Cloning and characterization of a novel aspartate/glutamate racemase from the acorn worm Saccoglossus kowalevskii.

Previously, we demonstrated that the animal aspartate racemase (AspR) gene has evolved from the serine racemase (SerR) gene by acquisition of three consecutive serine residues (Ser155-Ser156-Ser157) involved in the strong AspR activity, and this event has occurred independently and frequently during animal evolution. In the present study, we cloned and characterized two mammalian SerR homologous genes from the hemichordate acorn worm (Saccoglossus kowalevskii). The enzymes have been identified as an AspR and an aspartate/glutamate racemase (Asp/GluR) on the basis of their kinetic parameters. The S. kowalevskii Asp/GluR shows comparable substrate affinity and high catalytic efficiency (kcat/Km) for both aspartate and glutamate and is the first reported enzyme from animals that can synthesize d-glutamate. Amino acid sequence alignment analysis and site-directed mutagenesis studies have revealed that the amino acid residue at position 156, which is serine in AspR and alanine in Asp/GluR, is associated with binding and recognition of glutamate and aspartate. Phylogenetic analysis suggests that the S. kowalevskii AspR gene has evolved from the SerR gene after the divergence of hemichordata and vertebrate lineages by acquisition of the three serine residues at position 155 to 157 as in the case of other animal AspR genes. Furthermore, the S. kowalevskii Asp/GluR gene is the result of AspR gene duplication and several amino acid substitutions including that of the 156th serine residue with alanine. The fact that SerR has acquired substrate specificity towards aspartate or glutamate raises the possibility that synthesis of other d-amino acids is carried out by enzymes evolved from SerR.

Identification and bioactivity analysis of transthyretin-like protein in amphioxus: a case demonstrating divergent evolution from an enzyme to a hormone distributor.

Transthyretin-like proteins are a family of proteins that share remarkable structural similarities to transthyretin, that have been identified in a variety of taxa such as bacteria, fungi, plants, invertebrates and vertebrates. Despite the enormous progress in the study of transthyretin-like protein, little is known about it in amphioxus, a model organism for insights into the origin and evolution of vertebrates. Here we identified a transthyretin-like protein gene in Branchiostoma japonicum, named Bjtlp, which possessed a TLP-HIUase (an enzyme hydrolyzing 5-hydroxyisourate) domain and a consensus C-terminal tetrapeptide Tyr-Arg-Gly-Ser that are both characteristics of all known transthyretin-like proteins. Phylogenetic and intron-exon structure analyses support that TTR likely arose from a vertebrate specific duplication after vertebrates diverged from invertebrate chordates. Quantitative real-time PCR analysis revealed that Bjtlp was expressed in a tissue-specific fashion, with the transcript levels being most abundant in the hepatic caecum and hind gut. Enzymatic activity assays demonstrated that recombinant BjTLP had the capacity to hydrolyze 5-hydroxyisourate. Site-directed mutagenesis showed that both Y156 and R93 residues were critical for 5-hydroxyisourate hydrolase activity of recombinant BjTLP. Moreover, the single mutation, Y156T, at the active site of BjTLP caused approximately 97% loss of its enzymatic activity, and meanwhile gained the thyroxine binding activity. All these data together suggest that the single mutation Y156T is critical for converting BjTLP to a new transport protein capable of distributing thyroxine.

Putative chitin synthases from Branchiostoma floridae show extracellular matrix-related domains and mosaic structures.

The transition from unicellular to multicellular life forms requires the development of a specialized structural component, the extracellular matrix (ECM). In Metazoans, there are two main supportive systems, which are based on chitin and collagen/hyaluronan, respectively. Chitin is the major constituent of fungal cell walls and arthropod exoskeleton. However, presence of chitin/chitooligosaccharides has been reported in lower chordates and during specific stages of vertebrate development. In this study, the occurrence of chitin synthases (CHSs) was investigated with a bioinformatics approach in the cephalochordate Branchiostoma floridae, in which the presence of chitin was initially reported in the skeletal rods of the pharyngeal gill basket. Twelve genes coding for proteins containing conserved amino acid residues of processive glycosyltransferases from GT2 family were found and 10 of them display mosaic structures with novel domains never reported previously in a chitin synthase. In particular, the presence of a discoidin (DS) and a sterile alpha motif (SAM) domain was found in nine identified proteins. Sequence analyses and homology modelling suggest that these domains might interact with the extracellular matrix and mediate protein-protein interaction. The multi-domain putative chitin synthases from B. floridae constitute an emblematic example of the explosion of domain innovation and shuffling which predate Metazoans.

Amphioxus CaVPT and creatine kinase are crucial immune-related molecules in response to bacterial infection and immunization.

Although a great progress has been made, our understanding of innate immunity is incomplete. Here, we hypothesize that the innate immune response to pathogens is attributed into a group of functional proteins. The group contains information on host status post bacterial entry (infection or immunity) and bacterial species (Gram-positive or Gram-negative bacteria). Investigation of the group of proteins may result in disclosing of biomarkers identifying the status and species. For this regard, differential proteomics approach coupled with the pattern recognition methods are used to identify biomarkers from the proteins that being specifically regulated during the innate immune response of amphioxus to Gram-positive and Gram-negative bacteria with live or dead status. Four proteins, Calcium vector protein (CaVP), sarcoplasmic calcium-binding protein (SCP), CaVP-target protein (CaVPT) and creatine kinase (CK), are selected as the key biomarkers. Since immunoprotection of CaVP and SCP has been reported, the role of CaVPT and CK are further investigated. Gut CaVPT appears in dying amphioxus, whereas humoral fluid CK downregulates and gut CK keep no change in animals with immunity. The responses are stronger in Gram-negative than Gram-positive bacteria. These results indicate that CaVPT, CK, CaVP and SCP are the most important biomarkers to uncover amphioxus innate immunity to bacteria, and the approach is an efficient way to identify key biomarkers.

Induction of phenoloxidase and other immunological activities in the humoral fluids of amphioxus Branchiostoma belcheri challenged with Lipopolysaccharide (LPS).

The knowledge concerning the humoral immunity is scarce in amphioxus Branchiostoma belcheri. This study measured the humoral parameters including phenoloxidase (PO) activity, lysozyme activity, antimicrobial activity, microbial agglutinin, and hemagglutinin in amphioxus humoral fluids before and after lipopolysaccharide (LPS) challenge. Humoral fluids from unchallenged Branchiostoma belcheri (B. belcheri) had PO activity, lysozyme, antimicrobial, microbial agglutinating, and hemagglutinating activities, which may represent part of the baseline level of innate immunity in this organism. After challenge with LPS, many humoral parameters were all increased significantly including the PO activity, lysozyme activity, growth-inhibiting activities against Gram-negative bacteria Escherichia coli (E. coli) and Vibrio alginolyticus (V. alginolyticus), growth-inhibiting activities against Gram-positive bacteria Staphylococcus aureus (S. aureus) and Bacillus subtilis (B. subtilis), microbial agglutinating activities against Micrococcus lysodeikticus (M. lysodeikticus), B. subtilis, and S. aureus, and hemagglutinating activities against rabbit and human A and O erythrocytes. In contrast, the agglutinating activities against V. harveyi and E. coli and the hemagglutinating activity against human B erythrocytes in the humoral fluids were reduced in response to LPS challenge. It appears that the humoral fluids of B. belcheri contain components that are able to differentiate different microbes and different human blood cell types.

New insights into the evolution of metazoan tyrosinase gene family.

Tyrosinases, widely distributed among animals, plants and fungi, are involved in the biosynthesis of melanin, a pigment that has been exploited, in the course of evolution, to serve different functions. We conducted a deep evolutionary analysis of tyrosinase family amongst metazoa, thanks to the availability of new sequenced genomes, assessing that tyrosinases (tyr) represent a distinctive feature of all the organisms included in our study and, interestingly, they show an independent expansion in most of the analyzed phyla. Tyrosinase-related proteins (tyrp), which derive from tyr but show distinct key residues in the catalytic domain, constitute an invention of chordate lineage. In addition we here reported a detailed study of the expression territories of the ascidian Ciona intestinalis tyr and tyrps. Furthermore, we put efforts in the identification of the regulatory sequences responsible for their expression in pigment cell lineage. Collectively, the results reported here enlarge our knowledge about the tyrosinase gene family as valuable resource for understanding the genetic components involved in pigment cells evolution and development.

Identification and characterization of an amphioxus matrix metalloproteinase homolog BbMMPL2 responding to bacteria challenge.

Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases mainly involved in extracellular matrix (ECM) degradation. We have cloned and identified BbMMPL2 as homolog of MMPs from adult amphioxus. Recombinant BbMMPL2 proteins underwent self-processing during refolding in vitro. The final ~23 kDa polypeptide displayed proteolytic activity against ECM components like casein, gelatin, collagen IV and fibrinogen, but not laminin, fibronectin or α1-PI. This activity could be inhibited by GM6001 and TIMP-1/2. In addition, real-time RT-PCR analysis revealed that BbMMPL2 expressed in all issues/organs in adult amphioxus we tested. Its transcription was significantly up-regulated 12 h post immune challenge by Escherichia coli in epidermis and hepatic diverticulum but only slightly increased by Staphyloccocus aureus in epidermis. Furthermore, recombinant BbMMPL2-EGFP expressed in 293T and NIH/3T3 cells showed aggregation in cytoplasm and induced cell death. Our results provided new evidence that MMP was involved in immune response which could be conserved through evolution.

The carboxylesterase/cholinesterase gene family in invertebrate deuterostomes.

Carboxylesterase/cholinesterase family members are responsible for controlling the nerve impulse, detoxification and various developmental functions, and are a major target of pesticides and chemical warfare agents. Comparative structural analysis of these enzymes is thus important. The invertebrate deuterostomes (phyla Echinodermata and Hemichordata and subphyla Urochordata and Cephalochordata) lie in the transition zone between invertebrates and vertebrates, and are thus of interest to the study of evolution. Here we have investigated the carboxylesterase/cholinesterase gene family in the sequenced genomes of Strongylocentrotus purpuratus (Echinodermata), Saccoglossus kowalevskii (Hemichordata), Ciona intestinalis (Urochordata) and Branchiostoma floridae (Cephalochordata), using sequence analysis of the catalytic apparatus and oligomerisation domains, and phylogenetic analysis. All four genomes show blurring of structural boundaries between cholinesterases and carboxylesterases, with many intermediate enzymes. Non-enzymatic proteins are well represented. The Saccoglossus and Branchiostoma genomes show evidence of extensive gene duplication and retention. There is also evidence of domain shuffling, resulting in multidomain proteins consisting either of multiple carboxylesterase domains, or of carboxylesterase/cholinesterase domains linked to other domains, including RING finger, chitin-binding, immunoglobulin, fibronectin type 3, CUB, cysteine-rich-Frizzled, caspase activation and 7tm-1, amongst others. Such gene duplication and domain shuffling in the carboxylesterase/cholinesterase family appears to be unique to the invertebrate deuterostomes, and we hypothesise that these factors may have contributed to the evolution of the morphological complexity, particularly of the nervous system and neural crest, of the vertebrates.

Sialome analysis of the cephalochordate Branchiostoma belcheri, a key organism for vertebrate evolution.

Sialic acid, a common terminal substitution of glycoconjugates, has been so far consistently identified in all vertebrates as well as in a growing number of bacterial species. It is assumed to be widely distributed among animal species of the deuterostome phylum, based on its identification in few echinoderm and all vertebrate species. However, whole sections of deuterostome, especially those intermediate species between invertebrates and vertebrates including cephalochordates, urochordates and hemichordates, are still unexplored in term of sialylation capacities. The discovery of functional sialic acid machinery in some of these species may shed new light onto the evolution of glycosylation capacities in deuterostome lineage. In a first approach, we investigated the sialylation pattern of a cephalocordate species, Branchiostoma belcheri, which occupies a strategic phylogenetic position to understand the transition of invertebrates toward vertebrates. Structural analysis of B. belcheri glycoconjugates established that this organism synthesizes large quantities of various sialic acids, some of which present rare or novel structures such as methylated sialic acids. These sialic acids were shown to be mainly associated with mono- and disialylated core 1-type O-glycans. Moreover, screening of the animal organs revealed the existence of exquisite tissue specificity in the distribution of sialic acids. Description of sialylation profiles was then correlated with the expression patterns of key enzymes involved in the biosynthesis of major forms of sialic acids, which provides the first complete overview of the sialylation patterns in cephalochordates.

Evolution of AANAT: expansion of the gene family in the cephalochordate amphioxus.

BACKGROUND: The arylalkylamine N-acetyltransferase (AANAT) family is divided into structurally distinct vertebrate and non-vertebrate groups. Expression of vertebrate AANATs is limited primarily to the pineal gland and retina, where it plays a role in controlling the circadian rhythm in melatonin synthesis. Based on the role melatonin plays in biological timing, AANAT has been given the moniker "the Timezyme". Non-vertebrate AANATs, which occur in fungi and protists, are thought to play a role in detoxification and are not known to be associated with a specific tissue. RESULTS: We have found that the amphioxus genome contains seven AANATs, all having non-vertebrate type features. This and the absence of AANATs from the genomes of Hemichordates and Urochordates support the view that a major transition in the evolution of the AANATs may have occurred at the onset of vertebrate evolution. Analysis of the expression pattern of the two most structurally divergent AANATs in Branchiostoma lanceolatum (bl) revealed that they are expressed early in development and also in the adult at low levels throughout the body, possibly associated with the neural tube. Expression is clearly not exclusively associated with the proposed analogs of the pineal gland and retina. blAANAT activity is influenced by environmental lighting, but light/dark differences do not persist under constant light or constant dark conditions, indicating they are not circadian in nature. bfAANAT alpha and bfAANAT delta' have unusually alkaline (> 9.0) optimal pH, more than two pH units higher than that of vertebrate AANATs. CONCLUSIONS: The substrate selectivity profiles of bfAANAT alpha and delta' are relatively broad, including alkylamines, arylalkylamines and diamines, in contrast to vertebrate forms, which selectively acetylate serotonin and other arylalkylamines. Based on these features, it appears that amphioxus AANATs could play several roles, including detoxification and biogenic amine inactivation. The presence of seven AANATs in amphioxus genome supports the view that arylalkylamine and polyamine acetylation is important to the biology of this organism and that these genes evolved in response to specific pressures related to requirements for amine acetylation.

Identification and expression of an elastase homologue in Branchiostoma belcheri with implications to the origin of vertebrate pancreas.

Elastases have been identified in a variety of organisms ranging from bacteria to insects to mammals, yet little is known to date about them in amphioxus, a model animal for insights into the origin and evolution of vertebrates. In this study we demonstrate the presence of an elastase homologue, named BbElas, in Branchiostoma belcheri. The recombinant BbElas hydrolyses the elastase specific substrate N-succinyl-Ala-Ala-Ala p-nitroanilide, which can be inhibited by the serine proteinase inhibitor PMSF, the elastase-specific inhibitor elastatinal and the cysteine proteinase inhibitor PCMB. Phylogenetic analysis shows that BbElas represents the archetype of vertebrate elastases, hinting at the clues that the different isoforms of vertebrate elastases are originated from an ancestral gene like BbElas. Our results also suggest that the mid-gut in amphioxus is to homologous vertebrate pancreas, a novel proposal which deserves further study.

Creatine kinase is a bacteriostatic factor with a lectin-like activity.

Creatine kinase (CK), an enzyme catalyzing the conversion of adenosine diphosphate (ADP) to adenosine triphosphate (ATP), has been shown to be an acute phase reactant in the amphioxus Branchiostoma belcheri. However, immune and functional characterization of this protein remains lacking. Here we demonstrate clearly that the expression of B. belcheri gene, BbCK, was inducible by the challenge with LPS, and the recombinant BbCK was capable of binding to the Gram-negative bacterium Escherichia coli and inhibiting the bacterial growth. Moreover, the bacteriostatic activity of the recombinant BbCK against E. coli was able to be suppressed by some sugars including N-acetyl-d-mannosamine, N-acetyl-d-glucosamine, l-fucose, d-mannose, d-fructose and d-glucose. In contrast, BbCK exerted little effect on the growth of the Gram-positive bactterium Staphylococcus aureus. Taken together, these data suggest that CK is a bacteriostatic factor with a lectin-like activity, capable of specifically inhibiting the growth of the Gram-negative bacteria like E. coli. This is the first report exhibiting the integrative role of CK in immunity via its pleiotropic effects on recognizing the Gram-negative bacterium E. coli and inhibiting its growth.

Identification and functional characterization of legumain in amphioxus Branchiostoma belcheri.

Legumain has been reported from diverse sources such as plants, parasites (animals) and mammals, but little is known in the lower chordates. The present study reports the first characterization of legumain cDNA from the protochordate Branchiostoma belcheri. The deduced 435-amino-acid-long protein is structurally characterized by the presence of a putative N-terminal signal peptide, a peptidase_C13 superfamily domain with the conserved Lys123-Gly124-Asp125 motif and catalytic dyad His153 and Cys195 and two potential Asn-glycosylation sites at Asn85 and Asn270. Phylogenetic analysis demonstrates that B. belcheri legumain forms an independent cluster together with ascidian legumain, and is positioned at the base of vertebrate legumains, suggesting that B. belcheri legumain gene may represent the archetype of vertebrate legumain genes. Both recombinant legumain expressed in yeast and endogenous legumain are able to be converted into active protein of approximately 37 kDa via a C-terminal autocleavage at acid pH values. The recombinant legumain efficiently degrades the legumain-specific substrate Z-Ala-Ala-Asn-MCA (benzyloxycarbonyl-L-alanyl-L-alanyl-L-asparagine-4-methylcoumaryl-7-amide) at optimum pH 5.5; and the enzymatic activity is inhibited potently by iodoacetamide and N-ethylmaleimide, partially by hen's-egg white cystatin, but not by E-64 [trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane], PMSF and pepstatin A. In addition, legumain is expressed in vivo in a tissue-specific manner, with main expression in the hepatic caecum and hind-gut of B. belcheri. Altogether, these results suggest that B. belcheri legumain plays a role in the degradation of macromolecules in food.

Conservation of the glucan phosphatase laforin is linked to rates of molecular evolution and the glucan metabolism of the organism.

BACKGROUND: Lafora disease (LD) is a fatal autosomal recessive neurodegenerative disease. A hallmark of LD is cytoplasmic accumulation of insoluble glucans, called Lafora bodies (LBs). Mutations in the gene encoding the phosphatase laforin account for approximately 50% of LD cases, and this gene is conserved in all vertebrates. We recently demonstrated that laforin is the founding member of a unique class of phosphatases that dephosphorylate glucans. RESULTS: Herein, we identify laforin orthologs in a protist and two invertebrate genomes, and report that laforin is absent in the vast majority of protozoan genomes and it is lacking in all other invertebrate genomes sequenced to date. We biochemically characterized recombinant proteins from the sea anemone Nematostella vectensis and the amphioxus Branchiostoma floridae to demonstrate that they are laforin orthologs. We demonstrate that the laforin gene has a unique evolutionary lineage; it is conserved in all vertebrates, a subclass of protists that metabolize insoluble glucans resembling LBs, and two invertebrates. We analyzed the intron-exon boundaries of the laforin genes in each organism and determine, based on recently published reports describing rates of molecular evolution in Branchiostoma and Nematostella, that the conservation of laforin is linked to the molecular rate of evolution and the glucan metabolism of an organism. CONCLUSION: Our results alter the existing view of glucan phosphorylation/dephosphorylation and strongly suggest that glucan phosphorylation is a multi-Kingdom regulatory mechanism, encompassing at least some invertebrates. These results establish boundaries concerning which organisms contain laforin. Laforin is conserved in all vertebrates, it has been lost in the vast majority of lower organisms, and yet it is an ancient gene that is conserved in a subset of protists and invertebrates that have undergone slower rates of molecular evolution and/or metabolize a carbohydrate similar to LBs. Thus, the laforin gene holds a unique place in evolutionary biology and has yielded insights into glucan metabolism and the molecular etiology of Lafora disease.

Induction of phenoloxidases in the humoral fluids of amphioxus Branchiostoma belcheri by Vibrio alginolyticus and Escherichia coli.

The humoral immune responses of amphioxus Branchiostoma belcheri to microbial challenge remain open to date. Here we examined the changes in PO activity in the humoral fluids in amphioxus before and after challenge with Escherichia coli and Vibrio alginolyticus. It was found that PO activity in the humoral fluids is markedly increased by challenge with E. coli and V. alginolyticus; and the microbial challenge results in a significant rise in subunit 2 of the three PO subunits, making PO subunit 2 a marker enzyme responsive to microbial challenge. This is the first report on microbial induction of the immune-related molecules like PO in B. belcheri.

Identification, expression, function and localization of a DUF985 domain-containing hypothetical gene from amphioxus Branchiostoma belcheri.

The progress in genome sequencing has led to an increasing submission of uncharacterized hypothetical genes with the domain of unknown function, DUF985, in GenBank, and none of these genes is related to a known protein. We therefore underwent an experimental study to identify the function of a DUF985 domain-containing hypothetical gene BbDUF985 (GenBank Accession No. AY273818) isolated from amphioxus Branchiostoma belcheri (B. belcheri). BbDUF985 was successfully expressed in both prokaryotic and eukaryotic systems, and its recombinant proteins expressed in both systems definitely exhibited an activity of phosphoglucose isomerase (PGI). Both tissue-section in situ hybridization and immunohistochemistry demonstrated that BbDUF985 was expressed in a tissue-specific manner, with most abundant levels in the hepatic caecum and ovary. In CHO cells transfected with the expression plasmid pEGFP-N1/BbDUF985, the fusion protein was targeted in the cytoplasm of CHO cells, suggesting that BbDUF985 is a cytosolic protein. In contrast, Western blotting indicated that BbDUF985 was also present in amphioxus humoral fluids, suggesting that it exists as a secreted protein as well. Our study provided a framework for further understanding the biochemical properties and physiological function of DUF985-containing hypothetical proteins in other species.

Comments on 'Significance of developmental expression of amphioxus Branchiostoma belcheri and zebrafish Danio rerio Hsd17b10 in biological and medical research'.

The reported data on the developmental expression of Hsd17b10 gene in Danio rerio is crucial to the utilization of the D. rerio embryo as an animal model for human developmental disorders caused either by mutations on HSD17B10 (formerly HADH2) or by defective expression of the gene. Related diseases were summarized, and it was noticed that hyperinsulinaemic hypoglycaemia is not linked to HSD17B10. This inherited disease is actually caused by a deletion in the HADH gene on chromosome 4. Moreover, it was found by a revision of the reported phylogenetic tree that hydroxyacyl-CoA dehydrogenase II or rather hydroxysteroid (17beta) dehydrogenase 10 (HSD10) of amphioxus Branchiostoma belcheri-occupies a transition position from HSD10 orthologs of invertebrates to those of vertebrates.

The ycaC-related protein from the amphioxus Branchiostoma belcheri (BbycaCR) interacts with creatine kinase.

The ycaC-related gene, ycaCR, is uncharacterized, and has no assigned function to date. Here we clearly showed that the ycaC-related gene from the amphioxus Branchiostoma belcheri, BbycaCR, coded for a novel member of the isochorismatase superfamily, which is mainly localized in the mitochondrial fraction. Both pull-down and reverse pull-down analyses revealed that BbycaCR was able to interact with creatine kinase, an enzyme involved in energy transduction, in addition to binding to native ycaCR, forming a homopolymer. Surprisingly, neither isochorismatase, nicotinamidase nor N-carbamoylsarcosine amidohydrolase activity was detected for BbycaCR, although it possessed the putative catalytic triad of Asp19, Arg(Lys)84 and Cys118 that is found in ycaC proteins. Both tissue section in situ hybridization and immunohistochemistry showed that BbycaCR was ubiquitously expressed in amphioxus, although at different expression levels, suggesting that BbycaCR plays a conserved fundamental cellular role in amphioxus. It is proposed that BbycaCR may be indirectly involved in energy transduction.

Gene duplications and losses within the cyclooxygenase family of teleosts and other chordates.

Cyclooxygenase (COX) produces prostaglandins in animals via the oxidation and reduction of arachidonic acid. Different types and numbers of COX genes have been found in corals, sea squirts, fishes, and tetrapods, but no study has used a comparative phylogenetic approach to investigate the evolutionary history of this complex gene family. Therefore, to examine COX evolution in the teleosts and chordates, 9 novel COX sequences (possessing residues and domains critical to COX function) were acquired from the euryhaline killifish, longhorn sculpin, sea lamprey, Atlantic hagfish, and amphioxus using standard polymerase chain reaction (PCR) and cloning methods. Phylogenetic analyses of these and other COX sequences show a complicated history of COX duplications and losses. There are three main lineages of COX in the chordates corresponding to the three subphyla in the phylum Chordata, with each lineage representing an independent COX duplication. Hagfish and lamprey most likely have traditional COX-1/2 genes, suggesting that these genes originated with the first round of genome duplication in the vertebrates according to the 2R hypothesis and are not exclusively present in the gnathostomes. All teleosts examined have three COX genes due to a teleost-specific genome duplication followed by variable loss of a COX-1 (in the zebrafish and rainbow trout) or COX-2 gene (in the derived teleosts). Future studies should examine the functional ramifications of these differential gene losses.

Characterization, expression and localization of S-adenosylhomocysteine hydrolase from amphioxus Branchiostoma belcheri tsingtaunese.

A cDNA clone encoding AmphiSAHH [amphioxus SAHH (S-adenosylhomocysteine hydrolase)] protein was isolated from a cDNA library from the gut of Branchiostoma belcheri tsingtaunese. It contained a 1305 bp open reading frame corresponding to a deduced protein of 434 amino acid residues, with a predicted molecular mass of approx. 47.8 kDa. Phylogenetic analysis showed that AmphiSAHH and sea-urchin SAHH joined together and positioned at the base of the vertebrate SAHH clade, suggesting that both AmphiSAHH and sea-urchin SAHH might share some characteristics of the archetype of vertebrate SAHH proteins. The genomic DNA sequence of AmphiSAHH contained eight exons and seven introns, which was similar to B. floridae and sea-urchin SAHH exon/intron organization. Sequence comparison suggested the evolutionary appearance of the ten exon/nine intron organization of SAHH genes after the split of invertebrates and vertebrates, after which it has been highly conserved. AmphiSAHH has been successfully expressed in Escherichia coli and purified. Western blotting confirmed that the enzyme has a native molecular mass of approx. 48 kDa, and the catalytic activities and NAD(+)/NADH binding affinity of recombinant AmphiSAHH were measured. Immunohistochemistry analysis showed that SAHH was strongly expressed in hepatic caecum, gill, spermary and ovary of amphioxus.