothioneins.

To investigate the ability of cadmium to affect gene transcription in fish, the messenger RNA (mRNA) differential display technique was used to analyze gene expression in the Antarctic icefish Chionodraco hamatus exposed to sublethal doses of cadmium salt. Seven DNA complementary to RNA (cDNA) bands whose steady-state levels of expression significantly changed in response to cadmium exposure were identified. The results obtained show that two groups of genes are affected by cadmium in icefish liver. The first group comprises genes that are up-regulated by the metal: in particular, a gene encoding the heat-shock protein HSP70 and another encoding a protein homologous to GP49 of Sparus aurata egg envelope. The other group comprises genes down-regulated by cadmium. These are the transferrin gene and a gene encoding a protein presenting homology to mouse T2K, a kinase having a role in the prevention of apoptosis. Three cDNAs had no homology to known gene sequences, thus suggesting that may either encode not yet identified proteins, or correspond to untranslated regions of mRNA molecules.

The Arctic fish Anarhichas minor, a benthic sedentary species, displays high hemoglobin multiplicity. The three major hemoglobins (Hb 1, Hb 2, and Hb, 3) show important functional differences in pH and organophosphate regulation, subunit cooperativity, and response of oxygen binding to temperature. Hb 1 and Hb 2 display a low, effector-enhanced Bohr effect and no Root effect. In contrast, Hb 3 displays pronounced Bohr and Root effects, accompanied by strong organophosphate regulation. Hb 1 has the beta (beta(1)) chain in common with Hb 2; Hb 3 and Hb 2 share the alpha (alpha(2)) chain. The amino acid sequences have been established. Several substitutions in crucial positions were observed, such as Cys in place of C-terminal His in the beta(1) chain of Hb 1 and Rb 2. In Rb 3, Val E11 of the beta(2) chain is replaced by Ile. Homology modeling revealed an unusual structure of the Hb 3 binding site of inositol hexakisphoshate. Phylogenetic analysis indicated that only Hb 2 displays higher overall similarity with the major Antarctic hemoglobins. The oxygen transport system of A minor differs remarkably from those of Antarctic Notothenioidei, indicating distinct evolutionary pathways in the regulatory mechanisms of the fish respiratory system in the two polar environments.