Sequence and organization of coelacanth neurohypophysial hormone genes: Evolutionary history of the vertebrate neurohypophysial hormone gene locus

Background  

The mammalian neurohypophysial hormones, vasopressin and oxytocin are involved in osmoregulation and uterine smooth muscle contraction respectively. All jawed vertebrates contain at least one homolog each of vasopressin and oxytocin whereas jawless vertebrates contain a single neurohypophysial hormone called vasotocin. The vasopressin homolog in non-mammalian vertebrates is vasotocin; and the oxytocin homolog is mesotocin in non-eutherian tetrapods, mesotocin and [Phe²]mesotocin in lungfishes, and isotocin in ray-finned fishes. The genes encoding vasopressin and oxytocin genes are closely linked in the human and rodent genomes in a tail-to-tail orientation. In contrast, their pufferfish homologs (vasotocin and isotocin) are located on the same strand of DNA with isotocin gene located upstream of vasotocin gene separated by five genes, suggesting that this locus has experienced rearrangements in either mammalian or ray-finned fish lineage, or in both lineages. The coelacanths occupy a unique phylogenetic position close to the divergence of the mammalian and ray-finned fish lineages.     

Something fishy in the rat brain: molecular genetics of the hypothalamo-neurohypophysial system

The brain peptides vasopressin and oxytocin play crucial roles in the regulation of salt and water balance. The genes encoding these neurohormones are regulated by cell-specific and physiological cues, but the molecular mechanisms remain obscure. New strategies, involving the introduction of rat transgenes into rats, are being used to address these issues,⁽¹⁾ but the complexity of the rat genome has hampered progress. By contrast, the pufferfish, Fugu rubripes, has a “junk-free” genome.⁽²⁾ The oxytocin homologue from Fugu, isotocin, has been introduced into rats⁽³⁾ and is expressed in oxytocin neurons, where it is upregulated by physiological perturbations that upregulate the oxytocin gene. The Fugu and rat lineages separated 400 million years ago, yet the mechanisms that regulate the isotocin and oxytocin genes have been conserved. Fugu genome analysis and transgenesis in the physiologically tractable rat host are a powerful combination that will enable the identification of fundamental components of the neural systems that control homeostasis. BioEssays 20 :741–749, 1998. © 1998 John Wiley & Sons, Inc.     

Cloning and sequence analyses of cDNAs encoding vasotocin and isotocin precursors of chum salmon,Oncorhynchus keta: evolutionary relationships of neurohypophysial hormone precursors

Summary The nucleotide sequences of cloned cDNAs were used to determine the primary structures of the precursors of vasotocin (sVT) and isotocin (sIT) from the hypothalamus of the chum salmon,Oncorhynchus keta. Two different cDNAs were obtained for each of sVT and sIT precursors (sVT-I and sVT-II; sIT-I and sIT-II). Both sVT and sIT precursors were found to contain a signal peptide and hormone that is connected to a neurophysin by a Gly-Lys-Arg sequence. Northern and Southern blot analyses showed that the sVT and sIT genes are expressed by the same chum salmon hypothalamus, but not by the liver and kidney. Microheterogeneity was found in the nucleotide and amino acid sequences of sVT precursors between our results and the previously reported data (Heierhorst et al. 1990). The conspicuous difference is the occurrence of a stop codon in the middle of sVT-II cDNA. The carboxyl termini of both sVT and sIT neurophysins are about 30 amino acids longer than neurophysins of toad and mammalian neurohypophysial hormone precursors. Although these extended regions do not contain a glycosylation site, they show striking similarity with the glycopeptide moiety (copeptin) of toad vasotocin and mammalian vasopressin precursors. The central portion of the neurophysins shows highest homology among corresponding regions of sVT and sIT precursors. Moreover, calculation of nucleotide substitution rates suggests that a recent gene conversion may have occurred which encompasses the exon that encodes the central segment of the sVT and sIT precursors. A possible pathway for the evolution of precursor molecules of neurohypophysial hormones is discussed.Abbreviations AVP vasopressin - C carboxyl - h human - IT isotocin - MT mesotocin - N amino - OXT oxytocin - S chum salmon - SDS sodium dodecyl sulfate - t toad - VT vasotocin     

[3H]Adenine is a suitable radioligand for the labeling of G protein-coupled adenine receptors but shows high affinity to bacterial contaminations in buffer solutions

[³H]Adenine has previously been used to label the newly discovered G protein-coupled murine adenine receptors. Recent reports have questioned the suitability of [³H]adenine for adenine receptor binding studies because of curious results, e.g. high specific binding even in the absence of mammalian protein. In this study, we showed that specific [³H]adenine binding to various mammalian membrane preparations increased linearly with protein concentration. Furthermore, we found that Tris-buffer solutions typically used for radioligand binding studies (50 mM, pH 7.4) that have not been freshly prepared but stored at 4°C for some time may contain bacterial contaminations that exhibit high affinity binding for [³H]adenine. Specific binding is abolished by heating the contaminated buffer or filtering it through 0.2-μm filters. Three different, aerobic, gram-negative bacteria were isolated from a contaminated buffer solution and identified as Achromobacter xylosoxidans, A. denitrificans, and Acinetobacter lwoffii. A. xylosoxidans, a common bacterium that can cause nosocomial infections, showed a particularly high affinity for [³H]adenine in the low nanomolar range. Structure–activity relationships revealed that hypoxanthine also bound with high affinity to A. xylosoxidans, whereas other nucleobases (uracil, xanthine) and nucleosides (adenosine, uridine) did not. The nature of the labeled site in bacteria is not known, but preliminary results indicate that it may be a high-affinity purine transporter. We conclude that [³H]adenine is a well-suitable radioligand for adenine receptor binding studies but that bacterial contamination of the employed buffer solutions must be avoided. Anke C. Schiedel and Heiko Meyer contributed equally to this work.     

Structural and functional characterisation of the Toll like receptor 9 of Aotus nancymaae, a non-human primate model for malaria vaccine development

In the absence of suitable rodent animal models for Plasmodium falciparum malaria, the efficacy testing of asexual blood-stage vaccine candidates in Aotus nancymaae represents a tool to select between different formulations before conducting expensive human clinical trials. CpG oligonucleotides (ODN) specifically promote the production of pro-inflammatory and Th1-type cytokines and they enhance the immunogenicity of co-administered antigens. Toll like receptor 9 (TLR-9) binds directly and sequence-specifically to single-stranded un-methylated CpG-DNA mediating the biological effects of CpG ODN. We cloned and functionally characterised the TLR-9 cDNA of A. nancymaae. The cDNA encompassed 3,099 bp predicted to code for 1,032 amino acid residues. Results of homology searches to human TLR-9 suggested that the receptor is 93 and 94% identical at the nucleotide and amino acid sequence levels, respectively. Stimulation of splenocytes of A. nancymaae with CpG ODN resulted in proliferative responses in all animals analysed. FACS analysis of cultures incubated with CpG ODN 2006 indicated that the B cell marker CD20 was up-regulated consistent with B cell activation. The high level of sequence conservation of Aona-TLR-9 reinforces the suitability of A. nancymaae as animal model for malaria subunit vaccine development.The nucleotide sequence has been submitted to the GenBank nucleotide sequence database under the accession number AY788894.     

Chiloscyllium plagiosum low-density lipoprotein receptor: Evolutionary conservation of five different functional domains

All five functional domains of the low-density lipoprotein (LDL) receptor were assembled in their modern form more than 450 million years ago, as revealed from the cloning and sequencing of an LDL receptor cDNA fromChiloscyllium plagiosum (banded cat shark). The shark LDL receptor has the same overall architecture as the mammalian and amphibian counterparts. Each of the seven cysteine-rich repeats in the ligand binding domain resembles its counterpart in the human LDL receptor more than it does the other repeats in the shark receptor as suggested by the presence of unique signature sequences, indicating that these repeats had already acquired their independent structures by the time of shark development. Furthermore, amino acid sequences of the entire ligand binding domain of shark LDL receptor show 35% identity over a stretch of 294 residues with aLymnaea stagnalis G-protein-linked receptor (LSGLR). The region of homology between these unrelated proteins includes conservation of most of the unique characteristics of the cysteine-rich repeats of LDL receptor at the expected positions in LSGLR. The results presented are consistent with the hypothesis that all seven repeats in the ligand binding domain of LDL receptor may have been lifted directly from an ancestral gene instead of being evolutionary duplications of a single repeat recruited by the primitive LDL receptor from another gene.The nucleotide sequence reported will appear in GenBank under accession number L36118     

Molecular cloning of cDNA encoding N-acetylglucosaminyltransferase II from Arabidopsis thaliana

N-acetylglucosaminyltransferase II (GnTII, EC 2.4.1.143) is a Golgi enzyme involved in the biosynthesis of glycoprotein-bound N-linked oligosaccharides, catalysing an essential step in the conversion of oligomannose-type to complex N-glycans. GnTII activity has been detected in both animals and plants. However, while cDNAs encoding the enzyme have already been cloned from several mammalian sources no GnTII homologue has been cloned from plants so far. Here we report the molecular cloning of an Arabidopsis thalianaGnTII cDNA with striking homology to its animal counterparts. The predicted domain structure of A. thalianaGnTII indicates a type II transmembrane protein topology as it has been established for the mammalian variants of the enzyme. Upon expression of A. thalianaGnTII cDNA in the baculovirus/insect cell system, a recombinant protein was produced that exhibited GnTII activity.     

Molecular cloning and expression analysis of phospholipase Cdelta from mud loach, Misgurnus mizolepis

A gene encoding phosphoinositide-specific phospholipase C (PLC), designated ML-PLCδ, was cloned from mud loach (Misgurnus mizolepis) liver. A complete cDNA encoding ML-PLCδ was isolated by screening the cDNA library of mud loach liver and using the 5′-rapid amplification of cDNA ends (RACE) method. The full-length ML-PLCδ gene contains an open reading frame of 2325 base pairs encoding a 774 amino acid protein with a molecular mass of 88,072 Da; this corresponds to the size of the protein expressed in Escherichia coli BL21 (DE3) using pET28a vector. It contains all of the characteristic domains found in mammalian PLCδ isozymes (PH domain, EF-hands, X–Y catalytic region, and a C2 domain). A homology search revealed that ML-PLCδ shares relatively high sequence identity with mammalian PLCδ1 (51–52%) and catfish PLCδ (64%). The recombinant ML-PLCδ protein expressed as a histidine-tagged fusion protein in E. coli was purified to apparent homogeneity by Ni²⁺-NTA affinity chromatography. The recombinant ML-PLCδ showed a concentration-dependent PLC activity to phosphatidylinositol 4,5-bis-phosphate (PIP₂) and its activity was Ca²⁺-dependent, which was similar to mammalian PLCδ isozymes.     

Isolation and characterization of a cDNA from flowers of Cynara cardunculus encoding cyprosin (an aspartic proteinase) and its use to study the organ-specific expression of cyprosin

Poly(A)⁺ RNA isolated from flower buds of Cynara cardunculus has been used to prepare a cDNA library. Screening of the cDNA after expression of cloned DNA with antibodies raised against the large subunit of cyprosin 3 resulted in the isolation of six positive clones. One of these clones (cypro1s; a 1.7 kb Eco RI fragment) codes for cyprosin. The nucleotide sequence contain a 1419 bp open reading frame coding for 473 amino acids (aa) including a putative full-length mature protein (440 aa) and a partial prosequence (33 aa). Cypro1s contains a 162 bp 3 non-coding region followed by a poly(A) tail. The deduced amino acid sequence shows high homology to other plant aspartic proteinases. The homology to mammalian and microbial aspartic proteinases is somewhat lower. Plant aspartic proteinases contain an insert of around 100 aa. We are modelling where this plant-specific insert will appear in the structure of cyprosin. Using cypro1s as a probe in northern blot analysis, the expression of cyprosin in developing flowers and other tissues has been studied. The signal on the northern blot increased for RNA samples from early (flower buds 6 mm in length) to later stages of floral development (flower buds up to 40 mm in length). In late stages of floral development (open flowers 50 mm in length and styles from such flowers) no hybridization signal was visualized showing that the synthesis of mRNA encoding the cyprosin starts in early stages of floral development and switches off at maturation of the flower. Southern blot analysis of genomic DNA showed 4–5 strong hybridizing bands and several minor bands indicating that the cyprosin genes are organized as a multi-gene family in C. cardunculus.     

Biological studies and potential therapeutic applications of monoclonal antibodies and small molecules reactive with theneu/c-erbB-2 protein

The overexpression of the growth factor receptor p185 ^neu/c-erbB-2 has been observed in a number of human adenocarcinomas and is mechanistically linked to neoplastic growth. Monoclonal antibodies raised against extracellular domains of the p185 ^neu/c-erbB-2 receptor oncoprotein have been utilized to inhibit the pathway ofneu-induced tumor development. Our laboratory has demonstrated a direct effect of anti-p185 ^neu/c-erbB2 antibodies which requires receptor ligation. This induced aggregation causes the downmodulation of cell-surface expression and eventual degradation of p185 ^neu/c-erbB-2 protein. In cells transformed by theneu oncogene, the result of antibody-induced p185 ^neu/c-erbB-2 receptor modulation is the reversion of the malignant pheno-type. We are exploiting the direct efficacy of this monoclonal antibody by developing small molecules (peptides and organic mimietics) based on anti-p185 ^neu/c-erbB-2 antibody structure that can mediate similar receptor binding and biological effects.     

Agonist-induced Internalization of the Caenorhabditis elegans Muscarinic Acetylcholine Receptor GAR-3 in Chinese Hamster Ovary Cells

Many membrane-bound neurotransmitter receptors are known to be internalized by exposure to agonist. This agonist-induced receptor internalization is considered to play important roles in receptor-mediated signaling. Here we investigated the internalization of GAR-3, a Caenorhabditis elegans muscarinic acetylcholine receptor, using cultured mammalian cells. When Chinese hamster ovary cells stably expressing GAR-3 were treated with carbachol, GAR-3 was internalized in a dose- and time-dependent manner. Approximately 60% of the cell surface receptor was internalized by exposure to 1 mM carbachol for 1 h. Carbachol-induced GAR-3 internalization was suppressed by treatment with hypertonic sucrose, which blocks the formation of clathrin-coated pits. Overexpression of a dominant-negative dynamin mutant (DynK44A), but not of a dominant-negative β-arrestin mutant (Arr319–418), substantially inhibited carbachol-induced internalization of GAR-3. Thus, these data suggest that GAR-3 undergoes agonist-induced internalization via a clathrin- and dynamin-dependent but β-arrestin-independent pathway. Depletion of Ca²⁺ by simultaneous treatment of the cells with BAPTA/AM (Ca²⁺ mobilization blocker) and EGTA (Ca²⁺ influx blocker) almost completely blocked agonist-induced GAR-3 internalization. Moreover, treatment of the cells with the Ca²⁺ ionophore A23187 led to GAR-3 internalization in the absence of agonist. These results indicate that Ca²⁺ plays a critical role in GAR-3 internalization. We tested whether the third intracellular (i3) loop of GAR-3 is involved in agonist-stimulated receptor internalization. A GAR-3 deletion mutant lacking a large central portion of the i3 loop exhibited an internalization pattern comparable to that of the wild type, suggesting that the central i3 loop is not required for the internalization of GAR-3.     

Isolation of a cDNA clone (PcSrp) encoding serine-rich-protein from Porteresia coarctata T. and its expression in yeast and finger millet (Eleusine coracana L.) affording salt tolerance

A 1.4 Kb cDNA clone encoding a serine-rich protein has been isolated from the cDNA library of salt stressed roots of Porteresia coarctata, and designated as P. coarctata serine-rich-protein (PcSrp) encoding gene. Northern analysis and in situ mRNA hybridization revealed the expression of PcSrp in the salt stressed roots and rhizome of P. coarctata. However, no such expression was seen in the salt stressed leaves and in the unstressed tissues of root, rhizome and leaf, indicating that PcSrp is under the control of a salt-inducible tissue-specific promoter. In yeast, the PcSrp conferred increased NaCl tolerance, implicating its role in salinity tolerance at cellular level. Further, PcSrp was cloned downstream to rice Actin-1 promoter and introduced into finger millet through particle-inflow-gun method. Transgenic plants expressing PcSrp were able to grow to maturity and set seed under 250 mM NaCl stress. The untransformed control plants by contrast failed to survive under similar salt stress. The stressed roots of transgenic plants invariably accumulated higher Na⁺ and K⁺ ion contents compared to roots of untransformed plants; whereas, shoots of transgenics accumulated lower levels of both the ions than that of untransformed plants under identical stress, clearly suggesting the involvement of PcSrp in ion homeostasis contributing to salt tolerance.     

Cloning,sequence analysis and expression of a cDNA encoding active phosphoenolpyruvate carboxylase of the C3 plant Solanum tuberosum

A cDNA coding for phosphoenolpyruvate carboxylase (PEPC) was isolated from a cDNA library from Solanum tuberosum and the sequence of the cDNA was determined. It was inserted into a bacterial expression vector and a PEPC^- Escherichia coli mutant could be complemented by the cDNA construct. A functional fusion protein could be synthesized in E. coli. The properties of this PEPC protein clearly resembled those of typical C3 plant enzymes.     

Site-directed affinity-labeling of delta opioid receptors by

The S-3-nitro-2-pyridinesulfenyl (SNpys) group in an affinity ligand can bind to a free thiol group of a cysteine residue in a target receptor molecule, forming a disulfide bond via the thiol-disulfide exchange reaction. SNpys-containing Leu-enkephalin analogues of [-Ala², Leu⁵]-enkephalyl-Cys(Npys)⁶ and [-Ala²,Leu(CH₂SNpys)⁵]enkephalin, and dynorphin A analogues of [-Ala²,Cys(Npys)¹²]dynorphin A-(1-13) amide and [-Ala²,Cys(Npys)⁸]dynorphin A-(1-9) amide have been found to affinity-label all of the δ, μ (rat brain), and κ (guinea pig brain) opioid receptor subtypes. In this study, using these chemically synthesized SNpys-containing analogues, we attempted to identify the analogues that affinity-label the cysteine residue at position 60 of the δ opioid receptor. We first established the assay procedure, principally based on the receptor binding assay to use COS-7 cells expressing the δ opioid receptor. Then, using a mutant δ receptor with the Cys60→Ala substitution, we assayed the SNpys-containing analogues for their specific affinity-labeling. [-Ala²,Cys(Npys)¹²]dynorphin A-(1-13) amide was found to have drastically reduced labeling activity for this mutant receptor as compared to its activity for the wild-type δ receptor. Other analogues exhibited almost the same activity for both the wild-type and mutant δ receptors. These results indicate that the δ-Cys60 residue has a free thiol group, which is labeled by [-Ala²,Cys(Npys)¹²]dynorphin A-(1-13) amide.