Evidence for a period of directional selection following gene duplication in a neurally expressed locus of triosephosphate isomerase

A striking correlation between neural expression and high net negative charge in some teleost isozymes led to the interesting, yet untested, suggestion that negative charge represents an adaptation (via natural selection) to the neural environment. We examine the evolution of the triosephosphate isomerase (TPI) gene family in fishes for periods of positive selection. Teleost fish express two TPI proteins, including a generally expressed, neutrally charged isozyme and a neurally expressed, negatively charged isozyme; more primitive fish express only a single, generally expressed TPI isozyme. The TPI gene phylogeny constructed from sequences isolated from two teleosts, a single acipenseriform, and other TPI sequences from the databases, supports a single gene duplication event early in the evolution of bony fishes. Comparisons between inferred ancestral TPI sequences indicate that the neural TPI isozyme evolved through a period of positive selection resulting in the biased accumulation of negatively charged amino acids. Further, the number of nucleotide changes required for the observed amino acid substitutions suggests that selection acted on the overall charge of the protein and not on specific key amino acids.     

Evolution of the Vertebrate Cytosolic Malate Dehydrogenase Gene Family: Duplication and Divergence in Actinopterygian Fish

Abstract A general correlation between neural expression and negative charge in isozymes suggests charge represents an adaptation to the neural environment. Interestingly, a notable exception exists in teleost fish. Two cytosolic malate dehydrogenase (MDH) isozymes have different spatial expression patterns in certain fishes: one is expressed in all tissues and the second is expressed primarily in the eye and skeletal muscle. While the neural MDH isozyme is negatively charged, the difference in charge between the two isozymes is not as pronounced as that observed in other gene families (e.g., triosephosphate isomerase and lactate dehydrogenase). Most tetrapods express a single cytosolic MDH isozyme, and it has been demonstrated recently that the pair of isozymes found in teleosts results from a gene duplication sometime after the separation of teleosts and tetrapods, although the exact timing of this duplication has not been inferred. Phylogenetic analyses suggest that the duplication of teleost isozymes occurred during the radiation of actinopterygian fish, consistent with the timing of duplication at other loci. Using inferred amino acid sequences, we examine the pattern of change following the duplication and across the rest of the MDH gene tree. Comparison between the MDH gene family and another gene family that shows a larger charge differential among members (triosephosphate isomerase) indicates that the smaller charge difference between MDH isozymes is best explained by greater constraint on amino acid change directly following the duplication, not greater constraint across the entire gene tree. This difference in constraint might result from the wider pattern of expression of the “neural” MDH isozyme.     

Molecular Evolution of Teleost Neural Isozymes

Isozymes, homologous enzymes coded by separate loci within a genome, present interesting systems for examining molecular and functional divergence through natural selection. Isozyme pairs for a number of metabolic enzymes, including Triosephosphate isomerase (Tpi), Malate dehydrogenase (Mdh), Phosphoglucose isomerase (Pgi), and Guanylate kinase (Guk), appear to all result from a single, large duplication event early in teleost evolution. These small gene families include two forms, a generally expressed form with no apparent charge and a neurally expressed form with a pronounced negative charge although the canalization of expression of the second form varies across families. Using ancestral sequence reconstructions and standard comparisons of rates of nonsynonymous and synonymous change, combined with the examination of the specific amino acid changes observed and predicted we examined the evolution of the Tpi and Guk families using all available vertebrate sequences and all four families using a smaller, common, dataset. We find that post-duplication, the neural Tpi and Guk isozymes evolved through similar periods of positive selection as evidenced by elevated rates of nonsynonymous change and accumulation of negative amino acids. Over the same evolutionary period our analysis suggests that Mdh and Pgi isozymes appear to have evolved under a less divergent pattern of selection. These distinct results likely reflect functional differences between the isozymes, possibly a result of differences in expression patterns.     

Duplication of phospholipase C-delta gene family in fish genomes

Fishes possess more genes than other vertebrates, possibly because of a genome duplication event during the evolution of the teleost (ray-finned) fish lineage. To further explore this idea, we cloned five genes encoding phosphoinositide-specific phospholipase C-delta (PLC-delta), designated respectively PoPLC-deltas, from olive flounder (Paralichthys olivaceus), and we performed phylogenetic analysis and sequence comparison to compare our putative gene products (PoPLC-deltas) with the sequences of known human PLC isoforms. The deduced amino acid sequences shared high sequence identity with human PLC-delta1, -delta3, and -delta4 isozymes and exhibited similar primary structures. In phylogenetic analysis of PoPLC-deltas with PLC-deltas of five teleost fishes (zebrafish, stickleback, medaka, Tetraodon, and Takifugu), three tetrapods (human, chicken, and frog), and two tunicates (sea squirt and pacific sea squirt), whose putative sequences of PLC-delta are available in Ensembl genome browser, the result also indicated that the two paralogous genes corresponding to each PLC-delta isoform originated from fish-specific genome duplication prior to the divergence of teleost fish. Our analyses suggest that an ancestral PLC-delta gene underwent three rounds of genome duplication during the evolution of vertebrates, leading to the six genes of three PLC-delta isoforms in teleost fish.     

Coexpression of distinct eye- and liver-specific LDH isozymes in cichlid fish

In a recent electrophoretic survey of lactate dehydrogenase (LDH) in neotropical cichlid fishes (Perciformes, Cichlidae) we have discovered several species in which a cathodal liver-specific isozyme is expressed along with the highly-anodal eye-specific isozyme (LDH-C4) typically encountered in perciform fishes. We believe this fourth, liver-specific LDH isozyme to be real and not artifactual since homogenization of fresh liver from one of these species, the Basketmouth cichlid (Acaronia nassa), in either of two nondenaturing detergents or in the presence of the protease inhibitor phenylmethylsulfonylfluoride affects neither the presence nor mobility of this cathodal band. Moreover, it continues to be expressed in the captively bred F1 of these same wild fish. The discovery of several fish species, like the Basketmouth, in which biochemically distinct eye- and liver-specific LDH isozymes are coexpressed, is discussed in light of the currently accepted hypothesis that these two isozymes are encoded by a single locus (LDH-C) which has undergone divergent tissue expression in several other major teleost groups. Preliminary characterization of the liver-specific isozyme relative to the eye-specific LDH-C4 in the Basketmouth cichlid with respect to thermolability and NADH-induced binding to oxamate-sepharose columns suggests that the eye- and liver-specific LDH isozymes are biochemically quite distinct in this fish and that they are probably encoded by two distinct loci.     

Expression, purification and the 1.8 angstroms resolution crystal structure of human neuron specific enolase

Human neuron-specific enolase (NSE) or isozyme gamma has been expressed with a C-terminal His-tag in Escherichia coli. The enzyme has been purified, crystallized and its crystal structure determined. In the crystals the enzyme forms the asymmetric complex NSE x Mg2 x SO4/NSE x Mg x Cl, where "/" separates the dimer subunits. The subunit that contains the sulfate (or phosphate) ion and two magnesium ions is in the closed conformation observed in enolase complexes with the substrate or its analogues; the other subunit is in the open conformation observed in enolase subunits without bound substrate or analogues. This indicates negative cooperativity for ligand binding between subunits. Electrostatic charge differences between isozymes alpha and gamma, -19 at physiological pH, are concentrated in the regions of the molecular surface that are negatively charged in alpha, i.e. surface areas negatively charged in alpha are more negatively charged in gamma, while areas that are neutral or positively charged tend to be charge-conserved.     

Divergent Toll-like receptors in catfish (Ictalurus punctatus): TLR5S, TLR20, TLR21

Toll-like receptors (TLR) mediate pathogen recognition in vertebrate species through detection of conserved microbial ligands. Families of TLR molecules have been described from the genomes of the teleost fish model species zebrafish and Takifugu, but much research remains to characterize the full length sequences and pathogen specificities of individual TLR members in fish. While the majority of these pathogen receptors are conserved among vertebrate species with clear orthologues present in fish for most mammalian TLRs, several interesting differences are present in the TLR repertoire of teleost fish when compared to that of mammals. A soluble form of TLR5 has been reported from salmonid fish and Takifugu rubripes which is not present in mammals, and a large group of TLRs (arbitrarily numbered 19-23) was identified from teleost genomes with no easily discernible orthologues in mammals. To better understand these teleost adaptations to the TLR family, we have isolated, sequenced, and characterized the full-length cDNA and gene sequences of TLR5S, TLR20, and TLR21 from catfish as well as studied their expression pattern in tissues. We also mapped these genes to bacterial artificial chromosome (BAC) clones for genome analysis. While TLR5S appeared to be common in teleost fish, and TLR21 is common to birds, amphibians and fish, TLR20 has only been identified in zebrafish and catfish. Phylogenetic analysis of catfish TLR20 indicated that it is closely related to murine TLR11 and TLR12, two divergent TLRs about which little is known. All three genes appear to exist in catfish as single copy genes.     

Molecular characterization and expression pattern of the equine lactate dehydrogenase A and B genes

The species-specific properties of LDH isozymes are essentially determined by M (muscle) and H (heart) subunit proteins encoded by the LDHA and LDHB genes, respectively. In the present study, we molecularly characterized the full-length equine lactate dehydrogenase A (eLDHA) and B (eLDHB) cDNAs. The eLDHA cDNA consisted of a 999-bp open reading frame (ORF), while the eLDHB and newly acquired bat LDHB consisted of a 1002-bp ORF, which is 3 bp shorter than the LDHB ORF of other registered mammals. The alignment of amino acid sequences showed that eLDHA acquired positively charged His 88 and 226, and eLDHB lost negatively charged Glu 14, as compared to the highly conserved residues at these positions in the corresponding amino acid sequences of other mammals. These alterations were identified in six equine species by genomic DNA analysis. A comparison of the equine and human 3D structures revealed that the substituted His 88 and 226 of the eLDHA monomer and the deleted Glu 14 of the eLDHB monomer altered the surface charge of equine LDH tetramers and that these three residues were located in important regions affecting the catalytic kinetics. Also, RT-PCR amplification of the three myosin heavy chain isoforms corroborated that the cervical muscle as postural muscle of the thoroughbred horse was composed of more oxidative myofibers than the dynamic muscle. Based on this property, the mRNA expression patterns of eLDHA, eLDHB, and eGAPDH in various tissues were analyzed by using real-time PCR. The expression levels of these three genes in the cervical muscle were not always relatively higher than in the brain or heart.     

Spatial clustering of isozyme-specific residues reveals unlikely determinants of isozyme specificity in fructose-1,6-bisphosphate aldolase

Vertebrate fructose-1,6-bisphosphate aldolase exists as three isozymes (A, B, and C) that demonstrate kinetic properties that are consistent with their physiological role and tissue-specific expression. The isozymes demonstrate specific substrate cleavage efficiencies along with differences in the ability to interact with other proteins; however, it is unknown how these differences are conferred. An alignment of 21 known vertebrate aldolase sequences was used to identify all of the amino acids that are specific to each isozyme, or isozyme-specific residues (ISRs). The location of ISRs on the tertiary and quaternary structures of aldolase reveals that ISRs are found largely on the surface (24 out of 27) and are all outside of hydrogen bonding distance to any active site residue. Moreover, ISRs cluster into two patches on the surface of aldolase with one of these patches, the terminal surface patch, overlapping with the actin-binding site of aldolase A and overlapping an area of higher than average temperature factors derived from the x-ray crystal structures of the isozymes. The other patch, the distal surface patch, comprises an area with a different electrostatic surface potential when comparing isozymes. Despite their location distal to the active site, swapping ISRs between aldolase A and B by multiple site mutagenesis on recombinant expression plasmids is sufficient to convert the kinetic properties of aldolase A to those of aldolase B. This implies that ISRs influence catalysis via changes that alter the structure of the active site from a distance or via changes that alter the interaction of the mobile C-terminal portion with the active site. The methods used in the identification and analysis of ISRs discussed here can be applied to other protein families to reveal functionally relevant residue clusters not accessible by conventional primary sequence alignment methods.     

Patterns of gene expression during teleost embryogenesis: Lactate dehydrogenase isozyme ontogeny in the medaka (Oryzias iatipes)

The ontogeny of the lactate dehydrogenase (LDH; EC 1.1.1.27) isozymes during medaka (Oryzias latipes) embryogenesis was determined after the genetic and molecular bases of this multilocus isozyme system were established. Three LDH loci are differentially expressed among the tissues of the adult medaka. The LDH-A locus was expressed almost exclusively in the white skeletal muscle, the LDH-B locus in all tissues examined, and the LDH-C locus in the eye and brain. The contribution of each of these LDH loci was quantitatively determined throughout early medaka embryogenesis by using a combination of electrophoretic, immunochemical, and spectrophotometric procedures. LDH-B₄ is present throughout embryogenesis and is the predominant LDH isozyme during this period. LDH-C subunit activity was first detected 146 hr after fertilization (26°C), 142 hr prior to hatching. LDH-A subunit activity, however, was not detected until after hatching and, then, only as heterotetramers containing LDH-B subunits. The pattern of LDH gene expression during medaka embryogenesis was compared with the patterns of LDH gene expression during early development in five other teleost species. Some common patterns of differential LDH gene expression appear to exist among the teleosts. In all species examined, isozymes encoded in at least one LDH locus, A and/or B, were present throughout development. Those isozymes present continually during embryogenesis also tend to be active in a wide variety of differentiated tissues in the adult fish. Conversely, LDH isozymes which are active in a restricted number of adult tissues are detected only later in embryogenesis. The initiation of LDH-C gene expression, however, is closely coupled with morphological and functional differentiation of those cells in which this locus is predominantly expressed in the adult.     

Comparison of hypoxia-inducible factor-1 alpha in hypoxia-sensitive and hypoxia-tolerant fish species

Levels of oxygen can vary dramatically in aquatic environments. Aquatic organisms, including fishes, have adapted accordingly to survive. As there are both phylogenetically closely related fish species with differing oxygen requirements and distantly related species with similar oxygen requirements, fishes are good candidates for examining oxygen-related functions in vertebrates. We set out to investigate if sequence variation in the hypoxia-inducible factor-1 alpha (HIF-1α) gene is associated with variations in oxygen requirements. Since the teleost HIF-1α sequences available in databases represent a very limited dataset both phylogenetically and with regard to oxygen requirements, we have sequenced the protein coding sequence for HIF-1α from an additional 9 fish species. Our results indicate that the deduced HIF-1α proteins of teleost fishes are somewhat shorter than those of tetrapods. Additionally, the results suggest that tetrapod sequences more closely resemble the ancestral form of the protein than do teleost sequences. No clear signatures which could be associated with the oxygen requirements of the species were found. This study suggests that if species-specific differences in HIF-1α function with regards to oxygen dependence have evolved, they do not occur in the protein coding sequence but at other levels of the HIF-1α pathway.     

Stanniocalcin in terminally differentiated mammalian cells

Stanniocalcin (STC) is a glycoprotein hormone originally found in teleost fish, where it regulates the calcium/phosphate homeostasis, and protects the fish against toxic hypercalcemia. STC was considered an exclusive fish protein, until the cloning of cDNA for human (in 1995) and murine (in 1996) STC. We originally reported a high constitutive content of STC in mammalian brain neurons, and found that the expression of STC occurred concomitantly with terminal differentiation of neural cells. Since then, we have investigated the expression of STC in relation to terminal cell differentiation also in mammalian hematopoietic tissue, and fat tissues. In this review we summarize our findings on STC expression during postmitotic differentiation in three different cell systems; in neural cells, in megakaryocytes and in adipocytes. We also present findings, suggesting that STC plays a role for maintaining the integrity of terminally differentiated mammalian cells.     

Stimulation of dopamine D1 receptor improves learning capacity in cooperating cleaner fish

Accurate contextual decision-making strategies are important in social environments. Specific areas in the brain are tasked to process these complex interactions and generate correct follow-up responses. The dorsolateral and dorsomedial parts of the telencephalon in the teleost fish brain are neural substrates modulated by the neurotransmitter dopamine (DA), and are part of an important neural circuitry that drives animal behaviour from the most basic actions such as learning to search for food, to properly choosing partners and managing decisions based on context. The Indo-Pacific cleaner wrasse Labroides dimidiatus is a highly social teleost fish species with a complex network of interactions with its ‘client’ reef fish. We asked if changes in DA signalling would affect individual learning ability by presenting cleaner fish two ecologically different tasks that simulated a natural situation requiring accurate decision-making. We demonstrate that there is an involvement of the DA system and D₁ receptor pathways on cleaners'' natural abilities to learn both tasks. Our results add significantly to the growing literature on the physiological mechanisms that underlie and facilitate the expression of cooperative abilities.     

Characterization of the major histocompatibility complex class II genes in miiuy croaker

Major histocompatibility complex (MHC) has a central role in the adaptive immune system by presenting foreign peptide to the T-cell receptor. In order to study the molecular function and genomic characteristic of class II genes in teleost, the full lengths of MHC class IIA and IIB cDNA and genomic sequence were cloned from miiuy croaker (Miichthys miiuy). As in other teleost, four exons and three introns were identified in miiuy croaker class IIA gene; but the difference is that six exons and five introns were identified in the miiuy croaker class IIB gene. The deduced amino acid sequence of class IIA and class IIB had 26.3-85.7% and 11.0-88.8% identity with those of mammal and teleost, respectively. Real-time quantitative RT-PCR demonstrated that the MHC class IIA and IIB were ubiquitously expressed in ten normal tissues; expression levels of MHC genes were found first upregulated and then downregulated, and finally by a recovery to normal level throughout the pathogenic bacteria infection process. In addition, we report on the underlying mechanism that maintains sequences diversity among many fish species. A series of site-model tests implemented in the CODEML program revealed that positive Darwinian selection is likely the cause of the molecular evolution in the fish MHC class II genes.     

稀有鮈鲫脑芳香化酶cDNA片段的克隆与表达分析

芳香化酶（P450arom）是雌激素合成过程中的关键酶,在性别分化中起重要作用。鱼类存在卵巢性和脑型两种芳香化酶,分别由Cyp19a和Cyp19b编码。稀有鮈鲫作为我国特有的实验动物,尚无其芳香化酶的有关资料,其性别分化机制亦不清楚。本研究采用RT-PCR的方法以简并引物扩增了稀有鮈鲫脑型芳香化酶基因Cyp19b的部分序列,其长度为1070 bp, 编码357个氨基酸残基,具有典型的芳香化酶结构域。RT-PCR分析发现该基因主要在稀有鮈鲫的脑中表达,在性腺、肠、肾中也有表达;其在雌、雄脑中的表达差异不显著。在胚胎发育阶段,Cyp19b的表达从囊胚期开始,至神经胚期达到较高水平,随后下降,孵化期又有所增强,孵化10天后维持在高水平。这些结果说明Cyp19b以脑中表达为主,可能在稀有鮈鲫神经系统发育和脑的性别分化中有重要作用。     

Construction and expression of human aldolase A and B expression plasmids in Escherichia coli host

E. coli expression plasmids for human aldolases A and B (EC 4.1.2.13) have been constructed from the pIN-III expression vector and their cDNAs, and expressed in E. coli strain JM83. Enzymatically active forms of human aldolase have been generated in the cells when transfected with either pHAA47, a human aldolase A expression plasmid, or pHAB 141, a human aldolase B expression plasmid. These enzymes are indistinguishable from authentic enzymes with respect to molecular size, amino acid sequences at the NH2- and COOH-terminal regions, the Km for substrate, fructose 1,6-bisphosphate and the activity ratio of fructose 1,6-bisphosphate/fructose 1-phosphate (FDP/F1P), although net electric charge and the Km for FDP of synthetic aldolase B differed from those for a previously reported human liver aldolase B. In addition, both the expressed aldolases A and B complement the temperature-sensitive phenotype of the aldolase mutant of E. coli h8. These data argue that the expressed aldolases are structurally and functionally similar to the authentic human aldolases, and would provide a system for analysis of the structure-function relationship of human aldolases A and B.     

Basal lamina secreted by MDCK cells has size- and charge-selective properties

The role electrical charge plays in determining glomerular permeability to macromolecules remains unclear. If the glomerular basement membrane (GBM) has any significant role in permselectivity, physical principles would suggest a negatively charged GBM would reject similarly charged more than neutral species. However, recent in vivo studies with negative and neutral glomerular probes showed the opposite. Whether this observation is due to unique characteristics of the probes used or is a general physiological phenomenon remains to be seen. The goal of this study was to use the basement membrane deposited by Madin-Darby canine kidney epithelial cells as a simple model of a biologically derived, negatively charged filter to evaluate size- and charge-based sieving properties. Fluorescein isothiocyanate-labeled carboxymethylated Ficoll 400 (FITC-CM Ficoll 400) and amino-4-methyl-coumarin-labeled Ficoll 400 (AMC Ficoll 400) were used as negatively charged and neutral tracer molecules, respectively, during pressure-driven filtration. Streaming potential measurement indicated the presence of fixed, negative charge in the basal lamina. The sieving coefficient for neutral Ficoll 400 decreased by ～0.0013 for each 1-? increment in solute radius, compared with a decrease of 0.0023 per ? for the anionic Ficoll 400. In this system, molecular charge played a significant role in determining the sieving characteristics of the membrane, pointing to solute charge as a potential contributor to GBM permselectivity.     

Type I keratin cDNAs from the rainbow trout: independent radiation of keratins in fish

Five different type I keratins from a teleost fish, the rainbow trout Oncorhynchus mykiss, have been sequenced by cDNA cloning and identified at the protein level by peptide mass mapping using MALDI-MS. This showed that the entire range of type I keratins detected biochemically in this fish has now been sequenced. Three of the keratins are expressed in the epidermis (subtype Ie), whereas the other two occur in simple epithelia and mesenchymal cells (subtype Is). Among the Is keratins is an ortholog of human K18; the second Is polypeptide is clearly distinct from K18. We raised a new monoclonal antibody (F1F2, subclass IgG1) that specifically recognizes trout Is keratins, with negative reactions on zebrafish. A phylogenetic tree has been constructed from a multiple alignment of the rod domains of the new sequences together with type I sequences from other vertebrates such as shark, zebrafish, and human; a recently sequenced lamprey Is keratin was applied as outgroup. This tree shows one branch defining the K18 orthologs and a second branch containing all other type I keratins (mostly subtype Ie). Within this second branch, the teleost keratins form a separate, highly bootstrap-supported twig. This tree leaves little doubt that the teleost Ie keratins diversified independently from the mammalian Ie keratins.