Identification of cDNAs encoding two subtypes of vitamin D receptor in flounder, Paralichthys olivaceus

cDNAs encoding two subtypes of the vitamin D receptor (VDR) are cloned from a teleost (flounder, Paralichthys olivaceus). This is the first report of VDR subtypes in fish. Flounder VDR (fVDR) a and b share 86% identity at the amino acid level. With human (h), rat, mouse, quail, and Xenopus VDRs, fVDRa shares 72%, 71%, 71%, 69%, and 71% identity, and fVDRb shares 70%, 69%, 69%, 67%, and 68% identity, respectively. The peptide sequences of the DNA-binding domain (DBD) and hormone-binding domain (HBD) of both subtypes have particularly high homology to those of the tetrapods; e.g. 92% identity for DBP and 74% for HBD between fVDRa and hVDR. In an evolutionary tree constructed with peptide sequences of VDRs and related members of the nuclear receptor superfamily, fVDRa and b are more closely related to each other than to other molecules, and situated in the cluster of VDRs at a position which corresponds well with the evolutional position of fish in the vertebrates. Additional independent genome duplication which is thought to have occurred in ray-finned fish phylogeny may explain the existence of two subtypes of VDR in flounder.     

Isolation and characterization of human TR3 receptor: a member of steroid receptor superfamily

Complementary DNAs (cDNAs) encoding a member of steroid receptor super-family, named TR3 receptor, were isolated from a human prostate lambda gt11 cDNA library on the basis of homology of oligonucleotide probes to the DNA-binding domain common to members of the steroid receptor super-family. Expression of TR3 receptor cDNA produced a 64 kDa DNA-binding protein in a rabbit reticulocyte lysate. Nucleotide sequence analysis showed that TR3 receptor cDNA contains two regions of sequences which correspond to the DNA- and hormone-binding domains of members of the steroid receptor super-family. The amino acid sequences in the hormone-binding domain of the TR3 receptor shares about 20% homology with estrogen receptor and less than 15% homology with other known steroid receptors. The DNA-binding domain of the TR3 receptor has about 55% homology with all other known steroid receptors. TR3 receptor had 86% nucleotide and 91% amino acid sequence homology with mouse NUR/77, suggesting that TR3 receptor may be a human homologue of mouse NUR/77 gene product.     

Comparative aspects of adrenergic receptors in the hearts of lower vertebrates

The cardiac adrenoceptors of lower vertebrates were characterized in atrial preparations. Adrenaline (A) potentiated the force and frequency of contraction in the spontaneously beating atria of the frog, trout and flounder and in electrically paced atrial strips from the shark. The inotropic responses of A were most pronounced at the lower temperatures for the frog and trout, while A enhanced frequency to a greater extent at higher temperatures in the frog atria. Atrial alpha-receptors activated by A at 8 degrees C could not be detected in any of the species under study. The apparent affinities for the inotropic and chronotropic responses of agonist in the frog (15 degrees C) and trout (8 degrees C) atria were: Iso greater than Sal greater than or equal to A greater than NA. A cocaine-sensitive uptake for A and NA was apparent in these atria, consistent with sympathetic innervation. The affinities for the catecholamines in the flounder and shark atria were not increased by cocaine, in accordance with absence of sympathetic innervation of the atria in these species. These atria were also insensitive to corticosterone. The affinities for A and NA were on the other hand higher in the sympathetically non-innervated atria of the flounder than in the innervated atria of the frog and trout. The apparent orders of relative affinities for agonists were Iso greater than A = NA greater than Sal for the flounder, and of the relative potencies Iso = A greater than NA greater than Sal for the shark atrium. The results are consistent with the hypothesis that catecholamines enhance cardiac performance in lower vertebrates chiefly via "adrenaline" receptors which resemble the beta 2-type of mammalian adrenoceptors in many respects. Unlike that in mammals, cardiac adrenaline receptors in the frog and trout are activated by the sympathetic neurotransmitter ("innervated" receptors). On the other hand, the adrenaline receptors of the flounder and shark are responding to the circulating catecholamines ("humoral" receptors). However, the flounder atrium, with equal affinities for A and NA, appears as an exception to the rule by having a mixed population of humoral beta 1- and beta 2-adrenoceptors, indicating a role for circulating NA in cardiac regulation in this species.     

Molecular cloning,structural analysis,and expression of carp ZP2 gene

The cDNAs encoding carp ZP2 homologous to winter flounder and mammalian ZP2 were cloned. Carp ZP2 contains a tandemly repetitive domain and a nonrepetitive domain. A repeat is composed of 13 amino-acid residues whose consensus sequence is QQTSQQFQPQKPA/V. The length of the repetitive domain is highly variable, but that of the nonrepetitive domain is fairly constant among various cDNAs. The termination codons of various cDNAs appear at three different positions. Three groups of cDNAs were therefore categorized. Groups I–III encode a nonrepetitive domain of 356, 255, and 10 residues, respectively. A carp ZP2 gene corresponding to group II cDNA was cloned. It spans 2.4 kb and consists of eight exons and seven introns. Carp ZP2 mRNA was detected only in oocytes but not in other tissues. Carp ZP2 is heterogenous in size. The molecular weight ranges from 40–80 kDa. It is present in vitellogenic but not in previtellogenic oocytes, nor in other tissues. Carp ZP2 content in oocytes increases as vitellogenesis proceeds. Mol. Reprod. Dev. 46:258–267, 1997. © 1997 Wiley-Liss, Inc.     

Molecular analysis of complement component C8β and C9 cDNAs of Japanese flounder, Paralichthys olivaceus

 The amino acid sequences of the human terminal complement components show extensive structural similarity to each other. In this study the C8β and C9 cDNAs of Japanese flounder, Paralichthys olivaceus, were cloned and analyzed. The derived deduced amino acid sequences of the two terminal components were homologous to those of humans, in that the sequences of both species contained LDL receptor, EGF precursor, and two thrombospondin domains. Japanese flounder C9 was found to have a second thrombospondin region in the C-terminus, similar to that reported for rainbow trout and pufferfish. Moreover, these two complement component cDNAs of Japanese flounder had partial similarity to human perforin. These findings show that Japanese flounder C8β and C9 have similar structures, which supports the hypothesis that the terminal complement genes originated from the same ancestral gene. Collectively, these features emphasize the strong similarity among the members of the terminal complement family. Received: 23 March 1999 / Revised: 1 June 1999     

Stable Expression of Recombinant AMPA Receptor Subunits: Binding Affinities and Effects of Allosteric Modulators

Abstract: Homomeric AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid)-type glutamate receptors (GluRs) were stably expressed in kidney cells from cDNAs encoding GluR1 flop, GluR2 flip, GluR2 flop, and GluR3 flop subunits. The recombinant receptors were of the expected size and showed functional properties in whole-cell recording as previously reported. [³H]AMPA binding to all subunits was increased to a similar extent by the chaotropic ion thiocyanate (SCN^?). Significant differences were found in the Scatchard plots, however, which were linear and of high affinity for GluR1 and -3 receptors (K_D values of 33 and 52 nM, respectively) but showed curvature for GluR2 receptors, indicating the presence of two components with distinct affinities. As with brain AMPA receptors, solubilization of GluR2 receptors reduced the number of lower-affinity sites and correspondingly increased the number of higher-affinity sites. The sulfhydryl reagent p-chloromercuriphenylsulfonic acid, which increases binding to brain receptors, produced only minor changes except in the case of GluR2 flip. These results indicate that GluR2, among the subunits examined here, most closely resembles the native AMPA receptors in brain membranes. [³H]AMPA binding was inhibited in a noncompetitive manner by two drugs that change the desensitization kinetics of the AMPA receptor. In agreement with physiological observations, the apparent affinity of cyclothiazide for GluR2 flip (EC₅₀ = 7 µM) was higher than that for receptors made of flop subunits (49–130 µM). In contrast, BDP-37, a member of the benzamide family of drugs, exhibited a lower potency for GluR2 flip (58 µM) than for any of the flop isoforms (18–40 µM). These results predict that the action of centrally active AMPA-receptor modulators varies across brain regions depending on their flip/flop composition.     

Characterization of gene structure and expression of two toll-like receptors from Japanese flounder, <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis>

We cloned the cDNAs and genes of two different types of toll-like receptors from Japanese flounder. The results of homology searches suggested that these genes (designated JF-TLR2 and JF-TLR22) are homologues of human TLR2 and fugu TLR22, respectively. The cDNAs of JF-TLR2 and JF-TLR22 encoded 818 and 961 amino acid residues, respectively. JF-TLR2 and JF-TLR22 contained two distinct structural/functional motifs of the TLR family, such as a leucine-rich repeat (LRR) domain in the extracellular portion and a toll/interleukin-1 receptor (TIR) domain in the intracellular portion. The genes of JF-TLR2 and JF-TLR22 consisted of 12 exons (4.9 kb in total length) and four exons (4.3 kb in total length), respectively. The first exon of each gene is a non-coding exon. Southern blot hybridization indicated that both JF-TLR2 and JF-TLR22 exist as single copies in the genome. These genes were mainly expressed in peripheral blood leukocytes (PBLs) and weakly expressed in PBL-rich organs such as kidney, spleen and gill. Expression of these genes was induced by both peptidoglycan and polyI:C, although the number of JF-TLR-expressing cells were not changed after induction. All of these results suggest that they are involved in the innate immune system.     

Linking the Acetylcholine Receptor-Channel Agonist-Binding Sites with the Gate

The gating isomerization of neuromuscular acetylcholine receptors links the rearrangements of atoms at two transmitter-binding sites with those at a distant gate region in the pore. To explore the mechanism of this reversible process, we estimated the gating rate and equilibrium constants for receptors with point mutations of α-subunit residues located between the binding sites and the membrane domain (N95, A96, Y127, and I49). The maximum energy change caused by a side-chain substitution at αA96 was huge (∼8.6 kcal/mol, the largest value measured so far for any α-subunit amino acid). A Φ-value analysis suggests that αA96 experiences its change in energy (structure) approximately synchronously with residues αY127 and αI49, but after the agonist molecule and other residues in loop A. Double mutant-cycle experiments show that the energy changes at αA96 are strongly coupled with those of αY127 and αI49. We identify a column of mutation-sensitive residues in the α-subunit that may be a pathway for energy transfer through the extracellular domain in the gating isomerization.     

Complete sequencing and expression of three complement components,C1r,C4 and C1 inhibitor,of the classical activation pathway of the complement system in rainbow trout<Emphasis Type="Italic"> Oncorhynchus mykiss</Emphasis>

Three complement components, C1r, C4 and C1 inhibitor, of the classical activation pathway have been fully sequenced and their expression investigated in rainbow trout (Oncorhynchus mykiss). Trout C1r cDNA encodes a 707-amino-acid (aa) protein with a theoretical M _r of 77,200. The trout translation shows highest homology with carp C1r/s, and lower, equal homologies to mammalian C1r and C1s, and MASPs from other vertebrate species. However, phylogenetic analysis and structural features suggest that the trout sequence, together with the two carp sequences, are the orthologues of mammalian C1r. The trout C4 cDNA encodes a 1,724-aa protein with a theoretical M _r of 192,600. The trout translation shows higher homologies to the carp C4B and medaka C4, but lower homologies to C4 from other species and the carp C4A. It has a predicted signal peptide of 22 aa, a -chain of 773 aa, a -chain of 635 aa and a -chain of 288 aa. Trout C1 inhibitor cDNA encodes a 611-aa protein with a theoretical M _r of 68,700. The trout translation has a C-terminal serpin domain with high homologies with mammalian counterparts (~37% identities), and a longer N-terminus, with no significant homology to other serpins, which contains two Ig-like domains. A molecule containing two Ig-like domains followed by a serpin domain, has also been found in an EST clone from another bony fish, the Japanese flounder. This suggests a unique structural feature of C1 inhibitor in fish. The functional significance of the Ig domains is discussed. The liver is the major site of expression of the three trout complement components, C1r, C4 and C1 inhibitor, although their expression is also detectable in other tissues. The extra-hepatic expression of complement genes may be important for local protection and inflammatory responses. Low-level constitutive expression of the three components was also detectable in a trout monocyte/macrophage cell line RTS-11, but only the expression of C4 could be upregulated by LPS.The nucleotide sequence data will appear in the EMBL/DDBJ/GenBank nucleotide sequence database under the following accession numbers: AJ519929 (trout C1r), AJ519930 (trout C1 inhibitor), AJ544262 (trout C4) and BN000290 (flounder C1 inhibitor)     

Molecular cloning and mRNA tissue expression of thyroid hormone receptors in yellow catfish Pelteobagrus fulvidraco and Javelin goby Synechogobius hasta

Thyroid hormones (THs) play a pivotal role in many physiological functions in vertebrates, including fish. Their effects are mediated by thyroid hormone receptors (TRs), which are members of the nuclear hormone receptor superfamily. In this study, full-length cDNA sequences of TRs from yellow catfish Pelteobagrus fulvidraco and Javelin goby Synechogobius hasta were cloned and their mRNA tissue expression profiles were determined. In P. fulvidraco, the validated cDNAs encoding for TRα and TRβ were 1789 and 1848 bp in length, encoding peptides of 401 and 378 amino acid residues, respectively. In addition, a TRβ spliced variant (named P. fulvidraco-TRβv), containing a 60-bp insertion, was detected. In S. hasta, cDNAs encoding for TRαA, TRαB and TRβ were 1827, 2295 and 2258 bp in length, encoding peptides of 401, 409 and 393 amino acid residues, respectively. The phylogenetic analysis revealed that TRα and TRβ cDNAs grouped into two separate clusters with other vertebrate counterparts and two TRα sequences grouped separately, suggesting that the two TRαs derived from paralogous genes that might arise during a teleost-specific genome duplication event. All TR mRNAs were detected in various tissues sampled. The mRNA levels of both TRα and TRβ from P. fulvidraco were the highest in brain, followed by liver, and lowest in heart, intestine, muscle, gill and spleen. However, in S. hasta, TRαA, TRαB and TRβ showed the highest mRNA levels in brain and lowest in muscle. Identification and mRNA tissue expression of TR genes from P. fulvidraco and S. hasta provide an initial step towards understanding their biological roles in the two fish species.     

Signal transduction by steroid hormones: nuclear localization is differentially regulated in estrogen and glucocorticoid receptors.

The glucocorticoid receptor accumulates in nuclei only in the presence of bound hormone, whereas the estrogen receptor has been reported to be constitutively nuclear. To investigate this distinction, we compared the nuclear localization domains of the two receptors and the capacity of their respective hormone-binding regions to regulate nuclear localization activity. As with the glucocorticoid receptor, we showed that the human estrogen receptor contained a nuclear localization signal between the DNA-binding and hormone-binding regions (amino acids 256-303); however, in contrast to the glucocorticoid receptor, the estrogen receptor lacked a second nuclear localization domain within the hormone-binding region. Moreover, the hormone-binding domain of the unliganded estrogen receptor failed to regulate nuclear localization signals, although it efficiently regulated other receptor functions. We conclude that the two receptors employ a common mechanism for signal transduction involving a novel "inactivation" function, but that they differ in their control of nuclear localization. Thus, despite the strong relatedness of the estrogen and glucocorticoid receptors in structure and activity, certain differences in their properties could have important functional implications.     

Solution structure of peptides from HIV-1 Vpr protein that cause membrane permeabilization and growth arrest

Vpr, one of the accessory gene products encoded by HIV-1, is a 96-residue protein with a number of functions, including targeting of the viral pre-integration complex to the nucleus and inducing growth arrest of dividing cells. We have characterized by 2D NMR the solution conformations of bioactive synthetic peptide fragments of Vpr encompassing a pair of H(F/S)RIG sequence motifs (residues 71–75 and 78–82 of HIV-1 Vpr) that cause cell membrane permeabilization and death in yeast and mammalian cells. Due to limited solubility of the peptides in water, their structures were studied in aqueous trifluoroethanol. Peptide Vpr^59–86 (residues 59–86 of Vpr) formed an α-helix encompassing residues 60–77, with a kink in the vicinity of residue 62. The first of the repeated sequence motifs (HFRIG) participated in the well-defined α-helical domain whereas the second (HSRIG) lay outside the helical domain and formed a reverse turn followed by a less ordered region. On the other hand, peptides Vpr^71–82 and Vpr^71–96, in which the sequence motifs were located at the N-terminus, were largely unstructured under similar conditions, as judged by their C^αH chemical shifts. Thus, the HFRIG and HSRIG motifs adopt α-helical and turn structures, respectively, when preceded by a helical structure, but are largely unstructured in isolation. The implications of these findings for interpretation of the structure–function relationships of synthetic peptides containing these motifs are discussed. © 1998 European Peptide Society and John Wiley & Sons, Ltd.     

Cloning of two members of the calcitonin-family receptors from stingray, Dasyatis akajei: possible physiological roles of the calcitonin family in osmoregulation

In cartilaginous fish, two cDNAs encoding calcitonin-family receptors were isolated for the first time from the stingray brain. The open reading frame of one receptor cDNA coded a 525-amino acid protein. The amino acid identity of this receptor to human calcitonin-receptor-like receptor (CRLR) is 64.5%, frog CRLR is 64.7%, and flounder CRLR is 61.2% and this was higher than to human calcitonin receptor (CTR) (46.1%), frog CTR (54.7%), and flounder CTR (48.9%). We strongly suggested that this receptor is a ray CRLR based on phylogenetic analysis. In case of the second receptor, amino acid identity among CRLRs (human 50.5%, frog 50.7%, flounder 48.0%) and CTRs (human 43.2%, frog 49.1%, flounder 41.8%) was similar. From phylogenetic analysis of both CRLRs and CTRs, we believe that this receptor is ray CTR. The expression of ray CRLR mRNA was predominantly detected in the nervous system (brain) and vascular system (atrium, ventricle, and gill), which reflects the similar localization of CGRP in the nervous and vascular systems as mammals. It was observed that the second receptor was expressed in several tissues, namely cartilage, brain, pituitary gland, gill, atrium, ventricle, pancreas, spleen, liver, gall bladder, intestine, rectal gland, kidney, testis and ovary. This localization pattern was very similar to flounder CTR. Both receptor mRNAs were strongly expressed in the gill. This suggests that the calcitonin-family members are involved in the osmoregulation of stingray as this fish is known to be euryhaline. When a stingray was transferred to diluted seawater (20% seawater), the expression of both receptors significantly decreased in the gill. Similar results were obtained in the kidney of the stingray. Thus, our cloning and isolation of both receptors in the stingray will be helpful for elucidation of their physiological role(s) such as osmoregulation including calcium metabolism of cartilaginous fish.     

Molecular cloning and cDNA sequence analysis of carboxypeptidases A1, A2 and B from the Japanese flounder Paralichthys olivaceus

Although pancreatic serine proteases have been cloned in teleosts, no sequence data are currently available on members of the carboxypeptidase (CP) family. Here, we cloned cDNAs coding for two preproCPAs, corresponding to mammalian preproCPA1 and preproCPA2, and one preproCPB from a pancreatic cDNA library of the Japanese flounder, Paralichthys olivaceus. The activation peptides of flounder proCPs completely retained the sequences for inhibition of enzymatic activity of proCPs just like mammalian proCPs. Of 306–309 amino acids in total, 95 amino acids are completely conserved between bovine CPA1 and CPB and flounder CPs. Notably, amino acid residues for Zn²⁺ ligands, catalysis and substrate anchoring are completely conserved between flounder and bovine CPs. Three species of flounder preproCPs are all expressed in the pancreas of first feeding larvae.     

斜带石斑鱼2种胰脂肪酶基因全长cDNA序列的克隆与分析

利用RT-PCR和RACE方法克隆得到斜带石斑鱼（Epinephelus coioides）肝胰脏中胆盐活化的胰脂肪酶(bile salt-activated lipase,BSAL)和依赖于辅酶的胰脂肪酶(colipase-dependent pancreatic lipase,PL)基因的全长cDNA序列.BSAL基因全长cDNA序列1 796 bp,编码558个氨基酸,该蛋白序列含有BSAL的全部特征结构区,与其他脊椎动物BSAL的氨基酸序列同源性为49.9%～57.3%.PL基因的全长cDNA序列1 503bp,编码465个氨基酸,该蛋白序列含有PL全部的特征结构区,与其它脊椎动物PL的氨基酸同源性为49.1%～73.9%.系统树分析表明,斜带石斑鱼BSAL和PL与其它物种BSAL、PL和胰脂肪酶相关蛋白(PL-RP)聚于进化树的两个不同分支,属于2种不同的胰脂肪酶.结果证实,在同一鱼类体内也存在BSAL和PL两种胰脂肪酶基因.     

Analysis of an ascidian integrin provides new insight into early evolution of collagen recognition

αI domain integrins have been found in the ascidian Ciona intestinalis. We produced Ciona α1I domain as a recombinant protein. It did not recognize fibril-forming collagens or bind to GFOGER or other similar motifs in triple-helical peptides. No GFOGER motifs were found in Ciona collagens. As Ciona α1I bound to collagen IX, we propose that before the emergence of GFOGER-dependent collagen receptors in vertebrates, αI domain integrins might have been able to bind to collagen with alternative mechanisms.     

A steroid/thyroid hormone receptor superfamily member in Drosophila melanogaster that shares extensive sequence similarity with a mammalian homologue

A gene in Drosophila melanogaster that maps cytologically to 2C1-3 on the distal portion of the X-chromosome encodes a member of the steroid/thyroid hormone receptor superfamily. The gene was isolated from an embryonic cDNA library using an oligonucleotide probe that specifies the consensus amino acid sequence in the DNA-binding domain of several human receptors. The conceptual amino acid sequence of 2C reveals at least four regions of homology that are shared with all identified vertebrate receptors. Region I includes the two cysteine-cysteine zinc fingers that comprise a DNA-binding domain which typifies all members of the superfamily. In addition, three regions (Regions II-IV) in the carboxy-terminal portion of the protein that encode the putative hormone-binding domain of the 2C gene product resemble similar sequences in vertebrate steroid/thyroid hormone receptors. The similarity suggests that this Drosophila receptor possesses many of the regulatory functions attributed to these regions in vertebrate counterparts. A portion of Region II also resembles part of the human c-jun oncoprotein's leucine zipper, which in turn, has been demonstrated to be the heterodimerization site between the jun and fos oncoproteins. The 2C receptor-like protein most resembles the mouse H2RII binding protein, a member of the superfamily which has been implicated in the regulation of major histocompatibility complex (MHC) class I gene expression. These two gene products are 83% identical in the DNA-binding domain and 50% identical in the putative hormone-binding domain, although no ligand has been identified for either protein. The high degree of similarity in the hormone-binding domain between the 2C protein and the H2RII binding protein outside regions II-IV suggests specific functional roles which are not shared by other members of the superfamily.