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Sample records for receptors molecular cloning

  1. Gene Transfer and Molecular Cloning of the Human NGF Receptor

    NASA Astrophysics Data System (ADS)

    Chao, Moses V.; Bothwell, Mark A.; Ross, Alonzo H.; Koprowski, Hilary; Lanahan, Anthony A.; Buck, C. Randall; Sehgal, Amita

    1986-04-01

    Nerve growth factor (NGF) and its receptor are important in the development of cells derived from the neural crest. Mouse L cell transformants have been generated that stably express the human NGF receptor gene transfer with total human DNA. Affinity cross-linking, metabolic labeling and immunoprecipitation, and equilibrium binding with 125I-labeled NGF revealed that this NGF receptor had the same size and binding characteristics as the receptor from human melanoma cells and rat PC12 cells. The sequences encoding the NGF receptor were molecularly cloned using the human Alu repetitive sequence as a probe. A cosmid clone that contained the human NGF receptor gene allowed efficient transfection and expression of the receptor.

  2. Molecular cloning of prolactin receptor of the Peking duck.

    PubMed

    Wang, J; Hou, S S; Huang, W; Yang, X G; Zhu, X Y; Liu, X L

    2009-05-01

    Prolactin receptor (PRLR) cDNA of Peking duck was cloned using reverse transcription-PCR methodology (GenBank accession no. EF502054). The duck PRLR (dPRLR) cDNA contains 2,439 bp including the ATG start codon and TAA stop that encodes 832 amino acid residues. The putative receptor protein had 2 strong hydrophobic stretches: one was the signal sequence in N-terminal region and the other was the single transmembrane region of 24 amino acid residues in the central portion. Excluding the signal sequence, the mature dPRLR contained 808 amino acid residues with a theoretical molecular mass of 91.5 kDa. The mature protein could be divided into 3 domains, extracellular domain (ECD), transmembrane domain, and intracellular domain. The ECD contained 2 homologous repeat units with approximately 67% amino acid sequence identity to each other. The membrane distal unit and proximal unit consisted of 204 and 212 amino acids, respectively. Each unit was similar to the singular ECD of the mammalian PRLR. The characteristic extracellular cysteines and the WSXWS motif of the cytokine receptor were conserved in both repeat units. The intracellular domain of dPRLR consisted of 368 amino acids at the C-terminus and contained 2 conserved regions-box 1 and box 2. The aforementioned characters of duck were all similar to chicken and mammalians, which implied that the dPRLR had same mechanism with other species. Sequence similarity analysis shows that the dPRLR shares approximately 45 to 97% amino identity and approximately 30 to 96% nucleic acid identity as compared with other species. Overall, the greatest sequence identity was found with goose PRLR. In a phylogenetic analysis, the duck PRLR was found to cluster with the PRLR of goose, turkey, chicken, and pigeon. Semiquantitative reverse transcription-PCR indicated that dPRLR mRNA was expressed in all 8 tissues. Expression in the kidney was greatest in varietals tissues.

  3. Molecular cloning and characterization of hagfish estrogen receptors.

    PubMed

    Nishimiya, Osamu; Katsu, Yoshinao; Inagawa, Hiroyuki; Hiramatsu, Naoshi; Todo, Takashi; Hara, Akihiko

    2017-01-01

    One or more distinct forms of the nuclear estrogen receptor (ER) have been isolated from many vertebrates to date. To better understand the molecular evolution of ERs, we cloned and characterized er cDNAs from the inshore hagfish, Eptatretus burgeri, a modern representative of the most primitive vertebrates, the agnathans. Two er cDNAs, er1 and er2, were isolated from the liver of a reproductive female hagfish. A phylogenetic analysis placed hagfish ER1 into a position prior to the divergence of vertebrate ERs. Conversely, hagfish ER2 was placed at the base of the vertebrate ERβ clade. The tissue distribution patterns of both ER subtype mRNAs appeared to be different, suggesting that each subtype has different physiological roles associated with estrogen actions. An estrogen responsive-luciferase reporter assay using mammalian HEK293 cells was used to functionally characterize these hagfish ERs. Both ER proteins displayed estrogen-dependent activation of transcription. These results clearly demonstrate that the hagfish has two functional ER subtypes.

  4. Molecular cloning, characterization, and chromosome mapping of reptilian estrogen receptors.

    PubMed

    Katsu, Yoshinao; Matsubara, Kazumi; Kohno, Satomi; Matsuda, Yoichi; Toriba, Michihisa; Oka, Kaori; Guillette, Louis J; Ohta, Yasuhiko; Iguchi, Taisen

    2010-12-01

    In many vertebrates, steroid hormones are essential for ovarian differentiation during a critical developmental stage as well as promoting the growth and differentiation of the adult female reproductive system. Although studies have been extensively conducted in mammals and a few fish, amphibians, and bird species, the molecular mechanisms of sex steroid hormone (estrogens) action have been poorly examined in reptiles. Here, we evaluate hormone receptor and ligand interactions in two species of snake, the Okinawa habu (Protobothrops flavoviridis, Viperidae) and the Japanese four-striped rat snake (Elaphe quadrivirgata, Colubridae) after the isolation of cDNAs encoding estrogen receptor α (ESR1) and estrogen receptor β (ESR2). Using a transient transfection assay with mammalian cells, the transcriptional activity of reptilian (Okinawa habu, Japanese four-striped rat snake, American alligator, and Florida red-belly freshwater turtle) ESR1 and ESR2 was examined. All ESR proteins displayed estrogen-dependent activation of transcription via an estrogen-response element-containing promoter; however, the responsiveness to various estrogens was different. Further, we determined the chromosomal locations of the snake steroid hormone receptor genes. ESR1 and ESR2 genes were localized to the short and long arms of chromosome 1, respectively, whereas androgen receptor was localized to a pair of microchromosomes in the two snake species examined. These data provide basic tools that allow future studies examining receptor-ligand interactions and steroid endocrinology in snakes and also expands our knowledge of sex steroid hormone receptor evolution.

  5. Molecular Cloning, Characterization, and Chromosome Mapping of Reptilian Estrogen Receptors

    PubMed Central

    Katsu, Yoshinao; Matsubara, Kazumi; Kohno, Satomi; Matsuda, Yoichi; Toriba, Michihisa; Oka, Kaori; Guillette, Louis J.; Ohta, Yasuhiko; Iguchi, Taisen

    2010-01-01

    In many vertebrates, steroid hormones are essential for ovarian differentiation during a critical developmental stage as well as promoting the growth and differentiation of the adult female reproductive system. Although studies have been extensively conducted in mammals and a few fish, amphibians, and bird species, the molecular mechanisms of sex steroid hormone (estrogens) action have been poorly examined in reptiles. Here, we evaluate hormone receptor and ligand interactions in two species of snake, the Okinawa habu (Protobothrops flavoviridis, Viperidae) and the Japanese four-striped rat snake (Elaphe quadrivirgata, Colubridae) after the isolation of cDNAs encoding estrogen receptor α (ESR1) and estrogen receptor β (ESR2). Using a transient transfection assay with mammalian cells, the transcriptional activity of reptilian (Okinawa habu, Japanese four-striped rat snake, American alligator, and Florida red-belly freshwater turtle) ESR1 and ESR2 was examined. All ESR proteins displayed estrogen-dependent activation of transcription via an estrogen-response element-containing promoter; however, the responsiveness to various estrogens was different. Further, we determined the chromosomal locations of the snake steroid hormone receptor genes. ESR1 and ESR2 genes were localized to the short and long arms of chromosome 1, respectively, whereas androgen receptor was localized to a pair of microchromosomes in the two snake species examined. These data provide basic tools that allow future studies examining receptor-ligand interactions and steroid endocrinology in snakes and also expands our knowledge of sex steroid hormone receptor evolution. PMID:20926589

  6. Human GluR6 kainate receptor (GRIK2): Molecular cloning, expression, polymorphism, and chromosomal assignment

    SciTech Connect

    Paschen, W.; Blackstone, C.D.; Huganir, R.L. ); Ross, C.A. Max-Planck-Institute for Neurological Research, Koeln )

    1994-04-01

    Glutamate receptors mediate the majority of excitatory neurotransmission in the brain, and molecular cloning studies have revealed several distinct families. Because neuropathological states and possibly human disorders may involve kainate-preferring glutamate receptors, the authors have isolated a cDNA clone for the human GluR6 kainate-preferring receptor. This clone shows a very high sequence similarity with that of the rat, except for a part of the 3[prime] untranslated region in which there is a TAA triplet repeat. When the protein was overexpressed in human embryonic kidney 293 cells, it had a molecular weight, an antibody recognition, and a glutamate ligand-binding profile similar to those of the rate GluR6 receptor. Northern analysis showed expression in both human cerebral and cerebellar cortices. By PCR analysis of rodent-human monochromosomal cell lines, the human GluR6 could be assigned to chromosome 6. The length of the TAA triplet repeat was polymorphic in the normal population, with at least four alleles and an observed heterozygosity of about 45%. These studies should provide the basis for expression or linkage studies of the GluR6 kainate receptor in human disease or neuropathologic states. 53 refs., 7 figs.

  7. Molecular cloning.

    PubMed

    Lessard, Juliane C

    2013-01-01

    This protocol describes the basic steps involved in conventional plasmid-based cloning. The goals are to insert a DNA fragment of interest into a receiving vector plasmid, transform the plasmid into E. coli, recover the plasmid DNA, and check for correct insertion events.

  8. Molecular cloning and expression of a Toll receptor in the giant tiger shrimp, Penaeus monodon.

    PubMed

    Arts, Joop A J; Cornelissen, Ferry H J; Cijsouw, Tony; Hermsen, Trudi; Savelkoul, Huub F J; Stet, René J M

    2007-09-01

    Invertebrates rely completely for their protection against pathogens on the innate immune system. This non-self-recognition is activated by microbial cell wall components with unique conserved molecular patterns. Pathogen-associated molecular patterns (PAMPs) are recognised by pattern recognition receptors (PRRs). Toll and its mammalian homologs Toll-like receptors are cell-surface receptors acting as PRRs and involved in the signalling pathway implicated in their immune response. Here we describe a novel partial Toll receptor gene cloned from a gill library of the giant tiger shrimp, Penaeus monodon, using primers based on the highly conserved Toll/IL-1R (TIR) domain. The deduced amino acid sequence of the P. monodon Toll (PmToll) shows 59% similarity to a Toll-related protein of Apis mellifera. Analysis of the LRRs of shrimp Toll contained no obvious PAMP-binding insertions. Phylogenetic analysis with the insect Toll family shows clustering with Toll1 and Toll5 gene products, and it is less related to Toll3 and Toll4. Furthermore, RT-qPCR shows that PmToll is constitutively expressed in gut, gill and hepatopancreas. Challenge with white spot syndrome virus (WSSV) shows equal levels of expression in these organs. A role in the defence mechanism is discussed. In conclusion, shrimp possess at least one Toll receptor that might be involved in immune defence.

  9. Molecular cloning and expression of a GABA receptor subunit from the crayfish Procambarus clarkii.

    PubMed

    Jiménez-Vázquez, Eric N; Díaz-Velásquez, Clara E; Uribe, R M; Arias, Juan M; García, Ubaldo

    2016-02-01

    Molecular cloning has introduced an unexpected, large diversity of neurotransmitter hetero- oligomeric receptors. Extensive research on the molecular structure of the γ-aminobutyric acid receptor (GABAR) has been of great significance for understanding how the nervous system works in both vertebrates and invertebrates. However, only two examples of functional homo-oligomeric GABA-activated Cl(-) channels have been reported. In the vertebrate retina, the GABAρ1 subunit of various species forms homo-oligomeric receptors; in invertebrates, a cDNA encoding a functional GABA-activated Cl(-) channel has been isolated from a Drosophila melanogaster head cDNA library. When expressed in Xenopus laevis oocytes, these subunits function efficiently as a homo-oligomeric complex. To investigate the structure-function of GABA channels from the crayfish Procambarus clarkii, we cloned a subunit and expressed it in human embryonic kidney cells. Electrophysiological recordings show that this subunit forms a homo-oligomeric ionotropic GABAR that gates a bicuculline-insensitive Cl(-) current. The order of potency of the agonists was GABA > trans-4-amino-crotonic acid = cis-4-aminocrotonic acid > muscimol. These data support the notion that X-organ sinus gland neurons express at least two GABA subunits responsible for the formation of hetero-oligomeric and homo-oligomeric receptors. In addition, by in situ hybridization studies we demonstrate that most X-organ neurons from crayfish eyestalk express the isolated pcGABAA β subunit. This study increases the knowledge of the genetics of the crayfish, furthers the understanding of this important neurotransmitter receptor family, and provides insight into the evolution of these genes among vertebrates and invertebrates.

  10. Hepatic nuclear receptor PPARalpha in the koala (Phascolarctos cinereus): cloning and molecular characterisation.

    PubMed

    Ngo, Suong Ngoc Thi; McKinnon, Ross Allan; Stupans, Ieva

    2007-09-01

    Peroxisome proliferator-activated receptor alpha (PPARalpha) is a member of the nuclear/steroid receptor gene superfamily that plays an essential role in fatty acid metabolism. PPARalpha modulates the expression of genes encoding peroxisomal fatty acid beta-oxidation enzymes and microsomal fatty acid hydroxylases CYP4As. We have previously reported that the obligate Eucalyptus feeder koala (Phascolarctos cinereus) exhibits a higher hepatic CYP4A activity and an absence of peroxisomal palmitoyl-CoA oxidation as compared to non-Eucalyptus feeders human, rat or wallaby. Here we describe the cloning, expression and molecular characterisation of koala hepatic PPARalpha. A full-length PPARalpha cDNA of size 1515 bp was cloned by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The koala PPARalpha cDNA encodes a protein of 468 amino acids. Transfection of the koala PPARalpha cDNA into Cos-7 cells resulted in the expression of a protein recognised by a rabbit anti-human PPARalpha polyclonal antibody. PPARalpha immunoreactive bands of the same molecular mass were detected in nuclear extracts of koala livers. The results of this study demonstrate the presence of koala hepatic PPARalpha which shares several common features with other published PPARalphas; however, it exhibits important differences in both the DNA and ligand binding domains.

  11. Molecular cloning, chromosomal mapping, and functional expression of human brain glutamate receptors

    SciTech Connect

    Sun, W.; Ferrer-Montiel, A.V.; Schinder, A.F.; Montal, M. ); McPherson, J.P. ); Evans, G.A. )

    1992-02-15

    A full-length cDNA clone encoding a glutamate receptor was isolated from a human brain cDNA library, and the gene product was characterized after expression in Xenopus oocytes. Degenerate PCR primers to conserved regions of published rat brain glutamate receptor sequences amplified a 1-kilobase fragment from a human brain cDNA library. This fragment was used as a probe for subsequent hybridization screening. Two clones were isolated that, based on sequence information, code for different receptors: a 3-kilobase clone, HBGR1, contains a full-length glutamate receptor cDNA highly homologous to the rat brain clone GluR1, and a second clone, HBGR2, contains approximately two-thirds of the coding region of a receptor homologous to rat brain clone GluR2. Southern and PCr analysis of a somatic cell-hybrid panel mapped HBGR1 to human chromosome 5q31.3-33.3 and mapped HBGR2 to chromosome 4q25-34.3. Xenopus oocytes injected with in vitro-synthesized HBGR1 cRNA expressed currents activated by glutamate receptor agonists. These results indicate that clone HBGR1 codes for a glutamate receptor of the kainate subtype cognate to members of the glutamate receptor family from rodent brain.

  12. Molecular cloning and functional expression of a Drosophila receptor for the neuropeptides capa-1 and -2.

    PubMed

    Iversen, Annette; Cazzamali, Giuseppe; Williamson, Michael; Hauser, Frank; Grimmelikhuijzen, Cornelis J P

    2002-12-13

    The Drosophila Genome Project website contains an annotated gene (CG14575) for a G protein-coupled receptor. We cloned this receptor and found that the cloned cDNA did not correspond to the annotated gene; it partly contained different exons and additional exons located at the 5(')-end of the annotated gene. We expressed the coding part of the cloned cDNA in Chinese hamster ovary cells and found that the receptor was activated by two neuropeptides, capa-1 and -2, encoded by the Drosophila capability gene. Database searches led to the identification of a similar receptor in the genome from the malaria mosquito Anopheles gambiae (58% amino acid residue identities; 76% conserved residues; and 5 introns at identical positions within the two insect genes). Because capa-1 and -2 and related insect neuropeptides stimulate fluid secretion in insect Malpighian (renal) tubules, the identification of this first insect capa receptor will advance our knowledge on insect renal function.

  13. Molecular cloning and pharmacological characterization of giant panda (Ailuropoda melanoleuca) melanocortin-4 receptor.

    PubMed

    Wang, Zhi-Qiang; Wang, Wei; Shi, Lin; Chai, Ji-Tian; Zhang, Xin-Jun; Tao, Ya-Xiong

    2016-04-01

    The melanocortin-4 receptor (MC4R) is critical in regulating mammalian food intake and energy expenditure. Giant panda (Ailuropoda melanoleuca), famous as the living fossil, is an endangered species endemic to China. We are interested in exploring the functions of the giant panda MC4R (amMC4R) in regulating energy homeostasis and report herein the molecular cloning and pharmacology of the amMC4R. Sequence analysis revealed that amMC4R was highly homologous (>88%) at nucleotide and amino acid sequences to several mammalian MC4Rs. Western blot revealed that the expression construct myc-amMC4R in pcDNA3.1 was successfully constructed and expressed in HEK293T cells. With human MC4R (hMC4R) as a control, pharmacological characteristics of amMC4R were analyzed with binding and signaling assays. Four agonists, including [Nle(4), D-Phe(7)]-α-melanocyte stimulating hormone (NDP-MSH), α- and β-MSH, and a small molecule agonist, THIQ, were used in binding and signaling assays. We showed that amMC4R bound NDP-MSH with the highest affinity followed by THIQ, α-MSH, and β-MSH, with the same ranking order as hMC4R. Treatment of HEK293T cells expressing amMC4R with different concentrations of agonists resulted in dose-dependent increase of intracellular cAMP levels, with similar EC50s for the four agonists. The results suggested that the cloned amMC4R encoded a functional MC4R. The availability of amMC4R and its binding and signaling properties will facilitate the investigation of amMC4R in regulating food intake and energy homeostasis.

  14. Molecular cloning of a gene encoding the histamine H2 receptor

    SciTech Connect

    Gantz, I.; Schaeffer, M.; DelValle, J.; Logsdon, C.; Campbell, V.; Uhler, M.; Yamada, Tadataka )

    1991-01-15

    The H2 subclass of histamine receptors mediates gastric acid secretion, and antagonists for this receptor have proven to be effective therapy for acid peptic disorders of the gastrointestinal tract. The physiological action of histamine has been shown to be mediated via a guanine nucleotide-binding protein linked to adenylate cyclase activation and cellular cAMP generation. The authors capitalized on the technique of polymerase chain reaction, using degenerate oligonucleotide primers based on the known homology between cellular receptors linked to guanine nucleotide-binding proteins to obtain a partial-length clone from canine gastric parietal cell cDNA. This clone was used to obtain a full-length receptor gene from a canine genomic library. Histamine increased in a dose-dependent manner cellular cAMP content in L cells permanently transfected with this gene, and preincubation of the cells with the H2-selective antagonist cimetidine shifted the dose-response curve to the right. Cimetidine inhibited the binding of the radiolabeled H2 receptor-selective ligand (methyl-{sup 3}H)tiotidine to the transfected cells in a dose-dependent fashion, but the H1-selective antagonist diphenhydramine did not. These data indicate that they have cloned a gene that encodes the H2 subclass of histamine receptors.

  15. Molecular cloning and functional characterization of the diapause hormone receptor in the corn earworm Helicoverpa zea

    USDA-ARS?s Scientific Manuscript database

    The diapause hormone (DH) in the heliothine moth has shown its activity in termination of pupal diapause, while the orthology in the silkworm is known to induce embryonic diapause. In the current study, we cloned the diapause hormone receptor from the corn earworm Helicoverpa zea (HzDHr) and tested ...

  16. Molecular cloning and pharmacological characterization of rat melatonin MT1 and MT2 receptors.

    PubMed

    Audinot, Valérie; Bonnaud, Anne; Grandcolas, Line; Rodriguez, Marianne; Nagel, Nadine; Galizzi, Jean-Pierre; Balik, Ales; Messager, Sophie; Hazlerigg, David G; Barrett, Perry; Delagrange, Philippe; Boutin, Jean A

    2008-05-15

    In order to interpret the effects of melatonin ligands in rats, we need to determine their activity at the receptor subtype level in the corresponding species. Thus, the rat melatonin rMT(1) receptor was cloned using DNA fragments for exon 1 and 2 amplified from rat genomic DNA followed by screening of a rat genomic library for the full length exon sequences. The rat rMT(2) receptor subtype was cloned in a similar manner with the exception of exon 1 which was identified by screening a rat genomic library with exon 1 of the human hMT(2) receptor. The coding region of these receptors translates proteins of 353 and 364 amino acids, respectively, for rMT(1) and rMT(2). A 55% homology was observed between both rat isoforms. The entire contiguous rat MT(1) and MT(2) receptor coding sequences were cloned, stably expressed in CHO cells and characterized in binding assay using 2-[(125)I]-Iodomelatonin. The dissociation constants (K(d)) for rMT(1) and rMT(2) were 42 and 130 pM, respectively. Chemically diverse compounds previously characterized at human MT(1) and MT(2) receptors were evaluated at rMT(1) and rMT(2) receptors, for their binding affinity and functionality in [(35)S]-GTPgammaS binding assay. Some, but not all, compounds shared a similar binding affinity and functionality at both rat and human corresponding subtypes. A different pharmacological profile of the MT(1) subtype has also been observed previously between human and ovine species. These in vitro results obtained with the rat melatonin receptors are thus of importance to understand the physiological roles of each subtype in animal models.

  17. Molecular cloning and characterization of a nuclear androgen receptor activated by 11-ketotestosterone

    PubMed Central

    Olsson, Per-Erik; Berg, A Håkan; von Hofsten, Jonas; Grahn, Birgitta; Hellqvist, Anna; Larsson, Anders; Karlsson, Johnny; Modig, Carina; Borg, Bertil; Thomas, Peter

    2005-01-01

    Although 11-ketotestosterone is a potent androgen and induces male secondary sex characteristics in many teleosts, androgen receptors with high binding affinity for 11-ketotestosterone or preferential activation by 11-ketotestosterone have not been identified. So, the mechanism by which 11-ketotestosterone exhibits such high potency remains unclear. Recently we cloned the cDNA of an 11-ketotestosterone regulated protein, spiggin, from three-spined stickleback renal tissue. As spiggin is the only identified gene product regulated by 11-ketotestosterone, the stickleback kidney is ideal for determination of the mechanism of 11-ketotestosterone gene regulation. A single androgen receptor gene with two splicing variants, belonging to the androgen receptor-β subfamily was cloned from stickleback kidney. A high affinity, saturable, single class of androgen specific binding sites, with the characteristics of an androgen receptor, was identified in renal cytosolic and nuclear fractions. Measurement of ligand binding moieties in the cytosolic and nuclear fractions as well as to the recombinant receptor revealed lower affinity for 11-ketotestosterone than for dihydrotestosterone. Treatment with different androgens did not up-regulate androgen receptor mRNA level or increase receptor abundance, suggesting that auto-regulation is not involved in differential ligand activation. However, comparison of the trans-activation potential of the stickleback androgen receptor with the human androgen receptor, in both human HepG2 cells and zebrafish ZFL cells, revealed preferential activation by 11-ketotestosterone of the stickleback receptor, but not of the human receptor. These findings demonstrate the presence of a receptor preferentially activated by 11-ketotestosterone in the three-spined stickleback, so far the only one known in any animal. PMID:16107211

  18. Molecular cloning of natriuretic peptide receptor A from bullfrog (Rana catesbeiana) brain and its functional expression.

    PubMed

    Sekiguchi, T; Miyamoto, K; Mizutani, T; Yamada, K; Yazawa, T; Yoshino, M; Minegishi, T; Takei, Y; Kangawa, K; Minamino, N; Saito, Y; Kojima, M

    2001-08-08

    A comparative study of natriuretic peptide receptor (NPR) was performed by cloning the NPR-A receptor subtype from the bullfrog (Rana catesbeiana) brain and analyzing its functional expression. Like other mammalian NPR-A receptors, the bullfrog NPR-A receptor consists of an extracellular ligand binding domain, a hydrophobic transmembrane domain, a kinase-like domain and a guanylate cyclase domain. Sequence comparison among the bullfrog and mammalian receptors revealed a relatively low ( approximately 45%) similarity in the extracellular domain compared to a very high similarity ( approximately 92%) in the cytoplasmic regulatory and catalytic domains. Expression of NPR-A mRNA was detected in various bullfrog tissues including the brain, heart, lung, kidney and liver; highest levels were observed in lung. Functional expression of the receptor in COS-7 cells revealed that frog atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) elicited cyclic guanosine 3'5'-monophosphate production by stimulating the receptor in a dose-dependent manner from 10(-10) M concentrations. Rat ANP was also effective in stimulating the frog receptor whereas rat BNP and porcine BNP were less responsive to the receptor. On the other hand, frog C-type natriuretic peptide (CNP) as well as porcine CNP stimulated the receptor only at high concentrations (10(-7) M). This clearly indicates that the bullfrog receptor is a counterpart of mammalian NPR-A, and is specific for ANP or BNP but not for CNP.

  19. Molecular cloning, characterization and expression of goose Toll-like receptor 5.

    PubMed

    Fang, Qiang; Pan, Zhiming; Geng, Shizhong; Kang, Xilong; Huang, Jinlin; Sun, Xiaolin; Li, Qiuchun; Cai, Yinqiang; Jiao, Xinan

    2012-10-01

    Toll-like receptors (TLRs) are pattern recognition receptors (PRRs) that are vital to activation of the innate immune system in response to invading pathogens through their recognition of pathogen-associated molecular patterns (PAMPs). TLR5 is responsible for the recognition of bacterial flagellin in vertebrates. In this study, we cloned the goose TLR5 gene using rapid amplification of cDNA ends (RACE). The open reading frame (ORF) of goose TLR5 cDNA is 2583 bp in length and encodes an 860 amino acid protein. The entire coding region of the TLR5 gene was successfully amplified from genomic DNA and contained a single exon. The putative amino acid sequence of goose TLR5 consisted of a signal peptide sequence, 11 leucine-rich repeat (LRR) domains, a leucine-rich repeat C-terminal (LRR-CT) domain, a transmembrane domain and an intracellular Toll-interleukin-1 receptor (TIR) domain. The amino acid sequence of goose TLR5 shared 50.5% identity with human (Homo sapiens), 49.8% with mouse (Mus musculus) and 82.7% with chicken (Gallus gallus). The goose TLR5 gene was highly expressed in the spleen, liver and brain; moderately expressed in PBMCs, kidney, lung, heart, bone marrow, small intestine and large intestine; and minimally expressed in the cecum. HEK293 cells transfected with goose TLR5 and NF-κB-luciferase containing plasmids significantly responded to flagellin from Salmonella typhimurium indicating that it is a functional TLR5 homologue. In response to infection with S. enterica serovar Enteritidis (SE), the level of TLR5 mRNA significantly increased over the control in PBMCs at 1 d post infection (p.i.) and was slightly elevated in the spleen at 1 d or 3 d p.i. IL-6 was expressed below control levels in PBMCs but was upregulated in the spleen. In contrast to IL-6, an evident decrease in the expression level of IL-8 was observed in both PBMCs and spleens at 1 d or 3 d p.i. SE challenge also resulted in an increase in the mRNA expression of IL-18 and IFN-γ in PBMCs

  20. Molecular cloning, tissue distribution, and pharmacological characterization of melanocortin-4 receptor in grass carp (Ctenopharyngodon idella).

    PubMed

    Li, L; Yang, Z; Zhang, Y-P; He, S; Liang, X-F; Tao, Y-X

    2017-04-01

    Melanocortin-4 receptor (MC4R) plays a pivotal role in the mediation of leptin action on food intake and energy expenditure in mammals. The MC4R has also been identified in several teleosts, and its importance in the regulation of fish energy homeostasis is emerging. We herein reported on the molecular cloning, tissue distribution, and pharmacological characterization of MC4R in grass carp (Ctenopharyngodon idella), an economically and ecologically important fish. We showed that grass carp MC4R (ciMC4R) consisted of a 981 bp open reading frame encoding a protein of 326 amino acids, highly homologous (>95%) to several teleost MC4Rs. Phylogenetic and synteny analysis further indicated ciMC4R was closely related to piscine MC4Rs. Using reverse transcription PCR, we found that mc4r messenger RNA was expressed in the brain as well as various peripheral tissues in grass carp. The pharmacological properties of ciMC4R were investigated using 4 agonists, including α-melanocyte stimulating hormone (α-MSH), β-MSH, [Nle(4), D-Phe(7)]-MSH (NDP-MSH), and adrenocorticotropic hormone (ACTH). We showed that all 4 ligands could bind to ciMC4R and initiate dose-dependent intracellular cyclic adenosine monophosphate (cAMP) accumulation. Grass carp MC4R had the highest affinity for NDP-MSH. Both NDP-MSH and ACTH (1-24) exhibited higher potencies compared to the other 2 endogenous agonists. The ciMC4R was constitutively active, with significantly increased basal cAMP level compared with that of human MC4R (P < 0.01). The availability of ciMC4R and its pharmacologic characteristics provide a basis for future investigation of its functional roles in regulating diverse physiological processes and novel insights into understanding the mechanism of food habit transition in grass carp.

  1. Molecular cloning, characterization, and distribution of the gerbil angiotensin II AT2 receptor.

    PubMed

    Hoe, Kwang-Lae; Armando, Ines; Baiardi, Gustavo; Sreenath, Taduru; Kulkarni, Ashok; Martínez, Alfredo; Saavedra, Juan M

    2003-12-01

    We isolated a cDNA clone encoding the gerbil AT2 receptor (gAT2) gene from a gerbil adrenal gland cDNA library. The full-length cDNA contains a 1,089-bp open reading frame encoding 363 amino acid residues with 90.9, 96.1, and 95.6% identity with the human (hAT2), rat (rAT2), and mouse AT2 (mAT2) receptors, respectively. There are at least seven nonconserved amino acids in the NH2-terminal domain and in positions Val196, Val217, and Met293, important for angiotensin (ANG) II but not for CGP-42112 binding. Displacement studies in adrenal sections revealed that affinity of the gAT2 receptor was 10-20 times lower for ANG II, ANG III, and PD-123319 than was affinity of the rAT2 receptor. The affinity of each receptor remained the same for CGP-42112. When transfected into COS-7 cells, the gAT2 receptor shows affinity for ANG II that is three times lower than that shown by the hAT2 receptor, whereas affinities for ANG III and the AT2 receptor ligands CGP-42112 and PD-123319 were similar. Autoradiography in sections of the gerbil head showed higher binding in muscles, retina, skin, and molars at embryonic day 19 than at 1 wk of age. In situ hybridization and emulsion autoradiography revealed that at embryonic day 19 the gAT2 receptor mRNA was highly localized to the base of the dental papilla of maxillary and mandibular molars. Our results suggest selective growth-related functions in late gestation and early postnatal periods for the gAT2 receptor and provide an essential basis for future mutagenesis studies to further define structural requirements for agonist binding.

  2. Molecular cloning and expression of rat prostaglandin E receptor EP2 subtype.

    PubMed

    Sando, T; Usui, T; Tanaka, I; Mori, K; Sasaki, Y; Fukuda, Y; Namba, T; Sugimoto, Y; Ichikawa, A; Narumiya, S

    1994-05-16

    A cDNA clone encoding the rat prostaglandin (PG) E receptor EP2 subtype was cloned from a rat lung cDNA library. It encodes 488 amino acid residues with putative seven-transmembrane domains. Specific binding of [3H]PGE2 was found in COS-7 cells transfected with the cDNA and was displaced with unlabeled prostaglandins in the order of PGE2 = PGE1 > iloprost > or = PGF2 alpha > or = PGD2. The binding was also inhibited by misoprostol, an EP2 and EP3 agonist, but not by sulprostone, an EP1 and EP3 agonist. Northern blot analysis demonstrated that the EP2 mRNA is widely expressed in various tissues, the significant expression being observed in the thymus, lung, spleen, heart stomach, and pancreas.

  3. Molecular cloning of chicken FTZ-F1-related orphan receptors.

    PubMed

    Kudo, T; Sutou, S

    1997-09-15

    FTZ-F1 is a member of the orphan nuclear receptors, which belongs to the steroid hormone receptor superfamily, and plays a role in the blastoderm and nervous system development in Drosophila. Recently, several FTZ-F1 family genes have been cloned in several species. SF-1/Ad4BPs have been identified as master regulators controlling steroidogenic P-450 genes in mammals and are considered to be the mammalian homologues of FTZ-F1. Moreover, SF-1/Ad4BP plays a critical role in the sexual differentiation of gonads in mammals. In vertebrates, except for mammals, the functional homologue of SF-1/Ad4BP has not been identified before. Herein, we cloned two chicken cDNAs (OR2.0 and OR2.1), which encode putative FTZ-F1 family receptors, by reverse transcriptase-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). OR2.1 consists of 3255 bp, is expressed in the adrenal glands and gonads, and is considered to be the chicken counterpart of mammalian SF-1/Ad4BP. However, OR2.0 consists of 2945 bp, is expressed in the livers and the adrenal glands, and is considered to be the chicken counterpart of mouse LRH-1, which is a member of the FTZ-F1 family in mammals.

  4. Molecular cloning and functional analysis of Chinese sturgeon (Acipenser sinensis) growth hormone receptor.

    PubMed

    Liao, ZhiYong; Chen, XiaoLi; Wu, MingJiang

    2009-10-01

    A full length cDNA encoding the growth hormone receptor (GHR) of Chinese sturgeon was cloned in order to investigate the mechanism of growth hormone in regulating the growth of Chinese sturgeon. The open reading frame of the cloned Chinese sturgeon growth hormone receptor (csGHR) cDNA encodes a trans-membrane protein of 611 amino acids containing all the characteristic motifs of GHR. By sequence alignment, substitutions of amino acid residues highly conserved in other species were identified. Using the CHO cell culture system, the function of csGHR and the biological significance of the amino acid substitution in csGHR were examined. The promoter of serine protease inhibitor 2.1 (Spi2.1) was trans-activated upon stimulation of seabream GH (sbGH) in the csGHR-expressing CHO cells. Furthermore, CHO cells stably expressing csGHR were stimulated to proliferate by sbGH. In agreement with our previous report, Chinese sturgeon growth hormone-binding protein (csGHBP) was detected in the culture medium of CHO cells stably expressing csGHR. Mutation of Asp residue in the ligand binding motif in csGHR to Glu significantly enhanced csGHR's biological function, whereas mutation of Asp residue to Ala decreased its biological function. The results demonstrated that the cloned csGHR was of full biological function and the csGHBP could be generated through proteolysis of csGHR. These findings might provide new insights into thoroughly understanding the regulatory mechanism of Chinese sturgeon growth.

  5. Molecular and pharmacological characterization of a functional tachykinin NK3 receptor cloned from the rabbit iris sphincter muscle

    PubMed Central

    Medhurst, Andrew D; Hirst, Warren D; Jerman, Jeffery C; Meakin, Jacqueline; Roberts, Jennifer C; Testa, Tania; Smart, Darren

    1999-01-01

    A functional tachykinin NK3 receptor was cloned from the rabbit iris sphincter muscle and its distribution investigated in ocular tissues.Standard polymerase chain reaction (PCR) techniques were used to clone a full length rabbit NK3 receptor cDNA consisting of 1404 nucleotides. This cDNA encoded a protein of 467 amino acids with 91 and 87% homology to the human and rat NK3 receptors respectively.In CHO-K1 cells transiently expressing the recombinant rabbit NK3 receptor, the relative order of potency of NKB>>NKA⩾SP to displace [125I]-[MePhe7]-NKB binding and to increase intracellular calcium, together with the high affinity of NK3 selective agonists (e.g. senktide, [MePhe7]-NKB) and antagonists (e.g. SR 142801, SB 223412) in both assays was consistent with NK3 receptor pharmacology. In binding and functional experiments, agonist concentration response curves were shallow (0.7–0.8), suggesting the possibility of multiple affinity states of the receptor.Quantitative real time PCR analysis revealed highest expression of rabbit NK3 receptor mRNA in iris sphincter muscle, lower expression in retina and iris dilator muscle, and no expression in lens and cornea. In situ hybridization histochemistry revealed discrete specific localization of NK3 receptor mRNA in the iris muscle and associated ciliary processes. Discrete specific labelling of NK3 receptors with the selective NK3 receptor agonist [125I]-[MePhe7]-NKB was also observed in the ciliary processes using autoradiography.Our study reveals a high molecular similarity between rabbit and human NK3 receptor mRNAs, as predicted from previous pharmacological studies, and provide the first evidence that NK3 receptors are precisely located on ciliary processes in the rabbit eye. In addition, there could be two affinity states of the receptor which may correspond to the typical and ‘atypical' NK3 receptor subtypes previously reported. PMID:10516642

  6. Molecular cloning and characterization of the canine prostaglandin E receptor EP2 subtype.

    PubMed

    Hibbs, T A; Lu, B; Smock, S L; Vestergaard, P; Pan, L C; Owen, T A

    1999-05-01

    Prostaglandin E2 (PGE2) binds to four G-protein coupled cell surface receptors (EP1-EP4) and has been implicated as a local mediator of bone anabolism via a cyclic AMP mediated pathway following activation of the EP2 and/or EP4 receptor subtype. A canine kidney cDNA library was screened using a human EP2 probe, and a clone with an open reading frame of 1083 bp, potentially encoding a protein of 361 amino acids, was characterized. This open reading frame has 89% identity to the human EP2 cDNA at the nucleotide level and 87% identity at the predicted protein level. Scatchard analysis of a CHO cell line stably transfected with canine EP2 yielded a dissociation constant of 22 nM for PGE2. Competition binding studies, using 3H-PGE2 as ligand, demonstrated specific displacement by PGE2, Prostaglandin E1, Prostaglandin A3, and butaprost (an EP2 selective ligand), but not by ligands with selectivity for the related DP, FP, IP, or TP receptors. Specific ligand binding also resulted in increased levels of cAMP in EP2 transfected cells with no evidence of short-term, ligand-induced desensitization. Northern blot analysis revealed two transcripts of 3300 and 2400 bp in canine lung, and reverse-transcription polymerase chain reaction showed expression in all tissues examined. Southern blot analysis suggests the presence of a single-copy gene for EP2 in the dog.

  7. Molecular cloning and functional analysis of duck Toll-like receptor 5.

    PubMed

    Xiong, Dan; Pan, Zhiming; Kang, Xilong; Wang, Jing; Song, Li; Jiao, Xinan

    2014-08-01

    Toll-like receptor 5 (TLR5) is responsible for the recognition of bacterial flagellin in vertebrates. In this study, we cloned the single-exon TLR5 gene of the Maya breed of Common Shelduck (Tadorna tadorna). The TLR5 open reading frame is 2580 bp in length and encodes an 859-amino acid protein. The putative amino acid sequence of duck TLR5 consisted of a signal peptide sequence, 11 leucine-rich repeat domains, a leucine-rich repeat C-terminal domain, a transmembrane domain, and an intracellular Toll-interleukin-1 receptor domain. The duck TLR5 gene was highly expressed in the lung, bone marrow, spleen, and liver; moderately expressed in kidney, small intestine, large intestine, and brain. A plasmid expressing duck TLR5 was constructed and transfected into HEK293T cells, and expression was confirmed by indirect immunofluorescence assay. HEK293T cells transfected with duck TLR5- and NF-κB-luciferase-containing plasmids significantly responded to flagellin from Salmonella typhimurium, indicating that it is a functional TLR5 homolog.

  8. Molecular Cloning and Functional Characterization of a Zebrafish Nuclear Progesterone Receptor1

    PubMed Central

    Chen, Shi X.; Bogerd, Jan; García-López, Ángel; de Jonge, Hugo; de Waal, Paul P.; Hong, Wan S.; Schulz, Rüdiger W.

    2009-01-01

    Progestagenic sex steroid hormones play critical roles in reproduction across vertebrates, including teleost fish. To further our understanding of how progesterone modulates testis functions in fish, we set out to clone a progesterone receptor (pgr) cDNA exhibiting nuclear hormone receptor features from zebrafish testis. The open reading frame of pgr consists of 1854 bp, coding for a 617-amino acid-long protein showing the highest similarity with other piscine Pgr proteins. Functional characterization of the receptor expressed in mammalian cells revealed that zebrafish Pgr exhibited progesterone-specific, dose-dependent induction of reporter gene expression, with 17alpha,20beta-dihydroxy-4-pregnen-3-one (DHP), a typical piscine progesterone, showing the highest potency. Expression of pgr mRNA: 1) appeared in embryos at 8 h after fertilization; 2) was significantly higher in developing ovary than in early transforming testis at 4 wk of age but vice versa in young adults at 12 wk of age, and thus resembling the expression pattern of the germ cell marker piwil1; and, 3) was restricted to Leydig and Sertoli cells in adult testis. Zebrafish testicular explants released DHP concentration dependently in response to high concentrations of recombinant zebrafish gonadotropins. In addition, DHP stimulated 11-ketotestosterone release from zebrafish testicular explants, but only in the presence of its immediate precursor, 11beta-hydroxytestosterone. This stimulatory activity was blocked by a Pgr antagonist (RU486), suggesting that 11beta-hydroxysteroid dehydrogenase activity was stimulated by DHP via Pgr. Our data suggest that DHP contributes to the regulation of Leydig cell steroidogenesis, and potentially—via Sertoli cells—also to germ cell differentiation in zebrafish testis. PMID:19741208

  9. Molecular cloning and functional characterization of a zebrafish nuclear progesterone receptor.

    PubMed

    Chen, Shi X; Bogerd, Jan; García-López, Angel; de Jonge, Hugo; de Waal, Paul P; Hong, Wan S; Schulz, Rüdiger W

    2010-01-01

    Progestagenic sex steroid hormones play critical roles in reproduction across vertebrates, including teleost fish. To further our understanding of how progesterone modulates testis functions in fish, we set out to clone a progesterone receptor (pgr) cDNA exhibiting nuclear hormone receptor features from zebrafish testis. The open reading frame of pgr consists of 1854 bp, coding for a 617-amino acid-long protein showing the highest similarity with other piscine Pgr proteins. Functional characterization of the receptor expressed in mammalian cells revealed that zebrafish Pgr exhibited progesterone-specific, dose-dependent induction of reporter gene expression, with 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (DHP), a typical piscine progesterone, showing the highest potency. Expression of pgr mRNA: 1) appeared in embryos at 8 h after fertilization; 2) was significantly higher in developing ovary than in early transforming testis at 4 wk of age but vice versa in young adults at 12 wk of age, and thus resembling the expression pattern of the germ cell marker piwil1; and, 3) was restricted to Leydig and Sertoli cells in adult testis. Zebrafish testicular explants released DHP concentration dependently in response to high concentrations of recombinant zebrafish gonadotropins. In addition, DHP stimulated 11-ketotestosterone release from zebrafish testicular explants, but only in the presence of its immediate precursor, 11 beta-hydroxytestosterone. This stimulatory activity was blocked by a Pgr antagonist (RU486), suggesting that 11 beta-hydroxysteroid dehydrogenase activity was stimulated by DHP via Pgr. Our data suggest that DHP contributes to the regulation of Leydig cell steroidogenesis, and potentially--via Sertoli cells--also to germ cell differentiation in zebrafish testis.

  10. Molecular cloning and expression of thyroid hormone receptor alpha during salmonid development.

    PubMed

    Jones, Iwan; Rogers, Sarah A; Kille, Peter; Sweeney, Glen E

    2002-02-01

    Thyroid hormones have been implicated as important regulators of teleost development. To gain a better understanding of the potential roles of the thyroid system in salmonids a genomic clone which encoded rainbow trout TR-alpha was isolated. This clone exhibited highest amino acid identity to Japanese flounder TR-alphaB (94%) and zebrafish TR-alpha1 (94%). Oligonucleotides were designed against the rainbow trout sequence and the complete coding region of Atlantic salmon TR-alpha was isolated by RACE-PCR. The Atlantic salmon sequence exhibited highest amino acid identity to rainbow trout TR-alpha (98%), Japanese flounder TR-alphaB (93%), and zebrafish TR-alpha1 (90%). Atlantic salmon TR-alpha exhibited the classic modular structure associated with members of the nuclear receptor superfamily and consisted of a divergent A/B domain while the DNA and ligand-binding domains were highly conserved to other teleost TR proteins. Temporal expression from the rainbow trout TR-alpha gene was monitored by semiquantitative RT-PCR at selected stages during rainbow trout embryonic and larval development. High levels of maternal transcripts were present at cleavage (Stage 6) which were rapidly degraded by gastrulation (Stage 13). Low levels of TR-alpha expression were then detected during organogenesis (Stages 20, 24, 26, 29, and 31). A peak in mRNA levels was observed at hatch (Stage 32) after which levels rose in a gradual manner during larval development (Stages 33, 34, 35, and 36) to reach maximal values at first feeding (Stage 37). These results suggest that the thyroid axis is functional and that embryonic and larval rainbow trout are at least capable of responding to thyroid hormones. These observations implicate the thyroid system as being an important regulator of salmonid development.

  11. Molecular cloning and characterization of ligand- and species-specificity of amphibian estrogen receptors.

    PubMed

    Katsu, Yoshinao; Taniguchi, Ena; Urushitani, Hiroshi; Miyagawa, Shinichi; Takase, Minoru; Kubokawa, Kaoru; Tooi, Osamu; Oka, Tomohiro; Santo, Noriaki; Myburgh, Jan; Matsuno, Akira; Iguchi, Taisen

    2010-09-01

    Estrogens are essential for normal reproductive activity in both males and females as well as for ovarian differentiation during a critical developmental stage in most vertebrates. To understand the molecular mechanisms of estrogen action and to evaluate estrogen receptor ligand interactions in amphibians, we isolated cDNAs encoding the estrogen receptors (ERalpha and ERbeta) from the Japanese firebelly newt (Cynops pyrrhogaster), Tokyo salamander (Hynobius tokyoensis), axolotl (Ambystoma mexicanum), and Raucous toad (Bufo rangeri). Full-length amphibian ER cDNAs were obtained using 5' and 3' rapid amplification of cDNA ends. The predicted amino acid sequences of these amphibian ERs showed a high degree of amino acid sequence identity (over 70%) to each other. We analyzed the relationships of these amphibian ER sequences to other vertebrate ER sequences by constructing a phylogenetic tree. We verified that these were bona fide estrogen receptors using receptor dependent reporter gene assays. We analyzed the effects of natural estrogens, ethinylestradiol, and DDT and its metabolites on the transactivation of the four amphibian species listed above, and Xenopus tropicalis ERs and found that there were species-specific differences in the sensitivity of these ERs to hormones and environmental chemicals. These findings will expand our knowledge of endocrine-disrupting events in amphibians.

  12. Molecular cloning and characterization of a glycine-like receptor gene from the cattle tick Rhipicephalus (Boophilus) microplus (Acari: Ixodidae).

    PubMed

    Flores-Fernández, José Miguel; Gutiérrez-Ortega, Abel; Padilla-Camberos, Eduardo; Rosario-Cruz, Rodrigo; Hernández-Gutiérrez, Rodolfo; Martínez-Velázquez, Moisés

    2014-01-01

    The cattle tick Rhipicephalus (Boophilus) microplus is the most economically important ectoparasite affecting the cattle industry in tropical and subtropical areas around the world. The principal method of tick control has relied mainly on the use of chemical acaricides, including ivermectin; however, cattle tick populations resistant to ivermectin have recently been reported in Brazil, Mexico, and Uruguay. Currently, the molecular basis for ivermectin susceptibility and resistance are not well understood in R. microplus. This prompted us to search for potential molecular targets for ivermectin. Here, we report the cloning and molecular characterization of a R. microplus glycine-like receptor (RmGlyR) gene. The characterized mRNA encodes for a 464-amino acid polypeptide, which contains features common to ligand-gated ion channels, such as a large N-terminal extracellular domain, four transmembrane domains, a large intracellular loop and a short C-terminal extracellular domain. The deduced amino acid sequence showed around 30% identity to GlyRs from some invertebrate and vertebrate organisms. The polypeptide also contains the PAR motif, which is important for forming anion channels, and a conserved glycine residue at the third transmembrane domain, which is essential for high ivermectin sensitivity. PCR analyses showed that RmGlyR is expressed at egg, larval and adult developmental stages. Our findings suggest that the deduced receptor is an additional molecular target to ivermectin and it might be involved in ivermectin resistance in R. microplus.

  13. Molecular cloning and characterization of a glycine-like receptor gene from the cattle tick Rhipicephalus (Boophilus) microplus (Acari: Ixodidae)

    PubMed Central

    Flores-Fernández, José Miguel; Gutiérrez-Ortega, Abel; Padilla-Camberos, Eduardo; Rosario-Cruz, Rodrigo; Hernández-Gutiérrez, Rodolfo; Martínez-Velázquez, Moisés

    2014-01-01

    The cattle tick Rhipicephalus (Boophilus) microplus is the most economically important ectoparasite affecting the cattle industry in tropical and subtropical areas around the world. The principal method of tick control has relied mainly on the use of chemical acaricides, including ivermectin; however, cattle tick populations resistant to ivermectin have recently been reported in Brazil, Mexico, and Uruguay. Currently, the molecular basis for ivermectin susceptibility and resistance are not well understood in R. microplus. This prompted us to search for potential molecular targets for ivermectin. Here, we report the cloning and molecular characterization of a R. microplus glycine-like receptor (RmGlyR) gene. The characterized mRNA encodes for a 464-amino acid polypeptide, which contains features common to ligand-gated ion channels, such as a large N-terminal extracellular domain, four transmembrane domains, a large intracellular loop and a short C-terminal extracellular domain. The deduced amino acid sequence showed around 30% identity to GlyRs from some invertebrate and vertebrate organisms. The polypeptide also contains the PAR motif, which is important for forming anion channels, and a conserved glycine residue at the third transmembrane domain, which is essential for high ivermectin sensitivity. PCR analyses showed that RmGlyR is expressed at egg, larval and adult developmental stages. Our findings suggest that the deduced receptor is an additional molecular target to ivermectin and it might be involved in ivermectin resistance in R. microplus. PMID:25174962

  14. The murine ufo receptor: molecular cloning, chromosomal localization and in situ expression analysis.

    PubMed

    Faust, M; Ebensperger, C; Schulz, A S; Schleithoff, L; Hameister, H; Bartram, C R; Janssen, J W

    1992-07-01

    We have cloned the mouse homologue of the ufo oncogene. It encodes a novel tyrosine kinase receptor characterized by a unique extracellular domain containing two immunoglobulin-like and two fibronectin type III repeats. Comparison of the predicted ufo amino acid sequences of mouse and man revealed an overall identity of 87.6%. The ufo locus maps to mouse chromosome 7A3-B1 and thereby extends the known conserved linkage group between mouse chromosome 7 and human chromosome 19. RNA in situ hybridization analysis established the onset of specific ufo expression in the late embryogenesis at day 12.5 post coitum (p.c.) and localized ufo transcription to distinct substructures of a broad spectrum of developing tissues (e.g. subepidermal cells of the skin, mesenchymal cells of the periosteum). In adult animals ufo is expressed in cells forming organ capsules as well as in connective tissue structures. ufo may function as a signal transducer between specific cell types of mesodermal origin.

  15. Molecular cloning and further characterization of a probable plant vacuolar sorting receptor.

    PubMed Central

    Paris, N; Rogers, S W; Jiang, L; Kirsch, T; Beevers, L; Phillips, T E; Rogers, J C

    1997-01-01

    BP-80 is a type I integral membrane protein abundant in pea (Pisum sativum) clathrin-coated vesicles (CCVs) that binds with high affinity to vacuole-targeting determinants containing asparagine-proline-isoleucine-arginine. Here we present results from cDNA cloning and studies of its intracellular localization. Its sequence and sequences of homologs from Arabidopsis, rice (Oryza sativa), and maize (Zea mays) define a novel family of proteins unique to plants that is highly conserved in both monocotyledons and dicotyledons. The BP-80 protein is present in dilated ends of Golgi cisternae and in "prevacuoles," which are small vacuoles separate from but capable of fusing with lytic vacuoles. Its cytoplasmic tail contains a Tyr-X-X-hydrophobic residue motif associated with transmembrane proteins incorporated into CCVs. When transiently expressed in tobacco (Nicotiana tabacum) suspension-culture protoplasts, a truncated form lacking transmembrane and cytoplasmic domains was secreted. These results, coupled with previous studies of ligand-binding specificity and pH dependence, strongly support our hypothesis that BP-80 is a vacuolar sorting receptor that trafficks in CCVs between Golgi and a newly described prevacuolar compartment. PMID:9306690

  16. Type I interferon receptors in goose: molecular cloning, structural identification, evolutionary analysis and age-related tissue expression profile.

    PubMed

    Zhou, Hao; Chen, Shun; Qi, Yulin; Zhou, Qin; Wang, Mingshu; Jia, Renyong; Zhu, Dekang; Liu, Mafeng; Sun, Kunfeng; Liu, Fei; Chen, Xiaoyue; Cheng, Anchun

    2015-04-25

    The cDNAs encoding two distinct type I interferon receptors were firstly cloned from the spleen of white goose (the Chinese goose, Anser cygnoides). The cDNA of goose IFNAR1 consisted of 1616 bp and encoded 406 amino acids with a predicted molecular weight of 46.4 kDa, while the cDNA of goose IFNAR2 consisted of 1525 bp and encoded 294 amino acids with a predicted molecular weight of 32.6 kDa. The IFNAR1 shared 85.4% identity in deduced amino acid sequence with duck IFNAR1, while IFNAR2 amino acid sequence showed 86% identity with that of duck IFNAR2. The age-related analysis of gene expression revealed that goose IFNα and IFNARs were all highly transcribed in pancreas, which may due to a reasonable amount of dendritic cells aggregated in pancreas. And goose IFNα and its cognate receptors had different structural features and tissue expression patterns during the period from embryonic goose to adult goose, suggesting that IFNα and IFNARs may maintain a developmental dynamic immune competence in unstimulated states. The data provided in this study may contribute to future understanding of the interaction between interferon and interferon receptors in immune mechanism. And it also helps us to understand the age-related susceptibility to pathogens in birds better. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. Molecular cloning and characterization of the corticoid receptors from the American alligator.

    PubMed

    Oka, Kaori; Kohno, Satomi; Urushitani, Hiroshi; Guillette, Louis J; Ohta, Yasuhiko; Iguchi, Taisen; Katsu, Yoshinao

    2013-01-30

    Steroid hormones are essential for health in vertebrates. Corticosteroids, for example, have a regulatory role in many physiological functions, such as osmoregulation, respiration, immune responses, stress responses, reproduction, growth, and metabolism. Although extensively studied in mammals and some non-mammalian species, the molecular mechanisms of corticosteroid hormone (glucocorticoids and mineralocorticoids) action are poorly understood in reptiles. Here, we have evaluated hormone receptor-ligand interactions in the American alligator (Alligator mississippiensis), following the isolation of cDNAs encoding a glucocorticoid receptor (GR) and a mineralocorticoid receptor (MR). The full-length alligator GR (aGR) and aMR cDNAs were obtained using 5' and 3' rapid amplification cDNA ends (RACE). The deduced amino acid sequences exhibited high identity to the chicken orthologs (aGR: 83%; aMR: 90%). Using transient transfection assays of mammalian cells, both aGR and aMR proteins displayed corticosteroid-dependent activation of transcription from keto-steroid hormone responsive, murine mammary tumor virus promoters. We further compared the ligand-specifity of human, chicken, Xenopus, and zebrafish GR and MR. We found that the alligator and chicken GR/MR have very similar amino acid sequences, and this translates to very similar ligand specificity. This is the first report of the full-coding regions of a reptilian GR and MR, and the examination of their transactivation by steroid hormones. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  18. Molecular cloning, expression, and signaling pathway of four melanin-concentrating hormone receptors from Xenopus tropicalis.

    PubMed

    Kobayashi, Yuki; Hamamoto, Akie; Hirayama, Tomo; Saito, Yumiko

    2015-02-01

    Melanin-concentrating hormone (MCH) mainly regulates feeding in mammals and pigmentation in teleosts. It acts via two G-protein-coupled receptors, MCH receptor 1 (MCHR1) and MCHR2. Although many studies exploring the MCH system in teleosts and mammals have been carried out, studies on other organisms are limited. In this study, we cloned and characterized four MCHR subtypes from the diploid species Xenopus tropicalis (X-MCHRs; X-MCHR1a, R1b, R2a, and R2b). According to a phylogenetic tree of the X-MCHRs, X-MCHR1a and R2a are close to mammalian MCHRs, while X-MCHR1b and R2b are close to teleostean MCHRs. We previously reported that the G-protein coupling capacity of the MCHR subtypes differed between mammals (R1: Gαi/o and Gαq; R2: Gαq) and teleosts (R1: Gαq; R2: Gαi/o and Gαq) in mammalian cell-based assays. By using Ca(2+) mobilization assays with pertussis toxin in CHO dhfr(-) cells, we found that X-MCHR1a promiscuously coupled to both Gαi/o and Gαq, while X-MCHR1b and R2a exclusively coupled to Gαq. However, no Ca(2+) influx was detected in cells transfected with X-MCHR2b. Reverse transcription-PCR showed that the X-MCHR mRNAs were expressed in various tissues. In particular, both X-MCHR1b and R2b were exclusively found in melanophores of the dorsal skin. In skin pigment migration assays, melanophores were weakly aggregated at low concentrations but dispersed at high concentrations of MCH, suggesting possible interactions between X-MCHR1b and R2b for the regulation of body color. These findings demonstrate that X. tropicalis has four characteristic MCHRs and will be useful for elucidating the nature of MCHR evolution among vertebrates.

  19. Molecular biology of channel catfish gonadotropin receptors: 1. Cloning of a functional luteinizing hormone receptor and preovulatory induction of gene expression.

    PubMed

    Kumar, R S; Ijiri, S; Trant, J M

    2001-03-01

    There is little known about the molecular biology of piscine gonadotropin receptors, and information about gene expression during reproductive development is particularly lacking. We have cloned the LH receptor (LHR) in the channel catfish (cc), and examined its gene expression throughout a reproductive cycle. A cDNA encoding the receptor was isolated from the testis using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends procedures. It encoded a 696-amino acid protein that showed the greatest homology (46-50% identity) with the known LHRs and lesser similarity with FSH receptors and thyroid-stimulating hormone receptors (44-47% and 42-44% identity, respectively). In addition, two characteristics unique to the LHRs were conserved in the cloned receptor and the encoding gene: presence of an intron corresponding to intron 10 in mammals and turkey and occurrence of a double cysteine residue in the cytoplasmic tail for potential palmitoylation. The ccLHR gene was well expressed in the gonads and kidney and merely detectable in the gills, muscle, and spleen. The isolated cDNA encoded an active ccLHR protein, as the recombinant receptor expressed in COS7 cells activated a cAMP response element-driven reporter gene (luciferase) upon exposure to hCG in a dose-dependent manner. Seasonal changes in the ovarian expression of the ccLHR gene, as examined by measuring the transcript abundance by quantitative real-time RT-PCR, remained rather low during most of the reproductive cycle but was acutely induced around the time of spawning. This pattern of expression correlates well with the reported expression of its ligand (LH) in fishes and concurs with the notion that LH is a key regulator of the periovulatory maturational events.

  20. Molecular Cloning, Characterization, and Expression Analysis of an Ecdysone Receptor Homolog in Teleogryllus emma (Orthoptera: Gryllidae)

    PubMed Central

    He, Hui; Xi, Gengsi; Lu, Xiao

    2015-01-01

    Ecdysteroids are steroid hormones that play important roles in the regulation of Arthropoda animal growth development, larvae ecdysis, and reproduction. The effect of ecdysteroids is mediated by ecdysteroid receptor (EcR). The ecdysone receptor (EcR) belongs to the superfamily of nuclear receptors (NRs) that are ligand-dependent transcription factors. Ecdysone receptor is present only in invertebrates and plays a critical role in regulating the expression of a series of genes during development and reproduction. Here, we isolated and characterized cDNA of the cricket Teleopgryllus emma (Ohmachi & Matsuura) (Orthoptera: Gryllidae) and studied mRNA expression pattern using real time-polymerase chain reaction. The full-length cDNA of T. emma EcR, termed TeEcR, is 2,558 bp and contains a 5′-untranslated region of 555 bp and a 3′-untranslated region of 407 bp. The open reading frame of TeEcR encodes deduced 531-amino acid peptides with a predicted molecular mass of 60.7 kDa. The amino acid sequence of T. emma EcR was similar to that of known EcR especially in the ligand-binding domain of insect EcR. Real-time quantitative reverse transcription-polymerase chain reaction was performed to compare TeEcR mRNA expression level at the whole body and gonad during T. emma development. The data revealed that TeEcR mRNA is differentially expressed during T. emma development, with the highest expression level in late-instar larvae of the body and lowest in third instar. The levels of TeEcR transcripts also vary among gonads development, and levels in ovaries were higher than in testes at every developmental stage. These results suggest that TeEcR may have potential significance to regulate the morphological structure and gonad development of T. emma, due to its expression in different developmental periods. PMID:25797799

  1. Molecular cloning, characterization, and expression analysis of an ecdysone receptor homolog in Teleogryllus emma (Orthoptera: Gryllidae).

    PubMed

    He, Hui; Xi, Gengsi; Lu, Xiao

    2015-01-01

    Ecdysteroids are steroid hormones that play important roles in the regulation of Arthropoda animal growth development, larvae ecdysis, and reproduction. The effect of ecdysteroids is mediated by ecdysteroid receptor (EcR). The ecdysone receptor (EcR) belongs to the superfamily of nuclear receptors (NRs) that are ligand-dependent transcription factors. Ecdysone receptor is present only in invertebrates and plays a critical role in regulating the expression of a series of genes during development and reproduction. Here, we isolated and characterized cDNA of the cricket Teleopgryllus emma (Ohmachi & Matsuura) (Orthoptera: Gryllidae) and studied mRNA expression pattern using real time-polymerase chain reaction. The full-length cDNA of T. emma EcR, termed TeEcR, is 2,558 bp and contains a 5'-untranslated region of 555 bp and a 3'-untranslated region of 407 bp. The open reading frame of TeEcR encodes deduced 531-amino acid peptides with a predicted molecular mass of 60.7 kDa. The amino acid sequence of T. emma EcR was similar to that of known EcR especially in the ligand-binding domain of insect EcR. Real-time quantitative reverse transcription-polymerase chain reaction was performed to compare TeEcR mRNA expression level at the whole body and gonad during T. emma development. The data revealed that TeEcR mRNA is differentially expressed during T. emma development, with the highest expression level in late-instar larvae of the body and lowest in third instar. The levels of TeEcR transcripts also vary among gonads development, and levels in ovaries were higher than in testes at every developmental stage. These results suggest that TeEcR may have potential significance to regulate the morphological structure and gonad development of T. emma, due to its expression in different developmental periods.

  2. Molecular cloning of an unusual bicistronic cholecystokinin receptor mRNA expressed in chicken brain: a structural and functional expression study.

    PubMed

    Nilsson, Isabelle B M; Svensson, Samuel P S; Monstein, Hans-Jürg

    2003-06-15

    This report describes the molecular cloning and pharmacological characterization of a transiently expressed chicken brain cholecystokinin receptor (CCK-CHR) in COS-7 cells. A polymerase chain reaction (PCR)-based cloning strategy was applied using: (1) an initial PCR with deoxyinosine-containing primers designed to target conserved regions in CCK receptors, followed by (2) rapid amplification of cDNA ends (RACE), and (3) full-length PCR of the CCK-CHR cDNA. The full-length cloned bicistronic CCK-CHR cDNA contained a short upstream open reading frame (uORF) coding for a putative six-amino-acid-long peptide of unknown function, followed by a long open reading frame (lORF) encoding the 436-amino-acid-long CCK-CHR receptor protein. At the amino acid level, the CCK-CHR shared approximately 50% homology with mammalian and Xenopus laevis CCK receptors. The pharmacological profile of CCK-CHR resembled that of CCK-B receptors using agonists (CCK-8, CCK-4, gastrin-17), whereas CCK-CHR showed higher affinity for the CCK-A receptor antagonist, devazepide, than for the CCK-B receptor antagonist, L-365,260. To the best of our knowledge, this is the first description and functional expression study of a cloned chicken CCK receptor cDNA.

  3. Molecular cloning and characterization of a novel dopamine receptor (D3) as a target for neuroleptics

    NASA Astrophysics Data System (ADS)

    Sokoloff, Pierre; Giros, Bruno; Martres, Marie-Pascale; Bouthenet, Marie-Louise; Schwartz, Jean-Charles

    1990-09-01

    A dopamine receptor has been characterized which differs in its pharmacology and signalling system from the D1 or D2 receptor and represents both an autoreceptor and a postsynaptic receptor. The D3 receptor is localized to limbic areas of the brain, which are associated with cognitive, emotional and endocrine functions. It seems to mediate some of the effects of antipsychotic drugs and drugs used against Parkinson's disease, that were previously thought to interact only with D2 receptors.

  4. Molecular Cloning, Functional Characterization, and Evolutionary Analysis of Vitamin D Receptors Isolated from Basal Vertebrates

    PubMed Central

    Kollitz, Erin M.; Zhang, Guozhu; Hawkins, Mary Beth; Whitfield, G. Kerr; Reif, David M.; Kullman, Seth W.

    2015-01-01

    The vertebrate genome is a result of two rapid and successive rounds of whole genome duplication, referred to as 1R and 2R. Furthermore, teleost fish have undergone a third whole genome duplication (3R) specific to their lineage, resulting in the retention of multiple gene paralogs. The more recent 3R event in teleosts provides a unique opportunity to gain insight into how genes evolve through specific evolutionary processes. In this study we compare molecular activities of vitamin D receptors (VDR) from basal species that diverged at key points in vertebrate evolution in order to infer derived and ancestral VDR functions of teleost paralogs. Species include the sea lamprey (Petromyzon marinus), a 1R jawless fish; the little skate (Leucoraja erinacea), a cartilaginous fish that diverged after the 2R event; and the Senegal bichir (Polypterus senegalus), a primitive 2R ray-finned fish. Saturation binding assays and gel mobility shift assays demonstrate high affinity ligand binding and classic DNA binding characteristics of VDR has been conserved across vertebrate evolution. Concentration response curves in transient transfection assays reveal EC50 values in the low nanomolar range, however maximum transactivational efficacy varies significantly between receptor orthologs. Protein-protein interactions were investigated using co-transfection, mammalian 2-hybrid assays, and mutations of coregulator activation domains. We then combined these results with our previous study of VDR paralogs from 3R teleosts into a bioinformatics analysis. Our results suggest that 1, 25D3 acts as a partial agonist in basal species. Furthermore, our bioinformatics analysis suggests that functional differences between VDR orthologs and paralogs are influenced by differential protein interactions with essential coregulator proteins. We speculate that we may be observing a change in the pharmacodynamics relationship between VDR and 1, 25D3 throughout vertebrate evolution that may have been

  5. Molecular cloning and characterization of chicken interferon-gamma receptor alpha-chain.

    PubMed

    Han, Xue; Chen, Tong; Wang, Ming

    2008-07-01

    In this study, a cDNA sequence of Huiyang chicken interferon-gamma (IFN-gamma) receptor alpha-chain (chIFNGR-1) gene wasgenerated using rapid amplification of cDNA ends (RACE) method for the first time. The predicted 422 amino acids showed approximately 25%-29% sequence identity and 53%-55% similarity to mammalian homologues. There are two fibronectin type-III (FN-III) domains of about 110 residues in the extracellular domain, and LPKS and YDKPH motifs in the intracellular domain, which are conserved in the mammalian IFNGR-1 as the binding sites of JAK1 and STAT1. Expression analysis by Northern blot revealed that the chIFNGR-1 was highly expressed in spleen, thymus, peripheral blood lymphocytes (PBLs), lung, cecum tonsil, and liver. The extracellular region of chIFNGR-1 (chIFNGR-1EC) was expressed in Escherichia coli and purified. The purified IFNGR-1EC was further characterized by mass spectroscopy and circular dichroism (CD) spectroscopy. The molecular weight of the recombinant chIFNGR-1EC (rchIFNGR-1EC) was measured as 24 364 Da, and its secondary structure contained 17.6% alpha-helix, 36.4% beta-sheet, 17.2% turn, and 28.8% random coil. Furthermore, three-dimensional modeling presented the most probable structure of chIFNGR-1EC. These * ndings show that the identified chicken cDNA sequence encodes an IFNGR1 homologue, and the chIFNGR-1EC resembles the similar structure with other IFN receptors.

  6. Molecular cloning, expression, and regulation of estrogen receptors in pigeon oviduct epithelial cells.

    PubMed

    Zhang, H; Chen, F; Li, G L; Ding, Y Y; Tao, Z R; Li, J J; Zhong, S L; Lu, L Z

    2014-03-17

    Estrogen regulates reproductive behavior and drives the proliferation and differentiation of several cell types. These physiological functions of estrogen are mediated by estrogen receptors (ERs), and each ER isoform plays a distinct role. To clarify the molecular mechanism of estrogen action and to evaluate the effect of ERs on the secretion of ovalbumin (OVA) in pigeon oviduct epithelial cells (POECs), we determined the complete coding sequences encoding ER alpha (ERα) and ER beta (ERβ) in pigeons. The abundance of pigeon ERα and ERβ mRNA was detected using quantitative polymerase chain reaction. These results revealed that pigeon ERα is highly expressed in the oviduct, while pigeon ERb is highly expressed in the ovary and kidney. We hypothesize that ERα mRNA predominates over that of ERβ in the oviduct. The expression of ERα can be down-regulated by 17β-estradiol, and the knockdown of ERα promoted OVA mRNA expression in cultured POECs, indicating that ERα may play an important role in OVA secretion.

  7. Molecular cloning, characterization, and expression analysis of an estrogen receptor-related receptor homologue in the cricket, Teleogryllus emma.

    PubMed

    He, Hui; Xi, Gengsi; Lu, Xiao

    2010-01-01

    The estrogen receptor-related receptors (ERRs) are a group of nuclear receptors that were originally identified on the basis of sequence similarity to estrogen receptors. The three mammalian ERR genes have been implicated in diverse physiological processes ranging from placental development to maintenance of bone density, but the function and regulation of ERRs in invertebrates are not well understood. A homologue of human ERR was isolated from the cricket Teleogryllus emma (Ohmachi and Matsumura) (Orthoptera: Gryllidae). The full-length cDNA of T. emma ERR, termed TeERR, has 1618 base pair (bp) and contains a 5'-untranslated region of 140 bp and a 3'-untranslated region of 272 bp. The open reading frame of TeERR encodes a deduced 401 amino acid peptide with a predicted molecular mass of 45.75 kilodaltons. The results of sequence alignments indicate that the TeERR protein shares an overall identity of 65%-82% with other known ERR homologues, and is most closely related to that of Nasonia vitripennis (Hymenoptera: Pteromalidae) and Apis mellifera (Apidae). Real-time quantitative reverse transcription-polymerase chain reaction was performed to compare the TeERR mRNA expression level at the whole body and gonad during T. emma development. The data revealed that TeERR mRNA is differentially expressed during T. emma development, with the highest expression level in embryos and the lowest in the body of late-instar larvae. The levels of TeERR transcripts also varied throughout gonad development; interestingly testicles had higher higher expression levels than ovaries at every development stage. These results suggest that TeERR has potential significance in the regulation of development in T. emma, due to its expression during different developmental periods.

  8. Molecular Cloning, Characterization, and Expression Analysis of an Estrogen Receptor-Related Receptor Homologue in the Cricket, Teleogryllus emma

    PubMed Central

    He, Hui; Xi, Gengsi; Lu, Xiao

    2010-01-01

    The estrogen receptor-related receptors (ERRs) are a group of nuclear receptors that were originally identified on the basis of sequence similarity to estrogen receptors. The three mammalian ERR genes have been implicated in diverse physiological processes ranging from placental development to maintenance of bone density, but the function and regulation of ERRs in invertebrates are not well understood. A homologue of human ERR was isolated from the cricket Teleogryllus emma (Ohmachi and Matsumura) (Orthoptera: Gryllidae). The full-length cDNA of T. emma ERR, termed TeERR, has 1618 base pair (bp) and contains a 5′?-untranslated region of 140 bp and a 3′?-untranslated region of 272 bp. The open reading frame of TeERR encodes a deduced 401 amino acid peptide with a predicted molecular mass of 45.75 kilodaltons. The results of sequence alignments indicate that the TeERR protein shares an overall identity of 65%–82% with other known ERR homologues, and is most closely related to that of Nasonia vitripennis (Hymenoptera: Pteromalidae) and Apis mellifera (Apidae). Real-time quantitative reverse transcription-polymerase chain reaction was performed to compare the TeERR mRNA expression level at the whole body and gonad during T. emma development. The data revealed that TeERR mRNA is differentially expressed during T. emma development, with the highest expression level in embryos and the lowest in the body of late-instar larvae. The levels of TeERR transcripts also varied throughout gonad development; interestingly testicles had higher higher expression levels than ovaries at every development stage. These results suggest that TeERR has potential significance in the regulation of development in T. emma, due to its expression during different developmental periods. PMID:21265615

  9. Molecular cloning and properties of a full-length putative thyroid hormone receptor coactivator.

    PubMed

    Takeshita, A; Yen, P M; Misiti, S; Cardona, G R; Liu, Y; Chin, W W

    1996-08-01

    Thyroid hormone receptors (TRs) are ligand-dependent transcription factors that regulate target gene transcription. The conserved carboxy-terminal region of the ligand-binding domain (AF-2) has been thought to play a critical role in mediating ligand-dependent transactivation by the interaction with coactivator(s). Using bacterially-expressed TR as a probe, far-Western-based expression cDNA library screening identified cDNAs that encode, in part, the recently reported partial steroid receptor coactivator-1 (SRC-1) sequence. Additional work, including 5' RACE, has characterized a full-length cDNA that encodes a approximately 160 kD protein as a putative thyroid hormone receptor coactivator (F-SRC-1). In vitro binding studies show that F-SRC-1 binds to a variety of nuclear hormone receptors in a ligand-dependent manner, along with TBP and TFIIB, suggesting that F-SRC-1 may play a role as a bridging molecule between nuclear hormone receptors and general transcription factors. Interestingly, AF-2 mutants also retain ligand-dependent interaction with F-SRC-1. Although F-SRC-1 recognizes the ligand-induced conformational changes of nuclear hormone receptors, our observations suggest that F-SRC-1 may bind directly with subregion(s) in nuclear hormone receptors other than the AF-2 region.

  10. Molecular cloning and transcriptional analysis of a NPY receptor-like in common Chinese cuttlefish Sepiella japonica

    NASA Astrophysics Data System (ADS)

    Yang, Jingwen; Xu, Yuchao; Xu, Ke; Ping, Hongling; Shi, Huilai; Lü, Zhenming; Wu, Changwen; Wang, Tianming

    2017-08-01

    Neuropeptide Y (NPY) has a pivotal role in the regulation of many physiological processes. In this study, the gene encoding a NPY receptor-like from the common Chinese cuttlefish Sepiella japonica (SjNPYR-like) was identified and characterized. The full-length SjNPYR-like cDNA was cloned containing a 492-bp of 5' untranslated region (UTR), 1 182 bp open reading frame (ORF) encoding a protein of 393 amino acid residues, and 228 bp of 3' UTR. The putative protein was predicted to have a molecular weight of 45.54 kDa and an isoelectric point (pI) of 8.13. By informatic analyses, SjNPYR-like was identified as belonging to the class A G protein coupled receptor (GPCR) family (the rhodopsin-type). The amino acid sequence contained 12 potential phosphorylation sites and five predicted N-linked glycosylation sites. Multiple sequence alignment and 3D structure modeling were conducted to clarify SjNPYR bioinformatics characteristics. Phylogenetic analysis identifies it as an NPYR with identity of 33% to Lymnaea stagnalis NPFR. Transmembrane properties of SjNPYR-like were demonstrated in vitro using HEK293 cells and the pEGFP-N1 plasmid. Relative quantification of SjNPYR-like mRNA level confirmed a high level expression and broad distribution of SjNPYR - like in various tissues of female S. japonica. In addition, the transcriptional profile of SjNPYR - like in the brain, liver, and ovary during gonadal development was analyzed. The results provide basic understanding on the molecular characteristics of SjNPYR-like and its potentially physical functions.

  11. Molecular cloning and characterisation of a novel GABAB-related G-protein coupled receptor.

    PubMed

    Calver, A R; Michalovich, D; Testa, T T; Robbins, M J; Jaillard, C; Hill, J; Szekeres, P G; Charles, K J; Jourdain, S; Holbrook, J D; Boyfield, I; Patel, N; Medhurst, A D; Pangalos, M N

    2003-02-20

    Using a homology-based bioinformatics approach we have analysed human genomic sequence and identified the human and rodent orthologues of a novel putative seven transmembrane G protein coupled receptor, termed GABA(BL). The amino acid sequence homology of these cDNAs compared to GABA(B1) and GABA(B2) led us to postulate that GABA(BL) was a putative novel GABA(B) receptor subunit. The C-terminal sequence of GABA(BL) contained a putative coiled-coil domain, di-leucine and several RXR(R) ER retention motifs, all of which have been shown to be critical in GABA(B) receptor subunit function. In addition, the distribution of GABA(BL) in the central nervous system was reminiscent of that of the other known GABA(B) subunits. However, we were unable to detect receptor function in response to any GABA(B) ligands when GABA(BL) was expressed in isolation or in the presence of either GABA(B1) or GABA(B2). Therefore, if GABA(BL) is indeed a GABA(B) receptor subunit, its partner is a potentially novel receptor subunit or chaperone protein which has yet to be identified.

  12. Molecular cloning and characterization of the allatotropin precursor and receptor in the desert locust, Schistocerca gregaria

    PubMed Central

    Lismont, Els; Vleugels, Rut; Marchal, Elisabeth; Badisco, Liesbeth; Van Wielendaele, Pieter; Lenaerts, Cynthia; Zels, Sven; Tobe, Stephen S.; Vanden Broeck, Jozef; Verlinden, Heleen

    2015-01-01

    Allatotropins (ATs) are pleiotropic neuropeptides initially isolated from the tobacco hornworm, Manduca sexta. In 2008, the first receptor for AT-like peptides (ATR) was characterized in Bombyx mori. Since then, ATRs have also been characterized in M. sexta, Tribolium castaneum, Aedes aegypti and Bombus terrestris. These receptors show sequence similarity to vertebrate orexin (ORX) receptors. When generating an EST-database of the desert locust (Schistocerca gregaria) central nervous system, we found cDNA sequences encoding the Schgr-AT precursor and a fragment of its putative receptor. This receptor cDNA has now been completed and functionally expressed in mammalian cell lines. Activation of this receptor, designated as Schgr-ATR, by Schgr-AT caused an increase in intracellular calcium ions, as well as cyclic AMP (cAMP), with an EC50 value in the nanomolar range. In addition, the transcript distribution of both the Schgr-AT precursor and Schgr-ATR was investigated by means of quantitative real-time PCR. Moreover, we found more evidence for the myotropic and allatostimulatory actions of Schgr-AT in the desert locust. These data are discussed and situated in a broader context by comparison with literature data on AT and ATR in insects. PMID:25814925

  13. Molecular cloning and characterization of a human eotaxin receptor expressed selectively on eosinophils

    PubMed Central

    1996-01-01

    The chemokine eotaxin is unusual in that it appears to be a highly specific chemoattractant for eosinophils. Ligand-binding studies with radiolabeled eotaxin demonstrated a receptor on eosinophils distinct from the known chemokine receptors CKR-1 and -2. The distinct eotaxin binding site on human eosinophils also bound RANTES (regulated on activation T expressed and secreted) and monocyte chemotactic protein (MCP)3. We have now isolated a cDNA from eosinophils, termed CKR-3, with significant sequence similarity to other well characterized chemokine receptors. Cells transfected with CKR-3 cDNA bound radiolabeled eotaxin specifically and with high affinity, comparable to the binding affinity observed with eosinophils. This receptor also bound RANTES and MCP-3 with high affinity, but not other CC or CXC chemokines. Furthermore, receptor transfectants generated in a murine B cell lymphoma cell line migrated in transwell chemotaxis assays to eotaxin, RANTES, and MCP-3, but not to any other chemokines. A monoclonal antibody recognizing CKR-3 was used to show that eosinophils, but not other leukocyte types, expressed this receptor. This pattern of expression was confirmed by Northern blot with RNA from highly purified leukocyte subsets. The restricted expression of CKR-3 on eosinophils and the fidelity of eotaxin binding to CKR-3, provides a potential mechanism for the selective recruitment and migration of eosinophils within tissues. PMID:8676064

  14. Molecular cloning and expression of a chicken pituitary adenylate cyclase-activating polypeptide receptor.

    PubMed

    Peeters, K; Gerets, H H; Princen, K; Vandesande, F

    1999-08-25

    Although, since the isolation of pituitary adenylate cyclase-activating polypeptide (PACAP), a wealth of literature has been published describing its localization, binding sites, and biological activities in a variety of mammalian tissues, only very little is known about PACAP in avian species. Therefore, in order to find out the sites of actions of PACAP and to elucidate its physiological significance in birds, we identified a chicken PACAP receptor homologue of the mammalian type I receptors (PAC(1)-Rs). The chicken PACAP type I cDNA sequence was obtained using reverse transcriptase-polymerase chain reaction (RT-PCR) in combination with 3'- and 5'-RACE PCR. This cDNA encodes a 471 amino acid precursor protein, sharing 81-83% sequence identity with mammalian analogs and 76% amino acid identity with the goldfish type I PACAP receptor. Northern blot analysis of chicken brain poly(A)(+)-rich RNA revealed the presence of a 5.5 kb and 7.5 kb PAC(1) receptor transcript. RT-PCR revealed that the chicken PACAP receptor is mainly expressed in the brain and gonads. A smaller amount of the receptor mRNA was found in pituitary, adrenal gland, kidney, intestine, pancreas, lung, and heart tissue. In situ hybridization with specific antisense oligodeoxynucleotide probes showed a widespread distribution of PAC(1) receptor mRNA in the chicken brain, with the highest expression being found in the dorsal telencephalon, olfactory bulb, hypothalamus, optic tectum, and cerebellar cortex. These findings suggest that PACAP affect a variety of functions both in the brain and peripheral tissues of the chicken.

  15. Molecular cloning and functional analysis of an ethylene receptor gene from sugarcane (Saccharum spp.) by hormone and environmental stresses

    USDA-ARS?s Scientific Manuscript database

    Ethylene receptor (ethylene response sensor, ERS) is the primary component involving in the ethylene biosynthesis and ethylene signal transduction pathway. In the present study, a GZ-ERS gene encoding ERS was cloned from a sugarcane cv. YL17 (Saccharum spp.) using RT-PCR and ligation-mediated PCR wi...

  16. Molecular cloning of an orphan G-protein-coupled receptor that constitutively activates adenylate cyclase.

    PubMed Central

    Eggerickx, D; Denef, J F; Labbe, O; Hayashi, Y; Refetoff, S; Vassart, G; Parmentier, M; Libert, F

    1995-01-01

    A human gene encoding an orphan G-protein-coupled receptor named ACCA (adenylate cyclase constitutive activator) was isolated from a genomic library using as a probe a DNA fragment obtained by low-stringency PCR. Human ACCA (hACCA) is a protein of 330 amino acids that exhibits all the structural hallmarks of the main family of G-protein-coupled receptors. Expression of hACCA resulted in a dramatic stimulation of adenylate cyclase, similar in amplitude to that obtained with other Gs-coupled receptors fully activated by their respective ligands. This stimulation was obtained in a large variety of stable cell lines derived from various organs, and originating from different mammalian species. hACCA was found to be the human homologue of a recently reported mouse orphan receptor (GPCR21). The mouse ACCA (mACCA) was therefore recloned by PCR, and expression of mACCA in Cos-7 cells demonstrated that the mouse receptor behaved similarly as a constitutive activator of adenylate cyclase. It is not known presently whether the stimulation of adenylate cyclase is the result of a true constitutive activity of the receptor or, alternatively, is the consequence of a permanent stimulation by a ubiquitous ligand. The tissue distribution of mACCA was determined by RNase protection assay. Abundant transcripts were found in the brain, whereas lower amounts were detected in testis, ovary and eye. Various hypotheses concerning the constitutive activity of ACCA and their potential biological significance are discussed. Images Figure 4 Figure 5 PMID:7639700

  17. Guide to molecular cloning techniques

    SciTech Connect

    Berger, S.L.; Kimmel, A.R.

    1987-01-01

    This book includes the following selections: requirements for a molecular biology laboratory; general methods for isolating and characterizing nucleic acids; enzymatic techniques and recombinant DNA technology; restriction enzymes; growth and maintenance of bacteria; genetic cloning, preparation and characterization of RNA; preparation of cDNA and the generation of cDNA libraries; selections of clones from libraries; and identification and characterization of specific clones.

  18. Molecular Cloning and Characterization of Four Genes Encoding Ethylene Receptors Associated with Pineapple (Ananas comosus L.) Flowering.

    PubMed

    Li, Yun-He; Wu, Qing-Song; Huang, Xia; Liu, Sheng-Hui; Zhang, Hong-Na; Zhang, Zhi; Sun, Guang-Ming

    2016-01-01

    Exogenous ethylene, or ethephon, has been widely used to induce pineapple flowering, but the molecular mechanism behind ethephon induction is still unclear. In this study, we cloned four genes encoding ethylene receptors (designated AcERS1a, AcERS1b, AcETR2a, and AcETR2b). The 5' flanking sequences of these four genes were also cloned by self-formed adaptor PCR and SiteFinding-PCR, and a group of putative cis-acting elements was identified. Phylogenetic tree analysis indicated that AcERS1a, AcERS1b, AcETR2a, and AcETR2b belonged to the plant ERS1s and ETR2/EIN4-like groups. Quantitative real-time PCR showed that AcETR2a and AcETR2b (subfamily 2) were more sensitive to ethylene treatment compared with AcERS1a and AcERS1b (subfamily 1). The relative expression of AcERS1b, AcETR2a, and AcETR2b was significantly increased during the earlier period of pineapple inflorescence formation, especially at 1-9 days after ethylene treatment (DAET), whereas AcERS1a expression changed less than these three genes. In situ hybridization results showed that bract primordia (BP) and flower primordia (FP) appeared at 9 and 21 DAET, respectively, and flowers were formed at 37 DAET. AcERS1a, AcERS1b, AcETR2a, and AcETR2b were mainly expressed in the shoot apex at 1-4 DAET; thereafter, with the appearance of BP and FP, higher expression of these genes was found in these new structures. Finally, at 37 DAET, the expression of these genes was mainly focused in the flower but was also low in other structures. These findings indicate that these four ethylene receptor genes, especially AcERS1b, AcETR2a, and AcETR2b, play important roles during pineapple flowering induced by exogenous ethephon.

  19. Molecular Cloning and Characterization of Four Genes Encoding Ethylene Receptors Associated with Pineapple (Ananas comosus L.) Flowering

    PubMed Central

    Li, Yun-He; Wu, Qing-Song; Huang, Xia; Liu, Sheng-Hui; Zhang, Hong-Na; Zhang, Zhi; Sun, Guang-Ming

    2016-01-01

    Exogenous ethylene, or ethephon, has been widely used to induce pineapple flowering, but the molecular mechanism behind ethephon induction is still unclear. In this study, we cloned four genes encoding ethylene receptors (designated AcERS1a, AcERS1b, AcETR2a, and AcETR2b). The 5′ flanking sequences of these four genes were also cloned by self-formed adaptor PCR and SiteFinding-PCR, and a group of putative cis-acting elements was identified. Phylogenetic tree analysis indicated that AcERS1a, AcERS1b, AcETR2a, and AcETR2b belonged to the plant ERS1s and ETR2/EIN4-like groups. Quantitative real-time PCR showed that AcETR2a and AcETR2b (subfamily 2) were more sensitive to ethylene treatment compared with AcERS1a and AcERS1b (subfamily 1). The relative expression of AcERS1b, AcETR2a, and AcETR2b was significantly increased during the earlier period of pineapple inflorescence formation, especially at 1–9 days after ethylene treatment (DAET), whereas AcERS1a expression changed less than these three genes. In situ hybridization results showed that bract primordia (BP) and flower primordia (FP) appeared at 9 and 21 DAET, respectively, and flowers were formed at 37 DAET. AcERS1a, AcERS1b, AcETR2a, and AcETR2b were mainly expressed in the shoot apex at 1–4 DAET; thereafter, with the appearance of BP and FP, higher expression of these genes was found in these new structures. Finally, at 37 DAET, the expression of these genes was mainly focused in the flower but was also low in other structures. These findings indicate that these four ethylene receptor genes, especially AcERS1b, AcETR2a, and AcETR2b, play important roles during pineapple flowering induced by exogenous ethephon. PMID:27252725

  20. Molecular Cloning, Characterization, and Evolutionary Analysis of Estrogen Receptors from Phylogenetically Ancient Fish

    PubMed Central

    Katsu, Yoshinao; Kohno, Satomi; Hyodo, Susumu; Ijiri, Shigeho; Adachi, Shinji; Hara, Akihiko; Guillette, Louis J.; Iguchi, Taisen

    2008-01-01

    Estrogens are necessary for ovarian differentiation during a critical developmental stage in many vertebrates, and they promote the growth and differentiation of the adult female reproductive system. To understand the evolution of vertebrate estrogen receptors (ESRs) and to evaluate estrogen receptor-ligand interactions in phylogenetically ancient fish, we used PCR techniques to isolate the cDNA encoding ESRs from lungfish, sturgeon, and gar. Sequence analyses indicate that these fishes have two ESRs, ESR1 (ERα) and ESR2 (ERβ), as previously reported for other vertebrate species, but a second type of ESR2 (ERβ2) was not found as has been reported in a number of teleost fishes. Phylogenetic analysis of the ESR sequences indicated that the lungfish ESRs are classified to the tetrapod ESR group, not with the teleost fish ESRs as are the ESRs from gar and sturgeon. Using transient transfection assays of mammalian cells, ESR proteins from these three ancient fishes displayed estrogen-dependent activation of transcription from an estrogen-responsive-element containing promoter. We also examined the estrogenic potential of o,p′-dichloro-diphenyl-trichloroethane (o,p′-DDT) and p,p′-DDT as well as one of its common metabolites, p,p′-dichloro-diphenyl-ethylene (p,p′-DDE) on the ESRs from these fishes. Lungfish ESR1 was less sensitive to DDT/DDE than the ESR1 from the other two fishes. The response of lungfish ESR1 to these pesticides is similar to the pattern obtained from salamander ESR1. These data provide a basic tool allowing future studies examining the receptor-ligand interactions and endocrine-disrupting mechanisms in three species of phylogenetically ancient fish and also expands our knowledge of ESR evolution. PMID:18635653

  1. Molecular cloning and characterization of olive flounder (Paralichthys olivaceus) peroxisome proliferator-activated receptor gamma.

    PubMed

    Cho, Hyun Kook; Kong, Hee Jeong; Nam, Bo-Hye; Kim, Woo-Jin; Noh, Jae-Koo; Lee, Jeong-Ho; Kim, Young-Ok; Cheong, JaeHun

    2009-09-15

    Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that play key roles in lipid and energy homeostasis. Olive flounder (Paralichthys olivaceus) PPARgamma cDNA (olPPARgamma) was isolated by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The full-length cDNA is 1667-bp long and encodes a polypeptide with 532 amino acids containing a C4-type zinc finger and a ligand-binding domain. Quantitative RT-PCR revealed that olPPARgamma transcription was detected from 7days post-hatching, and its expression was increased under a starved condition. Overexpression of olPPARgamma stimulated PPAR response element (PPRE) activity, and treatment with rosiglitazone, a PPARgamma agonist, augmented olPPARgamma-stimulated PPRE activity in HINAE olive flounder cells. Cotransfection of olPPARgamma and olRXRbeta, in the absence or presence of rosiglitazone and ciglitazone, produced a synergistic effect on PPRE transactivation in 3T3L1 adipocytes. Moreover, olPPARgamma, in the presence or absence of rosiglitazone, regulated the expression of lipid synthesis- and adipogenesis-related proteins in NIH3T3 and 3T3L1 cells. Taken together, these results suggest that olPPARgamma is functionally and evolutionarily conserved in olive flounder and mammals.

  2. Molecular cloning and tissue distribution of a short form chicken leptin receptor mRNA.

    PubMed

    Liu, Xiaojun; Dunn, I C; Sharp, P J; Boswell, T

    2007-04-01

    In mammals, alternative splicing of the leptin receptor (LEPR) produces several C-terminal truncated isoforms that are believed to play a role in the transport, cellular internalisation and degradation of the hormone leptin. The chicken leptin receptor (chLEPR) is similar to its mammalian counterparts in terms of its intron/exon structure and conserved motifs. However, it is unknown whether the chLEPR also undergoes alternative splicing. To test this, structural analysis of intron 19 of the chLEPR, equivalent to the intron in which alternative splicing occurs in mammals, was combined with 3'-rapid amplification of cDNA ends (3'-RACE) to search for chLEPR splice variants. A 44-amino acid alternative exon 20 was identified that is spliced to generate a short isoform of the chLEPR (chLEPR-SF). Comparative sequence analysis of intron 19 identified two regions that are highly conserved between the chicken and mammals, indicating their possible importance as intronic elements in the regulation of alternative splicing of the LEPR in vertebrates. Tissue expression of the chLEPR-SF was lower and more restricted than that of the chLEPR long isoform. Collectively these data demonstrate that the chLEPR is alternatively spliced to produce at least one short isoform, as is the case in mammals.

  3. Molecular cloning and tissue-specific expression of Toll-like receptor 5 gene from turkeys.

    PubMed

    Gopinath, V P; Biswas, Moanaro; Raj, Gopal Dhinakar; Raja, A; Kumanan, A K; Elankumaran, Subbiah

    2011-09-01

    Toll-like receptors (TLRs), a family of transmembrane and cytosolic proteins, detect microbial patterns, initiating innate immune responses in various organisms. Although they are abundant, genetic characterization and functional differences of TLRs in economically important avian species such as chickens and turkeys have not been investigated in detail. In this study, the putative TLR5 coding region from turkey genome was sequenced, and its homology to other vertebrate species was analyzed. Secondary structure analysis revealed protein motifs typical of the chicken TLR5 protein structure, with 97% amino acid identity between them. mRNA expression profiling in adult turkeys revealed abundant TLR5 expression in a broad range of tissues. Stimulation with the TLR5 ligand flagellin resulted in the production of the inflammatory mediators interleukin (IL)-1beta, IL-6, and nitric oxide in peripheral blood mononuclear cells. To our knowledge, this is the first complete turkey TLR5 coding DNA sequence reported in sequence databases.

  4. Molecular cloning of mXCR1, the murine SCM-1/lymphotactin receptor.

    PubMed

    Yoshida, T; Izawa, D; Nakayama, T; Nakahara, K; Kakizaki, M; Imai, T; Suzuki, R; Miyasaka, M; Yoshie, O

    1999-09-10

    Single C motif-1 (SCM-1)/lymphotactin is a C-type member of the chemokine superfamily. Previously, we identified its specific receptor XCR1. Here we isolated the murine homologue of XCR1 (mXCR1). To demonstrate its biological activity, we produced recombinant mouse SCM-1 by the baculovirus expression system. B300-19 murine pre-B cells expressing mXCR1 responded to mSCM-1 in chemotactic and calcium-mobilization assays. mXCR1 mRNA was weakly expressed in spleen and lung of normal C57BL/6 mice. In spleen, CD8+ cells and NK1.1+ cells were found to express mXCR1. Identification of mXCR1 will now allow us to study the role of this unique cytokine system in the mouse models of inflammation and immunity.

  5. Molecular Cloning of Adenosinediphosphoribosyl Transferase.

    DTIC Science & Technology

    1987-09-08

    AD-RIB5 458 NOLECULNA CLONING OF AOENOSINEDXPNOSPHORIBOSyL 1/1 TRNSFERASEMU CAILIFORNIA UNIV SRN FRANCISCO E KUN US SEP 8? WFOSR-TR-87-0982 SWFOSR-B5...ACCESSION NO.D,. 03261102F 2312 A~5 11. TITLE (include Securqt Classification) 0 Molecular Cloning of Adenosinediphosphoribosyl Transferase 12. PERSONAL...I’:- AFOSR.Tlt. 8 7 - 0 9 8,2 0IL * pi AFOSR- 85 -0377 PROGRESS REPORT Molecular Cloning of Adenosinediphosphoribosyl Transferase 5." Period of

  6. Molecular cloning, expression and potential functions of the human proteinase-activated receptor-2.

    PubMed Central

    Bohm, S K; Kong, W; Bromme, D; Smeekens, S P; Anderson, D C; Connolly, A; Kahn, M; Nelken, N A; Coughlin, S R; Payan, D G; Bunnett, N W

    1996-01-01

    We used PCR to amplify proteinase activated receptor-2 (PAR-2) from human kidney cDNA. The open reading frame comprised 1191 bp and encoded a protein of 397 residues with 83% identity with mouse PAR-2. In KNRK cells (a line of kirsten murine sarcoma virus-transformed rat kidney epithelial cells) transfected with this cDNA, trypsin and activating peptide (AP) corresponding to the tethered ligand exposed by trypsin cleavage (SLIGKV-NH2) induced a prompt increase in cytosolic calcium ion concentration ([Ca2+]i). Human PAR-2 (hPAR-2) resided both on the plasma membrane and in the Golgi apparatus. hPAR-2 mRNA was highly expressed in human pancreas, kidney, colon, liver and small intestine, and by A549 lung and SW480 colon adenocarcinoma cells. Hybridization in situ revealed high expression in intestinal epithelial cells throughout the gut. Trypsin and AP stimulated an increase in [Ca2+]i in a rat intestinal epithelial cell line (hBRIE 380) and stimulated amylase secretion in isolated pancreatic acini. In A549 cells, which also responded to trypsin and AP with mobilization of cytosolic Ca2+, AP inhibited colony formation. Thus PAR-2 may serve as a trypsin sensor in the gut. Its expression by cells and tissues not normally exposed to pancreatic trypsin suggests that other proteases could serve as physiological activators. PMID:8615752

  7. Molecular cloning and expression analysis of the canine chemokine receptor CCR9.

    PubMed

    Maeda, Shingo; Ohno, Koichi; Tsukamoto, Atsushi; Nakashima, Ko; Fukushima, Kenjiro; Goto-Koshino, Yuko; Fujino, Yasuhito; Tsujimoto, Hajime

    2012-01-15

    The chemokine receptor CCR9, which interacts with the thymus-expressed chemokine TECK/CCL25, contributes to the localization of lymphocytes to the small intestine, and is implicated in the development of human inflammatory bowel disease (IBD); however, their role in canine IBD is unknown. The objective of this study was to isolate cDNA encoding CCR9 and to investigate CCR9 expression in normal canine tissues and lymphoid cell lines. The complete open reading frame contained 1104 bp, encoding 367 amino acids, with 85% and 81% identity to human and mouse homologs, respectively. CCR9 mRNA was detected in all tissues investigated with the highest expression level in the small intestine. CCR9 mRNA was also expressed in GL-1, a canine B cell leukemia cell line, but not in CLBL-1, a canine B cell lymphoma cell line. Immunoblot and flow cytometry analyses of these cell lines using an anti-human CCR9 monoclonal antibody revealed that CCR9 protein expression was detected only in GL-1, indicating the cross-reactivity of the antibody. Using the antibody, flow cytometry showed that the proportions of CCR9(+) cells were small (mean, 4.88%; SD, 2.15%) in the normal canine PBMCs. This study will be useful in understanding canine intestinal immunity and the immunopathogenesis of canine IBD. Copyright © 2011 Elsevier B.V. All rights reserved.

  8. The molecular cloning and characterisation of cDNA coding for the alpha subunit of the acetylcholine receptor.

    PubMed Central

    Sumikawa, K; Houghton, M; Smith, J C; Bell, L; Richards, B M; Barnard, E A

    1982-01-01

    A rare cDNA coding for most of the alpha subunit of the Torpedo nicotinic acetylcholine receptor has been cloned into bacteria. The use of a mismatched oligonucleotide primer of reverse transcriptase facilitated the design of an efficient, specific probe for recombinant bacteria. DNA sequence analysis has enabled the elucidation of a large part of the polypeptide primary sequence which is discussed in relation to its acetylcholine binding activity and the location of receptor within the plasma membrane. When used as a radioactive probe, the cloned cDNA binds specifically to a single Torpedo mRNA species of about 2350 nucleotides in length but fails to show significant cross-hybridisation with alpha subunit mRNA extracted from cat muscle. Images PMID:6183641

  9. Molecular cloning and tissue distribution of cholecystokinin-1 receptor (CCK-1R) in yellowtail Seriola quinqueradiata and its response to feeding and in vitro CCK treatment.

    PubMed

    Furutani, Takahiro; Masumoto, Toshiro; Fukada, Haruhisa

    2013-06-01

    In vertebrates, the peptide cholecystokinin (CCK) is one of the most important neuroregulatory digestive hormones. CCK acts via CCK receptors that are classified into two subtypes, CCK-1 receptor (CCK-1R; formally CCK-A) and CCK-2 receptor (formally CCK-B). In particular, the CCK-1R is involved in digestion and is regulated by CCK. However, very little information is known about CCK-1R in fish. Therefore, we performed molecular cloning of CCK-1R cDNA from the digestive tract of yellowtail Seriola quinqueradiata. Phylogenetic tree analysis showed a high sequence identity between the cloned yellowtail CCK receptor cDNA and CCK-1R, which belongs to the CCK-1R cluster. Furthermore, the expression of yellowtail CCK receptor mRNA was observed in gallbladder, pyloric caeca, and intestines, similarly to CCK-1R mRNA expression in mammals, suggesting that the cloned cDNA is of CCK-1R from yellowtail. In in vivo experiments, the CCK-1R mRNA levels increased in the gallbladder and pyloric caeca after feeding, whereas in vitro, mRNA levels of CCK-1R and digestive enzymes in cultured pyloric caeca increased by the addition of CCK. These results suggest that CCK-1R plays an important role in digestion stimulated by CCK in yellowtail.

  10. Molecular cloning and characterization of canine fractalkine and its receptor CX3CR1.

    PubMed

    Maeda, Shingo; Ohno, Koichi; Nakashima, Ko; Fukushima, Kenjiro; Tsukamoto, Atsushi; Suzuki, Hiyoshi; Fujiwara, Aki; Goto-Koshino, Yuko; Fujino, Yasuhito; Tsujimoto, Hajime

    2012-01-15

    Fractalkine, also known as CX(3)CL1, is a unique chemokine that mediates inflammatory responses and is involved in the pathogenesis of several inflammatory disorders, including inflammatory bowel disease (IBD) in humans. In this study, we isolated cDNAs encoding canine fractalkine and its receptor CX(3)CR1, and assessed the biological activity of these molecules. The deduced amino acid sequence of the canine fractalkine cDNA showed 66% and 57% identity to human and mouse homologs, respectively. The N-terminal chemokine domain of the canine fractalkine showed 68% and 65% identity to human and mouse counterparts, respectively. The canine CX(3)CR1 amino acid sequence showed close homology to its human (83% identity) and mouse (81% identity) counterparts. Fractalkine and CX(3)CR1 mRNA were detected in all tissues in this study. Relatively higher expression levels of fractalkine mRNA were observed in the brain, medulla spinalis, small intestine, and mesenteric lymph nodes (MLNs), whereas higher expression levels of CX(3)CR1 mRNA were observed in the medulla spinalis, brain, liver, small intestine, and MLNs. The cross-reactivities of anti-human fractalkine antibody and anti-rat CX(3)CR1 antibody to canine proteins were confirmed using recombinant canine fractalkine and a cell line overexpressing canine CX(3)CR1, respectively. A transwell chemotaxis assay showed that the recombinant canine fractalkine induced migration in canine lymphoid cells expressing CX(3)CR1. The present study will be useful in understanding the canine immune system and the immunopathogenesis of canine inflammatory diseases. Copyright © 2011 Elsevier B.V. All rights reserved.

  11. Molecular cloning of an anuran V(2) type [Arg(8)] vasotocin receptor and mesotocin receptor: functional characterization and tissue expression in the Japanese tree frog (Hyla japonica).

    PubMed

    Kohno, Satomi; Kamishima, Yoshihisa; Iguchi, Taisen

    2003-07-01

    In most amphibians, [Arg(8)] vasotocin (VT) has an antidiuretic effect that is coupled to the activation of adenylate cyclase. In contrast, mesotocin (MT) has a diuretic effect and acts via the inositol phosphate/calcium signaling pathway in amphibians. To further clarify the mechanisms of VT and MT activation, we report the molecular cloning of a VT receptor (VTR) and a MT receptor (MTR) from the Japanese tree frog, Hyla japonica. Tree frog VTR or MTR cDNA encoded 363 or 389 amino acids, and their amino acid sequences revealed close similarity to the mammalian vasopressin V(2) (51-52% identity) or toad MT (94% identity) receptors, respectively. Using CHO-K1 cells transfected with tree frog VTR, we observed elevated concentrations of intracellular cAMP following exposure of the cells to VT or other neurohypophysial hormones, whereas the cells transfected with MTR did not exhibit altered cAMP concentrations. The cells transfected with VTR exhibited the following efficiency for cAMP accumulation: VT = hydrin 1 > or = vasopressin > or = hydrin 2 > MT = oxytocin > isotocin. VTR or MTR mRNA exhibits a single 2.2- or 5.5-kb transcription band, respectively, and both are expressed in various tissues. VTR mRNA is clearly expressed in brain, heart, kidney, pelvic patch of skin, and urinary bladder, whereas brain, fat body, heart, kidney, and urinary bladder express MTR mRNA. Specifically, VTR mRNA in the pelvic patch or MTR mRNA in the dorsal skin is present at elevated levels in the skin. Characteristic distribution of VTR and MTR on osmoregulating organs indicates the ligands for these receptors would mediate a variety of functions. Further, the distribution of VTR in the skin would make the regional difference on cutaneous water absorption in response to VT in the Japanese tree frog.

  12. Molecular Mechanism of Dioxin Action: Molecular Cloning of the Ah Receptor Using a DNA Recognition Site Probe

    DTIC Science & Technology

    1992-01-13

    analysis of AhR binding to the DRE (see attached manuscript an the following brief description of these results) and have bequn the library screening . Although...relatively rapidly as to whether they represent AhR clones or not. As mentioned above, we have only recently begun the library screening . We have obtained a...DNA oligonucleotides, identify the DRE oligonucleotide with the highest binding affinity, optimize the screening protocol and begin the actual library

  13. Molecular cloning, characterization and expression analysis of TGF-β and receptor genes in the woodchuck model.

    PubMed

    Wang, Lu; Wang, Junzhong; Liu, Yana; Wang, Baoju; Yang, Shangqing; Yu, Qing; Roggendorf, Michael; Lu, Mengji; Liu, Jia; Yang, Dongliang

    2016-12-20

    Transforming growth factor beta (TGF-β) is an important cytokine with pleiotropic regulatory functions in the immune system and in the responses against viral infections. TGF-β acts on a variety of immune cells through the cell surface TGF-β receptor (University of Duisburg-EssenTGFBR). The woodchuck has been used as a biomedical model for studies of obesity and energy balance, endocrine and metabolic function, cardiovascular, cerebrovascular and neoplastic disease. Woodchucks infected with woodchuck hepatitis virus (WHV) represent an informative animal model to study hepatitis B virus (HBV) infection. In this study, the cDNA sequences of woodchuck TGF-β1, TGF-β2, TGFBR1 and TGFBR2 were cloned, sequenced and characterized. The full-length TGFBR1 cDNA sequence consisted of 1305bp coding sequence (CDS) that encoded 434 amino acids with a molecular weight of 48.9kDa. The phylogenetic tree analysis revealed that the woodchuck TGF-β family genes had a closer genetic relationship with Ictidomys tridecemlineatus. One antibody with cross-reactivity to woodchuck TGFBR1 was identified by flow cytometry. Moreover, the expression of these genes were analyzed at the transcriptional level. The quantitative PCR analysis showed that the TGF-β family transcripts were constitutively expressed in many tissues tested. Altered expression levels of the TGF-β family transcripts in the liver of WHV infected woodchucks were observed. These results serve as a foundation for further insight into the role of the TGF-β family in viral hepatitis in woodchuck model. Our work also possesses the potential value for characterizing the TGF-β family in other related diseases, such as obesity-related diseases, metabolic disorder, cardiovascular disease and cancer.

  14. Molecular cloning of canine protease-activated receptor-2 and its expression in normal dog tissues and atopic skin lesions.

    PubMed

    Maeda, Shingo; Maeda, Sadatoshi; Shibata, Sanae; Chimura, Naoki; Fukata, Tsuneo

    2009-05-01

    Protease-activated receptor-2 (PAR-2) belongs to a new G protein-coupled receptor subfamily and is activated by serine proteases. PAR-2 has been demonstrated to play an important role in inflammation and immune response in allergic diseases. In this study, we cloned canine PAR-2 cDNA from the canine kidney by RT-PCR. The canine PAR-2 clone contained a full-length open reading frame encoding 397 amino acids that had 84% and 80% homology with human and mouse homologues, respectively. Canine PAR-2 mRNA was detected in the heart, lung, liver, pancreas, stomach, small intestine, colon, kidney, adrenal gland, spleen, thyroid gland, thymus, skeletal muscle, lymph node, fat and skin of three healthy dogs. The expression pattern of PAR-2 mRNA in canine tissues was similar to that in humans. The expression level of PAR-2 mRNA in skin was not different between the atopic dermatitis (AD) and healthy dogs, suggesting that the level of PAR-2 mRNA transcription may not be associated with development of canine AD. The canine PAR-2 cDNA clone obtained in this study will be useful for further investigation of the immunopathogenesis of canine allergic diseases.

  15. Three additional inositol 1,4,5-trisphosphate receptors: molecular cloning and differential localization in brain and peripheral tissues.

    PubMed Central

    Ross, C A; Danoff, S K; Schell, M J; Snyder, S H; Ullrich, A

    1992-01-01

    Three inositol 1,4,5-trisphosphate receptor (IP3R) cDNAs, designated IP3R-II, -III, and -IV, were cloned from a mouse placenta cDNA library. All three display strong homology in membrane-spanning domains M7 and M8 to the originally cloned cerebellar IP3R-I, with divergences predominantly in cytoplasmic domains. Levels of mRNA for the three additional IP3Rs in general are substantially lower than for IP3R-I, though in the gastrointestinal tract the levels of IP3R-III may be comparable to IP3R-I. Cerebellar Purkinje cells express at least two and possibly three distinct IP3Rs, suggesting heterogeneity of IP3 action within a single cell. Images PMID:1374893

  16. Molecular characterization of two sea bass gonadotropin receptors: cDNA cloning, expression analysis, and functional activity.

    PubMed

    Rocha, Ana; Gómez, Ana; Zanuy, Silvia; Cerdá-Reverter, José Miguel; Carrillo, Manuel

    2007-06-30

    The follicle-stimulating hormone (FSH) and the luteinizing hormone (LH) play central roles in vertebrate reproduction. They act through their cognate receptors to stimulate testicular and ovarian functions. The present study reports the cloning and characterization of two sea bass (Dicentrarchus labrax) cDNAs encoding a FSH receptor (sbsFSHR) and a LH receptor (sbsLHR). The mature proteins display typical features of the glycoprotein hormone receptor family members, but the sbsFSHR also contains some remarkable differences when compared with other fish or mammalian FSHRs. Among them, a distinct extracellular N-terminal cysteine domain as regards to its length and cysteine number, and the presence of an extra leucine-rich repeat. Expression analysis revealed that the sbsFSHR is exclusively expressed in gonadal tissues, specifically in the follicular wall of previtellogenic and early-vitellogenic follicles. On the contrary, sbsLHR mRNA was found to be widely distributed in sea bass somatic tissues. When stably expressed in mammalian cell lines, sbsFSHR was specifically stimulated by bovine FSH, while sbsLHR was activated by both bovine LH and FSH. Nevertheless, specific stimulation of the sbsLHR was observed when recombinant sea bass gonadotropins were used. The isolation of a FSHR and a LHR in sea bass opens new ways to study gonadotropin action in this species.

  17. Molecular Cloning and Gene Expression Analysis of the Leptin Receptor in the Chinese Mitten Crab Eriocheir sinensis

    PubMed Central

    Jiang, Hui; Ren, Fei; Sun, Jiangling; He, Lin; Li, Weiwei; Xie, Yannan; Wang, Qun

    2010-01-01

    Background Leptin is an adipocyte-derived hormone with multiple functions that regulates energy homeostasis and reproductive functions. Increased knowledge of leptin receptor function will enhance our understanding of the physiological roles of leptin in animals. Methodology/Principal Findings In the present study, a full-length leptin receptor (lepr) cDNA, consisting of 1,353 nucleotides, was cloned from Chinese mitten crab (Eriocheir sinensis) using rapid amplification of cDNA ends (RACE) following the identification of a single expressed sequence tag (EST) clone in a cDNA library. The lepr cDNA consisted of a 22-nucleotide 5′-untranslated region (5′ UTR), a 402-nucleotide open reading frame (ORF) and a 929-nucleotide 3′ UTR. Multiple sequence alignments revealed that Chinese mitten crab lepr shared a conserved vacuolar protein sorting 55 (Vps55) domain with other species. Chinese mitten crab lepr expression was determined in various tissues and at three different reproductive stages using quantitative real-time RT-PCR. Lepr expression was highest in the intestine, thoracic ganglia, gonad, and accessory gonad, moderate in hepatopancreas and cranial ganglia, and low in muscle, gill, heart, haemocytes, and stomach. Furthermore, lepr expression was significantly higher in the intestine, gonad and thoracic ganglia in immature crabs relative to precocious and mature crabs. In contrast, lepr expression was significantly lower in the hepatopancreas of immature crabs relative to mature crabs. Conclusions/Significance We are the first to identify the lepr gene and to determine its gene expression patterns in various tissues and at three different reproductive stages in Chinese mitten crab. Taken together, our results suggest that lepr may be involved in the nutritional regulation of metabolism and reproduction in Chinese mitten crabs. PMID:20567508

  18. Molecular Cloning and Functional Characterization of Mannose Receptor in Zebra Fish (Danio rerio) during Infection with Aeromonas sobria

    PubMed Central

    Zheng, Feifei; Asim, Muhammad; Lan, Jiangfeng; Zhao, Lijuan; Wei, Shun; Chen, Nan; Liu, Xiaoling; Zhou, Yang; Lin, Li

    2015-01-01

    Mannose receptor (MR) is a member of pattern-recognition receptors (PRRs), which plays a significant role in immunity responses. Much work on MR has been done in mammals and birds while little in fish. In this report, a MR gene (designated as zfMR) was cloned from zebra fish (Danio rerio), which is an attractive model for the studies of animal diseases. The full-length cDNA of zfMR contains 6248 bp encoding a putative protein of 1428 amino acids. The predicted amino acid sequences showed that zfMR contained a cysteine-rich domain, a single fibronectin type II (FN II) domain, eight C-type lectin-like domains (CTLDs), a transmembrane domain and a short C-terminal cytoplasmic domain, sharing highly conserved structures with MRs from the other species. The MR mRNA could be detected in all examined tissues with highest level in kidney. The temporal expression patterns of MR, IL-1β and TNF-α mRNAs were analyzed in the liver, spleen, kidney and intestine post of infection with Aeromonas sobria. By immunohistochemistry assay, slight enhancement of MR protein was also observed in the spleen and intestine of the infected zebra fish. The established zebra fish-A. sobria infection model will be valuable for elucidating the role of MR in fish immune responses to infection. PMID:25988382

  19. Molecular Cloning and Functional Characterization of Mannose Receptor in Zebra Fish (Danio rerio) during Infection with Aeromonas sobria.

    PubMed

    Zheng, Feifei; Asim, Muhammad; Lan, Jiangfeng; Zhao, Lijuan; Wei, Shun; Chen, Nan; Liu, Xiaoling; Zhou, Yang; Lin, Li

    2015-05-15

    Mannose receptor (MR) is a member of pattern-recognition receptors (PRRs), which plays a significant role in immunity responses. Much work on MR has been done in mammals and birds while little in fish. In this report, a MR gene (designated as zfMR) was cloned from zebra fish (Danio rerio), which is an attractive model for the studies of animal diseases. The full-length cDNA of zfMR contains 6248 bp encoding a putative protein of 1428 amino acids. The predicted amino acid sequences showed that zfMR contained a cysteine-rich domain, a single fibronectin type II (FN II) domain, eight C-type lectin-like domains (CTLDs), a transmembrane domain and a short C-terminal cytoplasmic domain, sharing highly conserved structures with MRs from the other species. The MR mRNA could be detected in all examined tissues with highest level in kidney. The temporal expression patterns of MR, IL-1β and TNF-α mRNAs were analyzed in the liver, spleen, kidney and intestine post of infection with Aeromonas sobria. By immunohistochemistry assay, slight enhancement of MR protein was also observed in the spleen and intestine of the infected zebra fish. The established zebra fish-A. sobria infection model will be valuable for elucidating the role of MR in fish immune responses to infection.

  20. Molecular cloning, expression analysis and functional confirmation of ecdysone receptor and ultraspiracle from the Colorado potato beetle Leptinotarsa decemlineata.

    PubMed

    Ogura, Takehiko; Minakuchi, Chieka; Nakagawa, Yoshiaki; Smagghe, Guy; Miyagawa, Hisashi

    2005-08-01

    cDNA cloning of ecdysone receptor (EcR) and ultraspiracle (USP) of the coleopteran Colorado potato beetle Leptinotarsa decemlineata (LdEcR and LdUSP) was conducted. Amino-acid sequences of the proteins deduced from cDNA sequences showed striking homology to those of other insects, especially the coleopteran yellow mealworm Tenebrio molitor. Northern hybridization analysis showed a 12.4-kb message for the LdEcR A-isoform, a 10.5-kb message for the LdEcR B1-isoform and a 5.7-kb message for the LdUSP, in fat body, gut, integument, testis and ovaries. In developmental profile studies, expression of both the LdEcR and LdUSP transcript in integument changed dramatically. In gel mobility shift assays, in vitro translated LdEcR alone bound weakly to the pal1 ecdysone response element, although LdUSP alone did not, and this binding was dramatically enhanced by the addition of LdUSP. LdEcR/LdUSP complex also showed significant binding to an ecdysone agonist, ponasterone A (K(D) = 2.8 nm), while LdEcR alone showed only weak binding (K(D) = 73.4 nm), and LdUSP alone did not show any binding. The receptor-binding affinity of various ecdysone agonists to LdEcR/LdUSP was not correlated to their larvicidal activity to L. decemlineata. From these results, it was suggested that multiple factors including the receptor binding affinity are related to the determination of the larvicidal activity of nonsteroidal ecdysone agonists in L. decemlineata.

  1. Molecular cloning of the cDNA encoding the Epstein-Barr virus/C3d receptor (complement receptor type 2) of human B lymphocytes

    SciTech Connect

    Moore, M.D.; Cooper, N.R.; Tack, B.F.; Nemerow, G.R.

    1987-12-01

    Complementary DNA clones for complement receptor type 2 (CR2), the B-lymphocyte membrane protein that serves as the receptor for Epstein-Barr virus and the C3d complement fragment, were obtained by screening a lambda gt11 library generated from Raji B lymphoblastoid cell mRNA. A 4.2-kilobase (kb) clone, representing the entire coding sequence of the protein plus untranslated 5' and 3' nucleotide sequences was obtained and sequenced. The 4.2-kb clone, which contains all but about 500 base pairs (bp) of the 5' untranslated region of the full-length CR2 mRNA, consists of 63 bp of 5' untranslated nucleotide sequence followed successively by a start codon, a 20-amino acid hydrophobic signal peptide, 1005 amino acids having a repeating motif, a 28-amino acid probable transmembrane domain, and a 34-amino acid cytoplasmic tail. The deduced amino acid sequence of the protein indicates that the extracellular domain consists entirely of 16 tandemly arranged repeating elements, each 60-75 amino acids in length, which are identified by multiple conserved residues. This repeating motif also occurs in the C3b/C4b receptor, several complement proteins, and a number of noncomplement proteins. In CR2, the 16 repeats occur in four clusters of four repeats each. Approximately 10% of the deduced amino acid sequence, including the amino and carboxyl termini, was confirmed by amino acid sequencing of tryptic peptides derived from purified CR2. The nucleotide and derived amino acid sequence of CR2 and related studies are presented here.

  2. Molecular Cloning and Characterization of Estrogen, Androgen, and Progesterone Nuclear Receptors from a Freshwater Turtle (Pseudemys nelsoni)

    PubMed Central

    Katsu, Yoshinao; Ichikawa, Rie; Ikeuchi, Toshitaka; Kohno, Satomi; Guillette, Louis J.; Iguchi, Taisen

    2008-01-01

    Steroid hormones are essential for the normal function of many organ systems in vertebrates. Reproductive activity in females and males, such as the differentiation, growth, and maintenance of the reproductive system, requires signaling by the sex steroids. Although extensively studied in mammals and a few fish, amphibians, and bird species, the molecular mechanisms of sex steroid hormone (estrogens, androgens, and progestins) action are poorly understood in reptiles. Here we evaluate hormone receptor ligand interactions in a freshwater turtle, the red-belly slider (Pseudemys nelsoni), after the isolation of cDNAs encoding an estrogen receptor alpha (ERα), an androgen receptor (AR), and a progesterone receptor (PR). The full-length red-belly slider turtle (t)ERα, tAR, and tPR cDNAs were obtained using 5′ and 3′ rapid amplification cDNA ends. The deduced amino acid sequences showed high identity to the chicken orthologs (tERα, 90%; tAR, 71%; tPR, 71%). Using transient transfection assays of mammalian cells, tERα protein displayed estrogen-dependent activation of transcription from an estrogen-responsive element-containing promoter. The other receptor proteins, tAR and tPR, also displayed androgen- or progestin-dependent activation of transcription from androgen- and progestin-responsive murine mammary tumor virus promoters. We further examined the transactivation of tERα, tAR and tPR by ligands using a modified GAL4-transactivation system. We found that the GAL4-transactivation system was not suitable for the measurement of tAR and tPR transactivations. This is the first report of the full coding regions of a reptilian AR and PR and the examination of their transactivation by steroid hormones. PMID:17916628

  3. Identification of a novel class B scavenger receptor homologue in Portunus trituberculatus: Molecular cloning and microbial ligand binding.

    PubMed

    Yang, Ning; Zhang, Dan-Feng; Tao, Zhen; Li, Meng; Zhou, Su-Ming; Wang, Guo-Liang

    2016-11-01

    Class B scavenger receptors (SRBs), which are present in mammals and insects, have been implicated in a wide range of functions. Herein, a novel SRB homologue, PtSRB, was cloned from the swimming crab, Portunus trituberculatus. PtSRB has 538 amino acid residues, and it consists of two transmembrane regions, a large extracellular loop, and two intracellular tails. A phylogenetic analysis showed that PtSRB distinctly clustered with Marsupenaeus japonicas SRB-1 and most Drosophila SRB homologues, including Croquemort, Peste, NinaD, and Santa Maria, but was separate from the Drosophila sensory neuron membrane protein, MjSRB-2, and all vertebrate SRBs. Real-time quantitative PCR analyses showed that the PtSRB gene was constitutively expressed in all tissues tested. When PtSRB was overexpressed in human embryonic kidney 293T cells, it was distributed in the membrane and cytoplasm. Moreover, in vitro assays showed that rPtSRB bound microbial lipopolysaccharide with low affinity, and lipoteichoic acid and peptidoglycan with high affinity. PtSRB transcripts were down-regulated after challenge with Vibrio alginolyticus or white spot syndrome virus, but not after a Candida lusitaniae challenge. This study provides valuable data for understanding the role of SRBs in the host defense against microbial pathogens, which will facilitate future studies of host-pathogen interactions in crabs.

  4. Molecular cloning and functional analysis of the FSH receptor gene promoter from the volcano mouse (Neotomodon alstoni alstoni).

    PubMed

    Pérez-Solis, Marco Allán; Macías, Héctor; Acosta-MontesdeOca, Adriana; Pasapera, Ana María; Fierro, Reyna; Ulloa-Aguirre, Alfredo; Gutiérrez-Sagal, Rubén

    2010-02-01

    To gain further insights on the genetic divergence and the species-specific characteristics of the follicle-stimulating hormone receptor (FSHR), we cloned 946 bp of the 5'-flanking region of the FSHR gene from the volcano mouse (Neotomodon alstoni alstoni), and compared its features with those from other mammalian species. The sequence of neotomodon FSHR (nFSHR) gene from the translation initiation site to -946 is 74, 71, 64, and 59% homologous to rat, mouse (129/J), human, and sheep, respectively. The nFSHR 5'-flanking region exhibits new interesting putative cis-regulatory elements including those for the SRY transcription factor, which had not been previously related to the FSHR gene. The transcriptional regulation properties of nFSHR gene were studied in mouse Sertoli (MSC-1) and non-Sertoli (H441) cell lines, and compared with those obtained with similar 129/J constructs. All constructs tested were more active in H441 than in MSC-1 cells. The low transcription levels detected in MSC-1 cells probably reflect the recruitment of Sertoli cells-specific nuclear factors that repress transcription of the FSHR gene. In H441 cells, 129/J constructs were more active than their neotomodon counterparts, indicating important species-specific differences in their transcription pattern. Functional analysis of a series of progressive 5'-deletion mutants identified regions involved in positive and negative transcriptional regulation as well as the strongest minimal promoter spanning 260 bp upstream the translation initiation site. The identification of inhibitory nuclear transcription factors, which are apparently expressed in MSC-1 cells, may contribute to a better understanding of the transcriptional regulation of the FSHR gene.

  5. Molecular cloning, sequencing, and distribution of feline GnRH receptor (GnRHR) and resequencing of canine GnRHR.

    PubMed

    Samoylov, Alexandre M; Napier, India D; Morrison, Nancy E; Martin, Douglas R; Cox, Nancy R; Samoylova, Tatiana I

    2015-01-15

    GnRH receptors play vital roles in mammalian reproduction via regulation of gonadotropin secretion, which is essential for gametogenesis and production of gonadal steroids. GnRH receptors for more than 20 mammalian species have been sequenced, including human, mouse, and dog. This study reports the molecular cloning and sequencing of GnRH receptor (GnRHR) cDNA from the pituitary gland of the domestic cat, an important species in biomedical research. Feline GnRHR cDNA is composed of 981 nucleotides and encodes a 327 amino acid protein. Unlike the majority of mammalian species sequenced so far, but similar to canine GnRHR, feline GnRHR protein lacks asparagine in position three of the extracellular domain of the protein. At the amino acid level, feline GnRHR exhibits 95.1% identity with canine, 93.8% with human, and 88.9% with mouse GnRHR. Comparative sequence analysis of GnRHRs for multiple mammalian species led to resequencing of canine GnRHR, which differed from that previously published by a single base change that translates to a different amino acid in position 193. This single base change was confirmed in dogs of multiple breeds. Reverse transcriptase PCR analysis of GnRHR messenger RNA in different tissues from four normal cats indicated the presence of amplicons of varying lengths, including full-length as well as shortened GnRHR amplicons, pointing to the existence of truncated GnRHR transcripts in the domestic cat. This study is the first insight into molecular composition and expression of feline GnRHR and promotes better understanding of receptor organization, and distribution in various tissues of this species.

  6. Molecular cloning and evolutionary analysis of captive forest musk deer bitter taste receptor gene T2R16.

    PubMed

    Zhao, G J; Wu, N; Li, D Y; Zeng, D J; Chen, Q; Lu, L; Feng, X L; Zhang, C L; Zheng, C L; Jie, H

    2015-12-08

    Sensing bitter tastes is crucial for most animals because it can prevent them from ingesting harmful food. This process is mainly mediated by the bitter taste receptors (T2R) that are largely expressed in the taste buds. Previous studies have identified some T2R gene repertoires. Marked variation in repertoire size has been noted among species. However, research on T2Rs is still limited and the mechanisms underlying the evolution of vertebrate T2Rs remain poorly understood. In the present study, we analyzed the structure and features of the protein encoded by the forest musk deer (Moschus berezovskii) T2R16 and submitted the gene sequence to NCBI GenBank. The results showed that the full coding DNA sequence (CDS) of musk deer T2R16 (GenBank accession No. KP677279) was 906 bp, encoding 301 amino acids, which contained ATG start codon and TGA stop codon, with a calculated molecular weight of 35.03 kDa and an isoelectric point of 9.56. The T2R16 protein receptor had seven conserved transmembrane regions. Hydrophobicity analysis showed that most amino acid residues in T2R16 protein were hydrophobic, and the grand average of hydrophobicity (GRAVY) was 0.657. Phylogenetic analysis based on this gene revealed that forest musk deer had the closest association with sheep (Ovis aries), as compared to cow (Bos taurus), Tursiops truncatus, and other species, whereas it was genetically farthest from humans (Homo sapiens). We hope these results would complement the existing data on T2R16 and encourage further research in this respect.

  7. Molecular cloning, characterization and gene expression of murrel CXC chemokine receptor 3a against sodium nitrite acute toxicity and microbial pathogens.

    PubMed

    Bhatt, Prasanth; Chaurasia, Mukesh Kumar; Palanisamy, Rajesh; Kumaresan, Venkatesh; Arasu, Abirami; Sathyamoorthi, Akila; Gnanam, Annie J; Kasi, Marimuthu; Pasupuleti, Mukesh; Ramaswamy, Harikrishnan; Arockiaraj, Jesu

    2014-08-01

    CXCR3 is a CXC chemokine receptor 3 which binds to CXC ligand 4 (CXCL4), 9, 10 and 11. CXC chemokine receptor 3a (CXCR3a) is one of the splice variants of CXCR3. It plays crucial role in defense and other physiological processes. In this study, we report the molecular cloning, characterization and gene expression of CXCR3a from striped murrel Channa striatus (Cs). The full length CsCXCR3a cDNA sequence was obtained from the constructed cDNA library of striped murrel by cloning and sequencing using an internal sequencing primer. The full length sequence is 1425 nucleotides in length including an open reading frame of 1086 nucleotides which is encoded with a polypeptide of 361 amino acids (mol. wt. 40 kDa). CsCXCR3a domain analysis showed that the protein contains a G protein coupled receptor between 55 and 305 along with its family signature at 129-145. The transmembrane prediction analysis showed that CsCXCR3a protein contains 7 transmembrane helical regions at 34-65, 80-106, 113-146, 154-181, 208-242, 249-278 and 284-308. The 'DRY' motif from CsCXCR3a protein sequence at (140)Asp-(141)Arg-(142)Tyr which is responsible for G-protein binding is also highly conserved with CXCR3 from other species. Phylogenetic tree showed that the CXC chemokine receptors 3, 4, 5 and 6, each formed a separate clade, but 1 and 2 were clustered together, which may be due to the high similarity between these receptors. The predicted 3D structure revealed cysteine residues, which are responsible for 'CXC' motif at 116 and 198. The CsCXR3a transcript was found to be high in kidney, further its expression was up-regulated by sodium nitrite acute toxicity exposure, fungal, bacterial and poly I:C challenges. Overall, these results supported the active involvement of CsCXCR3a in inflammatory process of striped murrel during infection. However, further study is necessary to explore the striped murrel chemokine signaling pathways and their roles in defense system.

  8. Molecular cloning and partial characterization of an ovarian receptor with seven ligand binding repeats, an orthologue of low-density lipoprotein receptor, in the cutthroat trout (Oncorhynchus clarki).

    PubMed

    Luo, Wenshu; Ito, Yuta; Mizuta, Hiroko; Massaki, Kiyohiro; Hiramatsu, Naoshi; Todo, Takashi; Reading, Benjamin J; Sullivan, Craig V; Hara, Akihiko

    2013-10-01

    Teleost fish eggs contain a substantial yolk mass consisting of lipids and proteins that provides essential nutrients for embryonic and larval development. The polar lipid and protein components of the yolk are delivered to oocytes by circulating vitellogenins, however the source(s) of the neutral lipid remains unknown. We cloned a cDNA encoding an orthologue of low-density-lipoprotein receptor (LDLR) from the ovary of cutthroat trout, Oncorhynchus clarki (ct-Ldlr). Predominant expression of ct-ldlr mRNA was observed in the ovary and moderate expression was detected in intestine, gill and brain. The relative abundance of ct-ldlr transcripts was highest in early pre-vitellogenic ovaries and significantly decreased during vitellogenesis, followed by a slight increase during final maturation and in post-ovulatory follicles. In situ hybridization revealed an intense and evenly distributed localization of ct-ldlr transcripts in the ooplasm of pre-vitellogenic oocytes and these signals disappeared in vitellogenic follicles. Collectively, these results suggest that the Ldlr is involved in deposition of yolk lipids in cutthroat trout oocytes. The ct-ldlr transcripts also were detected in theca and granulosa cells, suggesting that this receptor may be involved in cholesterol uptake for ovarian steroidogenesis. This is the first report on partial characterization of an ldlr orthologue in any fish species.

  9. Toll-like receptor 22 of gilthead seabream, Sparus aurata: molecular cloning, expression profiles and post-transcriptional regulation.

    PubMed

    Muñoz, Iciar; Sepulcre, María Pilar; Meseguer, José; Mulero, Victoriano

    2014-05-01

    TLR22 is a fish-specific TLR that recognizes dsRNAs. In the present study, a TLR22 homologue gene from gilthead seabream (sbTLR22) was identified and characterized. The full coding sequence contained a single open-reading frame of 2895 nucleotides encoding a predicted protein of 964 amino acids in length. Its 3'-UTR was relatively long, 1380 nucleotides, and contained three AU-rich sequences frequently associated with mRNA instability. Functional studies showed that the sbTLR22 transcript had a short half-life, although the three AU-rich sequences in its 3'-UTR did not seem to be related with this fact. The sbTLR22 was highly expressed in the spleen, thymus and gills of healthy fish. After Vibrio anguillarum infection, the mRNA levels of sbTLR22 increased greatly in head kidney, blood and peritoneal exudate, but were only moderately induced in spleen and liver, suggesting the involvement of sbTLR22 in the immune response against bacterial infections. In addition, acidophilic granulocytes and macrophages, both considered professional phagocytes in seabream, displayed cell-type-specific sbTLR22 expression profiles when stimulated with different pathogen-associated molecular patterns (PAMPs). Although acidophilic granulocytes expressed sbTLR22, polyinosinic:polycytidylic acid (poly I:C) was unable to up-regulate the expression of this receptor. In contrast, poly I:C induced the expression of sbTLR22 in macrophages, in a process that was partially endosome-dependent. Taken together, our results suggest that sbTLR22 is involved in bacterial infection and might sense bacterial PAMPs. Copyright © 2013 Elsevier Ltd. All rights reserved.

  10. Receptors for parathyroid hormone and parathyroid hormone-related peptide: from molecular cloning to definition of diseases.

    PubMed

    Jüppner, H; Schipani, E

    1996-07-01

    The parathyroid hormone/parathyroid hormone-related peptide receptor belongs to a distinct family of G protein-coupled receptors, the members of which usually signal through at least two second messenger systems, adenylate cyclase and phospholipase C. The parathyroid hormone/ parathyroid hormone-related peptide receptor is most abundantly expressed in bone, kidney and growth-plate chondrocytes, and, at lower levels, in a variety of fetal and adult tissues. To search for human diseases that are caused by parathyroid hormone/parathyroid hormone-related peptide receptor defects, genomic DNA of patients with pseudohypoparathyroidism type Ib and of patients with Jansen's metaphyseal chondrodysplasia was screened for mutations in all coding exons of the receptor gene. Inactivating parathyroid hormone/parathyroid hormone-related peptide receptor mutations were excluded in patients with pseudohypoparathyroidism type Ib. However, a receptor mutation that causes agonist-independent, constitutive cAMP accumulation was identified in a patient with Jansen's metaphyseal chondrodysplasia, a rare form of short-limbed dwarfism associated with hypercalcemia despite normal or low concentrations of parathyroid hormone and parathyroid hormone-related peptide. These findings allow the conclusion to be drawn that parathyroid hormone/parathyroid hormone-related peptide receptors mediate the endocrine actions of parathyroid hormone, which are required for the control of calcium homeostasis and the autocrine-paracrine actions of parathyroid hormone-related peptide, which are required for normal growth-plate development.

  11. Molecular cloning and expression of prostaglandin F2α receptor isoforms during ovulation in the ovarian follicles of Xenopus laevis.

    PubMed

    Liu, Zhiming; Su, Xiurong; Li, Taiwu; Pan, Daodong; Sena, Johnny; Dhillon, Jasvinder

    2010-11-01

    Prostaglandins F2α levels increase during ovulatory period in Xenopus laevis in response to stimulation by gonadotropins and progesterone. PGF2α exerts its effects on ovulation through interaction with its receptor (FP) in ovaries. Little is known about the characteristics of the FP receptor and its regulation during the ovulatory period in non-mammalian species. In the present study, two isoforms of prostaglandin F receptor (FP A and B) cDNAs were isolated from Xenopus laevis ovarian tissues using reverse transcription-polymerase chain reaction (RT-PCR) followed by rapid amplification of cDNA ends (RACE). The cDNAs of FP A and FP B were sequenced. In Xenopus laevis ovary, FP A and B mRNA levels were up-regulated during gonadotropin- and progresterone-induced ovulation in vitro. The mRNA level of FP B was higher than that of FP A. Moreover, FP A and FP B mRNA levels were measured in various tissues including eye, liver, lungs, heart, muscle, ovary, and skin. Overall, FP B mRNA level was approximately 10- to 100-fold higher than that of FP A, except in the muscle and skin where FP A mRNA level was comparable to that of FP B. The results suggest that in Xenopus ovarian follicles FP receptors play an important role during gonadotropin- and progesterone-induced ovulation.

  12. Molecular Cloning and Pharmacological Characterization of Two Novel GnRH Receptors in the Lamprey (Petromyzon marinus)

    PubMed Central

    Joseph, Nerine T.; Aquilina-Beck, Allisan; MacDonald, Caryn; Decatur, Wayne A.; Hall, Jeffrey A.; Kavanaugh, Scott I.

    2012-01-01

    This paper reports the identification, expression, binding kinetics, and functional studies of two novel type III lamprey GnRH receptors (lGnRH-R-2 and lGnRH-R-3) in the sea lamprey, a basal vertebrate. These novel GnRH receptors share the structural features and amino acid motifs common to other known gnathostome GnRH receptors. The ligand specificity and activation of intracellular signaling studies showed ligands lGnRH-II and -III induced an inositol phosphate (IP) response at lGnRH-R-2 and lGnRH-R-3, whereas the ligand lGnRH-I did not stimulate an IP response. lGnRH-II was a more potent activator of lGnRH-R-3 than lGnRH-III. Stimulation of lGnRH-R-2 and lGnRH-R-3 testing all three lGnRH ligands did not elicit a cAMP response. lGnRH-R-2 has a higher binding affinity in response to lGnRH-III than lGnRH-II, whereas lGnRH-R-3 has a higher binding affinity in response to lGnRH-II than IGnRH-III. lGnRH-R-2 precursor transcript was detected in a wide variety of tissues including the pituitary whereas lGnRH-R-3 precursor transcript was not as widely expressed and primarily expressed in the brain and eye of male and female lampreys. From our phylogenetic analysis, we propose that lGnRH-R-1 evolved from a common ancestor of all vertebrate GnRH receptors and lGnRH-R-2 and lGnRH-R-3 likely occurred due to a gene duplication within the lamprey lineage. In summary, we propose from our findings of receptor subtypes in the sea lamprey that the evolutionary recruitment of specific pituitary GnRH receptor subtypes for particular physiological functions seen in later evolved vertebrates was an ancestral character that first arose in a basal vertebrate. PMID:22569788

  13. Molecular cloning and tissue distribution of peroxisome proliferator-activated receptor-alpha (PPARα) and gamma (PPARγ) in the pigeon (Columba livia domestica).

    PubMed

    Xie, P; Yuan, C; Wang, C; Zou, X-T; Po, Z; Tong, H-B; Zou, J-M

    2014-01-01

    1. Peroxisome proliferator-activated receptors (PPAR) are involved in lipid metabolism through transcriptional regulation of target gene expression. The objective of the current study was to clone and characterise the PPARα and PPARγ genes in pigeon. 2. The full-length of 1941-bp PPARα and 1653-bp PPARγ were cloned from pigeons. The two genes were predicted to encode 468 and 475 amino acids, respectively. Both proteins contained two C4-type zinc fingers, a nuclear hormone receptor DNA-binding region signature and a HOLI domain (ligand binding domain of hormone receptors), and had high identities with other corresponding avian genes. 3. Using quantitative real-time PCR, pigeon PPARα gene expression was shown to be high in kidney, liver, gizzard and duodenum whereas PPARγ was predominantly expressed in adipose tissue.

  14. Molecular cloning of a putative receptor protein kinase gene encoded at the self-incompatibility locus of Brassica oleracea

    SciTech Connect

    Stein, J.C.; Howlett, B.; Boyes, D.C.; Nasrallah, M.E.; Nasrallah, J.B. )

    1991-10-01

    Self-recognition between pollen and stigma during pollination in Brassica oleracea is genetically controlled by the multiallelic self-incompatibility locus (S). The authors describe the S receptor kinase (SRK) gene, a previously uncharacterized gene that residues at the S locus. The nucleotide sequences of genomic DNA and of cDNAs corresponding to SRK predict a putative transmembrane receptor having serine/threonine-specific protein kinase activity. Its extracellular domain exhibits striking homology to the secreted product of the S-locus genotypes are highly polymorphic and have apparently evolved in unison with genetically linked alleles of SLG. SRK directs the synthesis of several alternative transcripts, which potentially encode different protein products, and these transcripts were detected exclusively in reproductive organs. The identification of SRK may provide new perspectives into the signal transduction mechanism underlying pollen recognition.

  15. Molecular cloning and preliminary expression analysis of banded dogfish (Triakis scyllia) TNF decoy receptor 3 (TNFRSF6B).

    PubMed

    Inoue, Yuuki; Morinaga, Akihiro; Takizawa, Fumio; Saito, Tsubasa; Endo, Mariko; Haruta, Chiaki; Nakai, Takeshi; Moritomo, Tadaaki; Nakanishi, Teruyuki

    2008-03-01

    Decoy receptor 3 (DcR3), a member of TNF receptor superfamily, is a soluble receptor without death domain and cytoplasmic domain, and secreted by cells and binds with FasL, LIGHT and TL1A. The principal function of DcR3 is the inhibition of apoptosis by the binding cytotoxic ligands. Expression of DcR3 has been reported in a wide array of normal human tissues as well as tumors and tumor cell lines. Recently, DcR3 was reported to modulate a variety of immune responses in mammals. TNFR or DcR3 has been identified in some teleost fishes. However, DcR3 is not reported in cartilaginous fish which is the lowest vertebrate possessing the adaptive immune system. Here we identified DcR3 cDNA in shark (Trsc-DcR3) from an SSH library prepared from peripheral white blood cells stimulated with PMA. Four cysteine-rich domains (CRDs) in common with TNF receptor family members are present in the Trsc-DcR3 sequence. The deduced amino acid sequence of Trsc-DcR3 showed highest identity with the chicken (50.4%), followed by human (46.8%) and rainbow trout (36.5%) DcR3. In a phylogenetic tree of known TNFRSF sequences, the Trsc-DcR3 grouped with the chicken and human DcR3. Trsc-DcR3 mRNA was detected strongly in the gill, moderately in the brain, and weakly in the kidney, thymus and leydig. These data strongly suggest that the gene encoding Trsc-DcR3 in banded dogfish is a homolog of the human gene. mRNA expression of Trsc-DcR3 in the thymus and leydig suggests that DcR3 may act as a modulator in the immune system even at the phylogenetic level of cartilaginous fish.

  16. Toll-like receptor 22 in Labeo rohita: molecular cloning, characterization, 3D modeling, and expression analysis following ligands stimulation and bacterial infection.

    PubMed

    Samanta, Mrinal; Swain, Banikalyan; Basu, Madhubanti; Mahapatra, Girishbala; Sahoo, Bikash R; Paichha, Mahismita; Lenka, Saswati S; Jayasankar, Pallipuram

    2014-09-01

    Toll-like receptors (TLRs) are a class of innate immune receptors that sense pathogens or their molecular signatures and activate signaling cascades to induce a quick and non-specific immune response in the host. Among various types of TLRs, TLR22 is exclusively present in teleosts and amphibians and is expected to play the distinctive role in innate immunity. This report describes molecular cloning, three-dimensional (3D) modeling, and expression analysis of TLR22 in rohu (Labeo rohita), the most commercially important freshwater fish species in the Indian subcontinent. The open reading frame (ORF) of rohu TLR22 (LrTLR22) comprised of 2,838 nucleotides (nt), encoding 946 amino acid (aa) residues with the molecular mass of ∼ 107.6 kDa. The secondary structure of deduced LrTLR22 exhibited the presence of signal peptide (1-22 aa), 18 leucine-rich repeat (LRR) regions (79-736 aa), and TIR domain (792-935 aa). The 3D model of LrTLR22-LRR regions together elucidated the horse-shoe-shaped structure having parallel β-strands at the concave surface and few α-helices at the convex surface. The TIR domain structure revealed alternate presence of five α-helices and β-sheets. Phylogenetically, LrTLR22 was closely related to common carp and exhibited significant similarity (92.2 %) and identity (86.1 %) in their amino acids. In rohu, TLR22 was constitutively expressed in all embryonic developmental stages, and tissue-specific analysis illustrated its expression in all examined tissues, highest was in liver and lowest in brain. In vivo modulation of TLR22 gene expression was analyzed by quantitative real-time PCR (qRT-PCR) assay following stimulation with lipopolysaccharide (LPS), synthetic double stranded RNA (polyinosinic-polycytidylic acid), and bacterial (Aeromonas hydrophila) RNA. Among these ligands, bacterial RNA most significantly (p < 0.05) induced TLR22 gene expression in most of the tested tissues. In A. hydrophila infection, induction of TLR22 gene expression

  17. Molecular cloning, expression, and stress response of the estrogen-related receptor gene (AccERR) from Apis cerana cerana

    NASA Astrophysics Data System (ADS)

    Zhang, Weixing; Zhu, Ming; Zhang, Ge; Liu, Feng; Wang, Hongfang; Guo, Xingqi; Xu, Baohua

    2016-04-01

    Estrogen-related receptor (ERR), which belongs to the nuclear receptor superfamily, has been implicated in diverse physiological processes involving the estrogen signaling pathway. However, little information is available on ERR in Apis cerana cerana. In this report, we isolated the ERR gene and investigated its involvement in antioxidant defense. Quantitative real-time polymerase chain reaction (qPCR) revealed that the highest mRNA expression occurred in eggs during different developmental stages. The expression levels of AccERR were highest in the muscle, followed by the rectum. The predicted transcription factor binding sites in the promoter of AccERR suggested that AccERR potentially functions in early development and in environmental stress responses. The expression of AccERR was induced by cold (4 °C), heat (42 °C), ultraviolet light (UV), HgCl2, and various types of pesticides (phoxim, deltamethrin, triadimefon, and cyhalothrin). Western blot was used to measure the expression levels of AccERR protein. These data suggested that AccERR might play a vital role in abiotic stress responses.

  18. Molecular cloning and characterization of TNFSF14 (LIGHT) and its receptor TNFRSF14 (HVEM) in guinea pig (Cavia porcellus).

    PubMed

    Li, Chunlan; Chen, Shanshan; Song, Jinyun; Liu, Hongyan; Gu, Wei; Ai, Hongxin; Zhao, Bo; Zhang, Shuangquan

    2013-09-10

    LIGHT (lymphotoxin-related inducible ligand that competes with herpes simplex virus (HSV) glycoprotein D for herpesvirus entry mediator on T cells) is a member of the tumor necrosis factor (TNF) ligand superfamily, which plays important roles in inflammatory and immune responses. In the present study, the cDNAs of guinea pig (Cavia porcellus) LIGHT (designated as gpLIGHT) and its receptor herpes virus entry mediator (designated as gpHVEM) were amplified from spleen by reverse transcription polymerase chain reaction (RT-PCR). The ORFs of gpLIGHT and gpHVEM cover 726 and 861 bp, encoding predicted proteins with 241 and 286 aas, respectively. The three-dimensional (3D) structure, phylogenetic relationships, and characterization of both genes were also analyzed. We also generated a 3D model to verify interaction between the two proteins. Real-time quantitative PCR (qPCR) analysis revealed that both LIGHT and HVEM are constitutively expressed in guinea pig various tissues. A fusion protein SUMO (Small Ubiquitin-like Modifier)-gpsLIGHT (the soluble mature part of gpLIGHT) was efficiently expressed in Escherichia coli BL21 (DE3) and purified using metal chelate affinity chromatography (Ni-NTA). Laser scanning confocal microscopy (LSCM) showed that gpsLIGHT can bind its receptors on T cells. The LIGHT-HVEM signaling pathway plays an important role in the immune system, and our results might provide a platform for further research into the effects of LIGHT and HVEM.

  19. Molecular cloning, expression profile, polymorphism and the genetic effects of the dopamine D1 receptor gene on duck reproductive traits.

    PubMed

    Wang, Cui; Li, Shijun; Li, Chuang; Feng, Yanping; Peng, Xiuli; Gong, Yanzhang

    2012-09-01

    The dopamine D1 receptor (DRD1), a member of the dopamine receptor (DR) gene family, participates in the regulation of reproductive behaviors in birds. In this study, a 1,390 bp fragment covering the complete coding region (CDS) of duck DRD1 gene was obtained. The cDNA (GenBank: JQ346726) contains a 1,353 bp CDS and a 37 bp 3'- UTR including a TGA termination codon (nucleotides 1,354-1,356 bp). The duck DRD1 shares about 76-96 % nucleic acid identity and 82-98 % amino acid identity with their counterparts in other species. A phylogenetic tree based on amino acid sequences displays that duck DRD1 protein is closely related with those of chicken and zebra finch. The quantitative real-time PCR analysis indicates that the DRD1 mRNA is widely expressed in all examined tissues. Five single nucleotide polymorphisms (SNPs) (c.189A > T, c.507C > T, c.681C > T, c.765A > T, c.1044A > G) in the CDS of duck DRD1 gene were indentified, c.681C > T and c.765A > T were genotyped and analyzed in a two generations duck population by using of PCR-RFLP. Association analysis demonstrated that the c.681C > T genotypes were significantly associated with body weight at sexual maturity (when laying their first egg) (P < 0.01), egg production within 360 days (P < 0.05) and 420 days (P < 0.01); the c.765A > T genotypes were significantly associated with egg shape index and egg shell strength (P < 0.05). Those results suggest that the DRD1 gene may be a potential genetic marker to improve some reproductive traits in ducks.

  20. Ryanodine receptor genes of the rice stem borer, Chilo suppressalis: Molecular cloning, alternative splicing and expression profiling.

    PubMed

    Peng, Y C; Sheng, C W; Casida, John E; Zhao, C Q; Han, Z J

    2017-01-01

    The ryanodine receptor (RyR) of the calcium release channel is the main target of anthranilic and phthalic diamide insecticides which have high selective insecticidal activity relative to mammalian toxicity. In this study, the full-length cDNA of Chilo suppressalis RyR (CsRyR) was isolated and characterized. The CsRyR mRNA has an open reading frame (ORF) of 15,387bp nucleotides, which encodes 5128 amino acids with GenBank ID: KR088972. Comparison of protein sequences showed that CsRyR shared high identities with other insects of 77-96% and lower identity to mammals and nematodes with only 42-45%. One alternative splicing site (KENLG) unique to Lepidoptera was found and two exclusive exons of CsRyR (I /II) were revealed. Spatial and temporal expression of CsRyR mRNA was at the highest relative level in 3rd instar larvae and head (including brain and muscle), and at the lowest expression level in egg and fat body. The expression levels of whole body CsRyR mRNA were increased remarkably after injection of 4th instar larvae with chlorantraniliprole at 0.004 to 0.4μg/g. This structural and functional information on CsRyR provides the basis for further understanding the selective action of chlorantraniliprole and possibly other diamide insecticides.

  1. Molecular cloning and characterization of the human interleukin-11 receptor {alpha}-chain gene, IL11RA, located on chromosome 9p13

    SciTech Connect

    Van Leuven, F.; Stas, L.; Hilliker, C.

    1996-01-01

    The human gene coding for the interleukin-11 receptor (IL11RA) was cloned and its structure analyzed. The gene is composed of 13 exons comprising nearly 10 kb of DNA that was completely sequenced. The intron-exon boundaries were determined based on the mouse Etl2 and interleukin-11 receptor cDNAs that were recently cloned. The protein sequence predicted by the human gene was over 83% identical with its murine counterpart, with very strict conservation of functionally important domains and signatures. Fluorescence in situ hybridization showed the gene to be located on human chromosome 9p13, syntenic with the mouse etl2 gene on chromosome 4. The coding exons of the interleukin-11 gene were sequenced in a patient with the cartilage-hair hypoplasia syndrome, which has been linked to a gene on chromosome 9, but no functional mutations were detected. 26 refs., 4 figs., 2 tabs.

  2. Molecular cloning and functional analysis of a blue light receptor gene MdCRY2 from apple (Malus domestica).

    PubMed

    Li, Yuan-Yuan; Mao, Ke; Zhao, Cheng; Zhao, Xian-Yan; Zhang, Rui-Fen; Zhang, Hua-Lei; Shu, Huai-Rui; Hao, Yu-Jin

    2013-04-01

    MdCRY2 was isolated from apple fruit skin, and its function was analyzed in MdCRY2 transgenic Arabidopsis. The interaction between MdCRY2 and AtCOP1 was found by yeast two-hybrid and BiFC assays. Cryptochromes are blue/ultraviolet-A (UV-A) light receptors involved in regulating various aspects of plant growth and development. Investigations of the structure and functions of cryptochromes in plants have largely focused on Arabidopsis (Arabidopsis thaliana), tomato (Solanum lycopersicum), pea (Pisum sativum), and rice (Oryza sativa). However, no data on the function of CRY2 are available in woody plants. In this study, we isolated a cryptochrome gene, MdCRY2, from apple (Malus domestica). The deduced amino acid sequences of MdCRY2 contain the conserved N-terminal photolyase-related domain and the flavin adenine dinucleotide (FAD) binding domain, as well as the C-terminal DQXVP-acidic-STAES (DAS) domain. Relationship analysis indicates that MdCRY2 shows the highest similarity to the strawberry FvCRY protein. The expression of MdCRY2 is induced by blue/UV-A light, which represents a 48-h circadian rhythm. To investigate the function of MdCRY2, we overexpressed the MdCRY2 gene in a cry2 mutant and wild type (WT) Arabidopsis, assessed the phenotypes of the resulting transgenic plants, and found that MdCRY2 functions to regulate hypocotyl elongation, root growth, flower initiation, and anthocyanin accumulation. Furthermore, we examined the interaction between MdCRY2 and AtCOP1 using a yeast two-hybrid assay and a bimolecular fluorescence complementation assay. These data provide functional evidence for a role of blue/UV-A light-induced MdCRY2 in controlling photomorphogenesis in apple.

  3. Cloning, functional expression and characterization of a human olfactory receptor.

    PubMed

    Hatt, H; Gisselmann, G; Wetzel, C H

    1999-05-01

    The human olfactory system can recognize and discriminate a large number of different odorant molecules. The detection of chemically distinct odorants begins with the binding of an odorant ligand to a specific receptor protein on the olfactory neuron cell surface. To address the problem of olfactory perception at a molecular level, we have cloned, functionally expressed and characterized the first human olfactory receptor (OR 17-40). Application of a mixture of hundred different odorants elicited a transient increase in intracellular calcium at HEK 293-cells which were transfected with a plasmid containing the receptor encoding DNA and a membrane import sequence. By subdividing the odorant mixture in smaller groups we could identify a single component which represented the only effective substance: helional. Testing some structurally closely related molecules we found only one other compound which also could activate the receptor: heliotropyl acetone. All other compounds tested were completely ineffective. These findings represent the beginning of molecular understanding of odorant recognition in humans.

  4. Molecular cloning and pharmacological characterization of monkey MT1 and MT2 melatonin receptors showing high affinity for the agonist ramelteon.

    PubMed

    Nishiyama, Keiji; Shintani, Yasushi; Hirai, Keisuke; Yoshikubo, Shin-ichi

    2009-09-01

    Melatonin receptor agonists such as melatonin and ramelteon [(S)-N-[2-(1,6,7,8-tetrahydro-2H-indeno-[5,4-b]furan-8-yl)ethyl]-propionamide; TAK-375] have sleep-promoting effects in humans. In preclinical models, these effects are more similar to those observed in monkeys than in other species. However, in contrast to the human melatonin receptors, the pharmacological characteristics of the monkey melatonin receptors have yet to be elucidated. In this study, we cloned the cynomolgus monkey MT(1) and MT(2) melatonin receptors based on rhesus monkey genome sequences and then characterized the monkey melatonin receptors and compared their pharmacological properties with those of the human homologs. The overall amino acid sequences of the monkey MT(1) and MT(2) melatonin receptors showed high homology to the human MT(1) (95%) and MT(2) (96%) receptors, respectively. Saturation binding experiments with 2-[(125)I]iodomelatonin revealed that the dissociation constants (K(d)) for the monkey MT(1) and MT(2) melatonin receptors were 19.9 and 70.4 pM, respectively. In ligand competition assays using 2-[(125)I]iodomelatonin, ramelteon displayed approximately 3- to 7-fold higher affinities than melatonin for the recombinant monkey MT(1) and MT(2) melatonin receptors and monkey suprachiasmatic nucleus membranes. This higher affinity of ramelteon compared with melatonin has also been observed in human melatonin receptors. Furthermore, ramelteon inhibited pituitary adenylate cyclase-activating polypeptide-27-stimulated cAMP production with higher potency than melatonin. In conclusion, this information will help us to understand the pharmacological effects of melatonin receptor agonists in monkeys.

  5. Molecular cloning of the cDNA and chromosomal localization of the gene for a putative seven-transmembrane segment (7-TMS) receptor isolated from human spleen

    SciTech Connect

    Federsppiel, B.; Melhado, I.G.; Delaney, A.; Clark-Lewis, I. ); Duncan, A.M.V. ); Jirik, F.R. )

    1993-06-01

    A family of proinflammatory cytokines sharing several structural features has been described and includes, for example, interleukin-8, monocyte chemoattractant protein-1, and melanocyte growth stimulatory activity. Recently, the receptors for interleukin-8 have been isolated and found to belong to the seven-transmembrane domain class of G protein-coupled receptors. As other members of this cytokine family likely interact with similar receptors, the polymerase chain reaction was employed to isolate related receptors from human peripheral blood adherent cells. Degenerate oligonucleotide primers based on the rabbit interleukin-8 receptor sequence were used. The corresponding full-length cDNA was isolated from a human spleen cDNA library. The predicted protein sequence of this clone, designated pBE1.3, was 93% identical to that of a cDNA isolated from bovine locus coeruleus, which apparently encodes a neuropeptide Y receptor, and also shows similarity with the interleukin-8 receptor and the human cytomegalovirus US28 sequences. The gene, designated D2S201E, was localized to human chromosome 2q21. By Northern blotting, transcripts hybridizing to this cDNA were present in a variety of tissues and cells, including those of hemopoietic origin. 32 refs., 5 figs.

  6. The mouse insulin-like growth factor II/cation-independent mannose 6-phosphate (IGF-II/MPR) receptor gene: Molecular cloning and genomic organization

    SciTech Connect

    Szebenyi, G.; Rotwein, P. )

    1994-01-01

    The mammalian insulin-like growth factor III/cation-independent mannose 6-phosphate receptor (IGF-II/MPR) is a multifunctional protein that binds both IGF-II and ligands containing a mannose 6-phosphate recognition marker through distinct high-affinity sites. This receptor plays an integral part in lysosomal enzyme transport, has a potential role in growth factor maturation and clearance, and may mediate IGF-II-activated signal transduction through a G-protein-coupled mechanism. Recent studies have shown that production of IGF-II/MPR mRNA and protein begins in the mouse embryo soon after fertilization and have demonstrated that the receptor gene is on mouse chromosome 17 and is maternally imprinted. In this paper, the authors report the cloning and characterization of the mouse IGF-II/MPR gene. The gene is 93 kb long, is composed of 48 exons, and codes for a predicted protein of 2482 amino acids. The extracellular part of the receptor is encoded by exons 1-46, with each of 15 related repeating motifs being determined by parts of 3-5 exons. A single fibronectin type II-like element is found in exon 39. The transmembrane portion of the receptor also is encoded by exon 46, and the cytoplasmic region by exons 46-48. The positions of exon-intron splice junctions are conserved between several of the repeats in the IGF-II/MPR and the homologous extracellular region of the gene for the other known lysosomal sorting receptor, the cation-dependent mannose 6-phosphate receptor. The gene duplications that gave rise to the modern IGF-II/MPR probably occurred before the divergence of mammals, since there is more extensive protein sequence conservation between receptors from different species than between any pair of repeating motifs within a single receptor. 55 refs., 7 figs., 1 tab.

  7. Molecular cloning, expression and functional characterization of tumor necrosis factor (TNF) receptor-associated factor (TRAF)-interacting protein (TRIP) in grass carp, Ctenopharyngodon idella.

    PubMed

    Lu, R-H; Chang, Z-G; Sun, J; Yang, F; Nie, G-X; Ji, H

    2016-10-01

    TRIP (Tumor Necrosis Factor (TNF) Receptor-Associated Factor (TRAF)-Interacting Protein), a member of the TNF superfamily, plays a crucial role in the modulation of inflammation in vertebrates. However, no information about TRIP is available in teleosts. In this study, the full-length cDNA of TRIP, containing a 5'UTR of 112 bp, an ORF of 1359 bp, and a 3'UTR of 29 bp before the poly (A) tail, was cloned from grass carp, Ctenopharyngodon idella. The TRIP gene encoded a protein of 452 amino acids with an estimated molecular mass of 51.06 KD and a predicted theoretical isoelectric point (pI) of 9.11. Quantitative real-time PCR analysis revealed that TRIP mRNA was expressed in all the tissues examined in grass carp, with the highest expression in the kidney, followed by the intestine and thymus. However, lower levels of expression were also detected in fat, spleen, liver, gonad and heart. Subcellular localization and two-hybrid analysis revealed that TRIP was located in the nucleus and that it interacted with TRAF1 and TRAF2 in HEK293T cells. Furthermore, similar to TNF-α, IL-10 and TRIP mRNA expression was upregulated in the spleen of fish fed high-fat or high-carbohydrate diets, suggesting that TRIP might be associated with the response to excessive energy intake. The mRNA relative expression of TRIP was significantly reduced (P < 0.05) after hepatocyte of C. idella was treated with 2 μg/mL lipopolysaccharide (LPS) for 4 h, while the expression levels of inflammatory cytokines TNF-α and IL-10 were significantly increased (P < 0.05). Taken together, these results indicate that TRIP might play important roles in immune defense and has the potential to be used as a anti-inflammation target in grass carp.

  8. Molecular cloning and partial characterization of a low-density lipoprotein receptor-related protein 13 (Lrp13) involved in vitellogenin uptake in the cutthroat trout (Oncorhynchus clarki).

    PubMed

    Mushirobira, Yuji; Mizuta, Hiroko; Luo, Wenshu; Todo, Takashi; Hara, Akihiko; Reading, Benjamin J; Sullivan, Craig V; Hiramatsu, Naoshi

    2015-12-01

    Multiple ovarian membrane proteins that bind vitellogenin (Vtg) have been detected in teleosts. One of these Vtg receptors was recently identified as low-density lipoprotein receptor-related protein 13 (lrp13/Lrp13) in perciform species, but little is known about this Vtg receptor in salmonid fish. In this study, a cDNA encoding a putative Vtg receptor with 13+1 ligand binding repeats (lr13+1) was cloned from the ovary, and identified as an lrp13 ortholog for cutthroat trout (Oncorhynchus clarki). This lrp13 was predominantly expressed in the pre-vitellogenic stage ovary, and its expression decreased during vitellogenesis. Ovarian localization of Lrp13 was observed by immunohistochemistry using specific antiserum against recombinant Lrp13. Lrp13 immunoreactivity was observed at the oolemma, throughout the zona radiata, and within the perivitelline space between the zona radiata and granulosa cells in ovarian follicles at both the lipid-droplet and vitellogenic stages of growth-an expression pattern that mimics that of a lr8/LR8-type Vtg receptor in this species and of lrp13/Lrp13 in Morone species. Six discrete Vtg-binding proteins were detected in cutthroat trout ovarian membrane proteins when probing with a digoxygenin-labeled salmonid A-type Vtg (VtgAs) followed by chemiluminescent ligand detection. Western blotting using the anti-Lrp13 serum revealed a broad signal consisting of two proteins with masses ranging from ∼190 to ∼210 kDa, which corresponded with some of the VtgA-binding proteins. These findings suggest that, in addition to lr8/LR8, lrp13/Lrp13 acts as a VtgA receptor in trout.

  9. Molecular cloning and characterization of two novel retinoic acid-inducible orphan G-protein-coupled receptors (GPRC5B and GPRC5C).

    PubMed

    Robbins, M J; Michalovich, D; Hill, J; Calver, A R; Medhurst, A D; Gloger, I; Sims, M; Middlemiss, D N; Pangalos, M N

    2000-07-01

    Using homology searching of public databases with a metabotropic glutamate receptor sequence from Caenorhabditis elegans, two novel protein sequences (named RAIG-2 (HGMW-approved symbol GPRC5B) and RAIG-3 (HGMW-approved symbol GPRC5C) were identified containing seven putative transmembrane domains characteristic of G-protein-coupled receptors (GPCRs). RAIG-2 and RAIG-3 encode open reading frames of 403 and 442 amino acid polypeptides, respectively, and show 58% similarity to the recently identified retinoic acid-inducible gene-1 (RAIG-1, HGMW-approved symbol RAI3). Analysis of the three protein sequences places them within the type 3 GPCR family, which includes metabotropic glutamate receptors, GABA(B) receptors, calcium-sensing receptors, and pheromone receptors. However, in contrast to other type 3 GPCRs, RAIG-1, RAIG-2, and RAIG-3 have only short N-terminal domains. RAIG-2 and RAIG-3 cDNA sequences were cloned into the mammalian expression vector pcDNA3 with c-myc or HA epitope tags inserted at their N-termini, respectively. Transient transfection experiments in HEK239T cells using these constructs demonstrated RAIG-2 and RAIG-3 expression at the cell surface. Distribution profiles of mRNA expression obtained by semiquantitative Taq-Man PCR analysis showed RAIG-2 to be predominantly expressed in human brain areas and RAIG-3 to be predominantly expressed in peripheral tissues. In addition, expression of RAIG-2 and RAIG-3 mRNA was increased following treatment with all-trans-retinoic acid in a manner similar to that previously described for RAIG-1. Finally, RAIG-2 was mapped to chromosome 16p12 (D16S405-D16S3045) and RAIG-3 to chromosome 17q25 (D17S1352-D17S785). These results suggest that RAIG-1, RAIG-2, and RAIG-3 represent a novel family of retinoic acid-inducible receptors, most closely related to the type 3 GPCR subfamily, and provide further evidence for a linkage between retinoic acid and G-protein-coupled receptor signal transduction pathways.

  10. Cloning, molecular characterization, and chromosomal assignment of a gene encoding a second D1 dopamine receptor subtype: differential expression pattern in rat brain compared with the D1A receptor.

    PubMed Central

    Tiberi, M; Jarvie, K R; Silvia, C; Falardeau, P; Gingrich, J A; Godinot, N; Bertrand, L; Yang-Feng, T L; Fremeau, R T; Caron, M G

    1991-01-01

    Multiple D1 dopaminergic receptor subtypes have been postulated on the basis of pharmacological, biochemical, and genetic studies. We describe the isolation and characterization of a rat gene encoding a dopamine receptor that is structurally and functionally similar to the D1 dopamine receptor. The coding region, which is intronless, encodes a protein of 475 amino acids (Mr 52,834) with structural features that are consistent with receptors coupled to guanine nucleotide-binding regulatory proteins. The expressed protein binds dopaminergic ligands and mediates stimulation of adenylyl cyclase with pharmacological properties similar to those of the D1 dopamine receptor. The gene encoding the human homologue of this receptor subtype is located to the short arm of chromosome 4 (4p16.3), the same region as the Huntington disease gene. In striking contrast to the previously cloned D1 receptor, little or no mRNA for the receptor described here was observed in striatum, nucleus accumbens, olfactory tubercle, and frontal cortex. High levels of mRNA for this receptor were found in distinct layers of the hippocampus, the mammillary nuclei, and the anterior pretectal nuclei, brain regions that have been shown to exhibit little or no D1 dopamine receptor binding. On the basis of its properties we propose that this dopamine receptor subtype be called D1B. Images PMID:1831904

  11. Molecular cloning and expression analysis of estrogen receptor betas (ERβ1 and ERβ2) during gonad development in the Korean rockfish, Sebastes schlegeli.

    PubMed

    Mu, W J; Wen, H S; Shi, D; Yang, Y P

    2013-07-01

    Estrogen receptors (ER) play a crucial role in mediation of estrogen activities. Here we report the isolation and expression analysis of ERβ1 and ERβ2 from ovary Korean rockfish (Sebastes schlegeli). were isolated using reverse transcription-polymerase chain reaction (PCR) and rapid amplification of cDNA ends procedures. The cDNA of this study, ERβ1 (588 amino acids) and ERβ2 (659 amino acids) were identified using reverse-transcriptase PCR (RT-PCR) and rapid amplification of cDNA ends procedures. Structural analysis showed both ERβs contain six typical nuclear receptor-characteristic domains. Phylogenetic analysis indicated that Korean rockfish ERβs were highly conserved among teleost. RT-PCR confirmed that the ERβs were widely distributed in both gonads and extra gonadal tissues. Further, we analyzed the expression patterns of male and female S. schlegeli during the reproductive cycle using quantitative real-time PCR (qRT-PCR). The results showed that the highest expression levels were observed in testis at immature sperm stage for both of KrERβ1 and KrERβ2. For female, the expressions of KrERβ1 and KrERβ2 were significantly higher in the ovary at the early-oocyte stage. Cloning these two ERβ subtypes in the Korean rockfish, together with the information on expression levels in adult fish has given us the foundation to investigate their possible role in brain-pituitary-gonad neuroendocrine axis in future studies.

  12. Molecular cloning, characterization and expression analysis of Toll-like receptor 5M gene in Japanese sea perch (Lateolabrax japonicas) after bacterial infection.

    PubMed

    Wang, Chengyang; Zhao, Chao; Fu, Mingjun; Bao, Weiyang; Qiu, Lihua

    2016-09-01

    Toll-like receptor 5M belongs to Toll-like receptors (TLRs) family, which plays a crucial role in innate immunity due to its important role in the recognition of bacteria invasion and in the activation of immune related pathways downstream. In the present study, we firstly cloned the full-length cDNAs of TLR 5M (LjTLR 5M) from Japanese sea perch (Lateolabrax japonicas). The full-length cDNAs of LjTLR 5M include an open reading frame (ORF) of 2676 bp encoding a polypeptide of 891 amino acid residues. The deduced amino acid sequence analysis showed that LiTLR 5M contains LRRs (extracellular leucine rich repeats), transmembrane and TIR (Toll/interleukin-1 receptor) domain. Transcriptional expression analysis indicated that LiTLR 5M mRNAs were ubiquitously expressed in wide array of tissues and the peak level was observed in the head-kidney. The expression patterns of LjTLR 5M after Vibro harveyi and Streptococus agalactiae infection were detected by qRT-PCR, and the results showed that LjTLR 5M was significant up-regulated in spleen, liver and head-kidney. Additionally, the expression patterns of LjTLR 5M in infected spleen and head-kidney were further validated by in situ hybridization (ISH). In summary, these findings indicate that LjTLR 5M is significant induced after different bacterial infection and is involved in immune response. Furthermore, this study will provide foundational information for other TLRs research of L. japonicas against different bacterial pathogens invasion.

  13. Molecular cloning of the mouse grb2 gene: differential interaction of the Grb2 adaptor protein with epidermal growth factor and nerve growth factor receptors.

    PubMed Central

    Suen, K L; Bustelo, X R; Pawson, T; Barbacid, M

    1993-01-01

    We report the isolation and molecular characterization of the mouse grb2 gene. The product of this gene, the Grb2 protein, is highly related to the Caenorhabditis elegans sem-5 gene product and the human GRB2 protein and displays the same SH3-SH2-SH3 structural motifs. In situ hybridization studies revealed that the mouse grb2 gene is widely expressed throughout embryonic development (E9.5 to P0). However, grb2 transcripts are not uniformly distributed, and in certain tissues (e.g., thymus) they appear to be regulated during development. Recent genetic and biochemical evidence has implicated the Grb2 protein in the signaling pathways that link cell surface tyrosine kinase receptors with Ras. We have investigated the association of the Grb2 protein with epidermal growth factor (EGF) and nerve growth factor (NGF) receptors in PC12 pheochromocytoma cells. EGF treatment of PC12 cells results in the rapid association of Grb2 with the activated EGF receptors, an interaction mediated by the Grb2 SH2 domain. However, Grb2 does not bind to NGF-activated Trk receptors. Mitogenic signaling of NGF in NIH 3T3 cells ectopically expressing Trk receptors also takes place without detectable association between Grb2 and Trk. These results suggest that whereas EGF and NGF can activate the Ras signaling pathway in PC12 cells, only the EGF receptor is likely to do so through a direct interaction with Grb2. Finally, binding studies with glutathione S-transferase fusion proteins indicate that Grb2 binds two distinct subsets of proteins which are individually recognized by its SH2 and SH3 domains. These observations add further support to the concept that Grb2 is a modular adaptor protein. Images PMID:7689150

  14. Molecular cloning and characterization of two novel genes from hexaploid wheat that encode double PR-1 domains coupled with a receptor-like protein kinase.

    PubMed

    Lu, Shunwen; Faris, Justin D; Edwards, Michael C

    2017-04-01

    Hexaploid wheat (Triticum aestivum L.) contains at least 23 TaPr-1 genes encoding the group 1 pathogenesis-related (PR-1) proteins as identified in our previous work. Here, we report the cloning and characterization of TaPr-1-rk1 and TaPr-1-rk2, two novel genes closely related to the wheat PR-1 family. The two TaPr-1-rk genes are located on homoeologous chromosomes 3D and 3A, respectively, and each contains a large open reading frame (7385 or 6060 bp) that is interrupted by seven introns and subjected to alternative splicing (AS) with five or six isoforms of mRNA transcripts. The deduced full-length TaPR-1-RK1 and TaPR-1-RK2 proteins (95% identity) contain two repeat PR-1 domains, the second of which is fused via a transmembrane helix to a serine/threonine kinase catalytic (STKc) domain characteristic of receptor-like protein kinases. Phylogenetic analysis indicated that the two PR-1 domains of the TaPR-1-RK proteins form sister clades with their homologues identified in other monocot plants and are well separated from stand-alone PR-1 proteins, whereas the STKc domains may have originated from cysteine-rich receptor-like kinases (CRKs). Reverse-transcriptase-PCR analysis revealed that the TaPr-1-rk genes are predominantly expressed in wheat leaves and their expression levels are elevated in response to pathogen attack, such as infection by barley stripe mosaic virus (BSMV), and also to stress conditions, most obviously, to soil salinity. This is the first report of PR-1-CRK hybrid proteins in wheat. The data may shed new insights into the function/evolutionary origin of the PR-1 family and the STKc-mediated defense/stress response pathways in plants.

  15. Molecular cloning, characterization, tissue distribution and mRNA expression changes during the hibernation and reproductive periods of estrogen receptor alpha (ESR1) in Chinese alligator, Alligator sinensis.

    PubMed

    Zhang, Ruidong; Hu, Yuehong; Wang, Huan; Yan, Peng; Zhou, Yongkang; Wu, Rong; Wu, Xiaobing

    2016-10-01

    Chinese alligator, Alligator sinensis, is a critically endangered reptile species unique to China. Little is known about the mechanism of growth- and reproduction-related hormones gene expression in Chinese alligator. Estrogens play important roles in regulating multiple reproduction- and non-reproduction-related functions by binding to their corresponding receptors. Here, the full-length cDNA of estrogen receptor alpha (ERα/ESR1) was cloned and sequenced from Chinese alligator for the first time, which comprises 1764bp nucleotides and encodes a predicted protein of 587 amino acids. Phylogenetic analysis of ESR1 showed that crocodilians and turtles were the sister-group of birds. The results of real-time quantitative PCR indicated that the ESR1 mRNA was widely expressed in the brain and peripheral tissues. In the brain and pituitary gland, ESR1 was most highly transcribed in the cerebellum. But in other peripheral tissues, ESR1 mRNA expression level was the highest in the ovary. Compared with hibernation period, ESR1 mRNA expression levels were increased significantly in the reproductive period (P<0.05) in cerebellum, pituitary gland, liver, spleen, lung, kidney and ovary, while no significant change in other examined tissues (P>0.05). The ESR1 mRNA expression levels changes during the two periods of different tissues suggested that ESR1 might play an important role in mediation of estrogenic multiple reproductive effects in Chinese alligator. Furthermore, it was the first time to quantify ESR1 mRNA level in the brain of crocodilians, and the distribution and expression of ESR1 mRNA in the midbrain, cerebellum and medulla oblongata was also reported for the first time in reptiles.

  16. Molecular Cloning and Functional Characterization of Xenopus tropicalis Frog Transient Receptor Potential Vanilloid 1 Reveal Its Functional Evolution for Heat, Acid, and Capsaicin Sensitivities in Terrestrial Vertebrates*

    PubMed Central

    Ohkita, Masashi; Saito, Shigeru; Imagawa, Toshiaki; Takahashi, Kenji; Tominaga, Makoto; Ohta, Toshio

    2012-01-01

    The functional difference of thermosensitive transient receptor potential (TRP) channels in the evolutionary context has attracted attention, but thus far little information is available on the TRP vanilloid 1 (TRPV1) function of amphibians, which diverged earliest from terrestrial vertebrate lineages. In this study we cloned Xenopus tropicalis frog TRPV1 (xtTRPV1), and functional characterization was performed using HeLa cells heterologously expressing xtTRPV1 (xtTRPV1-HeLa) and dorsal root ganglion neurons isolated from X. tropicalis (xtDRG neurons) by measuring changes in the intracellular calcium concentration ([Ca2+]i). The channel activity was also observed in xtTRPV1-expressing Xenopus oocytes. Furthermore, we tested capsaicin- and heat-induced nocifensive behaviors of the frog X. tropicalis in vivo. At the amino acid level, xtTRPV1 displays ∼60% sequence identity to other terrestrial vertebrate TRPV1 orthologues. Capsaicin induced [Ca2+]i increases in xtTRPV1-HeLa and xtDRG neurons and evoked nocifensive behavior in X. tropicalis. However, its sensitivity was extremely low compared with mammalian orthologues. Low extracellular pH and heat activated xtTRPV1-HeLa and xtDRG neurons. Heat also evoked nocifensive behavior. In oocytes expressing xtTRPV1, inward currents were elicited by heat and low extracellular pH. Mutagenesis analysis revealed that two amino acids (tyrosine 523 and alanine 561) were responsible for the low sensitivity to capsaicin. Taken together, our results indicate that xtTRPV1 functions as a polymodal receptor similar to its mammalian orthologues. The present study demonstrates that TRPV1 functions as a heat- and acid-sensitive channel in the ancestor of terrestrial vertebrates. Because it is possible to examine vanilloid and heat sensitivities in vitro and in vivo, X. tropicalis could be the ideal experimental lower vertebrate animal for the study of TRPV1 function. PMID:22130664

  17. Molecular cloning and functional characterization of Xenopus tropicalis frog transient receptor potential vanilloid 1 reveal its functional evolution for heat, acid, and capsaicin sensitivities in terrestrial vertebrates.

    PubMed

    Ohkita, Masashi; Saito, Shigeru; Imagawa, Toshiaki; Takahashi, Kenji; Tominaga, Makoto; Ohta, Toshio

    2012-01-20

    The functional difference of thermosensitive transient receptor potential (TRP) channels in the evolutionary context has attracted attention, but thus far little information is available on the TRP vanilloid 1 (TRPV1) function of amphibians, which diverged earliest from terrestrial vertebrate lineages. In this study we cloned Xenopus tropicalis frog TRPV1 (xtTRPV1), and functional characterization was performed using HeLa cells heterologously expressing xtTRPV1 (xtTRPV1-HeLa) and dorsal root ganglion neurons isolated from X. tropicalis (xtDRG neurons) by measuring changes in the intracellular calcium concentration ([Ca(2+)](i)). The channel activity was also observed in xtTRPV1-expressing Xenopus oocytes. Furthermore, we tested capsaicin- and heat-induced nocifensive behaviors of the frog X. tropicalis in vivo. At the amino acid level, xtTRPV1 displays ∼60% sequence identity to other terrestrial vertebrate TRPV1 orthologues. Capsaicin induced [Ca(2+)](i) increases in xtTRPV1-HeLa and xtDRG neurons and evoked nocifensive behavior in X. tropicalis. However, its sensitivity was extremely low compared with mammalian orthologues. Low extracellular pH and heat activated xtTRPV1-HeLa and xtDRG neurons. Heat also evoked nocifensive behavior. In oocytes expressing xtTRPV1, inward currents were elicited by heat and low extracellular pH. Mutagenesis analysis revealed that two amino acids (tyrosine 523 and alanine 561) were responsible for the low sensitivity to capsaicin. Taken together, our results indicate that xtTRPV1 functions as a polymodal receptor similar to its mammalian orthologues. The present study demonstrates that TRPV1 functions as a heat- and acid-sensitive channel in the ancestor of terrestrial vertebrates. Because it is possible to examine vanilloid and heat sensitivities in vitro and in vivo, X. tropicalis could be the ideal experimental lower vertebrate animal for the study of TRPV1 function.

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

    PubMed

    Chen, Qi-Liang; Luo, Zhi; Tan, Xiao-Ying; Pan, Ya-Xiong; Zheng, Jia-Lang; Zou, Ming

    2014-02-25

    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.

  19. Molecular cloning, expression of, and regulation by thyroid-hormone receptor α A in the half-smooth tongue sole Cynoglossus semilaevis during metamorphosis.

    PubMed

    Zhang, W T; Liu, K; Xiang, J S; Zhang, L Y; Liu, W J; Dong, Z D; Li, Y Z; Li, H L; Chen, S L; Wang, N

    2016-05-01

    To elucidate the effect of thyroid hormone receptor α A (thraa) on metamorphosis, the full length cDNA of half-smooth tongue sole Cynoglossus semilaevis was cloned. The relative gene transcript level of thraa at different development stages was quantified using real-time PCR. Transcription of thraa increased and declined rapidly during metamorphosis. Hyperthyroidism was induced in juveniles and larvae with exposure to T3 and T4, and hypothyroidism with thiourea (TU), 2-mercapto-1-methylimidazole (MMI). thraa mRNA was higher in fish treated for 6 days with MMI than in untreated controls, although inhibited larvae did not complete metamorphosis. The addition of exogenous T4 reversed this effect in the MMI-treated group, but not in the TU-treated group. In situ hybridization revealed progressive tail end of body growth and change during developmental stages, with corresponding changes in thraa expression. This process may be induced by thyroid hormones with thraa as a major mediator. The morphological changes of tip of the tail may be associated with the development of lateral swimming.

  20. Molecular cloning, genomic structure, and tissue distribution of EW135, a novel chicken egg white protein with group B scavenger receptor cysteine-rich domains.

    PubMed

    Yoo, Whayoung; Nakamura, Tomohiro; Asanuma, Hideki; Matsushita, Misao

    2013-11-01

    Approximately 80 proteins are reported to be present in chicken egg white. The major function of egg white proteins isolated so far is to defend the egg yolk against infections. We recently isolated a novel protein termed EW135 from chicken egg white. In this paper, we have determined the complete amino acid sequence of EW135 based on cDNA cloning. EW135 consists of 970 amino acids with a putative signal peptide of 17 amino acids. It is composed exclusively of tandem repeats of nine group B scavenger receptor cysteine-rich (SRCR) domains separated by eight seven-amino acid peptides. The features of consensus sequences found in the group B SRCR domain were well conserved in EW135. The EW135 gene consists of putative 11 exons, with each SRCR domain being encoded by a single exon. Reverse transcription PCR showed that EW135 is expressed in only the oviduct among the 11 types of tissues tested. EW135 is a second soluble protein belonging to the group B SRCR domain superfamily identified in chickens. One of the important functions of proteins belonging to the group B SRCR domain superfamily is to recognize pathogens in innate immunity. It is, therefore, conceivable that EW135 could be involved in host defense in egg white.

  1. Canine prostaglandin F2alpha receptor (FP) and prostaglandin F2alpha synthase (PGFS): molecular cloning and expression in the corpus luteum.

    PubMed

    Kowalewski, Mariusz Pawel; Mutembei, Henry M'ikiugu; Hoffmann, Bernd

    2008-08-01

    In the dog luteolysis is not affected by hysterectomy. This observation led to the hypothesis that paracrine/autocrine rather than endocrine mechanisms of PGF2alpha are responsible for luteal regression in the dioestric bitch. The present experiments tested for the capacity of canine CL to produce and respond to PGF2alpha by qualitatively and quantitatively determining the expressions of PGFS, the enzyme converting PGH2 into PGF2alpha, and the PGF2alpha-receptor (FP) in CL of non-pregnant dogs during dioestrus. Canine PGFS and FP were isolated and cloned; both genes show a high homology (82-94%) when compared to those of other species. Relatively weak FP mRNA expression was detected on day 5 of dioestrus. It had increased by day 25 and remained constant thereafter. In situ hybridization (ISH) localized FP solely to the cytoplasm of the luteal cells, suggesting that these cells are the only luteal targets of PGF2alpha in this species. Only negative results were obtained for the expression of PGFS in canine CL by routine qualitative RT-PCR. When Real Time (TaqMan) PCR was applied, repetitively more negative than positive results were obtained at all timepoints. Any positive measurements observed at any point were neither repeatable nor related to the stage of dioestrus. This led us to conclude that expression of PGFS is either absent or present at very low level only. These data suggest that luteal regression in non-pregnant bitches is not modulated by PGF2alpha. However, the FP seems to be constitutionally expressed, explaining the receptivity of canine CL to exogenous PGF2alpha.

  2. Molecular cloning, characterization and expression profiles of multiple leptin genes and a leptin receptor gene in orange-spotted grouper (Epinephelus coioides).

    PubMed

    Zhang, Huixian; Chen, Huapu; Zhang, Yong; Li, Shuisheng; Lu, Danqi; Zhang, Haifa; Meng, Zining; Liu, Xiaochun; Lin, Haoran

    2013-01-15

    Leptin plays key roles in body weight regulation, energy metabolism, food intake, reproduction and immunity in mammals. However, its function in teleosts is still unclear. In the present study, two leptin genes (gLepA and gLepB) and one leptin receptor gene (gLepR) were cloned and characterized in orange-spotted grouper (Epinephelus coioides). The cDNAs of gLepA and gLepB were 671 bp and 684 bp in length, encoding for proteins of 161 amino acid (aa) and 158 aa, respectively. The three-dimensional (3D) structures modeling of gLepA and gLepB showed strong conservation of tertiary structure with that of other vertebrates. The total length of gLepR cDNA was 4242 bp, encoding a protein of 1169 aa which contained all functionally important domains conserved among vertebrate LEPR. Tissue distribution analysis showed that gLepA was highly expressed in cerebellum, liver and ovary, while gLepB mRNA abundantly in the brain regions, as well as in the ovary with some extend. The gLepR was mainly expressed in kidney, head kidney and most of brain regions. Analysis of expression profiles of gLep and gLepR genes during the embryonic stages showed that high expression of gLepR was observed in the brain vesicle stage, while neither gLepA nor gLepB mRNA was detected during different embryonic stages. Finally, fasting and refeeding experiments were carried out to investigate the possible function of leptin genes in food intake and energy metabolism, and the results showed that a significant increase of gLepA expression in the liver was induced by food deprivation in both short-term (7 days) and long-term (3 weeks) fasting and gLepA mRNA upregulation was eliminated after refeeding, while gLepB wasn't detected in the liver of grouper during fasting. No significant differences in hypothalamic leptin and leptin receptor expression were found during short-term fasting and refeeding. Hepatic expression of gLepA mRNA increased significantly 9h after a single meal. These results suggested g

  3. Molecular cloning and characterization of mouse Gipc3.

    PubMed

    Saitoh, Tetsuroh; Mine, Tetsuya; Katoh, Masaru

    2002-03-01

    GIPC1/GIPC interacts with GTPase-activating protein RGS-GAIP, transmembrane protein M-SemF, receptor tyrosine kinase TrkA, integrin alpha 6A subunit, and TGF beta type III receptor. Kermit, a Xenopus orthologue of human GIPC1, interacts with Frizzled-3 (FZD3) class of WNT receptor. We have recently cloned and characterized human GIPC2 and GIPC3. Here, we identified mouse Gipc3 gene fragments by using bioinformatics, and isolated mouse Gipc3 cDNAs by using cDNA-PCR. Mouse Gipc3 gene encoded a 297-amino-acid protein, showing 86.2% total-amino-acid identity with human GIPC3. In addition to the central PDZ domain, GIPC homologous domain 1 (GH1 domain) and GH2 domain were found to be conserved among mouse Gipc3, Gipc1, Gipc2, and Xenopus Kermit. Mouse Gipc3 gene was found to consist of 6 exons, and exon-intron structure was well conserved between mouse Gipc3 gene and human GIPC3 gene. Mouse Gipc3 mRNA was relatively highly expressed in adult lung, and was also expressed in brain and testis, but was almost undetectable in 7-, 11-, 15, and 17-day whole embryos. This is the first report on molecular cloning and initial characterization of mouse Gipc3.

  4. Molecular cloning, tissue expression and regulation of liver X receptor (LXR) transcription factors of Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss).

    PubMed

    Cruz-Garcia, L; Minghetti, M; Navarro, I; Tocher, D R

    2009-05-01

    Fish are important sources of high quality protein, essential minerals such as iodine and selenium, vitamins including A, D and E, and omega-3 fatty acids in the human diet. With declining fisheries worldwide, farmed fish constitute an ever-increasing proportion of fish in the food basket. Sustainable development of aquaculture dictates that diets will have to contain increasing levels of plant products that are devoid of cholesterol, but contain phytosterols that are known to have physiological effects in mammals. Liver X receptors (LXR) are transcription factors whose activity is modulated by sterols, with activation inducing cholesterol catabolism and de novo fatty acid biosynthesis in liver. Transcriptomic analysis has shown that substitution of fish meal and oil with plant products induces genes of cholesterol and fatty acid metabolism in salmonids. Here we report the cloning of LXR cDNAs from two species of salmonid fish that are important in aquaculture. The full-length cDNA (mRNA) of LXR obtained from salmon was shown to be 3766 bp, which included a 5'-untranslated region (UTR) of 412 bp and a 3'-UTR of 1960 bp and an open reading frame (ORF) of 1394 bp, which specified a protein of 462 amino acids. The trout LXR full-length cDNA was 2056 bp, including 5'- and 3'-UTRs of 219 and 547 bp, respectively, and an ORF of 1290 bp, which specified a protein of 427 amino acids. The protein sequences included characteristic features of mammalian LXRs, including the DNA binding (DBD), containing P-box, ligand binding (LBD) and activation function-2 (AF-2) domains, D-box, D (hinge) region, and eight cysteines that belong to the two zinc fingers. Phylogenetic analysis clustered the salmonid LXRs together, more closely with zebrafish and more distantly from medaka and stickleback. A pair-wise comparison among vertebrate LXR sequences showed the amino acid sequence predicted by the salmon LXR ORF showed greatest identity to that of trout 97%, and 97%, 87% and 81% identity

  5. Molecular cloning and expression analysis of the retinoid X receptor (RXR) gene in golden pompano Trachinotus ovatus fed Artemia nauplii with different enrichments.

    PubMed

    Yang, Qibin; Zheng, Panlong; Ma, Zhenhua; Li, Tao; Jiang, Shigui; Qin, Jian G

    2015-12-01

    The retinoid X receptors (RXRs) are involved in the skeletal development and other biological process such as blood vessel formation and metabolism. Partial sequences of RXRα and β genes were obtained, and their expressions were quantified on golden pompano Trachinotus ovatus at 28 days post hatching (DPH) to explore the molecular response to nutritional manipulation in fish larvae. As live food, Artemia nauplii were separately enriched with Nannochloropsis and Algamac 3080 and non-enriched Artemia nauplii (control) for fish feeding. The expressions of RXRs were detected in the embryos and fish larvae at early stages, suggesting that the skeletal development in golden pompano initiated before yolk re-sorption completion. Fish fed non-enriched Artemia nauplii ended up with higher jaw malformation. The highest specific growth rate was obtained when fish were fed with the Artemia nauplii enriched with Algamac 3080, and the lowest growth rate was observed when fish were fed with unenriched Artemia nauplii. The highest survival was obtained when fish were fed with non-enriched or Nannochloropsis-enriched Artemia nauplii. This study indicates that the use of enriched formula for Artemia nauplii can significantly affect the expression levels of RXRs and jaw malformation of golden pompano larvae, but there is no clear correlation between RXRs expressions and malformation rates when fish are subjected to nutrient challenge.

  6. A modified version of the digestion-ligation cloning method for more efficient molecular cloning.

    PubMed

    Gao, Song; Li, Yanling; Zhang, Jiannan; Chen, Hongman; Ren, Daming; Zhang, Lijun; An, Yingfeng

    2014-05-15

    Here we describe a modified version of the digestion-ligation approach for efficient molecular cloning. In comparison with the original method, the modified method has the additional steps of gel purification and a second ligation after the first ligation of the linearized vector and DNA insert. During this process, the efficiency and reproducibility could be significantly improved for both stick-end cloning and blunt-end cloning. As an improvement of the very important molecular cloning technique, this method may find a wide range of applications in bioscience and biotechnology.

  7. Molecular cloning and characterization of two novel genes from hexaploid wheat that encode double PR-1 domains coupled with a receptor-like protein kinase

    USDA-ARS?s Scientific Manuscript database

    Hexaploid wheat (Triticum aestivum L.) contains at least 23 TaPr-1 genes encoding the group 1 pathogenesis-related (PR-1) proteins as identified in our previous work. Here we report the cloning and characterization of TaPr-1-rk1 and TaPr-1-rk2, two novel genes closely related to the wheat PR-1 famil...

  8. Cloning and characterization of a Drosophila tyramine receptor.

    PubMed Central

    Saudou, F; Amlaiky, N; Plassat, J L; Borrelli, E; Hen, R

    1990-01-01

    Receptors for biogenic amines such as dopamine, serotonin and epinephrine belong to the family of receptors that interact with G proteins and share a putative seven transmembrane domain structure. Using a strategy based on nucleotide sequence homology between the corresponding genes, we have isolated Drosophila cDNA clones encoding a new member of the G protein-coupled receptor family. This protein exhibits highest homology to the human alpha 2 adrenergic receptors, the human 5HT1A receptor and a recently cloned Drosophila serotonin receptor. The corresponding mRNA is found predominantly in adult Drosophila heads. Membranes from mammalian cells expressing this receptor displayed high affinity binding sites for [3H]yohimbine, an alpha 2 adrenergic receptor antagonist (Kd = 4.45 x 10(-9) M). Tyramine was the most efficient of the putative Drosophila neurotransmitters at displacing [3H]yohimbine binding (EC50 = 1.25 x 10(-6) M). Furthermore tyramine induced an inhibition of adenylate cyclase activity in NIH 3T3 cells expressing this receptor. The Drosophila tyramine receptor that we have isolated might therefore be an invertebrate equivalent of the mammalian alpha 2 adrenergic receptors. Images Fig.2 Fig.5 PMID:2170118

  9. Cloning and expression of a novel neuropeptide Y receptor.

    PubMed

    Weinberg, D H; Sirinathsinghji, D J; Tan, C P; Shiao, L L; Morin, N; Rigby, M R; Heavens, R H; Rapoport, D R; Bayne, M L; Cascieri, M A; Strader, C D; Linemeyer, D L; MacNeil, D J

    1996-07-12

    The neuropeptide Y family of peptides, which includes neuropeptide Y (NPY), peptide YY (PYY), and pancreatic polypeptide (PP), are found in the central and peripheral nervous system and display a wide array of biological activities. These actions are believed to be mediated through pharmacologically distinct G protein-coupled receptors, and, to date, three members of the NPY receptor family have been cloned. In this study we describe the cloning and expression of a novel NPY receptor from mouse genomic DNA. This receptor, designated NPY Y5, shares 60% amino acid identity to the murine NPY Y1 receptor. The pharmacology of this novel receptor resembles that of the NPY Y1 receptor and is distinct from that described for the NPY Y2, Y3, and Y4 receptors. In situ hybridization of mouse brain sections reveals expression of this receptor within discrete regions of the hypothalamus including the suprachiasmatic nucleus, anterior hypothalamus, bed nucleus stria terminalis, and the ventromedial nucleus with no localization apparent elsewhere in the brain.

  10. Molecular cloning of chicken aggrecan. Structural analyses.

    PubMed Central

    Chandrasekaran, L; Tanzer, M L

    1992-01-01

    The large, aggregating chondroitin sulphate proteoglycan of cartilage, aggrecan, has served as a generic model of proteoglycan structure. Molecular cloning of aggrecans has further defined their amino acid sequences and domain structures. In this study, we have obtained the complete coding sequence of chicken sternal cartilage aggrecan by a combination of cDNA and genomic DNA sequencing. The composite sequence is 6117 bp in length, encoding 1951 amino acids. Comparison of chicken aggrecan protein primary structure with rat, human and bovine aggrecans has disclosed both similarities and differences. The domains which are most highly conserved at 70-80% identity are the N-terminal domains G1 and G2 and the C-terminal domain G3. The chondroitin sulphate domain of chicken aggrecan is smaller than that of rat and human aggrecans and has very distinctive repeat sequences. It has two separate sections, one comprising 12 consecutive Ser-Gly-Glu repeats of 20 amino acids each, adjacent to the other which has 23 discontinuous Ser-Gly-Glu repeats of 10 amino acids each; this latter region, N-terminal to the former one, appears to be unique to chicken aggrecan. The two regions contain a total of 94 potential chondroitin sulphate attachment sites. Genomic comparison shows that, although chicken exons 11-14 are identical in size to the rat and human exons, chicken exon 10 is the smallest of the three species. This is also reflected in the size of its chondroitin sulphate coding region and in the total number of Ser-Gly pairs. The putative keratan sulphate domain shows 31-45% identity with the other species and lacks the repetitive sequences seen in the others. In summary, while the linear arrangement of specific domains of chicken aggrecan is identical to that in the aggrecans of other species, and while there is considerable identity of three separate domains, chicken aggrecan demonstrates unique features, notably in its chondroitin sulphate domain and its keratan sulphate

  11. Cloning and molecular characterization of an ethylene receptor gene, MiERS1, expressed during mango fruitlet abscission and fruit ripening.

    PubMed

    Ish-Shalom, Mazal; Dahan, Yardena; Maayan, Inbar; Irihimovitch, Vered

    2011-08-01

    We isolated and characterized a mango (Mangifera indica L.) cDNA homolog of the ethylene receptor gene ERS1, designated MiERS1. Genomic Southern blot analysis suggested the existence of a second gene with homology to MiERS1. Spatial and temporal expression patterns of MiERS1 were first studied during fruitlet drop and compared with those of a previously identified MiETR1 gene that encodes an ETR1-type ethylene receptor. Experiments were conducted on developing fruitlet explants in which fruitlet abscission was induced by ethephon treatment. Northern analysis revealed a notable increase in MiERS1 mRNA levels in the fruitlet's activated abscission zone within 24 h of ethephon application, followed by a decreasing pattern 48 h post-treatment. A transient, albeit lesser, increase in MiERS1 mRNA levels was also observed in treated fruitlet seed and mesocarp tissues. In contrast, in the abscission zone, accumulation of MiETR1 transcript remained unchanged; a temporal increase in MiETR1 transcript level was observed in the fruitlet mesocarp, whereas in the seed, MiETR1 expression had already dropped by 24 h. Expression profiles of MiERS1 and MiETR1 were then studied during fruit ripening. In agreement with a previous study and coinciding with the climacteric rise in ethylene production, RNA blot analysis revealed that during fruit ripening, MiETR1 mRNA level increases in both mesocarp and seed tissues. Unexpectedly, however, in those same tissues, MiERS1 transcript accumulation was barely detected. Collectively, our data highlight MiERS1's possible specific function in regulating fruitlet abscission rather than fruit ripening.

  12. Cloning and expression of the human vasoactive intestinal peptide receptor.

    PubMed Central

    Sreedharan, S P; Robichon, A; Peterson, K E; Goetzl, E J

    1991-01-01

    Vasoactive intestinal peptide (VIP) is a neuroendocrine mediator found in the central and peripheral nervous system. Distinct subsets of neural, respiratory, gastrointestinal, and immune cells bear specific high-affinity receptors for VIP, which are associated with a guanine nucleotide-binding (G) protein capable of activating adenylate cyclase. A cDNA clone (GPRN1) encoding the human VIP receptor was identified in libraries prepared from the Nalm 6 line of leukemic pre-B lymphoblasts and the HT-29 line of colon carcinoma cells. The deduced 362-amino acid polypeptide sequence encoded by GPRN1 shares a seven-transmembrane-segment hydropathicity profile with other G protein-coupled receptors. Northern blot analyses identified a 2.7-kilobase transcript of the VIP receptor in Nalm 6 and HT-29 cells as well as in tissues from rat brain, colon, heart, lung, kidney, spleen, and small intestine. COS-6 cells transfected with GPRN1 bound 125I-labeled VIP specifically with a dissociation constant (Kd) of 2.5 nM. VIP--and less effectively secretin, peptide histidine isoleucine (PHI), and glucagon competitively displaced bound 125I-VIP from transfected COS-6 cells, with potencies in the order VIP greater than secretin = PHI much greater than glucagon. VIP stimulated adenylate cyclase activity in stably transfected Chinese hamster ovary K1 cells, inducing a 3-fold increase in the intracellular level of cAMP. When the antisense orientation of the VIP receptor clone was introduced into HT-29 cells, there was a 50% suppression of the specific binding of 125I-VIP and of the VIP-induced increase in cAMP level, relative to untransfected cells. The VIP receptor cloned exhibits less than or equal to 24% homology with other receptors in the same superfamily and thus represents a subset of G protein-coupled receptors for peptide ligands. Images PMID:1675791

  13. Molecular cloning, pathologically-correlated expression and functional characterization of the colonystimulating factor 1 receptor (CSF-1R) gene from a teleost, Plecoglossus altivelis

    PubMed Central

    CHEN, Qiang; LU, Xin-Jiang; LI, Ming-Yun; CHEN, Jiong

    2016-01-01

    Colony-stimulating factor 1 receptor (CSF-1R) is an important regulator of monocytes/macrophages (MO/MΦ). Although several CSF-1R genes have been identified in teleosts, the precise role of CSF- 1R in ayu (Plecoglossus altivelis) remains unclear. In this study, we characterized the CSF-1R homologue from P. altivelis, and named it PaCSF-1R. Multiple sequence alignment and phylogenetic tree analysis showed that PaCSF-1R was most closely related to that of Japanese ricefish (Oryzias latipes). Tissue distribution and expression analysis showed that the PaCSF-1R transcript was mainly expressed in the head kidney-derived MO/MΦ, spleen, and head kidney, and its expression was significantly altered in various tissues upon Vibrio anguillarum infection. After PaCSF-1R neutralization for 48 h, the phagocytic activity of MO/MΦ was significantly decreased, suggesting that PaCSF-1R plays a role in regulating the phagocytic function of ayu MO/MΦ. PMID:27029867

  14. Molecular cloning and expression analysis of toll-like receptor genes (TLR7, TLR8 and TLR9) of golden pompano (Trachinotus ovatus).

    PubMed

    Wei, Youchuan; Hu, Shu; Sun, Baobao; Zhang, Qihuan; Qiao, Guo; Wang, Zisheng; Shao, Rong; Huang, Guoqiang; Qi, Zhitao

    2017-02-20

    Toll like receptor (TLR) 7, 8 and 9 are intracellular TLRs which play important roles in host immune defense against bacterial or virus pathogens. In this study, TLR7, 8 and 9 were identified from golden pompano (Trachinotus ovatus), a marine teleost with great economic values. Sequence analysis revealed that the three TLRs contained several conserved characteristic features, including signal peptides, 25 leucine-rich repeat (LRR) motifs, a transmembrane domain and a TIR motif. These three TLRs shared high sequence identity and similarity with their counterparts from other teleosts. The phylogenetic tree analysis showed the three TLRs were clustered well with their piscine counterparts, confirming the correctness of their nomenclatures and closed relationships during evolution. Quantitative real-time PCR revealed that the three TLRs were ubiquitously expressed in all the tested tissues from normal pompano, with high expression in spleen and head kidney, indicating their role in immune reaction. Further, pompano TLR7 and TLR8 was up-regulated in spleen and head kidney from 12 h to 48 h following polyI:C challenge, but remained no changes to Vibrio alginilyticus infection. In contrast, pompano TLR9 could be induced by V. alginilyticus infection but remained apathetic to polyI:C challenge. These results indicated that pompano TLR7, 8 and 9 might have distinct roles in response to bacterial or virus pathogens. Our results provided the basis for further study on ligand specificity and signaling pathways of fish TLRs which are required for elucidating the immune functions of fish TLRs.

  15. Two leptin genes and a leptin receptor gene of female chub mackerel (Scomber japonicus): Molecular cloning, tissue distribution and expression in different obesity indices and pubertal stages.

    PubMed

    Ohga, Hirofumi; Matsumori, Kojiro; Kodama, Ryoko; Kitano, Hajime; Nagano, Naoki; Yamaguchi, Akihiko; Matsuyama, Michiya

    2015-10-01

    Leptin is a hormone produced by fat cells that regulates the amount of fat stored in the body and conveys nutritional status to the reproductive axis in mammals. In the present study we identified two subtypes of leptin genes (lepa and lepb) and a leptin receptor gene (lepr) from chub mackerel (Scomber japonicus) and there gene expression under different feeding conditions (control and high-feed) and pubertal development stages was analyzed using quantitative real-time PCR. The protein lengths of LepA, LepB and LepR were 161 amino acids (aa), 163 aa and 1149 aa, respectively and both leptin subtypes shared only 15% similarity in aa sequences. In pubertal females, lepa was expressed in the brain, pituitary gland, liver, adipose tissue and ovary; however, in adult (gonadal maturation after the second in the life) females, lepa was expressed only in the liver. lepb was expressed primarily in the brain of all fish tested and was expressed strongly in the adipose tissue of adults. lepr was characterized by expression in the pituitary. The high-feed group showed a high conditioning factor level; unexpectedly, hepatic lepa and brain lepr were significantly more weakly expressed compared with the control-feed group. Furthermore, the expression levels of lepa, lepb and lepr genes showed no significant differences between pre-pubertal and post-pubertal fish. On the other hand, pituitary fshβ and lhβ showed no significant differences between different feeding groups of pre-pubertal fish. In contrast, fshβ and lhβ expressed abundantly in the post-pubertal fish of control feed group. Based on these results, whether leptin plays an important role in the nutritional status and pubertal onset of chub mackerel remains unknown.

  16. Expression cloning of a receptor for C5a anaphylatoxin on differentiated HL-60 cells

    SciTech Connect

    Boulay, F.; Mery, L.; Tardif, M.; Brouchon, L.; Vignais, P. )

    1991-03-26

    A cDNA clone encoding the human C5a anaphylatoxin receptor has been isolated by expression cloning from a CDM8 expression library prepared from mRNA of human myeloid HL-60 cells differentiated to the granulocyte phenotype with dibutyryladenosine cyclic monophosphate. The cDNA clone was able to transfer to COS-7 cells the capacity to specifically bind iodinated human recombinant C5a. The cDNA was 2.3 kb long, with an open reading frame encoding a 350-residue polypeptide. Cross-linking of iodinated C5a to the plasma membrane of transfected COS cells revealed a complex with an apparent molecular mass of 52-55 kDa, similar to that observed for the constitutively expressed receptor in differentiated HL-60 cells or human neutrophils. Although differentiated HL-60 cells display a single class of binding sites, with a dissociation constant of approximately 800-900 pM, the C5a-R cDNA, expressed in COS cells, generates both high-affinity (1.7 nM) and low-affinity (20-25 nM) receptors. Sequence comparison established that the degree of sequence identity between the C5a receptor and the N-formlypeptide receptor is 34%.

  17. Pathogenicity of molecularly cloned bovine leukemia virus.

    PubMed Central

    Rovnak, J; Boyd, A L; Casey, J W; Gonda, M A; Jensen, W A; Cockerell, G L

    1993-01-01

    To delineate the mechanisms of bovine leukemia virus (BLV) pathogenesis, four full-length BLV clones, 1, 8, 9, and 13, derived from the transformed cell line FLK-BLV and a clone construct, pBLV913, were introduced into bovine spleen cells by microinjection. Microinjected cells exhibited cytopathic effects and produced BLV p24 and gp51 antigens and infectious virus. The construct, pBLV913, was selected for infection of two sheep by inoculation of microinjected cells. After 15 months, peripheral blood mononuclear cells from these sheep served as inocula for the transfer of infection to four additional sheep. All six infected sheep seroconverted to BLV and had detectable BLV DNA in peripheral blood mononuclear cells after amplification by polymerase chain reaction. Four of the six sheep developed altered B/T-lymphocyte ratios between 33 and 53 months postinfection. One sheep died of unrelated causes, and one remained hematologically normal. Two of the affected sheep developed B lymphocytosis comparable to that observed in animals inoculated with peripheral blood mononuclear cells from BLV-infected cattle. This expanded B-lymphocyte population was characterized by elevated expression of B-cell surface markers, spontaneous blastogenesis, virus expression in vitro, and increased, polyclonally integrated provirus. One of these two sheep developed lymphocytic leukemia-lymphoma at 57 months postinfection. Leukemic cells had the same phenotype and harbored a single, monoclonally integrated provirus but produced no virus after in vitro cultivation. The range in clinical response to in vivo infection with cloned BLV suggests an important role for host immune response in the progression of virus replication and pathogenesis. Images PMID:8230433

  18. Molecular cloning of human terminal deoxynucleotidyltransferase.

    PubMed Central

    Peterson, R C; Cheung, L C; Mattaliano, R J; Chang, L M; Bollum, F J

    1984-01-01

    A cDNA of the human terminal deoxynucleotidyltransferase (TdT; "terminal transferase," EC 2.7.7.31) was isolated from a human lymphoblastoid cell cDNA library in lambda gt 11 by using immunological procedures. Four inserts containing 723 to 939 base pairs were recloned in pBR322 for hybridization and preliminary sequence studies. mRNA selected by hybridization to recombinant DNA was translated to a 58-kDa peptide that specifically immunoprecipitated with rabbit antibodies to calf terminal transferase and mouse monoclonal antibody to human terminal transferase. Blot hybridization of total poly(A)+ RNA from KM3 (TdT+) cells with nick-translated pBR322 recombinant DNA detected a message of about 2000 nucleotides, sufficient to code for the 580 amino acids in the protein. mRNA from terminal transferase- cells gave no signal in hybrid selection or RNA blot hybridization. The complete sequence of the 939-base-pair insert sequence was obtained from deletions cloned in pUC8. The DNA sequence contains an open reading frame coding for 238 amino acids, about 40% of the protein. Three peptides isolated by HPLC from tryptic digests of succinylated 58-kDa calf thymus terminal transferase were sequenced, providing 20, 18, and 22 residues of peptide sequence. A search of the translated sequence of the 939-base-pair insert shows three regions beginning after arginine that have greater than 90% homology with the sequence determined from the calf thymus terminal transferase peptides. These results provide unambiguous evidence that the human terminal transferase sequence has been cloned. Images PMID:6087320

  19. Actions of tilidine and nortilidine on cloned opioid receptors.

    PubMed

    Thierry, Christophe; Boeynaems, Jean-Marie; Paolo, Meoni

    2005-01-04

    Tilidine, alone or combined with naloxone to prevent drug abuse, is used as an oral opioid analgesic. Although the analgesic action of tilidine and its active metabolite nortilidine is reversed by naloxone and therefore believed to involve the activation of the Mu opioid (MOP, OP3, mu) receptor, this has never been studied in recombinant systems. We have measured the selectivity of tilidine and nortilidine for human opioid and opioid-like receptors stably expressed in CHO-K1 cells, using the inhibition of the forskolin (FK)-induced accumulation of cAMP as endpoint. In cells expressing the MOP receptor, tilidine and nortilidine inhibited cAMP accumulation with IC50 of 11 microM and 110 nM, respectively. The agonist effects of nortilidine and [D-Ala2-MePhe4-Gly5-ol]enkephalin (DAMGO) on the MOP receptor were reversed by naloxone with very similar IC50 (1.2 versus 1.8 nM). At concentrations up to 100 microM, tilidine and nortilidine had no agonist effect on the DOP, KOP and NOP receptors. In conclusion, this study on cloned human receptors demonstrates that nortilidine is a selective agonist of the MOP receptor.

  20. Endomorphins fully activate a cloned human mu opioid receptor.

    PubMed

    Gong, J; Strong, J A; Zhang, S; Yue, X; DeHaven, R N; Daubert, J D; Cassel, J A; Yu, G; Mansson, E; Yu, L

    1998-11-13

    Endomorphins were recently identified as endogenous ligands with high selectivity for mu opioid receptors. We have characterized the ability of endomorphins to bind to and functionally activate the cloned human mu opioid receptor. Both endomorphin-1 and endomorphin-2 exhibited binding selectivity for the mu opioid receptor over the delta and kappa opioid receptors. Both agonists inhibited forskolin-stimulated increase of cAMP in a dose-dependent fashion. When the mu opioid receptor was coexpressed in Xenopus oocytes with G protein-activated K+ channels, application of either endomorphin activated an inward K+ current. This activation was dose-dependent and blocked by naloxone. Both endomorphins acted as full agonists with efficacy similar to that of [D-Ala2,N-Me-Phe4,Gly-ol5]enkephalin (DAMGO). These data indicate that endomorphins act as full agonists at the human mu opioid receptor, capable of stimulating the receptor to inhibit the cAMP/adenylyl cyclase pathway and activate G-protein-activated inwardly rectifying potassium (GIRK) channels.

  1. Molecular cloning and characterization of multidomain xylanase from manure library

    USDA-ARS?s Scientific Manuscript database

    The gene (manf-x10) encoding xylanase from an environmental genomic DNA library was cloned and expressed in Escherichia coli. The encoded enzyme was predicted to be 467 amino acids with a molecular mass of 50.3 kD. The recombinant ManF-X10 was purified by HisTrap affinity column and showed activit...

  2. Advances and applications of molecular cloning in clinical microbiology.

    PubMed

    Sharma, Kamal; Mishra, Ajay Kumar; Mehraj, Vikram; Duraisamy, Ganesh Selvaraj

    2014-10-01

    Molecular cloning is based on isolation of a DNA sequence of interest to obtain multiple copies of it in vitro. Application of this technique has become an increasingly important tool in clinical microbiology due to its simplicity, cost effectiveness, rapidity, and reliability. This review entails the recent advances in molecular cloning and its application in the clinical microbiology in the context of polymicrobial infections, recombinant antigens, recombinant vaccines, diagnostic probes, antimicrobial peptides, and recombinant cytokines. Culture-based methods in polymicrobial infection have many limitation, which has been overcome by cloning techniques and provide gold standard technique. Recombinant antigens produced by cloning technique are now being used for screening of HIV, HCV, HBV, CMV, Treponema pallidum, and other clinical infectious agents. Recombinant vaccines for hepatitis B, cholera, influenza A, and other diseases also use recombinant antigens which have replaced the use of live vaccines and thus reduce the risk for adverse effects. Gene probes developed by gene cloning have many applications including in early diagnosis of hereditary diseases, forensic investigations, and routine diagnosis. Industrial application of this technology produces new antibiotics in the form of antimicrobial peptides and recombinant cytokines that can be used as therapeutic agents.

  3. Molecular cloning of a second subunit of the receptor for human granulocyte - macrophage colony-stimulating factor (GM-CSF): Reconstitution of a high-affinity GM-CSF receptor

    SciTech Connect

    Hayashida, Kazuhiro; Kitamura, Toshio; Gorman, D.M.; Miyajima, Atsushi ); Arai, Kenichi; Yokota, Takashi )

    1990-12-01

    Using the mouse interleukin 3 (IL-3) receptor cDNA as a probe, the authors obtained a monologous cDNA (KH97) from a cDNA library of a human hemopoietic cell line, TF-1. The protein encoded by the KH97 cDNA has 56% amino acid sequence identity with the mouse IL-3 receptor and retains features common to the family of cytokine receptors. Fibroblasts transfected with the KH97 cDNA expressed a protein of 120 kDa but did not bind any human cytokines, including IL-3 and granulocyte - macrophage colony-stimulating factor (GM-CSF). Interestingly, cotransfection of cDNAs for KH97 and the low-affinity human GM-CSF receptor in fibroblasts resulted in formation of a high-affinity receptor for GM-CSF. The dissociation rate of GM-CSF from the reconstituted high-affinity receptor was slower than that from the low-affinity site, whereas the association rate was unchanged. Cross-linking of {sup 125}I-labeled GM-CSF to fibroblasts cotransfected with both cDNAs revealed the same cross-linking patterns as in TF-1 cells - i.e., two major proteins of 80 and 120 kDa which correspond to the low-affinity GM-CSF receptor and the KH97 protein, respectively. These results indicate that the high-affinity GM-CSF receptor is composed of at least two components in a manner analogous to the IL-2 receptor. They therefore propose to designate the low-affinity GM-CSF receptor and the KH97 protein as the {alpha} and {beta} subunits of the GM-CSF receptor, respectively.

  4. Molecular characterization of opioid receptors

    SciTech Connect

    Howard, A.D.

    1986-01-01

    The aim of this research was to purify and characterize active opioid receptors and elucidate molecular aspects of opioid receptor heterogeneity. Purification to apparent homogeneity of an opioid binding protein from bovine caudate was achieved by solubilization in the non-ionic detergent, digitonin, followed by sequential chromatography on the opiate affinity matrix, ..beta..-naltrexylethylenediamine-CH-Sepharose 4B, and on the lectine affinity matrix, wheat germ agglutinin-agarose. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) followed by autoradiography revealed that radioiodinated purified receptor gave a single band. Purified receptor preparations showed a specific activity of 12,000-15,000 fmol of opiate bound per mg of protein. Radioiodinated human beta-endorphin (/sup 125/I-beta-end/sub H/) was used as a probe to investigate the ligand binding subunits of mu and delta opioid receptors. /sup 125/I-beta-end/sub H/ was shown to bind to a variety of opioid receptor-containing tissues with high affinity and specificity with preference for mu and delta sites, and with little, if any, binding to kappa sites. Affinity crosslinking techniques were employed to covalently link /sup 125/I-beta-end/sub H/ to opioid receptors, utilizing derivatives of bis-succinimidyl esters that are bifunctional crosslinkers with specificities for amino and sulfhydryl groups. This, and competition experiments with high type-selective ligands, permitted the assignment of two labeled peptides to their receptor types, namely a peptide of M/sub r/ = 65,000 for mu receptors and one of M/sub r/ = 53,000 for delta receptors.

  5. Molecular cloning of kisspeptin receptor genes (gpr54-1 and gpr54-2) and their expression profiles in the brain of a tropical damselfish during different gonadal stages.

    PubMed

    Imamura, Satoshi; Hur, Sung-Pyo; Takeuchi, Yuki; Bouchekioua, Selma; Takemura, Akihiro

    2017-01-01

    The kisspeptin receptor (GPR54) mediates neuroendocrine control of kisspeptin in the brain and acts as a gateway for a pulsatile release of hypothalamic gonadotropin-releasing hormone. This study aimed to clone two GPR54 genes (gpr54-1 and gpr54-2) from the brain of the sapphire devil Chrysiptera cyanea, a tropical damselfish, and to study their involvement in reproduction. The partial sequences of the sapphire devil gpr54-1 cDNA (1059bp) and gpr54-2 cDNA (1098bp) each had an open reading frame encoding a protein of 353 and 366 amino acids, respectively, both of which had structural features of a G-protein-coupled receptor. The expression of gpr54-1 mRNA was observed in the diencephalon and telencephalon, and gpr54-2 mRNA was found in the optic tectum of sapphire devil. When gpr54-1 and gpr54-2 mRNA levels were examined in the brain of sapphire devil by real-time quantitative polymerase chain reaction (qPCR), they were found to increase during late vitellogenesis and post-spawning. Treatment of fish with estradiol-17β (Ε2) resulted in an increase in gpr54-1 and gpr54-2 expression in the brain of sapphire devil. Thus, kisspeptin receptors likely mediate the activity of kisspeptin in the brain and are involved in controlling reproductive events in a tropical damselfish.

  6. Molecular and cytogenetic characterization of expanded B-cell clones from multiclonal versus monoclonal B-cell chronic lymphoproliferative disorders

    PubMed Central

    Henriques, Ana; Rodríguez-Caballero, Arancha; Criado, Ignacio; Langerak, Anton W.; Nieto, Wendy G.; Lécrevisse, Quentin; González, Marcos; Cortesão, Emília; Paiva, Artur; Almeida, Julia; Orfao, Alberto

    2014-01-01

    Chronic antigen-stimulation has been recurrently involved in the earlier stages of monoclonal B-cell lymphocytosis, chronic lymphocytic leukemia and other B-cell chronic lymphoproliferative disorders. The expansion of two or more B-cell clones has frequently been reported in individuals with these conditions; potentially, such coexisting clones have a greater probability of interaction with common immunological determinants. Here, we analyzed the B-cell receptor repertoire and molecular profile, as well as the phenotypic, cytogenetic and hematologic features, of 228 chronic lymphocytic leukemia-like and non-chronic lymphocytic leukemia-like clones comparing multiclonal (n=85 clones from 41 cases) versus monoclonal (n=143 clones) monoclonal B-cell lymphocytosis, chronic lymphocytic leukemia and other B-cell chronic lymphoproliferative disorders. The B-cell receptor of B-cell clones from multiclonal cases showed a slightly higher degree of HCDR3 homology than B-cell clones from mono clonal cases, in association with unique hematologic (e.g. lower B-lymphocyte counts) and cytogenetic (e.g. lower frequency of cytogenetically altered clones) features usually related to earlier stages of the disease. Moreover, a subgroup of coexisting B-cell clones from individual multiclonal cases which were found to be phylogenetically related showed unique molecular and cytogenetic features: they more frequently shared IGHV3 gene usage, shorter HCDR3 sequences with a greater proportion of IGHV mutations and del(13q14.3), than other unrelated B-cell clones. These results would support the antigen-driven nature of such multiclonal B-cell expansions, with potential involvement of multiple antigens/epitopes. PMID:24488564

  7. Molecular cloning and expression analysis of adiponectin and its receptors (AdipoR1 and AdipoR2) in the hypothalamus of the Huoyan goose during different stages of the egg-laying cycle.

    PubMed

    Cao, Zhongzan; Li, Juan; Luo, Lina; Li, Xiaoshuang; Liu, Mei; Gao, Ming; Yin, Yunhou; Luan, Xinhong

    2015-08-07

    Adiponectin and its receptors (AdipoR1 and AdipoR2) are novel endocrine systems that act at various levels to regulate metabolic homeostasis and reproductive processes. We cloned and characterized the cDNA of adiponectin and its receptors from the hypothalamus of the Huoyan goose to reveal the influence of these factors on the process of goose egg-laying. We also determined the mRNA and protein expression profiles during different stages of the egg-laying cycle. Hypothalamus tissues were obtained from 36 Huoyan geese in the pre-laying, early-laying, peak-laying, and ceased periods. The cDNA sequences of goose adiponectin and its receptors (AdipoR1 and AdipoR2) were cloned and characterized using the 5'-RACE and 3'-RACE methods. Multiple alignments and phylogenetic analyses of the deduced amino acid sequence were conducted using bioinformatics tools. The expression profiles of mRNA and protein in the hypothalamus during the pre-laying, early-laying, peak-laying and ceased periods were examined using real-time PCR (qRT-PCR) and Western blotting techniques. The cDNA of adiponectin, AdipoR1 and AdipoR2 consisted of 738, 1131 and 1161 bp open reading frame encoding 245, 376 and 386 amino acids, respectively. The deduced amino acid sequence of goose adiponectin, as well as AdipoR1 and AdipoR2 showed a closer genetic relationship to the avian species than to other mammal species. The expression level of adiponectin mRNA and protein increased from the pre-laying period to the peak-laying period, reached its peak in the peak-laying period, and then decreased during the ceased period. Conversely, the expression levels of AdipoR1 and AdipoR2 mRNA and protein decreased in the early-laying period, peak-laying period, and ceased period compared with the pre-laying period. This study is the first to obtain full-length cDNA sequences of goose adiponectin and the genes of its receptors from the hypothalamus, and demonstrate that the egg-laying cycle affects the expression of the

  8. Monkey corticotropin-releasing factor1 receptor: Complementary DNA cloning and pharmacological characterization.

    PubMed

    Oshida, Yuichi; Ikeda, Yoko; Chaki, Shigeyuki; Okuyama, Shigeru

    2004-02-27

    The full-length complementary DNA (cDNA) of monkey corticotropin-releasing factor type 1 (CRF1) receptor was isolated from a rhesus monkey (Macaca mulatta) amygdala cDNA library. The cloned monkey CRF1 receptor cDNA has 2,374 bp with an open reading frame encoding a 415-amino acid protein. The sequence of the monkey CRF1 receptor cDNA showed a high degree of sequence identity with other species of CRF1 receptors, and being 99.5% identical to human CRF1 receptors. When monkey CRF1 was expressed into COS-7 cells, high specific binding of [125I]-ovine CRF was observed. CRF and CRF-related peptides inhibited [125I]-ovine CRF binding in a concentration-dependent manner. IC50 values of ovine CRF, human/rat CRF, sauvagine and urotensin I were 23.5 +/- 7.4, 22.7 +/- 10.8, 27.5 +/- 12.3 and 14.2 +/- 7.0 nM, respectively. CRF1 receptor specific antagonists, such as CP-154,526, SC241 and CRA1000, also inhibited the [125I]-ovine CRF binding, with IC50 values of 3.9 +/- 0.4, 43.5 +/- 8.0 and 19.8 +/- 2.0 nM, respectively. GTP and its nonhydrolyzed analogue, GTPgammaS, reduced [125I]-ovine CRF binding, while ATP had a negligible effect, thereby indicating that the monkey CRF1 receptor belongs to a family of G-protein coupled receptors. CRF and its related peptides increased cyclic AMP formation concentration-dependently in COS-7 cells transiently expressing the monkey CRF1 receptor. Monkey CRF1 was expressed abundantly in the pituitary, cerebral cortex, hippocampus, amygdala and cerebellum. Thus the monkey CRF1 receptor and the human CRF1 receptor have similar molecular and pharmacological characteristics.

  9. Cloning and characterization of the ionotropic GABA receptor subunit ρ1 from pig (Sus scrofa).

    PubMed

    Reyes-Ruiz, Jorge Mauricio; Limon, Agenor; Miledi, Ricardo

    2014-01-13

    Since human and pig eyes have remarkably anatomical and physiological similitudes swine models have been broadly used for functional studies and therapeutic research. Recently, a GABAρ-mediated relaxation of retinal vascularity suggested that GABAρ signaling may be used to improve retinal blood flow in vascular-driven impaired vision, and a further molecular characterization of GABAρ receptors would be beneficial. However, none of the GABAρ type subunits from pigs has been yet cloned; Among the 19 subunits that compose the family of GABAA receptors, ρ1-3 subunits are capable of forming homomeric channels. These homomeric receptors are particularly interesting because their pharmacological and kinetic properties are notably different from receptors composed by other GABAA subunits. Here we report the cloning of the GABAρ1subunit from the pig and the functional expression of homomeric channels in Xenopus oocytes. The most notable difference found in the pig GABAρ1 receptor was the absence of a stretch of 17 amino acids near the amino terminus (R41-V58) conserved in the rat and the human. This sequence has a higher nucleotidic match with the transcript variant 2 of the human GABAρ1 subunit. Xenopus oocytes injected with cRNA from the receptor generated currents when exposed to GABA that shared all the characteristics of other GABAρ1 subunits in mammals, including its modulation by dopamine. This study will help to increase the knowledge of the genetics of the pig, further the understanding of this important neurotransmitter receptor family and will shed some light in the evolution of these genes among mammals.

  10. Molecular cloning and amino acid sequence of human 5-lipoxygenase

    SciTech Connect

    Matsumoto, T.; Funk, C.D.; Radmark, O.; Hoeoeg, J.O.; Joernvall, H.; Samuelsson, B.

    1988-01-01

    5-Lipoxygenase (EC 1.13.11.34), a Ca/sup 2 +/- and ATP-requiring enzyme, catalyzes the first two steps in the biosynthesis of the peptidoleukotrienes and the chemotactic factor leukotriene B/sub 4/. A cDNA clone corresponding to 5-lipoxygenase was isolated from a human lung lambda gt11 expression library by immunoscreening with a polyclonal antibody. Additional clones from a human placenta lambda gt11 cDNA library were obtained by plaque hybridization with the /sup 32/P-labeled lung cDNA clone. Sequence data obtained from several overlapping clones indicate that the composite DNAs contain the complete coding region for the enzyme. From the deduced primary structure, 5-lipoxygenase encodes a 673 amino acid protein with a calculated molecular weight of 77,839. Direct analysis of the native protein and its proteolytic fragments confirmed the deduced composition, the amino-terminal amino acid sequence, and the structure of many internal segments. 5-Lipoxygenase has no apparent sequence homology with leukotriene A/sub 4/ hydrolase or Ca/sup 2 +/-binding proteins. RNA blot analysis indicated substantial amounts of an mRNA species of approx. = 2700 nucleotides in leukocytes, lung, and placenta.

  11. Cloning

    MedlinePlus

    Cloning describes the processes used to create an exact genetic replica of another cell, tissue or organism. ... named Dolly. There are three different types of cloning: Gene cloning, which creates copies of genes or ...

  12. Entamoeba invadens: cloning and molecular characterization of chitinases.

    PubMed

    Dey, Tuli; Basu, Raunak; Ghosh, Sudip K

    2009-11-01

    Entamoeba histolytica, the causative agent of amebiasis infects through its cyst form and this transmission may be blocked using encystation specific protein as drug target. In this study, we have characterized the enzyme chitinase which express specifically during encystation. The reptilian parasite Entamoeba invadens, used as a model for encystation study contain three chitinases. We report the molecular cloning, over-expression and biochemical characterization of all three E. invadens chitinase. Cloned chitinases were over-expressed in bacterial system and purified by affinity chromatography. Their enzymatic profiles and substrate cleaving patterns were characterized. All of them showed binding affinity towards insoluble chitin though two of them lack the chitin binding domain. All the chitinases cleaved and released dimmers from the insoluble substrate and act as an exochitinase. Homology modeling was also done to understand the substrate binding and cleavage pattern.

  13. Molecular cloning of a glycosylphosphatidylinositol-anchored molecule CDw108.

    PubMed

    Yamada, A; Kubo, K; Takeshita, T; Harashima, N; Kawano, K; Mine, T; Sagawa, K; Sugamura, K; Itoh, K

    1999-04-01

    CDw108, also known as the John-Milton-Hagen human blood group Ag, is an 80-kDa glycosylphosphatidylinositol (GPI)-anchored membrane glycoprotein that is preferentially expressed on activated lymphocytes and E. The molecular characteristics and biological function of the CDw108 were not clarified previously. In this manuscript, we identify the cDNA clone containing the entire coding sequence of the CDw108 gene and report its molecular characteristics. The 1998-base pairs of the open reading frame of the cloned cDNA encoded a protein of 666 amino acids (aa), including the 46 aa of the signal peptide and the 19 aa of the GPI-anchor motif. Thus, the membrane-anchoring form of CDw108 was the 602 aa, and the estimated molecular mass of the unglycosylated form was 68 kDa. The RGD (Arg-Gly-Asp) cell attachment sequence and the five potential N-linked glycosylation sites were located on the membrane-anchoring form. Flow cytometric and immunoprecipitation analyses of the CDw108 cDNA transfectants confirmed that the cloned cDNA encoded the native form of CDw108. The CDw108 mRNA was expressed in activated PBMCs as well as in the spleen, thymus, testis, placenta, and brain, but was not expressed in any other tissues tested. Radiation hybrid mapping indicated that the CDw108 gene was located in the middle of the long arm of chromosome 15 (15q23-24). This molecular information will be critical for understanding the biological function of the CDw108 Ag.

  14. Teaching molecular genetics: chapter 4-positional cloning of genetic disorders.

    PubMed

    Puliti, Aldamaria; Caridi, Gianluca; Ravazzolo, Roberto; Ghiggeri, Gian Marco

    2007-12-01

    Positional cloning is the approach of choice for the identification of genetic mutations underlying the pathological development of diseases with simple Mendelian inheritance. It consists of different consecutive steps, starting with recruitment of patients and DNA collection, that are critical to the overall process. A genetic analysis of the enrolled patients and their families is performed, based on genetic recombination frequencies generated by meiotic cross-overs and on genome-wide molecular studies, to define a critical DNA region of interest. This analysis culminates in a statistical estimate of the probability that disease features may segregate in the families independently or in association with specific molecular markers located in known regions. In this latter case, a marker can be defined as being linked to the disease manifestations. The genetic markers define an interval that is a function of their recombination frequencies with the disease, in which the disease gene is localised. The identification and characterisation of chromosome abnormalities as translocations, deletions and duplications by classical cytogenetic methods or by the newly developed microarray-based comparative genomic hybridisation (array CGH) technique may define extensions and borders of the genomic regions involved. The step following the definition of a critical genomic region is the identification of candidate genes that is based on the analysis of available databases from genome browsers. Positional cloning culminates in the identification of the causative gene mutation, and the definition of its functional role in the pathogenesis of the disorder, by the use of cell-based or animal-based experiments. More often, positional cloning ends with the generation of mice with homologous mutations reproducing the human clinical phenotype. Altogether, positional cloning has represented a fundamental step in the research on genetic renal disorders, leading to the definition of several

  15. Ancient DNA: extraction, characterization, molecular cloning, and enzymatic amplification.

    PubMed

    Pääbo, S

    1989-03-01

    Several chemical and enzymatic properties were examined in the DNA extracted from dry remains of soft tissues that vary in age from 4 to 13,000 years and represent four species, including two extinct animals (the marsupial wolf and giant ground sloth). The DNA obtained was invariably of a low average molecular size and damaged by oxidative processes, which primarily manifest themselves as modifications of pyrimidines and sugar residues as well as baseless sites and intermolecular cross-links. This renders molecular cloning difficult. However, the polymerase chain reaction can be used to amplify and study short mitochondrial DNA sequences that are of anthropological and evolutionary significance. This opens up the prospect of performing diachronical studies of molecular evolutionary genetics.

  16. Cloning and identification of an oxytocin/vasopressin-like receptor and its ligand from insects.

    PubMed

    Stafflinger, Elisabeth; Hansen, Karina K; Hauser, Frank; Schneider, Martina; Cazzamali, Giuseppe; Williamson, Michael; Grimmelikhuijzen, Cornelis J P

    2008-03-04

    knowledge, this is the first report on the molecular cloning of an oxytocin/vasopressin-like receptor and its ligand from arthropods.

  17. Isolation of rat genomic clones encoding subtypes of the alpha 2-adrenergic receptor. Identification of a unique receptor subtype.

    PubMed

    Lanier, S M; Downing, S; Duzic, E; Homcy, C J

    1991-06-05

    alpha 2-Adrenergic receptors (alpha 2-AR) exist as subtypes that are expressed in a tissue-specific manner and differ in 1) their ligand recognition properties, 2) their extent of receptor protein glycosylation, and possible 3) their mechanism of signal transduction. Genomic or cDNA clones encoding three receptor subtypes have been characterized; however, both functional and radioligand binding studies in rodents suggest the existence of a fourth receptor subtype. To isolate the rat genes encoding receptor subtypes we screened a rat genomic library with an oligonucleotide probe encompassing the third membrane span of the human C-4 alpha 2-AR. Two intronless rat genes were isolated that encode distinct receptor subtypes (RG10, RG20). RG10 and RG20 encode proteins of 458 and 450 amino acids, respectively, that are 56% homologous and possess the structural features expected of this class of membrane-bound receptors. RG10 identifies a mRNA species of approximately 2500 nucleotides that is found primarily in brain, whereas RG20 identifies a larger mRNA species (approximately 4000 nucleotides) that is found in several tissues including brain, kidney, and salivary gland. RG10 is 88% homologous to the human C-4 alpha 2-AR and exhibits similar binding properties ( [3H]rauwolscine KD = 0.7 +/- 0.3 nM) as determined following transient expression of the receptor in COS-1 cells. RG20 exhibits ligand binding properties distinct from the three receptor subtypes identified by molecular cloning. Saturation binding studies indicate an affinity constant of 15 +/- 1.2 nM for the alpha 2-AR antagonist [3H]rauwolscine, a value 6-20 times higher than that observed for the three cloned receptor subtypes. In competition binding studies the potency order of competing ligands for RG20 is phentolamine greater than idazoxan greater than yohimbine greater than rauwolscine greater than prazosin. Of the three previously cloned alpha 2-AR, RG20 is most closely related to the human C-10 alpha 2-AR

  18. Cloning and preliminary pharmacological characterization of the anaphylatoxin C5a receptor in the rabbit.

    PubMed

    Bachvarov, D R; Houle, S; Bachvarova, M; Bouthillier, J; St-Pierre, S A; Fukuoka, Y; Ember, J A; Marceau, F

    1999-09-01

    1 The rabbit receptor for C5a was cloned from a genomic library and found to be 79.5% identical to the human homologue, the highest degree of similarity found so far in nonprimate laboratory animals. 2 The rabbit C5a receptor stably expressed in RBL cells binds human 125I-C5a (2 nM). Unlabelled C5a and the C-terminal analogue N-acetyl-Tyr-Ser-Phe-Lys-Pro-Met-Pro-Leu-D-Ala-Arg (Ac-YSFKPMPLaR) were found to be competitors of that binding, the peptide analogue retaining approximately 0.1% of the affinity of human C5a. 3 The order of potency human C5a>Ac-YSFKPMPLaR was conserved in bioassays based on rabbits (relaxation of the isolated portal vein and pulmonary artery; acute in vivo neutropenia), but with a decreasing potency gap between the two compounds, a likely consequence of the resistance to peptidases of the analogue. 4 The molecular definition of the rabbit C5a receptor evidenced a high preservation degree of sequence and pharmacologic properties relative to the human ortholog receptor, thus defining a set of molecular tools for the investigation of the role of C5a in physiologic and pathologic models based on the rabbit (e.g. atherosclerosis, inflammation).

  19. Cloning of the mouse GABA-benzodiazepine receptor. alpha. 1 subunit in a study of alcohol neurosensitivity

    SciTech Connect

    Keir, W.J.; Deitrich, R.A.; Sikela, J.M. )

    1989-02-09

    The inhibitory action of gamma amino butyric acid (GABA) is mediated by its binding to the benzodiazepine (BDZ) receptor and opening of a chloride channel. This receptor contains a variety of binding sites for several behavorially active drugs. Recent studies with SS and LS mice which were selected for differential neurosensitivity to ethanol, suggest that the GABAergic system plays a role in this differential sensitivity. Thus genes controlling the GABAergic system may also influence the acute hypnotic actions of ethanol. As a fist step towards verifying this hypothesis we have cloned and partially sequenced the mouse GABA-BDZ {alpha}1 subunit cDNA using a 40 bp oligonucleotide derived from the N terminus of a published bovine {alpha} subunit cDNA. A positive clone from a mouse brain cDNA library was identified and contains an insert of approximately 2.5 Kb. Partial sequence analysis indicates that this clone corresponds to the mouse homolog of the {alpha}1 subunit of the GABA-BDZ receptor. This clone is being used as a probe to identify restriction fragment length polymorphisms in several mouse genotypes which differ in their neurosensitivity to ethanol in an attempt to identify molecular genetic changes in the GABA-BDZ receptor that are related to differential ethanol neurosensitivity.

  20. Molecular cloning of canine co-chaperone small glutamine-rich tetratricopeptide repeat-containing protein α (SGTA) and investigation of its ability to suppress androgen receptor signalling in androgen-independent prostate cancer.

    PubMed

    Kato, Yuiko; Ochiai, Kazuhiko; Michishita, Masaki; Azakami, Daigo; Nakahira, Rei; Morimatsu, Masami; Ishiguro-Oonuma, Toshina; Yoshikawa, Yasunaga; Kobayashi, Masato; Bonkobara, Makoto; Kobayashi, Masanori; Takahashi, Kimimasa; Watanabe, Masami; Omi, Toshinori

    2015-11-01

    Although the morbidity of canine prostate cancer is low, the majority of cases present with resistance to androgen therapy and poor clinical outcomes. These pathological conditions are similar to the signs of the terminal stage of human androgen-independent prostate cancer. The co-chaperone small glutamine-rich tetratricopeptide repeat-containing protein α (SGTA) is known to be overexpressed in human androgen-independent prostate cancer. However, there is little information about the structure and function of canine SGTA. In this study, canine SGTA was cloned and analysed for its ability to suppress androgen receptor signalling. The full-length open reading frame (ORF) of the canine SGTA gene was amplified by RT-PCR using primers designed from canine-expressed sequence tags that were homologous to human SGTA. The canine SGTA ORF has high homology with the corresponding human (89%) and mouse (81%) sequences. SGTA dimerisation region and tetratricopeptide repeat (TPR) domains are conserved across the three species. The ability of canine SGTA to undergo homodimerisation was demonstrated by a mammalian two-hybrid system and a pull-down assay. The negative impact of canine SGTA on androgen receptor (AR) signalling was demonstrated using a reporter assay in androgen-independent human prostate cancer cell lines. Pathological analysis showed overexpression of SGTA in canine prostate cancer, but not in hyperplasia. A reporter assay in prostate cells demonstrated suppression of AR signalling by canine SGTA. Altogether, these results suggest that canine SGTA may play an important role in the acquisition of androgen independence by canine prostate cancer cells. Copyright © 2015 Elsevier Ltd. All rights reserved.

  1. Molecular cloning, characterization and expression profile of kisspeptin1 and kisspeptin1 receptor at brain-pituitary-gonad (BPG) axis of golden mahseer, Tor putitora (Hamilton, 1822) during gonadal development.

    PubMed

    Shahi, Neetu; Singh, Atul Kumar; Sahoo, Monalisa; Mallik, Sumanta Kumar; Thakuria, Dimpal

    2017-03-01

    Complete cDNA sequences of kiss1 (gmkiss1) and its receptor kiss1r (gmkiss1r) were cloned and characterized from brain tissue of adult golden mahseer (Tor putitora). Thereafter, quantification of gmkiss1 and gmkiss1r mRNA expression in brain-pituitary-gonad (BPG) axis of male and female golden mahseer was carried out using quantitative real-time (qRT)-PCR assay during an annual reproductive cycle, at different gonadal development stages. The gmkiss1 cDNA was 508bp, with 330bp open reading frame (ORF), encoding a precursor protein of 109 amino acids, whereas gmkiss1r cDNA was 1383bp with an ORF of 1004bp, which encodes a 334 amino acid protein residue. The qRT-PCR study shows that gmkiss1 and gmkiss1r are expressed in brain, pituitary and gonads of both the sexes of golden mahseer. An apparent sexual dimorphism in transcript level of gmkiss1 and gmkiss1r in brain and gonads was evident during the reproductive cycle. Overall, in brain, testis and ovary, the gmkiss1 and gmkiss1r mRNA expression level was comparatively higher during the initial stages of gonadal development, than that of spermiation or ovulation stage. In pituitary of both the sexes, throughout the gonadal development, consistently low transcript level of gmkiss1 and gmkiss1r was observed. The gmkiss1 mRNA expression level in brain and ovary of female golden mahseer was several folds higher than the brain and testis of male fish. In conclusion, we confirm the presence of kiss1 and its receptor in golden mahseer, and results of our study strongly suggested the involvement of kisspeptin1 system in gonadal development and annual reproductive cycle of this species.

  2. Cloning, functional expression, and characterization of the human prostaglandin E2 receptor EP2 subtype.

    PubMed

    Bastien, L; Sawyer, N; Grygorczyk, R; Metters, K M; Adam, M

    1994-04-22

    A cDNA clone encoding the human prostaglandin (PG) E2 receptor EP2 subtype has been isolated from a human lung cDNA library. The 1.9-kilobase pair cDNA, hEP2, encodes for a 488-amino acid protein with a predicted molecular mass of 53,115 and has the seven putative transmembrane domains characteristic of G protein-coupled receptors. The specific binding of [3H]PGE2 to COS cell membranes transfected with the hEP2 cDNA was of high affinity with an equilibrium dissociation constant (Kd) of 1 nM and the rank order of potency for prostaglandins in competition for [3H]PGE2 specific binding was PGE1 = PGE2 > iloprost > PGF2 alpha > PGD2. In competition studies using more selective prostanoid-receptor agonist and antagonists, the [3H]PGE2 specific binding was competed by MB28767, an EP3 agonist, but not by the EP1-preferring antagonists AH6809 and SC19220, or by the EP2 agonist butaprost. Electrophysiological studies of Xenopus oocytes co-injected with hEP2 and cystic fibrosis transmembrane conductance regulator (cAMP-activated Cl- channel) cDNAs detected PGE2-specific inward Cl- currents, demonstrating that the hEP2 cDNA encoded a functional receptor which produced an increase in cAMP levels. Thus, we have cloned the human EP2 receptor subtype which is functionally coupled to increase in cAMP. Northern blot analysis showed that hEP2 is expressed as a 3.8-kilobase mRNA in a number of human tissues with the highest expression levels present in the small intestine.

  3. Cloning and Characterisation of Schistosoma japonicum Insulin Receptors

    PubMed Central

    You, Hong; Zhang, Wenbao; Jones, Malcolm K.; Gobert, Geoffrey N.; Mulvenna, Jason; Rees, Glynn; Spanevello, Mark; Blair, David; Duke, Mary; Brehm, Klaus; McManus, Donald P.

    2010-01-01

    Background Schistosomes depend for growth and development on host hormonal signals, which may include the insulin signalling pathway. We cloned and assessed the function of two insulin receptors from Schistosoma japonicum in order to shed light on their role in schistosome biology. Methodology/Principal Findings We isolated, from S. japonicum, insulin receptors 1 (SjIR-1) and 2 (SjIR-2) sharing close sequence identity to their S. mansoni homologues (SmIR-1 and SmIR-2). SjIR-1 is located on the tegument basal membrane and the internal epithelium of adult worms, whereas SjIR-2 is located in the parenchyma of males and the vitelline tissue of females. Phylogenetic analysis showed that SjIR-2 and SmIR-2 are close to Echinococcus multilocularis insulin receptor (EmIR), suggesting that SjIR-2, SmIR-2 and EmIR share similar roles in growth and development in the three taxa. Structure homology modelling recovered the conserved structure between the SjIRs and Homo sapiens IR (HIR) implying a common predicted binding mechanism in the ligand domain and the same downstream signal transduction processing in the tyrosine kinase domain as in HIR. Two-hybrid analysis was used to confirm that the ligand domains of SjIR-1 and SjIR-2 contain the insulin binding site. Incubation of adult worms in vitro, both with a specific insulin receptor inhibitor and anti-SjIRs antibodies, resulted in a significant decrease in worm glucose levels, suggesting again the same function for SjIRs in regulating glucose uptake as described for mammalian cells. Conclusions Adult worms of S. japonicum possess insulin receptors that can specifically bind to insulin, indicating that the parasite can utilize host insulin for development and growth by sharing the same pathway as mammalian cells in regulating glucose uptake. A complete understanding of the role of SjIRs in the biology of S. japonicum may result in their use as new targets for drug and vaccine development against schistosomiasis. PMID:20352052

  4. cDNA cloning, molecular characterization, and chromosomal localization of NET(EPHT2), a human EPH-related receptor protein-tyrosine kinase gene preferentially expressed in brain

    SciTech Connect

    Tang, X.X.; Yoshioka, A.; Pleasure, D.E.

    1995-09-20

    By screening a human fetal brain cDNA expression library using a monoclonal anti-phosphotyrosine antibody , we have isolated a cDNA clone encoding a receptor type protein-tyrosine kinase belonging to the EPH family, NET (neuronally expressed EPH-related tyrosine kinase). NET shows 87% homology in nucleotide sequence and 99% homology in the deduced amino acid sequence to rat elk, suggesting that NET is the human homologue of elk. The NET gene is mapped to human chromosome 3q21-q23 by PCR screening of a human rodent somatic cell hybrid panel and by fluorescence in situ hybridization. Examination of NET mRNA expression in several human tissues has shown that the NET gene is expressed preferentially in brain as a 5-kb transcript. Steady-state levels of NET mRNA in human brain are greater in the midterm fetus than in the adult. Lower levels of NET mRNA are found in fetal kidney and adult skeletal muscle. The expression pattern of NET mRNA thus differs from that of elk, suggesting that these two gene products may preform distinct roles in human and rat. NET transcripts are detected in human acid-induced neuronal differentiation. Several human tumor cell lines derived from neuroectoderm including primitive neuroblastoma also express NET transcripts. Since the NET mRNA expression in human brain is developmentally regulated and is induced during neuronal differentiation, NET potentially plays important roles in human neurogenesis. 89 refs., 7 figs.

  5. Molecular cloning, mapping to human chromosome 1 q21-q23, and cell binding characteristics of Spalpha, a new member of the scavenger receptor cysteine-rich (SRCR) family of proteins.

    PubMed

    Gebe, J A; Kiener, P A; Ring, H Z; Li, X; Francke, U; Aruffo, A

    1997-03-07

    CD5 and CD6, two type I cell surface antigens predominantly expressed by T cells and a subset of B cells, have been shown to function as accessory molecules capable of modulating T cell activation. Here we report the cloning of a cDNA encoding Spalpha, a secreted protein that is highly homologous to CD5 and CD6. Spalpha has the same domain organization as the extracellular region of CD5 and CD6 and is composed of three SRCR (scavenger receptor cysteine rich) domains. Chromosomal mapping by fluorescence in situ hybridization and radiation hybrid panel analysis indicated that the gene encoding Spalpha is located on the long arm of human chromosome 1 at q21-q23 within contig WC1.17. RNA transcripts encoding Spalpha were found in human bone marrow, spleen, lymph node, thymus, and fetal liver but not in non-lymphoid tissues. Cell binding studies with an Spalpha immunoglobulin (Spalpha-mIg) fusion protein indicated that Spalpha is capable of binding to peripheral monocytes but not to T or B cells. Spalpha-mIg was also found to bind to the monocyte precursor cell lines K-562 and weakly to THP-1 but not to U937. Spalpha-mIg also bound to the B cell line Raji and weakly to the T cell line HUT-78. These findings indicate that Spalpha, a novel secreted protein produced in lymphoid tissues, may regulate monocyte activation, function, and/or survival.

  6. Rapid extraction and purification of environmental DNA for molecular cloning applications and molecular diversity studies.

    PubMed

    Santosa, D A

    2001-01-01

    A rapid method for the extraction and purification of DNA from environmental samples for molecular cloning applications was developed. The indigenous cells from plant debris, organic materials, sediments, and soils were lysed directly by using DAS-IZ solution and the nucleic acids were precipitated with isopropanol. A simple purification step using DAS-IIZ solution without binding matrix produced highly pure, colorless and undegraded DNA with molecular weight of more than 20 kb. The superiority of this method was tested for wide applications in molecular cloning, i.e., construction of genomic library by using Lambda DASHII Vector and GigapackIII XL, plasmid library, cloning of gene encoding protease, and molecular microbial diversity analysis. An additional advantage of this method is that only 0.1 g of sample is required, if analysis of many samples in short time should be done. To extract large amounts of environmental DNA for molecular cloning lasts only 30 min and to purify it less than 1 h.

  7. Dopamine receptor genes: new tools for molecular psychiatry.

    PubMed Central

    Niznik, H B; Van Tol, H H

    1992-01-01

    For over a decade it has been generally assumed that all the pharmacological and biochemical actions of dopamine within the central nervous system and periphery were mediated by two distinct dopamine receptors. These receptors, termed D1 and D2, were defined as those coupled to the stimulation or inhibition of adenylate cyclase, respectively, and by their selectivity and avidity for various drugs and compounds. The concept that two dopamine receptors were sufficient to account for all the effects mediated by dopamine was an oversimplification. Recent molecular biological studies have identified five distinct genes which encode at least eight functional dopamine receptors. The members of the expanded dopamine receptor family, however, can still be codifed by way of the original D1 and D2 receptor dichotomy. These include two genes encoding dopamine D1-like receptors (D1 [D1A]/D5 [D1B]) and three genes encoding D2-like receptors (D2/D3/D4). We review here our recent work on the cloning and characterization of some of the members of the dopamine receptor gene family (D1, D2, D4, D5), their relationship to neuropsychiatric disorders and their potential role in antipsychotic drug action. Images Fig. 1 PMID:1450188

  8. Functional properties of a cloned 5-hydroxytryptamine ionotropic receptor subunit: comparison with native mouse receptors.

    PubMed Central

    Hussy, N; Lukas, W; Jones, K A

    1994-01-01

    1. A comparative study of the whole-cell and single-channel properties of cloned and native mouse 5-hydroxytryptamine ionotropic receptors (5-HT3) was undertaken using mammalian cell lines expressing the cloned 5-HT3 receptor subunit A (5-HT3R-A), superior cervical ganglia (SCG) neurones and N1E-115 cells. 2. No pharmacological difference was found in the sensitivity to the agonists 5-HT and 2-methyl-5-HT, or to the antagonists d-tubocurare and 3-tropanyl-3,5-dichlorobenzoate (MDL-72222). 3. Current-voltage (I-V) relationships of whole-cell currents showed inward rectification in the three preparations. Rectification was stronger both in cells expressing the 5-HT3R-A subunit and in N1E-115 cells when compared with SCG neurones. 4. No clear openings could be resolved in 5-HT-activated currents in patches excised from cells expressing the 5-HT3R-A subunit or N1E-115 cells. Current fluctuation analysis of whole-cell and excised-patch records revealed a slope conductance of 0.4-0.6 pS in both preparations. Current-voltage relationships of these channels showed strong rectification that fully accounted for the whole-cell voltage dependence. 5. In contrast, single channels of about 10 pS were activated by 5-HT in patches excised from SCG neurones. The weak voltage dependence of their conductance did not account completely for the rectification of whole-cell currents. A lower unitary conductance (3.4 pS) was inferred from whole-cell noise analysis. 6. We conclude that the receptor expressed from the cloned cDNA is indistinguishable from the 5-HT3 receptor of N1E-115 cells, suggesting an identical structure for these two receptors. The higher conductance and different voltage dependence of the 5-HT3 receptor in SCG neurones might indicate the participation of an additional subunit in the structure of native ganglionic 5-HT3 receptors. Homo-oligomeric 5-HT3R-A channels may also be present as suggested by the lower conductance estimated by whole-cell noise analysis. PMID

  9. A highly efficient molecular cloning platform that utilises a small bacterial toxin gene.

    PubMed

    Mok, Wendy W K; Li, Yingfu

    2013-04-15

    Molecular cloning technologies that have emerged in recent years are more efficient and simpler to use than traditional strategies, but many have the disadvantages of requiring multiple steps and expensive proprietary enzymes. We have engineered cloning vectors containing variants of IbsC, a 19-residue toxin from Escherichia coli K-12. These toxic peptides offer selectivity to minimise the background, labour, and cost associated with conventional molecular cloning. As demonstrated with the cloning of reporter genes, this "detox cloning" system consistently produced over 95 % positive clones. Purification steps between digestion and ligation are not necessary, and the total time between digestion and plating of transformants can be as little as three hours. Thus, these IbsC-based cloning vectors are as reliable and amenable to high-throughput cloning as commercially available systems, and have the advantage of being more time-efficient and cost-effective.

  10. Molecular identification, cloning and characterization of transmitted/founder HIV-1 subtype A, D and A/D infectious molecular clones

    PubMed Central

    Baalwa, Joshua; Wang, Shuyi; Parrish, Nicholas; Decker, Julie M.; Keele, Brandon F.; Learn, Gerald H.; Yue, Ling; Ruzagira, Eugene; Ssemwanga, Deogratius; Kamali, Anatoli; Amornkul, Pauli N.; Price, Matt A.; Kappes, John C.; Karita, Etienne; Kaleebu, Pontiano; Sanders, Eduard; Gilmour, Jill; Allen, Susan; Hunter, Eric; Montefiori, David C.; Haynes, Barton F.; Cormier, Emmanuel; Hahn, Beatrice H.; Shaw, George M.

    2012-01-01

    We report the molecular identification, cloning and initial biological characterization of 12 full-length HIV-1 subtype A, D and A/D recombinant transmitted/founder (T/F) genomes. T/F genomes contained intact canonical open reading frames and all T/F viruses were replication competent in primary human T-cells, although subtype D virus replication was more efficient (p<0.05). All 12 viruses utilized CCR5 but not CXCR4 as a co-receptor for entry and exhibited a neutralization profile typical of tier 2 primary virus strains, with significant differences observed between subtype A and D viruses with respect to sensitivity to monoclonal antibodies VRC01, PG9 and PG16 and polyclonal subtype C anti-HIV IgG (p<0.05 for each). The present report doubles the number of T/F HIV-1 clones available for pathogenesis and vaccine research and extends their representation to include subtypes A, B, C and D. PMID:23123038

  11. Ab initio multiple cloning algorithm for quantum nonadiabatic molecular dynamics

    NASA Astrophysics Data System (ADS)

    Makhov, Dmitry V.; Glover, William J.; Martinez, Todd J.; Shalashilin, Dmitrii V.

    2014-08-01

    We present a new algorithm for ab initio quantum nonadiabatic molecular dynamics that combines the best features of ab initio Multiple Spawning (AIMS) and Multiconfigurational Ehrenfest (MCE) methods. In this new method, ab initio multiple cloning (AIMC), the individual trajectory basis functions (TBFs) follow Ehrenfest equations of motion (as in MCE). However, the basis set is expanded (as in AIMS) when these TBFs become sufficiently mixed, preventing prolonged evolution on an averaged potential energy surface. We refer to the expansion of the basis set as "cloning," in analogy to the "spawning" procedure in AIMS. This synthesis of AIMS and MCE allows us to leverage the benefits of mean-field evolution during periods of strong nonadiabatic coupling while simultaneously avoiding mean-field artifacts in Ehrenfest dynamics. We explore the use of time-displaced basis sets, "trains," as a means of expanding the basis set for little cost. We also introduce a new bra-ket averaged Taylor expansion (BAT) to approximate the necessary potential energy and nonadiabatic coupling matrix elements. The BAT approximation avoids the necessity of computing electronic structure information at intermediate points between TBFs, as is usually done in saddle-point approximations used in AIMS. The efficiency of AIMC is demonstrated on the nonradiative decay of the first excited state of ethylene. The AIMC method has been implemented within the AIMS-MOLPRO package, which was extended to include Ehrenfest basis functions.

  12. Ab initio multiple cloning algorithm for quantum nonadiabatic molecular dynamics.

    PubMed

    Makhov, Dmitry V; Glover, William J; Martinez, Todd J; Shalashilin, Dmitrii V

    2014-08-07

    We present a new algorithm for ab initio quantum nonadiabatic molecular dynamics that combines the best features of ab initio Multiple Spawning (AIMS) and Multiconfigurational Ehrenfest (MCE) methods. In this new method, ab initio multiple cloning (AIMC), the individual trajectory basis functions (TBFs) follow Ehrenfest equations of motion (as in MCE). However, the basis set is expanded (as in AIMS) when these TBFs become sufficiently mixed, preventing prolonged evolution on an averaged potential energy surface. We refer to the expansion of the basis set as "cloning," in analogy to the "spawning" procedure in AIMS. This synthesis of AIMS and MCE allows us to leverage the benefits of mean-field evolution during periods of strong nonadiabatic coupling while simultaneously avoiding mean-field artifacts in Ehrenfest dynamics. We explore the use of time-displaced basis sets, "trains," as a means of expanding the basis set for little cost. We also introduce a new bra-ket averaged Taylor expansion (BAT) to approximate the necessary potential energy and nonadiabatic coupling matrix elements. The BAT approximation avoids the necessity of computing electronic structure information at intermediate points between TBFs, as is usually done in saddle-point approximations used in AIMS. The efficiency of AIMC is demonstrated on the nonradiative decay of the first excited state of ethylene. The AIMC method has been implemented within the AIMS-MOLPRO package, which was extended to include Ehrenfest basis functions.

  13. Ab initio multiple cloning algorithm for quantum nonadiabatic molecular dynamics

    SciTech Connect

    Makhov, Dmitry V.; Shalashilin, Dmitrii V.; Glover, William J.; Martinez, Todd J.

    2014-08-07

    We present a new algorithm for ab initio quantum nonadiabatic molecular dynamics that combines the best features of ab initio Multiple Spawning (AIMS) and Multiconfigurational Ehrenfest (MCE) methods. In this new method, ab initio multiple cloning (AIMC), the individual trajectory basis functions (TBFs) follow Ehrenfest equations of motion (as in MCE). However, the basis set is expanded (as in AIMS) when these TBFs become sufficiently mixed, preventing prolonged evolution on an averaged potential energy surface. We refer to the expansion of the basis set as “cloning,” in analogy to the “spawning” procedure in AIMS. This synthesis of AIMS and MCE allows us to leverage the benefits of mean-field evolution during periods of strong nonadiabatic coupling while simultaneously avoiding mean-field artifacts in Ehrenfest dynamics. We explore the use of time-displaced basis sets, “trains,” as a means of expanding the basis set for little cost. We also introduce a new bra-ket averaged Taylor expansion (BAT) to approximate the necessary potential energy and nonadiabatic coupling matrix elements. The BAT approximation avoids the necessity of computing electronic structure information at intermediate points between TBFs, as is usually done in saddle-point approximations used in AIMS. The efficiency of AIMC is demonstrated on the nonradiative decay of the first excited state of ethylene. The AIMC method has been implemented within the AIMS-MOLPRO package, which was extended to include Ehrenfest basis functions.

  14. Cloned M1 muscarinic receptors mediate both adenylate cyclase inhibition and phosphoinositide turnover.

    PubMed Central

    Stein, R; Pinkas-Kramarski, R; Sokolovsky, M

    1988-01-01

    The rat M1 muscarinic receptor gene was cloned and expressed in a rat cell line lacking endogenous muscarinic receptors. Assignment of the cloned receptors to the M1 class was pharmacologically confirmed by their high affinity for the M1-selective muscarinic antagonist pirenzepine and low affinity for the M2-selective antagonist AF-DX-116. Guanylyl imidodiphosphate [Gpp(NH)p] converted agonist binding sites on the receptor, from high-affinity to the low-affinity state, thus indicating that the cloned receptors couple to endogenous G-proteins. The cloned receptors mediated both adenylate cyclase inhibition and phosphoinositide hydrolysis, but by different mechanisms. Pertussis toxin blocked the inhibition of adenylate cyclase (indicating coupling of the receptor to inhibitory G-protein), but did not affect phosphoinositide turnover. Furthermore, the stimulation of phosphoinositide hydrolysis was less efficient than the inhibition of adenylate cyclase. These findings demonstrate that cloned M1 receptors are capable of mediating multiple responses in the cell by coupling to different effectors, possibly to different G-proteins. Images PMID:2846274

  15. Attachment of an anti-receptor antibody to non-target cells renders them susceptible to lysis by a clone of cytotoxic T lymphocytes.

    PubMed

    Kranz, D M; Tonegawa, S; Eisen, H N

    1984-12-01

    The molecular basis for the dependence of antigen recognition by T cells on products of the major histocompatibility complex (MHC) is unknown, and the antigenic structures that are actually bound by T-cell receptors are ill-defined. In this study, we asked whether a monoclonal antibody (mAb) that reacts with the T-cell receptor of a clone of murine cytotoxic T lymphocytes (CTL) and not with the receptors of other CTL clones can substitute for that clone's natural ligand in specific cytolytic reactions. To answer the question, a mAb (1B2) to the receptor of a CTL clone (2C) was attached covalently to 51Cr-labeled cells that were not otherwise susceptible to lysis by clone 2C, and the cells thus modified were then tested as targets for clone 2C and other CTL clones of similar specificity. All labeled cells modified in this way, including a murine cell line that expresses no cell-surface MHC class I molecules and a human cell line, were lysed by clone 2C but not by other CTL clones. If, however, instead of attaching the mAb to the receptor of clone 2C, the cells were modified by attaching to them mAbs to other surface antigens on CTL [lymphocyte function-associated antigen (LFA-1), Thy-1.2], they were not lysed. In cytolytic titrations, the cells that had been converted by attachment of mAb 1B2 into specific targets for clone 2C were just as susceptible to lysis by that clone as the clone's natural H-2d targets (e.g., P815 cells). However, some accessory surface molecules (LFA-1, Lyt-2) that are required for clone 2C to lyse its natural H-2d targets seemed not to be required for this clone to lyse the mAb-converted target cells. By demonstrating that a variety of different cell types can be thus converted into target cells for CTL, the approach described in this study may provide opportunities to analyze further the mechanisms by which CTL destroy target cells.

  16. Using "Pseudomonas Putida xylE" Gene to Teach Molecular Cloning Techniques for Undergraduates

    ERIC Educational Resources Information Center

    Dong, Xu; Xin, Yi; Ye, Li; Ma, Yufang

    2009-01-01

    We have developed and implemented a serial experiment in molecular cloning laboratory course for undergraduate students majored in biotechnology. "Pseudomonas putida xylE" gene, encoding catechol 2, 3-dioxygenase, was manipulated to learn molecular biology techniques. The integration of cloning, expression, and enzyme assay gave students…

  17. Using "Pseudomonas Putida xylE" Gene to Teach Molecular Cloning Techniques for Undergraduates

    ERIC Educational Resources Information Center

    Dong, Xu; Xin, Yi; Ye, Li; Ma, Yufang

    2009-01-01

    We have developed and implemented a serial experiment in molecular cloning laboratory course for undergraduate students majored in biotechnology. "Pseudomonas putida xylE" gene, encoding catechol 2, 3-dioxygenase, was manipulated to learn molecular biology techniques. The integration of cloning, expression, and enzyme assay gave students…

  18. Expression of cloned α6* nicotinic acetylcholine receptors.

    PubMed

    Wang, Jingyi; Kuryatov, Alexander; Lindstrom, Jon

    2015-09-01

    Nicotinic acetylcholine receptors (AChRs) are ACh-gated ion channels formed from five homologous subunits in subtypes defined by their subunit composition and stoichiometry. Some subtypes readily produce functional AChRs in Xenopus oocytes and transfected cell lines. α6β2β3* AChRs (subtypes formed from these subunits and perhaps others) are not easily expressed. This may be because the types of neurons in which they are expressed (typically dopaminergic neurons) have unique chaperones for assembling α6β2β3* AChRs, especially in the presence of the other AChR subtypes. Because these relatively minor brain AChR subtypes are of major importance in addiction to nicotine, it is important for drug development as well as investigation of their functional properties to be able to efficiently express human α6β2β3* AChRs. We review the issues and progress in expressing α6* AChRs. This article is part of the Special Issue entitled 'The Nicotinic Acetylcholine Receptor: From Molecular Biology to Cognition'.

  19. Cloning

    MedlinePlus

    ... 2001, researchers produced the first clone of an endangered species: a type of Asian ox known as a ... few days after its birth. In 2003, another endangered type of ox, called the ... many species that would otherwise disappear, others argue that cloning ...

  20. Molecular cloning and expression of rat liver aminopeptidase B.

    PubMed

    Fukasawa, K M; Fukasawa, K; Kanai, M; Fujii, S; Harada, M

    1996-11-29

    We isolated, by immunological screening of a Uni-ZAP XR cDNA library constructed from rat liver mRNAs, a cDNA clone with 2212 base pairs encoding aminopeptidase B (EC 3.4.11.6). The open reading frame encodes a 649-amino acid protein with a theoretical molecular mass of 72,545 Da and bears the consensus sequence of the zinc metalloexopeptidases, indicating that the enzyme belongs to this family, which includes aminopeptidase A, aminopeptidase N, and leukotriene-A4 hydrolase. Escherichia coli SOLR cells infected with the pBluescript phagemid excised from the Uni-ZAP XR vector containing the aminopeptidase B cDNA had a high L-arginyl-beta-naphthylamidase activity. The recombinant protein was purified to homogeneity from the recombinant E. coli extracts. The enzyme had Cl--dependent aminopeptidase activity specifically restricted to the Arg and Lys derivatives and contained 1 mol of zinc per mol of the enzyme.

  1. Molecular cloning, purification and characterization of Brugia malayi phosphoglycerate kinase.

    PubMed

    Kumar, Ranjeet; Doharey, Pawan Kumar; Saxena, Jitendra Kumar; Rathaur, Sushma

    2017-04-01

    Phosphoglycerate kinase (PGK) is a glycolytic enzyme present in many parasites. It has been reported as a candidate molecule for drug and vaccine developments. In the present study, a full-length cDNA encoding the Brugia malayi 3-phosphoglycerate kinase (BmPGK) with an open reading frame of 1.3 kb was isolated and PCR amplified and cloned. The exact size of the BmPGK's ORF is 1377 bps. The BmPGK gene was subcloned into pET-28a (+) expression vector, the expressed enzyme was purified by affinity column and characterized. The SDS-PAGE analysis revealed native molecular weight of recombinant Brugia malayi 3-phosphoglycerate kinase (rBmPGK) to be ∼45 kDa. The enzyme was found sensitive to temperature and pH, it showed maximum activity at 25 °C and pH 8.5. The Km values for PGA and ATP were 1.77 and 0.967 mM, respectively. The PGK inhibitor, clorsulon and antifilarial drugs albendazole and ivermectin inhibited the enzyme. The specific inhibitor of PGK, clorsulon, competitively inhibited enzyme with Ki value 1.88 μM. Albendazole also inhibited PGK competitively with Ki value 35.39 μM. Further these inhibitory studies were confirmed by docking and molecular simulation of drugs with enzyme. Clorsulon interacted with substrate binding site with glutamine 37 as well as in hinge regions with aspartic acid 385 and valine 387 at ADP binding site. On the other hand albendazole interacted with asparagine 335 residues. These effects were in good association with binding interactions. Thus current study might help in designing and synthesis of effective inhibitors for this novel drug target and understanding their mode of interaction with the potent anthelmintic drugs.

  2. Cloning and Characterization of the Mouse Hepatitis Virus Receptor

    DTIC Science & Technology

    1991-02-11

    interactions, the cell surface receptor is a protein with a binding domain that interacts with a soluble ligand. For virus-receptor interactions, the...cell surface receptor is either a protein, a carbohydrate , or a lipid molecule. The ligand is the viral attachment protein. Three major criteria...The examples of utilization of viral receptors to block infection are the use of the soluble , secreted forms of the CD4 molecule and fragments of

  3. Quantum dot-based molecular imaging of cancer cell growth using a clone formation assay.

    PubMed

    Geng, Xia-Fei; Fang, Min; Liu, Shao-Ping; Li, Yan

    2016-10-01

    This aim of the present study was to investigate clonal growth behavior and analyze the proliferation characteristics of cancer cells. The MCF‑7 human breast cancer cell line, SW480 human colon cancer cell line and SGC7901 human gastric cancer cell line were selected to investigate the morphology of cell clones. Quantum dot‑based molecular targeted imaging techniques (which stained pan‑cytokeratin in the cytoplasm green and Ki67 in the cell nucleus yellow or red) were used to investigate the clone formation rate, cell morphology, discrete tendency, and Ki67 expression and distribution in clones. From the cell clone formation assay, the MCF‑7, SW480 and SGC7901 cells were observed to form clones on days 6, 8 and 12 of cell culture, respectively. These three types of cells had heterogeneous morphology, large nuclear:cytoplasmic ratios, and conspicuous pathological mitotic features. The cells at the clone periphery formed multiple pseudopodium. In certain clones, cancer cells at the borderline were separated from the central cell clusters or presented a discrete tendency. With quantum dot‑based molecular targeted imaging techniques, cells with strong Ki67 expression were predominantly shown to be distributed at the clone periphery, or concentrated on one side of the clones. In conclusion, cancer cell clones showed asymmetric growth behavior, and Ki67 was widely expressed in clones of these three cell lines, with strong expression around the clones, or aggregated at one side. Cell clone formation assay based on quantum dots molecular imaging offered a novel method to study the proliferative features of cancer cells, thus providing a further insight into tumor biology.

  4. Quantum dot-based molecular imaging of cancer cell growth using a clone formation assay

    PubMed Central

    Geng, Xia-Fei; Fang, Min; Liu, Shao-Ping; Li, Yan

    2016-01-01

    This aim of the present study was to investigate clonal growth behavior and analyze the proliferation characteristics of cancer cells. The MCF-7 human breast cancer cell line, SW480 human colon cancer cell line and SGC7901 human gastric cancer cell line were selected to investigate the morphology of cell clones. Quantum dot-based molecular targeted imaging techniques (which stained pan-cytokeratin in the cytoplasm green and Ki67 in the cell nucleus yellow or red) were used to investigate the clone formation rate, cell morphology, discrete tendency, and Ki67 expression and distribution in clones. From the cell clone formation assay, the MCF-7, SW480 and SGC7901 cells were observed to form clones on days 6, 8 and 12 of cell culture, respectively. These three types of cells had heterogeneous morphology, large nuclear:cytoplasmic ratios, and conspicuous pathological mitotic features. The cells at the clone periphery formed multiple pseudopodium. In certain clones, cancer cells at the borderline were separated from the central cell clusters or presented a discrete tendency. With quantum dot-based molecular targeted imaging techniques, cells with strong Ki67 expression were predominantly shown to be distributed at the clone periphery, or concentrated on one side of the clones. In conclusion, cancer cell clones showed asymmetric growth behavior, and Ki67 was widely expressed in clones of these three cell lines, with strong expression around the clones, or aggregated at one side. Cell clone formation assay based on quantum dots molecular imaging offered a novel method to study the proliferative features of cancer cells, thus providing a further insight into tumor biology. PMID:27572664

  5. Molecular Cloning of Actinomyces Bacteriophage DNA in E. Coli.

    DTIC Science & Technology

    2007-11-02

    recombinant clones revealed the presence of the expected phi63 DNA fragments that were used in the subcloning and they were stably maintained in E . coli . Further...feasibility of cloning of Actinomyces phage DNA fragments onto an E . coli expression vector.

  6. Databasing Molecular Identities of Louisiana, Florida, and Texas Sugarcane Clones

    USDA-ARS?s Scientific Manuscript database

    Sugarcane (Saccharum spp. hybrids) clones (cultivars and superior breeding lines) are routinely exchanged across geographic locations for field-testing or crossing. It is crucial to maintain the genetic identity of these clones during field collection, shipping, and quarantine. Traditionally, suga...

  7. Cloning and expression of prostaglandin E2 receptor subtype 1 (ep 1 ) in Bostrichthys sinensis.

    PubMed

    Lai, Xiao Jian; Hong, Wan Shu; Liu, Fang; Zhang, Yu Ting; Chen, Shi Xi

    2014-08-01

    Our previous studies suggested that prostaglandin E2 (PGE2) is a putative sex pheromone in Chinese black sleeper Bostrichthys sinensis, a fish species that inhabits intertidal zones and mates and spawns inside a muddy burrow. We found immunoreactivities of PGE2 receptor subtypes (Ep1-3) expressed in the olfactory sac, but only Ep1 presented higher density of immunoreactivity in mature fish than that in immature fish in both sexes. To gain a better understanding of the underlying molecular mechanism for the detection of PGE2 in the olfactory system, we cloned an ep 1 cDNA from the adult olfactory sac. The open-reading frame of the ep 1 consisted of 1,134-bp nucleotides that encoded a 378-amino acid-long protein with a seven-transmembrane domain, typical for the G protein-coupled receptors superfamily. Expression of ep 1 mRNA was observed in all tissues examined, with higher levels obtained in the olfactory sacs and testes. The expression of ep 1 mRNA in the olfactory sacs and gonads was significantly higher in both sexes of mature fish than in those of immature ones. Taken together, our results suggested that Ep1, which is highly expressed in the olfactory sacs and gonads of mature fish, is important for the control of reproduction and may be involved in PGE2-initiated spawning behavior in B. sinensis.

  8. Molecular Mechanisms of Prolactin and Its Receptor

    PubMed Central

    2012-01-01

    Prolactin and the prolactin receptors are members of a family of hormone/receptor pairs which include GH, erythropoietin, and other ligand/receptor pairs. The mechanisms of these ligand/receptor pairs have broad similarities, including general structures, ligand/receptor stoichiometries, and activation of several common signaling pathways. But significant variations in the structural and mechanistic details are present among these hormones and their type 1 receptors. The prolactin receptor is particularly interesting because it can be activated by three sequence-diverse human hormones: prolactin, GH, and placental lactogen. This system offers a unique opportunity to compare the detailed molecular mechanisms of these related hormone/receptor pairs. This review critically evaluates selected literature that informs these mechanisms, compares the mechanisms of the three lactogenic hormones, compares the mechanism with those of other class 1 ligand/receptor pairs, and identifies information that will be required to resolve mechanistic ambiguities. The literature describes distinct mechanistic differences between the three lactogenic hormones and their interaction with the prolactin receptor and describes more significant differences between the mechanisms by which other related ligands interact with and activate their receptors. PMID:22577091

  9. Cloning of Octopus cephalotocin receptor, a member of the oxytocin/vasopressin superfamily.

    PubMed

    Kanda, Atsuhiro; Takuwa-Kuroda, Kyoko; Iwakoshi-Ukena, Eiko; Furukawa, Yasuo; Matsushima, Osamu; Minakata, Hiroyuki

    2003-11-01

    We reported that the common octopus, Octopus vulgaris, in common with vertebrates, possesses two members of the oxytocin/vasopressin superfamily: octopressin (OP) and cephalotocin (CT). This was the first observation of its kind in invertebrates. As OP and CT have different biological activities, the presence of specific receptors has been proposed. We cloned the cDNA of an orphan receptor from Octopus brain and found it to encode a polypeptide of 397 amino acids that displays sequences characteristic of G-protein coupled receptors. The orphan receptor showed high homology to receptors of the oxytocin/vasopressin superfamily and seemed to conserve the agonist-binding pocket common to the oxytocin and vasopressin receptors. Xenopus oocytes that express the orphan receptor responded to the application of CT by an induction of membrane Cl(-) currents coupled to the inositol phosphate/Ca(2+) pathway. OP and the other members of the oxytocin/vasopressin superfamily did not activate this receptor. HPLC fractionation of the Octopus brain extract combined with an oocyte assay yielded a single substance that was identical to CT. On the basis of these results, we conclude that the cloned receptor is the CT receptor (CTR). Expression of CTR mRNA in Octopus was detected in the central and the peripheral nervous systems, the pancreas, the oviduct and the ovary. This receptor may mediate physiological functions of CT in Octopus such as neurotransmission, reproduction and metabolism.

  10. Human pyridoxal phosphatase. Molecular cloning, functional expression, and tissue distribution.

    PubMed

    Jang, Young Min; Kim, Dae Won; Kang, Tae-Cheon; Won, Moo Ho; Baek, Nam-In; Moon, Byung Jo; Choi, Soo Young; Kwon, Oh-Shin

    2003-12-12

    Pyridoxal phosphatase catalyzes the dephosphorylation of pyridoxal 5'-phosphate (PLP) and pyridoxine 5'-phosphate. A human brain cDNA clone was identified to the PLP phosphatase on the basis of peptide sequences obtained previously. The cDNA predicts a 296-amino acid protein with a calculated Mr of 31698. The open reading frame is encoded by two exons located on human chromosome 22q12.3, and the exon-intron junction contains the GT/AG consensus splice site. In addition, a full-length mouse PLP phosphatase cDNA of 1978 bp was also isolated. Mouse enzyme encodes a protein of 292 amino acids with Mr of 31512, and it is localized on chromosome 15.E1. Human and mouse PLP phosphatase share 93% identity in protein sequence. A BLAST search revealed the existence of putative proteins in organism ranging from bacteria to mammals. Catalytically active human PLP phosphatase was expressed in Escherichia coli, and characteristics of the recombinant enzyme were similar to those of erythrocyte enzyme. The recombinant enzyme displayed Km and kcat values for pyridoxal of 2.5 microM and 1.52 s(-1), respectively. Human PLP phosphatase mRNA is differentially expressed in a tissue-specific manner. A single mRNA transcript of 2.1 kb was detected in all human tissues examined and was highly abundant in the brain. Obtaining the molecular properties for the human PLP phosphatase may provide new direction for investigating metabolic pathway involving vitamin B6.

  11. Molecular genetics: DNA analysis of a putative dog clone.

    PubMed

    Parker, Heidi G; Kruglyak, Leonid; Ostrander, Elaine A

    2006-03-09

    In August 2005, Lee et al. reported the first cloning of a domestic dog from adult somatic cells. This putative dog clone was the result of somatic-cell nuclear transfer from a fibroblast cell of a three-year-old male Afghan hound into a donor oocyte provided by a dog of mixed breed. In light of recent concerns regarding the creation of cloned human cell lines from the same institution, we have undertaken an independent test to determine the validity of the claims made by Lee et al..

  12. [Parathyroid hormone receptors: from cloning to physiological, physiopathological and clinical implications].

    PubMed

    Mannstadt, M; Drüeke, T B

    1997-01-01

    It has long been known that parathyroid hormone (PTH) exerts its effects on target tissues via its binding to a membrane receptor. Recently, several types of PTH receptors have been identified. The first receptor which has been cloned and well characterized is "PTH/PTHrP receptor-1". It is activated not only by PTH, but also by PTH-related peptide (PTHrP), via a signal transduction system involving G-proteins, adenylate cyclase and phospholipase C. It is expressed in many tissues, in addition to kidney and bone. The results of recent studies are suggestive of the existence of additional PTH receptors. One or several receptors are probably expressed in the keratinocyte and the glomerular podocyte which are not identical with PTH/ PTHrP receptor-1. A third receptor, which has been cloned recently and called "PTH2 receptor", recognizes solely PTH. It is expressed in brain, pancreas, testis and placenta. Its function is unknown. There is also evidence for a fourth receptor, called "C-PTH receptor", recognizing C-terminal PTH fragments which are generally considered to be biologically inactive. The regulation of these receptors is subject to intensive research. Down-regulation of PTH/PTHrP receptor-1 mRNA expression could explain the well-known resistance to the action of PTH in chronic renal failure. In contrast, the receptor mRNA is up-regulated in vitamin D deficiency, despite a similar tissue resistance to PTH. A mutation of PTH/PTHrP receptor-1 causes Jansen-type metaphyseal chondrodysplasia. However, no alteration of the PTH/PTHrP receptor-1 gene structure has been found in type 1b pseudohypoparathyroidism.

  13. Molecular characterization of an. alpha. sub 2B -adrenergic receptor

    SciTech Connect

    Harrison, J.K.; Dewan Zeng; D'Angelo, D.D.; Tucker, A.L.; Zhihong Lu; Barber, C.M.; Lynch, K.R. )

    1990-02-26

    {alpha}{sub 2}-Adrenergic receptors comprise a heterogeneous population based on pharmacologic and molecular evidence. The authors have isolated a cDNA clone (pRNG{alpha}2) encoding a previously undescribed third subtype of an {alpha}{sub 2}-adrenergic receptor from a rat kidney cDNA library. The library was screened with an oligonucleotide encoding a highly conserved region found in all biogenic amine receptors described to date. The deduced amino acid sequence displays many features of G-protein coupled receptors with exception of the absence of the consensus N-linked glycosylation site at the amino terminus. Membranes prepared from COS-1 cells transfected with pRNG{alpha}2 display high affinity and saturable binding to {sup 3}H-rauwolscine (K{sub d}=2 nM).Competition curve data analysis shows that pRNG{alpha}2 protein binds to a variety of adrenergic drugs with the following rank order of potency: yohimbine {ge} cholorpromazine > prazosin {ge} clonidine > norepinephrine {ge} oxymetazoline. pRNG{alpha}2 RNA accumulates in both adult rat kidney and rat neonatal lung (predominant species is 4.0 kb). They conclude that pRNG{alpha}2 likely represents a cDNA for the {alpha}{sub 2B}-adrenergic receptor.

  14. Construction of a molecular clone of ovine enzootic nasal tumor virus.

    PubMed

    Walsh, Scott R; Gerpe, María Carla Rosales; Wootton, Sarah K

    2016-12-30

    Enzootic nasal tumor virus (ENTV-1) is an ovine betaretrovirus that has been linked to enzootic nasal adenocarcinoma (ENA), a contagious tumor of the ethmoid turbinates of sheep. Transmission experiments performed using virus isolated from cell free nasal tumor homogenates suggest that ENTV-1 is the causative agent of ENA; however, this etiological relationship has not been conclusively proven due to the fact that the virus cannot be propagated in vitro nor is there an infectious molecular clone of the virus. Here we report construction of a molecular clone of ENTV-1 and demonstrate that transfection of this molecular clone into HEK 293T cells produces mature virus particles. Analysis of recombinant virus particles derived from the initial molecular clone revealed a defect in the proteolytic processing of Gag; however, this defect could be corrected by co-expression of the Gag-Pro-Pol polyprotein from the highly related Jaagsiekte sheep retrovirus (JSRV) suggesting that the polyprotein cleavage sites in the ENTV-1 molecular clone were functional. Mutagenesis of the molecular clone to correct amino acid variants identified within the pro gene did not restore proteolytic processing; whereas deletion of one proline residue from a polyproline tract located in variable region 1 (VR1) of the matrix resulted in production of CA protein of the mature (cleaved) size strongly suggesting that normal virion morphogenesis and polyprotein cleavage took place. Finally, electron microscopy revealed the presence of spherical virus particles with an eccentric capsid and an average diameter of about 100 nm. In summary, we have constructed the first molecular clone of ENTV-1 from which mature virus particles can be produced. Future experiments using virus produced from this molecular clone can now be conducted to fulfill Koch's postulates and demonstrate that ENTV-1 is necessary and sufficient to induce ENA in sheep.

  15. Molecular cloning of Taenia taeniaeformis oncosphere antigen genes.

    PubMed

    Cougle, W G; Lightowlers, M W; Bogh, H O; Rickard, M D; Johnson, K S

    1991-03-01

    Infection of mice with the cestode Taenia taeniaeformis exhibits several important features common to other cestode infections, including the ability to vaccinate with crude antigen mixtures. Partial purification of the protective oncosphere antigens has been reported with a cutout from deoxycholate (DOC) acrylamide gels; this cutout was called fraction II (FII), and comprises approximately 10% of total DOC-soluble oncosphere antigen. Western blots of DOC gels probed with anti-FII antisera revealed a series of 3-5 discrete bands within the FII region. Further fractionation of the FII antigens on DOC gels was impractical due to limitations in supply of oncospheres, so a cDNA library was constructed from 150 ng of oncosphere mRNA and screened with alpha-FII antisera. Two distinct clone families were identified, oncA and oncB. Antibodies affinity-purified on either of two representative members, oncA1 and oncB1, recognised all the FII bands. Individual FII bands excised from a DOC gel resolved into an overlapping series of molecules when re-run on SDS-PAGE, indicating that each FII band consisted of several polypeptides of differing molecular weight. Immunoprecipitates resolved on SDS-PAGE revealed that alpha-FII recognised 3 major oncosphere antigens, of 62, 34 and 25 kDa; antisera against oncB precipitated both the 34- and 25-kDa antigens, whereas alpha-oncA antisera precipitated the 62-kDa antigen. We conclude that oncA and oncB encode the major antigens in the FII complex. The 62-kDa antigen encoded by oncA1 was the only common antigen precipitated by anti-FII and two other antisera raised against different protective extracts, suggesting that it may be a protective component in all three. Southern blot results indicate that oncA and oncB are distinct genes present at low copy number in the genome. Evidence is also presented suggesting that some cestode mRNAs, including oncA, may use variant polyadenylation signals.

  16. Cloning and expression of an A1 adenosine receptor from rat brain

    SciTech Connect

    Mahan, L.C.; McVittie, L.D.; Smyk-Randall, E.M.; Nakata, H.; Monsma, F.J. Jr.; Gerfen, C.R.; Sibley, D.R. )

    1991-07-01

    The authors have used the polymerase chain reaction technique to selectively amplify guanine nucleotide-binding regulatory protein (G protein)-coupled receptor cDNA sequences from rat striatal mRNA, using sets of highly degenerate primers derived from transmembrane sequences of previously cloned G protein-coupled receptors. A novel cDNA fragment was identified, which exhibits considerable homology to various members of the G protein-coupled receptor family. This fragment was used to isolate a full-length cDNA from a rat striatal library. A 2.2-kilobase clone was obtained that encodes a protein of 326 amino acids with seven transmembrane domains, as predicted by hydropathy analysis. Stably transfected mouse A9-L cells and Chinese hamster ovary cells that expressed mRNA for this clone were screened with putative receptor ligands. Saturable and specific binding sites for the A1 adenosine antagonist (3H)-1,3-dipropyl-8-cyclopentylxanthine were identified on membranes from transfected cells. The rank order of potency and affinities of various adenosine agonist and antagonist ligands confirmed the identity of this cDNA clone as an A1 adenosine receptor. The high affinity binding of A1 adenosine agonists was shown to be sensitive to the nonhydrolyzable GTP analog guanylyl-5{prime}-imidodiphosphate. In adenylyl cyclase assays, adenosine agonists inhibited forskolin-stimulated cAMP production by greater than 50%, in a pharmacologically specific fashion. Northern blot and in situ hybridization analyses of receptor mRNA in brain tissues revealed two transcripts of 5.6 and 3.1 kilobases, both of which were abundant in cortex, cerebellum, hippocampus, and thalamus, with lower levels in olfactory bulb, striatum, mesencephalon, and retina. These regional distribution data are in good agreement with previous receptor autoradiographic studies involving the A1 adenosine receptor.

  17. Cloning and characterization of 5'-end alternatively spliced human cholecystokinin-B receptor mRNAs.

    PubMed

    Monstein, H J; Nilsson, I; Ellnebo-Svedlund, K; Svensson, S P

    1998-01-01

    We report here the cloning and characterization of a 5'-end alternatively spliced human cholecystokinin-B (CCK-B) receptor mRNA. The 5'-end of this CCK-B receptor transcript (termed CCK-BRtx) consisted of exon Ia, present in the ordinary full-length CCK-B receptor mRNA (CCK-BRwt), and exon Ib, present in a previously described 5'-end alternatively spliced CCK-B receptor mRNA (CCK-BRt). A short open reading frame preceded the AUG translation initiation codon of the CCK-BRtx. Transfection of COS-7 cells with the CCK-BRtx or CCK-BRt cDNAs did not lead to the appearance of peptidergic and non-peptidergic binding sites. Cell free in vitro translation yielded proteins of approximately 44 kDa (CCK-B receptor) and 40 kDa (CCK-BRt receptor) whereas no 40 kDa product was detected from the cloned CCK-BRtx cDNA. Instead, a protein product of approximately 9 kDa was visualized, the size corresponding to the predicted protein encoded by the short open reading frame. The alternatively spliced CCK-B receptor transcripts were concomitantly expressed with the ordinary full-length CCK-B receptor mRNA in the brain, pancreas, and stomach. The possibility that such transcripts are translated in vivo into truncated CCK-B receptors is discussed.

  18. Identification of putative human T cell receptor delta complementary DNA clones

    SciTech Connect

    Hata, S.; Brenner, M.B.; Krangel, M.S.

    1987-10-30

    A novel T cell receptor (TCR) subunit termed TCR delta, associated with TCY ..gamma.. and CD3 polypeptides, were recently found on a subpopulation of human T lymphocytes. T cell-specific complementary DNA clones present in a human TCR..gamma..delta T cell complementary DNA library were obtained and characterized in order to identify candidate clones encoding TCR delta. One cross-hybridizing group of clones detected transcripts that are expressed in lymphocytes bearing TCR ..gamma..delta but not in other T lymphocytes and are encoded by genes that are rearranged in TCR ..gamma..delta lymphocytes but deleted in other T lymphocytes. Their sequences indicate homology to the variable, joining, and constant elements of other TCR and immunoglobulin genes. These characteristics are strong evidence that the complementary DNA clones encode TCR delta.

  19. Functional analysis and molecular modeling of a cloned urate transporter/channel.

    PubMed

    Leal-Pinto, E; Cohen, B E; Abramson, R G

    1999-05-01

    Recombinant protein, designated UAT, prepared from a cloned rat renal cDNA library functions as a selective voltage-sensitive urate transporter/channel when fused with lipid bilayers. Since we previously suggested that UAT may represent the mammalian electrogenic urate transporter, UAT has been functionally characterized in the presence and absence of potential channel blockers, several of which are known to block mammalian electrogenic urate transport. Two substrates, oxonate (a competitive uricase inhibitor) and pyrazinoate, that inhibit renal electrogenic urate transport also block UAT activity. Of note, oxonate selectively blocks from the cytoplasmic side of the channel while pyrazinoate only blocks from the channel's extracellular face. Like oxonate, anti-uricase (an electrogenic transport inhibitor) also selectively blocks channel activity from the cytoplasmic side. Adenosine blocks from the extracellular side exclusively while xanthine blocks from both sides. These effects are consistent with newly identified regions of homology to uricase and the adenosine A1/A3 receptor in UAT and localize these homologous regions to the cytoplasmic and extracellular faces of UAT, respectively. Additionally, computer analyses identified four putative alpha-helical transmembrane domains, two beta sheets, and blocks of homology to the E and B loops of aquaporin-1 within UAT. The experimental observations substantiate our proposal that UAT is the molecular representation of the renal electrogenic urate transporter and, in conjunction with computer algorithms, suggest a possible molecular structure for this unique channel.

  20. Molecular cloning and biochemical characterization of a Drosophila phosphatidylinositol-specific phosphoinositide 3-kinase.

    PubMed

    Linassier, C; MacDougall, L K; Domin, J; Waterfield, M D

    1997-02-01

    Molecular, biochemical and genetic characterization of phosphoinositide 3-kinases (PI3Ks) have identified distinct classes of enzymes involved in processes mediated by activation of cell-surface receptors and in constitutive intracellular protein trafficking events. The latter process appears to involve a PtdIns-specific PI3K first described in yeast as a mutant, vps34, defective in the sorting of newly synthesized proteins from the Golgi to the vacuole. We have identified a representative member of each class of PI3Ks in Drosophila using a PCR-based approach. In the present paper we describe the molecular cloning of a PI3K from Drosophila, P13K_59F, that shows sequence similarity to Vps34. PI3K_59F encodes a protein of 108 kDa co-linear with Vps34 homologues, and with three regions of sequence similarity to other PI3Ks. Biochemical characterization of the enzyme, by expression of the complete coding sequence as a glutathione S-transferase fusion protein in Sf9 cells, demonstrates that PI3K_59F is a PtdIns-specific PI3K that can utilize either Mg2+ or Mn2+. This activity is sensitive to inhibition both by non-ionic detergent (Nonidet P40) and by wortmannin (IC50 10 nM). PI3K_59F, therefore, conserves both the structural and biochemical properties of the Vps34 class of enzymes.

  1. Cloning of a novel G protein-coupled receptor, SLT, a subtype of the melanin-concentrating hormone receptor.

    PubMed

    Mori, M; Harada, M; Terao, Y; Sugo, T; Watanabe, T; Shimomura, Y; Abe, M; Shintani, Y; Onda, H; Nishimura, O; Fujino, M

    2001-05-25

    A DNA fragment encoding an amino acid sequence possessing common features to the G protein-coupled receptor (GPCR) superfamily was found in the human genomic sequence, and from this information, the full-length cDNA of a novel GPCR, designated SLT, was cloned from the human hippocampus cDNA library. SLT showed the highest homology to the melanin-concentrating hormone (MCH) receptor, SLC-1 (31.5% identity), and to a lesser extent, to the somatostatin (SST) receptor subtypes. MCH exhibited agonistic behavior when applied to the SLT-expressing CHO cells at subnanomolar doses whereas more than 200 known peptides, including SST and cortistatin, did not. These results indicated that MCH is the cognate ligand of the SLT receptor and that this newly cloned GPCR is the second subtype of the MCH receptor. Quantitative polymerase chain reaction analysis of the SLT gene expression in human tissues showed that the SLT receptor is expressed mainly in brain areas including the cerebral cortex, amygdala, hippocampus, and corpus callosum, as well as in a limited number of peripheral tissues. The distribution of the SLT nearly overlapped that of SLC-1, suggesting that some of the neural functions of MCH may be mediated by both of these receptor subtypes.

  2. Molecular cloning of the 8000-base thyroglobulin structural gene.

    PubMed

    Christophe, D; Mercken, L; Brocas, H; Pohl, V; Vassart, G

    1982-03-01

    Bovine thyroglobulin mRNA was reverse-transcribed into full-length double-stranded cDNA. The existence of three HindIII restriction endonuclease sites in the 8000-base thyroglobulin structural gene had allowed the easy cloning of the two internal HindIII fragments [Christophe et al. (1980) Eur. J. Biochem. 111, 419-423]. In the present study, the central portion of the structural gene was cloned in Escherichia coli as two individual recombinant plasmids containing 2000-base-pair and 4700-base-pair segments located respectively 5' and 3' relative to the unique BamHI site of the cDNA. BamHI linkers were added to the double-stranded cDNA and, following restriction with HindIII, selective cloning of the 5' (2600-base-pair) and 3' (1000-base-pair) terminal HindIII fragments was achieved by inserting them between the HindIII and BamHI sites of the plasmid pBR322. Partial sequencing of the 1000-base-pair 3'-terminal fragment demonstrated the presence of an A-A-U-A-A-A sequence in the mRNA 14 bases upstream from a poly(A) tract corresponding to the 3' end of the mRNA. Together, the four clones represent about 99% of the thyroglobulin structural gene and provide the starting material for the determination of thyroglobulin primary structure.

  3. Molecular cloning of gluconobacter oxydans DSM 2003 xylitol dehydrogenase gene.

    PubMed

    Sadeghi, H Mir Mohammad; Ahmadi, R; Aghaabdollahian, S; Mofid, M R; Ghaemi, Y; Abedi, D

    2011-01-01

    Due to the widespread applications of xylitol dehydrogenase, an enzyme used for the production of xylitol, the present study was designed for the cloning of xylitol dehydrogenase gene from Glcunobacter oxydans DSM 2003. After extraction of genomic DNA from this bacterium, xylitol dehydrogenase gene was replicated using polymerase chain reaction (PCR). The amplified product was entered into pTZ57R cloning vector by T/A cloning method and transformation was performed by heat shocking of the E. coli XL1-blue competent cells. Following plasmid preparation, the cloned gene was digested out and ligated into the expression vector pET-22b(+). Electrophoresis of PCR product showed a 789 bp band. Recombinant plasmid (rpTZ57R) was then constructed. This plasmid was double digested with XhoI and EcoRI resulting in 800 bp and 2900 bp bands. The obtained insert was ligated into pET-22b(+) vector and its orientation was confirmed with XhoI and BamHI restriction enzymes. In conclusion, in the present study the recombinant expression vector containing xylitol dehydrogenase gene has been constructed and can be used for the production of this enzyme in high quantities.

  4. Molecular cloning and characterization of duck interleukin-17

    USDA-ARS?s Scientific Manuscript database

    Interleukin-17 (IL-17) belonging to the Th17 family is a proinflammatory cytokine produced by activated T cells. A 1034-bp cDNA encoding duck IL-17 (duIL-17) was cloned from ConA-activated splenic lymphocytes of ducks. The encoded protein, predicted to consisted of 169 amino acids, displayed a molec...

  5. Molecular cloning and functional characterization of avian interleukin-19

    USDA-ARS?s Scientific Manuscript database

    The present study describes the cloning and functional characterization of avian interleukin (IL)-19, a cytokine that, in mammals, alters the balance of Th1 and Th2 cells in favor of the Th2 phenotype. The full-length avian IL-19 gene, located on chromosome 26, was amplified from LPS-stimulated chi...

  6. Cloning and characterization of the ecdysone receptor and ultraspiracle protein from the water flea Daphnia magna.

    PubMed

    Kato, Yasuhiko; Kobayashi, Kaoru; Oda, Shigeto; Tatarazako, Norihisa; Watanabe, Hajime; Iguchi, Taisen

    2007-04-01

    cDNAs encoding the ecdysone receptor (EcR) and ultra spiracle (USP) protein were cloned from the water flea Daphnia magna (Crustacea: Cladocera). The deduced EcR and USP amino acid sequences showed a high degree of homology to those of other crustaceans as well as insects. We isolated three isoforms of EcR that differ in the A/B domain. Quantitative PCR analysis indicated differing temporal expression patterns of the EcR isoforms during the molting period and demonstrated that the expression of one subtype correlated well with the timing of molt. Using cDNAs encoding EcR and USP, we constructed a Daphnia EcR/USP reporter based on a two-hybrid system. The gene fusions encoded the EcR ligand-binding domain (LBD) fused to the Gal4 DNA-binding domain, and the USP-LBD fused to the Vp16 activation domain. These chimeric genes were transfected with a luciferase reporter gene. Dose-dependent activation of the reporter gene could be observed when transfectants were exposed to Ec and other chemicals known to have Ec-like activities. This two-hybrid system may represent a useful reporter system for further examination of hormonal and chemical effects on Daphnia at the molecular level.

  7. Problem-Solving Test: Expression Cloning of the Erythropoietin Receptor

    ERIC Educational Resources Information Center

    Szeberenyi, Jozsef

    2008-01-01

    Terms to be familiar with before you start to solve the test: cytokines, cytokine receptors, cDNA library, cDNA synthesis, poly(A)[superscript +] RNA, primer, template, reverse transcriptase, restriction endonucleases, cohesive ends, expression vector, promoter, Shine-Dalgarno sequence, poly(A) signal, DNA helicase, DNA ligase, topoisomerases,…

  8. Problem-Solving Test: Expression Cloning of the Erythropoietin Receptor

    ERIC Educational Resources Information Center

    Szeberenyi, Jozsef

    2008-01-01

    Terms to be familiar with before you start to solve the test: cytokines, cytokine receptors, cDNA library, cDNA synthesis, poly(A)[superscript +] RNA, primer, template, reverse transcriptase, restriction endonucleases, cohesive ends, expression vector, promoter, Shine-Dalgarno sequence, poly(A) signal, DNA helicase, DNA ligase, topoisomerases,…

  9. Characterization of ligand binding properties of the 5-HT1D receptors cloned from chimpanzee, gorilla and rhesus monkey in comparison with those from the human and guinea pig receptors.

    PubMed

    Pregenzer, J F; Alberts, G L; Bock, J H; Slightom, J L; Im, W B

    1997-10-17

    The 5-HT1D receptor is a potential target of anti-migraine drugs, and here its genes were cloned from chimpanzee, gorilla and rhesus monkey, via polymerase chain reactions with their genomic DNAs and the primers designed from the 5' and 3' untranslated regions of the human receptor. Direct sequencing of the polymerase chain reaction (PCR) products revealed high degrees of identity between their deduced amino acid sequences (the chimpanzee, gorilla and rhesus monkey) and that of human, differing by two, four and 11 residues, respectively. The binding properties of the receptors, as expressed in human embryonic kidney 293 cells, were compared to those obtained with the human and guinea pig receptors, the latter differing by 33 residues from the human receptor. Standard serotonergic ligands including several indoles, ergots and methiothepin bound all the cloned primate and guinea pig receptors with comparable, low nanomolar affinities, leading to high correlation coefficients among their Ki values. R(+)-8-Hydroxydipropylaminotetralin, on the other hand, bound the human receptor with the affinity higher than those for the primates and guinea pig receptors. This indicates that certain chemical templates may differentiate the molecular divergences among the 5-HT1D receptors of various animal species, and the use of the non-human primates will be beneficial for pharmacological characterizations, more relevant to the human receptor, of future novel ligands for the 5-HT1D receptor, which are potential anti-migraine drugs.

  10. Cloning and characterization of the A-factor receptor gene from Streptomyces griseus.

    PubMed Central

    Onaka, H; Ando, N; Nihira, T; Yamada, Y; Beppu, T; Horinouchi, S

    1995-01-01

    A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone) and its specific receptor protein control streptomycin production, streptomycin resistance, and aerial mycelium formation in Streptomyces griseus. The A-factor receptor protein (ArpA) was purified from a cell lysate of S. griseus IFO 13350. The NH2-terminal amino acid sequences of ArpA and lysyl endopeptidase-generated fragments were determined for the purpose of preparing oligonucleotide primers for cloning arpA by the PCR method. The arpA gene cloned in this way directed the synthesis of a protein having A-factor-specific binding activity when expressed in Escherichia coli under the control of the T7 promoter. The arpA gene was thus concluded to encode a 276-amino-acid protein with a calculated molecular mass of 29.1 kDa, as determined by nucleotide sequencing. The A-factor-binding activity was observed with a homodimer of ArpA. The NH2-terminal portion of ArpA contained an alpha-helix-turn-alpha-helix DNA-binding motif that showed great similarity to those of many DNA-binding proteins, which suggests that it exerts its regulatory function for the various phenotypes by directly binding to a certain key gene(s). Although a mutant strain deficient in both the ArpA protein and A-factor production overproduces streptomycin and forms aerial mycelium and spores earlier than the wild-type strain because of repressor-like behavior of ArpA, introduction of arpA into this mutant abolished simultaneously its streptomycin production and aerial mycelium formation. All of these data are consistent with the idea that ArpA acts as a repressor-type regulator for secondary metabolite formation and morphogenesis during the early growth phase and A-factor at a certain critical intracellular concentration releases the derepression, thus leading to the onset of secondary metabolism and aerial mycelium formation. The presence of ArpA-like proteins among Streptomyces spp., as revealed by PCR, together with the presence of A

  11. Molecular Cloning and Analysis of a DNA Repetitive Element from the Mouse Genome

    ERIC Educational Resources Information Center

    Geisinger, Adriana; Cossio, Gabriela; Wettstein, Rodolfo

    2006-01-01

    We report the development of a 3-week laboratory activity for an undergraduate molecular biology course. This activity introduces students to the practice of basic molecular techniques such as restriction enzyme digestion, agarose gel electrophoresis, cloning, plasmid DNA purification, Southern blotting, and sequencing. Students learn how to carry…

  12. Molecular Cloning and Analysis of a DNA Repetitive Element from the Mouse Genome

    ERIC Educational Resources Information Center

    Geisinger, Adriana; Cossio, Gabriela; Wettstein, Rodolfo

    2006-01-01

    We report the development of a 3-week laboratory activity for an undergraduate molecular biology course. This activity introduces students to the practice of basic molecular techniques such as restriction enzyme digestion, agarose gel electrophoresis, cloning, plasmid DNA purification, Southern blotting, and sequencing. Students learn how to carry…

  13. Cloning and expression of the mouse histamine H3 receptor: evidence for multiple isoforms.

    PubMed

    Rouleau, Agnès; Héron, Anne; Cochois, Véronique; Pillot, Catherine; Schwartz, Jean-Charles; Arrang, Jean-Michel

    2004-09-01

    The existence of mouse H3-receptor isoforms was investigated by PCR analysis and cDNA cloning. Splicing mechanisms previously reported in various species are conserved in the mouse. The retention/deletion of a fragment in the third intracellular loop of the mouse receptor leads to the existence of three isoforms designated mH(3(445)), mH(3(413)) and mH(3(397)) according to the length of their deduced amino acid sequence. PCR analysis showed that mouse H3-receptor isoforms display different expression patterns in the brain. Following expression in Cos-1 cells, [125I]iodoproxyfan binding indicated similar pharmacological profiles of the mH(3(445)), mH(3(413)) and mH(3(397)) isoforms. The pharmacological profile of the mouse H3 receptor is more similar to the rat receptor than to the human receptor, although some differences were also observed between the mouse and rat receptors. For example, the potency of thioperamide and ciproxifan is slightly higher at the mouse receptor than at the rat receptor but 40-100-fold higher than at the human receptor. In situ hybridization histochemistry showed that the distribution of H3-receptor mRNAs in the mouse brain is rather similar to that previously reported in the rat brain. However, the autoradiographic and cellular expression patterns observed in several brain areas such as the thalamus or hippocampus reveal important differences between the two species.

  14. Expression cloning of a high-affinity melatonin receptor from Xenopus dermal melanophores.

    PubMed Central

    Ebisawa, T; Karne, S; Lerner, M R; Reppert, S M

    1994-01-01

    Using an expression cloning strategy, a high-affinity melatonin receptor cDNA has been isolated from Xenopus laevis dermal melanophores. Transient expression of the cDNA in COS-7 cells resulted in high-affinity 2-[125I]-iodomelatonin binding (Kd = 6.3 +/- 0.3 x 10(-11) M). In addition, six ligands exhibited a rank order of inhibition of specific 2-[125I]iodomelatonin binding that was identical to that reported for endogenous high-affinity receptors. Functional studies of CHO cells stably expressing the receptor cDNA showed that melatonin acting through the cloned receptor inhibited forskolin-stimulated cAMP accumulation in a dose-dependent manner. Northern blot analysis showed that melatonin receptor transcripts are moderately expressed in Xenopus dermal melanophores. The cDNA encodes a protein of 420 amino acids, which contains seven hydrophobic segments. Structural analysis revealed that the receptor protein is a newly discovered member of the guanine nucleotide binding protein-coupled receptor family. Images PMID:7517042

  15. Molecular cloning of mannose-binding lectins from Clivia miniata.

    PubMed

    Van Damme, E J; Smeets, K; Van Leuven, F; Peumans, W J

    1994-03-01

    Screening of a cDNA library constructed from total RNA isolated from young developing ovaries of Clivia miniata Regel with the amaryllis lectin cDNA clone resulted in the isolation of four different isolectin clones which clearly differ from each other in their nucleotide sequences and hence also in their deduced amino acid sequences. Apparently the lectin is translated from an mRNA of ca. 800 nucleotides encoding a precursor polypeptide of 163 amino acids. Northern blot analysis of total RNA isolated from different tissues of Clivia miniata has shown that the lectin is expressed in most plant tissues with very high lectin mRNA concentrations in the ovary and the seed endosperm.

  16. Molecular cloning of seal myoglobin mRNA.

    PubMed Central

    Wood, D; Blanchetot, A; Jeffreys, A J

    1982-01-01

    Grey seal skeletal muscle containing high levels of myoglobin was used to prepare poly(A)+ RNA. In vitro translation of this RNA produced a range of polypeptides including myoglobin. cDNA was prepared by reverse transcription of muscle poly(A)+ RNA and cloned into the plasmid pAT 153. 4% of cDNA recombinants were shown to contain myoglobin cDNA inserts. DNA sequence analysis of one clone (pSM 178) which contained a relatively large myoglobin cDNA insert showed an incomplete cDNA comprising the terminal 293 nucleotides of 3' non-translated mRNA sequences. Hybridization experiments using this myoglobin cDNA indicated that seal myoglobin is coded by a single gene which is transcribed to give a 1400 nucleotide mRNA considerably longer than related haemoglobin mRNAs. Images PMID:6185919

  17. Molecular cloning and characterisation of banana fruit polyphenol oxidase.

    PubMed

    Gooding, P S; Bird, C; Robinson, S P

    2001-09-01

    Polyphenol oxidase (PPO; EC 1.10.3.2) is the enzyme thought to be responsible for browning in banana [Musa cavendishii (AAA group, Cavendish subgroup) cv. Williams] fruit. Banana flesh was high in PPO activity throughout growth and ripening. Peel showed high levels of activity early in development but activity declined until ripening started and then remained constant. PPO activity in fruit was not substantially induced after wounding or treatment with 5-methyl jasmonate. Banana flowers and unexpanded leaf roll had high PPO activities with lower activities observed in mature leaves, roots and stem. Four different PPO cDNA clones were amplified from banana fruit (BPO1, BPO11, BPO34 and BPO35). Full-length cDNA and genomic clones were isolated for the most abundant sequence (BPO1) and the genomic clone was found to contain an 85-bp intron. Introns have not been previously found in PPO genes. Northern analysis revealed the presence of BPO1 mRNA in banana flesh early in development but little BPO1 mRNA was detected at the same stage in banana peel. BPO11 transcript was only detected in very young flesh and there was no detectable expression of BPO34 or BPO35 in developing fruit samples. PPO transcripts were also low throughout ripening in both flesh and peel. BPO1 transcripts were readily detected in flowers, stem, roots and leaf roll samples but were not detected in mature leaves. BPO11 showed a similar pattern of expression to BPO1 in these tissues but transcript levels were much lower. BPO34 and BPO35 mRNAs were only detected at a low level in flowers and roots and BPO34 transcript was detected in mature leaves, the only clone to do so. The results suggest that browning of banana fruit during ripening results from release of pre-existing PPO enzyme, which is synthesised very early in fruit development.

  18. Molecular cloning and analysis of functional cDNA and genomic clones encoding bovine cellular retinoic acid-binding protein.

    PubMed

    Shubeita, H E; Sambrook, J F; McCormick, A M

    1987-08-01

    A recombinant cDNA clone, pCRABP-HS1, encoding cellular retinoic acid-binding protein was isolated from a bovine adrenal cDNA library. COS-7 cells transfected with pCRABP-HS1 produced a biologically active retinoic acid-binding protein molecule of the expected molecular mass (15.5 kDa). RNA blot hybridization analysis using pCRABP-HS1 as a probe revealed a single 1050-nucleotide mRNA species in bovine adrenal, uterus, and testis, tissues that contain the highest levels of retinoic acid-binding activity. No hybridization was detected in RNA extracted from ovary, spleen, kidney, or liver, which contain relatively low levels of cellular retinoic acid-binding protein activity. Analysis of genomic clones isolated from an EcoRI bovine genomic library demonstrated that the bovine cellular retinoic acid-binding protein gene is composed of four exons and three introns. Two putative promoter sequences were identified in the cloned 5' sequence of the gene.

  19. Molecular cloning and analysis of functional cDNA and genomic clones encoding bovine cellular retinoic acid-binding protein.

    PubMed Central

    Shubeita, H E; Sambrook, J F; McCormick, A M

    1987-01-01

    A recombinant cDNA clone, pCRABP-HS1, encoding cellular retinoic acid-binding protein was isolated from a bovine adrenal cDNA library. COS-7 cells transfected with pCRABP-HS1 produced a biologically active retinoic acid-binding protein molecule of the expected molecular mass (15.5 kDa). RNA blot hybridization analysis using pCRABP-HS1 as a probe revealed a single 1050-nucleotide mRNA species in bovine adrenal, uterus, and testis, tissues that contain the highest levels of retinoic acid-binding activity. No hybridization was detected in RNA extracted from ovary, spleen, kidney, or liver, which contain relatively low levels of cellular retinoic acid-binding protein activity. Analysis of genomic clones isolated from an EcoRI bovine genomic library demonstrated that the bovine cellular retinoic acid-binding protein gene is composed of four exons and three introns. Two putative promoter sequences were identified in the cloned 5' sequence of the gene. Images PMID:3039499

  20. Purification, molecular cloning, and expression of the mammalian sigma1-binding site.

    PubMed

    Hanner, M; Moebius, F F; Flandorfer, A; Knaus, H G; Striessnig, J; Kempner, E; Glossmann, H

    1996-07-23

    Sigma-ligands comprise several chemically unrelated drugs such as haloperidol, pentazocine, and ditolylguanidine, which bind to a family of low molecular mass proteins in the endoplasmic reticulum. These so-called sigma-receptors are believed to mediate various pharmacological effects of sigma-ligands by as yet unknown mechanisms. Based on their opposite enantioselectivity for benzomorphans and different molecular masses, two subtypes are differentiated. We purified the sigma1-binding site as a single 30-kDa protein from guinea pig liver employing the benzomorphan(+)[3H]pentazocine and the arylazide (-)[3H]azidopamil as specific probes. The purified (+)[3H]pentazocine-binding protein retained its high affinity for haloperidol, pentazocine, and ditolylguanidine. Partial amino acid sequence obtained after trypsinolysis revealed no homology to known proteins. Radiation inactivation of the pentazocine-labeled sigma1-binding site yielded a molecular mass of 24 +/- 2 kDa. The corresponding cDNA was cloned using degenerate oligonucleotides and cDNA library screening. Its open reading frame encoded a 25.3-kDa protein with at least one putative transmembrane segment. The protein expressed in yeast cells transformed with the cDNA showed the pharmacological characteristics of the brain and liver sigma1-binding site. The deduced amino acid sequence was structurally unrelated to known mammalian proteins but it shared homology with fungal proteins involved in sterol synthesis. Northern blots showed high densities of the sigma1-binding site mRNA in sterol-producing tissues. This is also in agreement with the known ability of sigma1-binding sites to interact with steroids, such as progesterone.

  1. Human self-reactive T cell clones expressing identical T cell receptor beta chains differ in their ability to recognize a cryptic self-epitope

    PubMed Central

    1996-01-01

    Recognition of self-antigens by T lymphocytes is a central event in autoimmunity. Understanding of the molecular interactions between T cell receptors (TCR) and self-epitopes may explain how T cells escape thymic education and initiate an autoimmune reaction. We have studied five human in vivo activated T cell clones specific for the region 535- 551 of human thyroid peroxidase (TPO) established from a Graves' patient. Three clones (37, 72, and 73) expressed identical TCR beta and alpha chains rearranging V beta 1.1 and V alpha 15.1, and were considered sister clones. Clone 43 differed from clone 37 and its sisters in the J alpha region only. Clone NP-7 expressed V beta 6.5 but rearranged two in-frame TCR alpha chain, both using the V alpha 22.1 segment. Fine epitope mapping using nested peptides showed that clones using identical TCR beta chains, identical V alpha, but a different J alpha recognized distinct, nonoverlapping epitopes in the TPO 535-551 region. This finding shows that a different J alpha region alone leads to a heterogeneous pattern of recognition. This indicates that the "restricted" TCR V region usage sometimes found in autoimmune diseases may not always correspond to identical epitope recognition. To confirm that clones 37 (and its sisters) and 43 recognize different epitopes, the T cell clones were stimulated with a TPO-transfected autologous Epstein-Barr virus (EBV) cell line (TPO-EBV) that presents TPO epitopes afer endogenous processing. Only clone 37 and its sisters recognizes the TPO-EBV cell line, suggesting that the epitope recognized by clone 43 is not presented upon endogenous processing. We have shown that thyroid epithelial cells (TEC), the only cells that produce TPO, express HLA class II molecules in Graves' disease and can act as an antigen-presenting cells, presenting TPO after endogenous processing to autoantigen-reactive T cell clones. We tested, therefore, whether autologous TEC induced the same pattern of stimulation as TPO

  2. Molecular piracy of chemokine receptors by herpesviruses.

    PubMed

    Murphy, P M

    1994-01-01

    To succeed as a biological entity, viruses must exploit normal cellular functions and elude the host immune system; they often do so by molecular mimicry. One way that mimicry may occur is when viruses copy and modify host genes. The best studied examples of this are the oncogenes of RNA retroviruses, but a growing number of examples are also known for DNA viruses. So far they all come from just two groups of DNA viruses, the herpesviruses and poxviruses, and the majority of examples are for genes whose products regulate immune responses, such as cytokines, cytokine receptors, and complement control proteins. This review will focus on human and herpesvirus receptors for chemokines, a family of leukocyte chemoattractant and activating factors that are thought to be important mediators of inflammation. Although the biological roles of the viral chemokine receptor homologues are currently unknown, their connection to specific sets of chemokines has suggested a number of possible functions.

  3. Molecular cloning and analysis of a C-type lectin from silkworm Bombyx mori.

    PubMed

    Shahzad, Toufeeq; Zhan, Ming-Yue; Yang, Pei-Jin; Yu, Xiao-Qiang; Rao, Xiang-Jun

    2017-07-01

    C-type lectins (CTLs) play a variety of roles in plants and animals. They are involved in animal development, pathogen recognition, and the activation of immune responses. CTLs carry one or more non-catalytic carbohydrate-recognition domains (CRDs) to bind specific carbohydrates reversibly. Here, we report the molecular cloning and functional analysis of a single-CRD CTL, named C-type lectin-S2 (BmCTL-S2) from the domesticated silkmoth Bombyx mori (Lepidoptera: Bombycidae). The ORF of CTL-S2 is 666 bp, which encodes a putative protein of 221 amino acids. BmCTL-S2 is expressed in a variety of immune-related tissues, including hemocytes and fat body among others. BmCTL-S2 mRNA level in the midgut and the fat body was significantly increased by bacterial challenges. The recombinant protein (rBmCTL-S2) bound different bacterial cell wall components and bacterial cells. rBmCTL-S2 also inhibited the growth of Bacillus subtilis and Staphylococcus aureus. Taken together, we infer that BmCTL-S2 is a pattern-recognition receptor with antibacterial activities. © 2017 Wiley Periodicals, Inc.

  4. Molecular cloning and characterization of mouse aquaporin 6.

    PubMed

    Nagase, Hiroaki; Agren, Johan; Saito, Akiko; Liu, Kun; Agre, Peter; Hazama, Akihiro; Yasui, Masato

    2007-01-05

    In the rat kidney, aquaporin (AQP) 6 is localized in the intracellular vesicle membranes of type-A intercalated cells of the collecting duct; mouse AQP6 (mAQP6) has not been characterized. Although mAQP6 was originally cloned from cDNA in a mouse cerebellum library (GenBank NM 175087), we have independently cloned a cDNA encoding mAQP6 from an adult kidney cDNA library (C57BL/6J strain). We identified two different spliced variants of mAQP6: mAQP6a and mAQP6b. The mAQP6a isoform is almost identical to that of rat AQP6, whereas mAQP6b is identical to that reported in the mouse cerebellum library mentioned above. We found that the mRNA expression of these two spliced variants is regulated in a tissue-specific and age-dependent manner. Functional analyses of water and ion permeation revealed that mAQP6a functions like rat AQP6 and that mAQP6b does not function as either a water channel or an ion channel under our experimental conditions.

  5. Molecular cloning and characterization of mouse aquaporin 6

    PubMed Central

    Nagase, Hiroaki; Agren, Johan; Saito, Akiko; Liu, Kun; Agre, Peter; Hazama, Akihiro; Yasui, Masato

    2007-01-01

    In the rat kidney, aquaporin (AQP) 6 is localized in the intracellular vesicle membranes of type-A intercalated cells of the collecting duct; mouse AQP6 (mAQP6) has not been characterized. Although mAQP6 was originally cloned from cDNA in a mouse cerebellum library (GenBank NM 175087), we have independently cloned a cDNA encoding mAQP6 from an adult kidney cDNA library (C57BL/6J strain). We identified two different spliced variants of mAQP6: mAQP6a and mAQP6b. The mAQP6a isoform is almost identical to rat AQP6, whereas mAQP6b is identical to that reported in the mouse cerebellum library mentioned above. We found that the mRNA expression of these two spliced variants is regulated in a tissue-specific and age-dependent manner. Functional analyses of water and ion permeation revealed that mAQP6a functions like rat AQP6 and that mAQP6b does not function as either a water channel or an ion channel under our experimental conditions. PMID:17112474

  6. Molecular cloning and characterization of an extracellular protease gene from Aeromonas hydrophila.

    PubMed Central

    Rivero, O; Anguita, J; Paniagua, C; Naharro, G

    1990-01-01

    A structural gene which codes for an extracellular protease in Aeromonas hydrophilia SO2/2 and D13 was cloned in Escherichia coli C600-1 by using pBR322 as a vector. The gene codes for a temperature-stable protease with a molecular mass of approximately 38,000 daltons. The protein was secreted to the periplasm of E. coli C600-1 and purified by osmotic shock. Cloned protease (P3) was identical in molecular mass and properties to the one purified from A. hydrophila SO2/2 culture supernatant as an extracellular product. Images PMID:2193924

  7. Anti-idiotypes, receptors, and molecular mimicry

    SciTech Connect

    Linthicum, D.S.; Farid, N.R.

    1987-01-01

    This book provides a review of new methods and results in anti-idiotypes, receptors, and molecular mimicry. It begins with a discussion of the theoretical background of the anti-idiotypic network, it's role in the regulation of immune response, and the physical characteristics of anti-idiotypic antibodies. It then goes on to explore many applications in such areas as insulin action, thyroid cell function, the neurosciences, cardiology, virology, pharmacology, and reproduction.

  8. Cloning and characterization of a Drosophila serotonin receptor that activates adenylate cyclase.

    PubMed Central

    Witz, P; Amlaiky, N; Plassat, J L; Maroteaux, L; Borrelli, E; Hen, R

    1990-01-01

    Using a strategy based on nucleotide sequence homology between genes encoding receptors that interact with guanine nucleotide-binding proteins, we have isolated Drosophila genomic and cDNA clones encoding a functional serotonin receptor (5HT-dro receptor). This protein is expressed predominantly in Drosophila heads and exhibits highest homology with the human 5HT1A receptor. The predicted structure of the 5HT-dro receptor reveals two unusual features: (i) eight putative transmembrane domains instead of the expected seven and (ii) a Gly-Ser repeat that is a potential glycosaminoglycan attachment site. When stably introduced into mouse NIH 3T3 cells, the 5HT-dro receptor activates adenylate cyclase in response to serotonin and is inhibited by serotonin receptor antagonists such as dihydroergocryptine. The 5HT-dro receptor or closely related receptors might be responsible for the serotonin-sensitive cyclase that has been suggested to play a role in learning and modulation of circadian rhythm in a number of invertebrate systems. Images PMID:2174167

  9. Cloning of two adenosine receptor subtypes from mouse bone marrow-derived mast cells.

    PubMed

    Marquardt, D L; Walker, L L; Heinemann, S

    1994-05-01

    Adenosine potentiates the stimulated release of mast cell mediators. Pharmacologic studies suggest the presence of two adenosine receptors, one positively coupled to adenylate cyclase and the other coupled to phospholipase C activation. To identify mast cell adenosine receptor subtypes, cDNAs for the A1 and A2a adenosine receptors were obtained by screening a mouse brain cDNA library with the use of PCR-derived probes. Mouse bone marrow-derived mast cell cDNA libraries were constructed and screened with the use of A1 and A2a cDNA probes, which revealed the presence of A2a, but not A1, receptor clones. A putative A2b receptor was identified by using low stringency mast cell library screening. Northern blotting of mast cell poly(A)+ RNA with the use of receptor subtype probes labeled single mRNA bands of 2.4 kb and 1.8 kb for the A2a and A2b receptors, respectively. In situ cells. An A2a receptor-specific agonist failed to enhance mast cell mediator release, which suggests that the secretory process is modulated through the A2b and/or another receptor subtype. By using RNase protection assays, we found that mast cells that had been cultured in the presence of N-ethylcarboxamidoadenosine for 24 h exhibited a decrease in both A2a and A2b receptor RNA levels. Cells that had been cultured for 1 to 2 days in the presence of dexamethasone demonstrated increased amounts of A2a receptor mRNA, but no identifiable change in A2b receptor mRNA. Mast cells possess at least two adenosine receptor subtypes that may be differentially regulated.

  10. Cloning and functional characterization of Chondrichthyes, cloudy catshark, Scyliorhinus torazame and whale shark, Rhincodon typus estrogen receptors.

    PubMed

    Katsu, Yoshinao; Kohno, Satomi; Narita, Haruka; Urushitani, Hiroshi; Yamane, Koudai; Hara, Akihiko; Clauss, Tonya M; Walsh, Michael T; Miyagawa, Shinichi; Guillette, Louis J; Iguchi, Taisen

    2010-09-15

    Sex-steroid hormones are essential for normal reproductive activity in both sexes in all vertebrates. Estrogens are required for ovarian differentiation during a critical developmental stage and promote the growth and differentiation of the female reproductive system following puberty. Recent studies have shown that environmental estrogens influence the developing reproductive system as well as gametogenesis, especially in males. To understand the molecular mechanisms of estrogen actions and to evaluate estrogen receptor-ligand interactions in Elasmobranchii, we cloned a single estrogen receptor (ESR) from two shark species, the cloudy catshark (Scyliorhinus torazame) and whale shark (Rhincodon typus) and used an ERE-luciferase reporter assay system to characterize the interaction of these receptors with steroidal and other environmental estrogens. In the transient transfection ERE-luciferase reporter assay system, both shark ESR proteins displayed estrogen-dependent activation of transcription, and shark ESRs were more sensitive to 17beta-estradiol compared with other natural and synthetic estrogens. Further, the environmental chemicals, bisphenol A, nonylphenol, octylphenol and DDT could activate both shark ESRs. The assay system provides a tool for future studies examining the receptor-ligand interactions and estrogen disrupting mechanisms in Elasmobranchii. Copyright (c) 2010 Elsevier Inc. All rights reserved.

  11. Molecular cloning and amino acid sequence of leukotriene A4 hydrolase.

    PubMed Central

    Funk, C D; Rådmark, O; Fu, J Y; Matsumoto, T; Jörnvall, H; Shimizu, T; Samuelsson, B

    1987-01-01

    A cDNA clone corresponding to leukotriene A4 hydrolase was isolated from a human lung lambda gt11 expression library by immunoscreening with a polyclonal antiserum. Several additional clones from human lung and placenta cDNA lambda g11 libraries were obtained by plaque hybridization with the 32P-labeled lung cDNA clone. One of these clones has an insert of 1910 base pairs that contains the complete protein-coding region. From the deduced primary structure, leukotriene A4 hydrolase is a 610 amino and protein with a calculated molecular weight of 69,140. No apparent homologies with microsomal epoxide hydrolases were found. RNA blot analysis indicated substantial amounts of a discrete mRNA of approximately equal to 2250 nucleotides in lung tissue and leukocytes. Images PMID:2821541

  12. Mega primer-mediated molecular cloning strategy for chimaeragenesis and long DNA fragment insertion.

    PubMed

    Zhang, Hui; Liu, Chang-Jun; Jiang, Hui; Zhou, Lu; Li, Wen-Ying; Zhu, Ling-Yun; Wu, Lei; Meng, Er; Zhang, Dong-Yi

    2017-04-30

    Molecular cloning methods based on primer and overlap-extension PCR are widely used due to their simplicity, reliability, low cost and high efficiency. In this article, an efficient mega primer-mediated (MP) cloning strategy for chimaeragenesis and long DNA fragment insertion is presented. MP cloning is a seamless, restriction/ligation-independent method that requires only three steps: (i) the first PCR for mega primer generation; (ii) the second PCR for exponential amplification mediated by the mega primers and (iii) DpnI digestion and transformation. Most importantly, for chimaeragenesis, genes can be assembled and constructed into the plasmid vector in a single PCR step. By employing this strategy, we successfully inserted four DNA fragments (approximately 500 bp each) into the same vector simultaneously. In conclusion, the strategy proved to be a simple and efficient tool for seamless cloning.

  13. Molecular intermediates of fitness gain of an RNA virus: characterization of a mutant spectrum by biological and molecular cloning.

    PubMed

    Arias, A; Lázaro, E; Escarmís, C; Domingo, E

    2001-05-01

    The mutant spectrum of a virus quasispecies in the process of fitness gain of a debilitated foot-and-mouth disease virus (FMDV) clone has been analysed. The mutant spectrum was characterized by nucleotide sequencing of three virus genomic regions (internal ribosome entry site; region between the two AUG initiation codons; VP1-coding region) from 70 biological clones (virus from individual plaques formed on BHK-21 cell monolayers) and 70 molecular clones (RT--PCR products cloned in E. coli). The biological and molecular clones provided statistically indistinguishable definitions of the mutant spectrum with regard to the distribution of mutations among the three genomic regions analysed and with regard to the types of mutations, mutational hot-spots and mutation frequencies. Therefore, the molecular cloning procedure employed provides a simple protocol for the characterization of mutant spectra of viruses that do not grow in cell culture. The number of mutations found repeated among the clones analysed was higher than expected from the mean mutation frequencies. Some components of the mutant spectrum reflected genomes that were dominant in the prior evolutionary history of the virus (previous passages), confirming the presence of memory genomes in virus quasispecies. Other components of the mutant spectrum were genomes that became dominant at a later stage of evolution, suggesting a predictive value of mutant spectrum analysis with regard to the outcome of virus evolution. The results underline the observation that greater insight into evolutionary processes of viruses may be gained from detailed clonal analyses of the mutant swarms at the sequence level.

  14. Molecular modulators of benzodiazepine receptor ligand binding

    SciTech Connect

    Villar, H.O.; Loew, G.H. )

    1989-01-01

    Ten derivatives of {beta}-carbolines with known affinities to the GABA{sub A}/BDZ (benzodiazepine) receptor were studied using the Am 1 and MNDO/H Semiempirical techniques to identify and characterize molecular modulators of receptor recognition. Steric, lipophilic, and electrostatic properties of these compounds were calculated and examined for their possible role in recognition. Particular attention was paid to the regions around the two most favorable proton-accepting sites, the ON and the substituent at the C{sub 3} position, already implicated in recognition, as well as to the acidic N9H group that could be a proton donating center. To probe further the role of these three ligand sites in receptor interactions, a model of the receptor using three methanol molecules was made and optimum interactions of these three sites with them characterized. The results indicate some similarity in the shape of these ligands, which could reflect a steric requirement. The receptor affinity appears to be modulated to some extent by the ratio of lipophilic to hydrophilic surface, the negative potential at the {beta}N, provided there is also one at the C{sub 3} substituent confirming the importance of two accepting sites in recognition. The acidic N9H does not appear to be a modulator of affinity or does it form a stable H-bond with methanol as acceptor. The two proton donating molecules do form such a stable complex, and both are needed for high affinity.

  15. Cloning, sequencing, and expression of the gene coding for the human platelet. cap alpha. /sub 2/-adrenergic receptor

    SciTech Connect

    Kobilka, B.K.; Matsui, H.; Kobilka, T.S.; Yang-Feng, T.L.; Francke, U.; Caron, M.G.; Lefkowitz, R.J.; Regan, J.W.

    1987-10-30

    The gene for the human platelet ..cap alpha../sub 2/-adrenergic receptor has been cloned with oligonucleotides corresponding to the partial amino acid sequence of the purified receptor. The identity of this gene has been confirmed by the binding of ..cap alpha../sub 2/-adrenergic ligands to the cloned receptor expressed in Xenopus laevis oocytes. The deduced amino acid sequence is most similar to the recently cloned human ..beta../sub 2/- and ..beta../sub 1/-adrenergic receptors; however, similarities to the muscarinic cholinergic receptors are also evident. Two related genes have been identified by low stringency Southern blot analysis. These genes may represent additional ..cap alpha../sub 2/-adrenergic receptor subtypes.

  16. Transgenic cloned sheep overexpressing ovine toll-like receptor 4.

    PubMed

    Deng, Shoulong; Li, Guiguan; Zhang, Jinlong; Zhang, Xiaosheng; Cui, Maosheng; Guo, Yong; Liu, Guoshi; Li, Guangpeng; Feng, Jianzhong; Lian, Zhengxing

    2013-07-01

    An ovine fetal fibroblast cell line highly expressing TLR4 was established by inserting TLR4 into a reconstructive p3S-LoxP plasmid. Transgenic sheep overexpressing TLR4 were produced by transferring TLR4-transfected fetal fibroblasts into metaphase (M)II-stage enucleated oocytes (using SCNT). Because reconstructed embryos derived from MII-stage enucleated oocytes matured in vivo using a delayed-activated method had a higher pregnancy rate (18.52%) than that from MII-stage enucleated oocytes matured in vitro, the former procedure was used. Nine TLR4-transgenic live births were confirmed using polymerase chain reaction and Southern blot analysis. Increased expression of TLR4 at mRNA and protein levels in ear tissues of transgenic lambs were verified using reverse transcription polymerase chain reaction and immunohistochemistry, respectively. More toll-like receptor 4 protein was expressed by peripheral blood monocytes and/or macrophages collected from 3-month-old TLR4-transgenic than nontransgenic lambs at 0, 1, and 4 hours after lipopolysaccharide stimulation. Furthermore, interferon-γ and tumor necrosis factor α secreted by monocytes and/or macrophages of TLR4-transgenic lambs were significantly higher at 1 hour. Therefore, lipopolysaccharide-induced inflammatory responses from monocytes and/or macrophages occurred sooner in TLR4-transgenic lambs, consistent with an enhanced host immune response. In conclusion, transgenic sheep overexpressing TLR4 are a primary model to investigate the role of transgenic animals in disease resistance and have potential for breeding sheep with disease resistance.

  17. Molecular cloning and evolutionary analysis of GJB6 in mammals.

    PubMed

    Ru, Binghua; Han, Naijian; He, Guimei; Brayer, Kathryn; Zhang, Shuyi; Wang, Zhe

    2012-04-01

    GJB6 plays a crucial role in hearing. In mammals, bats use ultrasonic echolocation for orientation and locating prey. To investigate the evolution of GJB6 in mammals, we cloned the full-length coding region of GJB6 from 16 species of bats and 4 other mammal species and compared them with orthologous sequences in 11 other mammals. The results show purifying selection on GJB6 in mammals, as well as in the bat lineage, which indicates an important role for GJB6 in mammal hearing. We also found one unique amino acid substitution shared by 16 species of bats and 10 shared by two species of artiodactyls. This positioned the artiodactyls at an abnormal location in the gene tree. In addition, the cytoplasmic loop and carboxy terminus were more variable than other domains in all the mammals. These results demonstrate that GJB6 is basically conserved in mammals but has undergone relatively rapid evolution in particular lineages and domains.

  18. Molecular cloning of genes that specify virulence in Pseudomonas solanacearum.

    PubMed

    Xu, P L; Leong, S; Sequeira, L

    1988-02-01

    The suicide plasmid pSUP2021 was used to introduce Tn5 into the Pseudomonas solanacearum wild-type strain K60. We isolated eight avirulent mutants after screening 6,000 kanamycin-resistant transconjugants by inoculating eggplant (Solanum melongena L. cv. Black Beauty) and tobacco (Nicotiana tabacum L. cv. Bottom Special) seedlings. The Tn5-containing EcoRI fragments from the eight mutants were unique, suggesting that numerous genes specify virulence in this species. These EcoRI fragments were cloned into pBR322 or pUC12, and one of the clones, pKD810, was transformed into K60. All of the kanamycin-resistant, ampicillin-sensitive transformants were avirulent. Three randomly selected avirulent transformants were shown to carry the Tn5-containing fragment in place of the wild-type fragment and to exhibit the same hybridization pattern as the original KD810 mutant did. With pKD810 as a probe, we identified cosmids carrying the wild-type virulence genes by using a genomic library of K60 prepared in pLAFR3. Two of the homologous cosmids, pL810A and pL810C, when introduced into KD810 by transformation, restored virulence and normal growth of this mutant in tobacco. Altogether, these data indicate that the gene(s) interrupted by Tn5 insertion in KD810 is essential for the virulence of P. solanacearum. Further characterization of this gene is now being completed by subcloning, transposon mutagenesis, and complementation analysis.

  19. Molecular cloning, tissue distribution, and immune function of goose TLR7.

    PubMed

    Qi, Yulin; Chen, Shun; Zhao, Qiurong; Wang, Mingshu; Jia, Renyong; Zhu, Dekang; Liu, Mafeng; Liu, Fei; Chen, Xiaoyue; Cheng, Anchun

    2015-02-01

    TLR7 is a transmembrane endosomal protein that plays an essential role in innate antiviral responses via the recognition of conserved viral molecular patterns. Here, we cloned the full-length cDNA of goose TLR7 and carried out a molecular characterization of goose TLR7. The goose TLR7 gene is 3900 bp and encodes a 1045 amino acid protein with high homology to poultry (93% to duck and 83% to chicken). Similar conclusions were made by phylogenetic analysis. The predicted protein secondary structure of goose TLR7 contained a conserved Toll/interleukin-1 receptor domain and characteristic leucine-rich repeat regions, which has also been reported for duck TLR7. Additionally, the tissue distribution of goose TLR7 suggests that immune-associated tissues, especially the cecal tonsil and bursa of Fabricius, have high goose TLR7 expression levels. Goose TLR7 is abundantly expressed in lung tissues, which is distinct from its expression in chickens. Similar to duck TLR7, goose spleen mononuclear cells (MNCs) exposed to the mammalian TLR7 agonists R848 and Imiquimod showed significant induction of the production of proinflammatory cytokines and IFN-α. New type gosling viral enteritis virus (NGVEV) infection resulted in high mRNA expression levels of goose TLR7 in the spleen. By contrast, no direct interaction between NGVEV and goose TLR7 was detected after infecting goose spleen MNCs with NGVEV in vitro. However, triggering of goose TLR7 resulted in the rapid up-regulation of proinflammatory cytokines and anti-viral molecules, suggesting that goose TLR7 plays an important role in anti-viral defense. Copyright © 2014 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

  20. Expression of a mutant androgen receptor in cloned fibroblasts derived from a heterozygous carrier for the syndrome of testicular feminization.

    PubMed Central

    Elawady, M K; Allman, D R; Griffin, J E; Wilson, J D

    1983-01-01

    Thermolability of androgen binding was compared in fibroblasts cloned from normal female skin, skin from a subject with testicular feminization whose mutation is known to be associated with a thermolabile androgen receptor, and from the mother of the subject with testicular feminization. Seven of 28 clones studied from the mother exhibited thermolability of binding, indicating that the mutant gene that causes thermolability of binding, like the gene responsible for the normal androgen receptor, is X-linked. PMID:6602545

  1. Infectious virus replication in papillomas induced by molecularly cloned cottontail rabbit papillomavirus DNA.

    PubMed Central

    Brandsma, J L; Xiao, W

    1993-01-01

    The ability to obtain infectious papillomavirus virions from molecularly cloned DNA has not been previously reported. We demonstrate here that viral genomes isolated from a recombinant++ DNA clone of cottontail rabbit papillomavirus (CRPV) gave rise to infectious virus when inoculated into cottontail rabbit skin. Replication occurred in papillomas that formed at inoculation sites. Extract of a DNA-induced papilloma was serially passaged to naive rabbits with high efficiency. Complete virus was fractionated on cesium chloride density gradients, and papillomavirus particles were visualized by electron microscopy. CRPV DNA isolated from virions contained DNA sequence polymorphisms that are characteristic of the input CRPV-WA strain of virus, thereby proving that the newly generated virus originated from the molecularly cloned viral genome. These findings indicate that this will be a useful system in which to perform genetic analysis of viral gene functions involved in replication. Images PMID:8380092

  2. Cloning and expression pattern of the ecdysone receptor and retinoid X receptor from the centipede Lithobius peregrinus (Chilopoda, Lithobiomorpha).

    PubMed

    Bortolin, Francesca; Piulachs, Maria-Dolors; Congiu, Leonardo; Fusco, Giuseppe

    2011-10-01

    In arthropods, molting events are mediated by the binding of the ecdysone hormone to a heterodimer of two nuclear receptors: the ecdysone receptor (EcR) and the retinoid X receptor (RXR), a homolog of ultraspiracle (USP). We have cloned partial sequences of several isoforms for EcR and RXR genes from the centipede Lithobius peregrinus, and studied their expression profile during the second post-embryonic stage. LpEcR and LpRXR inferred amino acid sequences are very similar to other arthropod orthologs, especially to those of chelicerates and hemimetabolous insects, and their expression levels are significantly higher during the 48 h that precede the molt. Results obtained in this study represent the first data on the genetic basis of the ecdysone signal pathway for a myriapod, and in particular for an animal that, through a stereotyped developmental schedule paced by the molt cycle, completes trunk segmentation during post-embryonic life.

  3. Molecular cloning of a hyaluronidase from Bothrops pauloensis venom gland

    PubMed Central

    2014-01-01

    Background Hyaluronate is one of the major components of extracellular matrix from vertebrates whose breakdown is catalyzed by the enzyme hyaluronidase. These enzymes are widely described in snake venoms, in which they facilitate the spreading of the main toxins in the victim’s body during the envenoming. Snake venoms also present some variants (hyaluronidases-like substances) that are probably originated by alternative splicing, even though their relevance in envenomation is still under investigation. Hyaluronidases-like proteins have not yet been purified from any snake venom, but the cDNA that encodes these toxins was already identified in snake venom glands by transcriptomic analysis. Herein, we report the cloning and in silico analysis of the first hyaluronidase-like proteins from a Brazilian snake venom. Methods The cDNA sequence of hyaluronidase was cloned from the transcriptome of Bothrops pauloensis venom glands. This sequence was submitted to multiple alignment with other related sequences by ClustalW. A phylogenetic analysis was performed using MEGA 4 software by the neighbor joining (NJ) method. Results The cDNA from Bothrops pauloensis venom gland that corresponds to hyaluronidase comprises 1175 bp and codifies a protein containing 194 amino acid residues. The sequence, denominated BpHyase, was identified as hyaluronidase-like since it shows high sequence identities (above 83%) with other described snake venom hyaluronidase-like sequences. Hyaluronidases-like proteins are thought to be products of alternative splicing implicated in deletions of central amino acids, including the catalytic residues. Structure-based sequence alignment of BpHyase to human hyaluronidase hHyal-1 demonstrates a loss of some key secondary structures. The phylogenetic analysis indicates an independent evolution of BpHyal when compared to other hyaluronidases. However, these toxins might share a common ancestor, thus suggesting a broad hyaluronidase-like distribution among

  4. Molecular Pharmacology of δ-Opioid Receptors

    PubMed Central

    Gendron, Louis; Cahill, Catherine M.; von Zastrow, Mark; Schiller, Peter W.

    2016-01-01

    Opioids are among the most effective analgesics available and are the first choice in the treatment of acute severe pain. However, partial efficacy, a tendency to produce tolerance, and a host of ill-tolerated side effects make clinically available opioids less effective in the management of chronic pain syndromes. Given that most therapeutic opioids produce their actions via µ-opioid receptors (MOPrs), other targets are constantly being explored, among which δ-opioid receptors (DOPrs) are being increasingly considered as promising alternatives. This review addresses DOPrs from the perspective of cellular and molecular determinants of their pharmacological diversity. Thus, DOPr ligands are examined in terms of structural and functional variety, DOPrs’ capacity to engage a multiplicity of canonical and noncanonical G protein–dependent responses is surveyed, and evidence supporting ligand-specific signaling and regulation is analyzed. Pharmacological DOPr subtypes are examined in light of the ability of DOPr to organize into multimeric arrays and to adopt multiple active conformations as well as differences in ligand kinetics. Current knowledge on DOPr targeting to the membrane is examined as a means of understanding how these receptors are especially active in chronic pain management. Insight into cellular and molecular mechanisms of pharmacological diversity should guide the rational design of more effective, longer-lasting, and better-tolerated opioid analgesics for chronic pain management. PMID:27343248

  5. [Molecular cloning and structural characteristics of the R complex of maize]. Annual progress report

    SciTech Connect

    Not Available

    1992-07-01

    Studies on the R complex in Maize continued Progress is discussed in the following areas: Establishing identity of R components and cloning of R components; CO allele origin; molecular organization of R-r complex; NCO allele origin; genetic analysis of R-r complex; studies of the Sn locus and reverse paramutation.

  6. (Molecular cloning and structural characteristics of the R complex of maize)

    SciTech Connect

    Not Available

    1992-01-01

    Studies on the R complex in Maize continued Progress is discussed in the following areas: Establishing identity of R components and cloning of R components; CO allele origin; molecular organization of R-r complex; NCO allele origin; genetic analysis of R-r complex; studies of the Sn locus and reverse paramutation.

  7. Molecular cloning, characterization, and expression of wheat cystatins.

    PubMed

    Kuroda, M; Kiyosaki, T; Matsumoto, I; Misaka, T; Arai, S; Abe, K

    2001-01-01

    We cloned four kinds of cDNAs of wheat cystatins (WCs), WC1, WC2, WC3, and WC4, from the seed. They had 47-68% amino acid sequence similarities to other plant cystatins. WC1, WC2, and WC4 had 63-67% similalities to one another while 93% of amino acids were identical between WC1 and WC3. This suggested that WCI, WC2, and WC4 should be regarded as the isoforms of wheat cystatins. The mRNAs for WC1, WC2, and WC4 were all expressed in seed at an early stage of maturation and, after that, their quantities decreased gradually. However, each of the mRNAs was again expressed one day after the start of germination and the expression continued for the following five days. WC1 seemed to be expressed at a higher level than WC2 and WC4. Immunostaining for looking at site-specific expression of each WC demonstrated that both WC1 and WC4 existed in the aleuron layer and embryo, but in the endosperm the only existing species was WC1. Differences in mRNA level and tissue localization found for the WCs may suggest their differential physiological roles.

  8. Molecular cloning and characterization of the Candida albicans enolase gene.

    PubMed Central

    Mason, A B; Buckley, H R; Gorman, J A

    1993-01-01

    A DNA clone containing the putative Candida albicans enolase gene (ENO1) was isolated from a genomic DNA library. The sequenced insert contained a continuous open reading frame of 1,320 bp. The predicted 440-amino-acid protein is 78 and 76% identical, respectively, to Saccharomyces cerevisiae enolase proteins 1 and 2. Only one enolase gene could be detected in C. albicans genomic DNA by Southern analysis with a homologous probe. Northern (RNA) analysis detected a single, abundant C. albicans ENO1 transcript of approximately 1,600 nucleotides. When cells were grown on glucose, levels of ENO1 mRNA were markedly increased by comparison with ENO1 mRNA levels in cells grown on ethanol, a gluconeogenic carbon source. In contrast to this glucose-mediated transcriptional induction, the carbon source had no dramatic effect on the levels of enolase protein or enzyme activity in the C. albicans strains tested. These results suggest that posttranscriptional mechanisms are responsible for modulating expression of the C. albicans enolase gene. Images PMID:8478328

  9. Molecular cloning and expression of the mouse ornithine decarboxylase gene.

    PubMed Central

    McConlogue, L; Gupta, M; Wu, L; Coffino, P

    1984-01-01

    We used mRNA from a mutant S49 mouse lymphoma cell line that produces ornithine decarboxylase (OrnDCase) as its major protein product to synthesize and clone cDNA. Plasmids containing OrnDCase cDNA were identified by hybrid selection of OrnDCase mRNA and in vitro translation. The two of these with the largest inserts together span 2.05 kilobases of cDNA. Southern blot analysis of DNA from wild-type or mutant S49 cells, cleaved with EcoRI or with BamHI, revealed multiple bands homologous to OrnD-Case cDNA, only one of which was amplified in the mutant cells. RNA transfer blot analysis showed that the major OrnD-Case mRNA in the mouse lymphoma cells is 2.0 kilobases long. A similar size mRNA was found in mouse kidney and was more abundant in the kidneys of mice treated with testosterone, an inducer of OrnDCase activity in that tissue. Images PMID:6582509

  10. Molecular cloning and functional expression of soybean allene oxide synthases.

    PubMed

    Kongrit, Darika; Jisaka, Mitsuo; Iwanaga, Chitose; Yokomichi, Hiroshi; Katsube, Takuya; Nishimura, Kohji; Nagaya, Tsutomu; Yokota, Kazushige

    2007-02-01

    A plant allene oxide synthase (AOS) reacting with 13S-hydroperoxy-9Z,11E,15Z-octadecatrienoic acid (13-HPOT), a lipoxygenase product of alpha-linolenic acid, provides an allene oxide which functions as an intermediate for jasmonic acid (JA) synthesis, making AOS a key enzyme regulating the JA level in plants. Although AOSs in various plants have been investigated, there is only limited information about AOSs in soybean (Glycine max). In this study, we cloned and characterized two soybean AOSs, GmAOS1 and GmAOS2, sharing 95% homology in the predicted amino acid sequences. GmAOS1 and GmAOS2 were composed of 564 and 559 amino acids respectively, with predicted N-terminal chloroplast-targeting signal peptides. Both AOSs expressed in Escherichia coli were selective for 13S-hydroperoxides of alpha-linolenic and linoleic acids, suggesting the potential of GmAOS1 and GmAOS2 to contribute to JA synthesis. GmAOS1 and GmAOS2 were expressed in leaves, stems, and roots, suggesting broad distribution in a soybean plant.

  11. Molecular cloning of a human macrophage lectin specific for galactose

    SciTech Connect

    Cherayil, B.J.; Chairovitz, S.; Wong, C.; Pillai, S. Harvard Medical School, Boston )

    1990-09-01

    The murine Mac-2 protein is a galactose- and IgE-binding lectin secreted by inflammatory macrophages. The authors describe here the cloning an dcharacterization of cDNA representing the human homolog of Mac-2 (hMac-2). The amino acid sequence derived from the hMac-2 cDNA indicates that the protein is evolutionarily highly conserved, with 85% of its amino acid residues being similar to those in the murine homolog. This conservation is especially marked in the carboxyl-terminal lectin domain. The amino-terminal half of the protein is less conserved but still contains the repetitive proline-glycine-rich motif seen in the mouse protein. hMac-2 synthesized in vitro is recognized by the M3/38 monoclonal antibody to Mac-2 and binds to the desialylated glycoprotein asialofetuin and to laminin, a major component of basement membranes. These findings are discussed in the context of the potential functions of hMac-2.

  12. Molecular cloning of cecropin B responsive endonucleases in Yersinia ruckeri

    USDA-ARS?s Scientific Manuscript database

    We have previously demonstrated that Yersinia ruckeri resists cecropin B in an inducible manner. In this study, we sought to identify the molecular changes responsible for the inducible cecropin B resistance of Y. ruckeri. Differences in gene expression associated with the inducible resistance were ...

  13. Distinguishing features of an infectious molecular clone of the highly divergent and noncytopathic human immunodeficiency virus type 2 UC1 strain.

    PubMed Central

    Barnett, S W; Quiroga, M; Werner, A; Dina, D; Levy, J A

    1993-01-01

    A full-length infectious molecular clone was derived from the noncytopathic human immunodeficiency virus type 2 UC1 strain (HIV-2UC1) that was originally recoverd from an individual from the Ivory Coast. Like the parental isolate, the molecularly cloned virus (HIV-2UC1mc or UC1 mc) demonstrates a reduced ability to induce syncytium formation, to kill cells, and to down-modulate the cell surface CD4 receptor in infected cells. Phylogenetic analysis of the DNA sequence of UC1mc revealed that it is the first full-length infectious molecular clone in the second HIV-2 subgroup previously identified by partial sequence analysis of the HIV-2D205 and HIV-2GH-2 strains. These highly divergent HIV-2 strains appear to be genetically equidistant from other HIV-2 and simian immunodeficiency virus SIVmac/sm strains. UC1mc is unlike any other HIV-2 or SIVmac/sm strain in that it lacks a cysteine residue at the proposed signal peptide cleavage site in Env. However, site-directed mutagenesis experiments indicate that this missing cysteine is not alone important in the noncytopathic phenotype of UC1mc. Like other HIV-2 and SIV strains, the UC1mc Env transmembrane protein (gp43) is mutated to a truncated form (gp34) after passage in certain T-cell lines. The UC1 molecular clone should be helpful in determining the genetic sequences associated with HIV-2 cytopathicity. Images PMID:8419635

  14. Molecular cloning and biological characterization of full-length HIV-1 subtype C from Botswana.

    PubMed

    Ndung'u, T; Renjifo, B; Novitsky, V A; McLane, M F; Gaolekwe, S; Essex, M

    2000-12-20

    Human immunodeficiency virus type 1 (HIV-1) subtype C is now responsible for more than half of all HIV-1 infections in the global epidemic and for the high levels of HIV-1 prevalence in southern Africa. To facilitate studies of the biological nature and the underlying molecular determinants of this virus, we constructed eight full-length proviral clones from two asymptomatic and three AIDS patients infected with HIV-1 subtype C from Botswana. Analysis of viral lysates showed that Gag, Pol, and Env structural proteins were present in the virions. In four clones, the analysis suggested inefficient envelope glycoprotein processing. Nucleotide sequence analysis of the eight clones did not reveal frameshifts, deletions, premature truncations, or translational stop codons in any structural, regulatory, or accessory genes. None of the subtype C clones were replication competent in donor peripheral blood mononuclear cells (PBMCs), macrophages, Jurkat(tat) cells, or U87. CD4.CCR5 cells. However, infection by two clones could be rescued by complementation with a functional subtype C envelope clone, resulting in a productive infection of PBMCs, macrophages, and U87. CD4.CCR5 cells.

  15. Cloning and pharmacological characterisation of the guinea pig 5-ht5A receptor.

    PubMed

    Thomas, David R; Larminie, Christopher G; Lyons, Helen R; Fosberry, Andrew; Hill, Matthew J; Hayes, Philip D

    2004-06-28

    The guinea pig 5-hydroxytryptamine(5A) (gp5-ht(5A)) receptor was cloned from guinea pig brain using degenerate polymerase chain reaction (PCR) and shows 88%, 85% and 84% amino acid sequence identity versus the human, rat and mouse 5-ht(5A) receptors, respectively. The receptor was transiently expressed in human embryonic kidney (HEK) 293 cells. [(3)H]-Lysergic acid diethylamide (LSD) bound saturably to gp5-ht(5A)/HEK293 membranes with a K(d) of 2.3+/-0.1 nM and B(max) of 15.7+/-3.4 pmol/mg protein. The receptor binding profile, determined by competition with [(3)H]LSD, correlated well with that for the human 5-ht(5A) receptor. 5-HT stimulated [(35)S]GTPgammaS binding to gp5-ht(5A)/HEK293 membranes (pEC(50) 8.1+/-0.2), and the response was surmountably antagonised by methiothepin and ritanserin, giving apparent pK(B) values of 8.0 and 7.2, respectively. The 5-HT response was absent using membranes prepared from gp5-ht(5A)/HEK293 cells pretreated with pertussis toxin (PTX). These data suggest that the gp5-ht(5A) receptor couples to G(i)-proteins in this expression system and shows a similar pharmacological profile to that for the human 5-ht(5A) receptor.

  16. Molecularly imprinted polymers: synthetic receptors in bioanalysis.

    PubMed

    Tse Sum Bui, Bernadette; Haupt, Karsten

    2010-11-01

    Molecularly imprinted polymers (MIPs) are tailor-made synthetic materials possessing specific cavities designed for a target molecule. Since they recognise their target analyte with affinities and selectivities comparable to those of antibody-antigen, enzyme-substrate and ligand-receptor interactions, they are often referred to as synthetic receptors or plastic antibodies. In this review, we describe the great potential and recent developments of MIPs in affinity separations, with emphasis on their application to the solid-phase extraction (SPE) of analytes from complex matrices. Research efforts made in this field to obtain water-compatible polymers for their applicability in aqueous environments are described. We particularly discuss problems encountered in the use of MIPs in SPE and the attempts carried out to improve their efficiency.

  17. Characterization of a highly pathogenic molecular clone of feline immunodeficiency virus clade C.

    PubMed

    de Rozières, Sohela; Mathiason, Candace K; Rolston, Matthew R; Chatterji, Udayan; Hoover, Edward A; Elder, John H

    2004-09-01

    We have derived and characterized a highly pathogenic molecular isolate of feline immunodeficiency virus subtype C (FIV-C) CABCpady00C. Clone FIV-C36 was obtained by lambda cloning from cats that developed severe immunodeficiency disease when infected with CABCpady00C (Abbotsford, British Columbia, Canada). Clone FIV-C36 Env is 96% identical to the noninfectious FIV-C isolate sequence deposited in GenBank (FIV-Cgb; GenBank accession number AF474246) (A. Harmache et al.) but is much more divergent in Env when compared to the subgroup A clones Petaluma (34TF10) and FIV-PPR (76 and 78% divergence, respectively). Clone FIV-C36 was able to infect freshly isolated feline peripheral blood mononuclear cells and primary T-cell lines but failed to productively infect CrFK cells, as is typical of FIV field isolates. Two-week-old specific-pathogen-free cats infected with FIV-C36 tissue culture supernatant became PCR positive and developed severe acute immunodeficiency disease similar to that caused by the uncloned CABCpady00C parent. At 4 to 5 weeks postinfection (PI), 3 of 4 animals developed CD4(+)-T-cell depletion, fever, weight loss, diarrhea, and opportunistic infections, including ulcerative stomatitis and tonsillitis associated with abundant bacterial growth, pneumonia, and pyelonephritis, requiring euthanasia. Histopathology confirmed severe thymic and systemic lymphoid depletion. Interestingly, the dam also became infected with a high viral load at 5 weeks PI of the kittens and developed a similar disease syndrome, requiring euthanasia at 11 weeks PI of the kittens. This constitutes the first report of a replication-competent, infectious, and pathogenic molecular clone of FIV-C. Clone FIV-C36 will facilitate dissection of the pathogenic determinants of FIV.

  18. A novel IL-1 receptor, cloned from B cells by mammalian expression, is expressed in many cell types.

    PubMed Central

    McMahan, C J; Slack, J L; Mosley, B; Cosman, D; Lupton, S D; Brunton, L L; Grubin, C E; Wignall, J M; Jenkins, N A; Brannan, C I

    1991-01-01

    cDNA clones corresponding to an Mr approximately 80,000 receptor (type I receptor) for interleukin-1 (IL-1) have been isolated previously by mammalian expression. Here, we report the use of an improved expression cloning method to isolate human and murine cDNA clones encoding a second type (Mr approximately 60,000) of IL-1 receptor (type II receptor). The mature type II IL-1 receptor consists of (i) a ligand binding portion comprised of three immunoglobulin-like domains; (ii) a single transmembrane region; and (iii) a short cytoplasmic domain of 29 amino acids. This last contrasts with the approximately 215 amino acid cytoplasmic domain of the type I receptor, and suggests that the two IL-1 receptors may interact with different signal transduction pathways. The type II receptor is expressed in a number of different tissues, including both B and T lymphocytes, and can be induced in several cell types by treatment with phorbol ester. Both IL-1 receptors appear to be well conserved in evolution, and map to the same chromosomal location. Like the type I receptor, the human type II IL-1 receptor can bind all three forms of IL-1 (IL-1 alpha, IL-1 beta and IL-1ra). Vaccinia virus contains an open reading frame bearing strong resemblance to the type II IL-1 receptor. Images PMID:1833184

  19. Molecular cloning, genomic organization and cell‐binding characteristics of mouse Spα

    PubMed Central

    Gebe, J A; Llewellyn, M‐B C; Hoggatt, H; Aruffo, A

    2000-01-01

    Several group B scavenger receptor cysteine‐rich (SRCR) proteins have been shown to function as modulators in the immune response. Recently, we reported the cloning of a new member of this family, human Spα (hSpα). Herein we report the cloning and characterization of the mouse homologue of hSpα. Like its human counterpart, mouse Spα (mSpα), is a secreted protein containing three SRCR domains. Most lymphoid tissues express RNA transcripts encoding mSpα. Characterization of a genomic clone encoding the mature mSpα protein showed that each of the SRCR domains of mSpα is encoded by a single exon. Comparison of the sequence of mSPα with those of other published proteins indicates that it is the same as the recently reported protein named AIM (apoptosis inhibitor expressed by macrophages). Cell‐binding studies with a mSpα immunoglobulin (mSpα‐Rγ) fusion protein indicated that mSpα is capable of binding to spleen‐derived CD19+ B cells and minimally to peritoneal cavity‐derived CD19+ B cells but not to peripheral blood‐derived B cells. Spleen‐derived CD3+ T cells also bound mSpα‐Rγ; however, no binding was observed to either peripheral blood mononuclear cells or peritoneal cavity‐derived CD3+ T cells. The mSpα‐Rγ fusion protein was also shown to bind to the mouse cell lines WEHI3 (monocytic) and EL‐4 (thymoma, T cell). The cloning of cDNA and genomic clones encoding mSpα and the identification of cells expressing a putative mSpα receptor(s) should facilitate in vivo studies designed to investigate the function of Spα in the immune compartment. PMID:10651944

  20. Molecular cloning, genomic organization and cell-binding characteristics of mouse Spalpha.

    PubMed

    Gebe, J A; Llewellyn, M; Hoggatt, H; Aruffo, A

    2000-01-01

    Several group B scavenger receptor cysteine-rich (SRCR) proteins have been shown to function as modulators in the immune response. Recently, we reported the cloning of a new member of this family, human Spalpha (hSpalpha). Herein we report the cloning and characterization of the mouse homologue of hSpalpha. Like its human counterpart, mouse Spalpha (mSpalpha), is a secreted protein containing three SRCR domains. Most lymphoid tissues express RNA transcripts encoding mSpalpha. Characterization of a genomic clone encoding the mature mSpalpha protein showed that each of the SRCR domains of mSpalpha is encoded by a single exon. Comparison of the sequence of mSPalpha with those of other published proteins indicates that it is the same as the recently reported protein named AIM (apoptosis inhibitor expressed by macrophages). Cell-binding studies with a mSpalpha immunoglobulin (mSpalpha-Rgamma) fusion protein indicated that mSpalpha is capable of binding to spleen-derived CD19+ B cells and minimally to peritoneal cavity-derived CD19+ B cells but not to peripheral blood-derived B cells. Spleen-derived CD3+ T cells also bound mSpalpha-Rgamma; however, no binding was observed to either peripheral blood mononuclear cells or peritoneal cavity-derived CD3+ T cells. The mSpalpha-Rgamma fusion protein was also shown to bind to the mouse cell lines WEHI3 (monocytic) and EL-4 (thymoma, T cell). The cloning of cDNA and genomic clones encoding mSpalpha and the identification of cells expressing a putative mSpalpha receptor(s) should facilitate in vivo studies designed to investigate the function of Spalpha in the immune compartment.

  1. Molecular cloning, expression and characterization of pyridoxamine–pyruvate aminotransferase

    PubMed Central

    Yoshikane, Yu; Yokochi, Nana; Ohnishi, Kouhei; Hayashi, Hideyuki; Yagi, Toshiharu

    2006-01-01

    Pyridoxamine–pyruvate aminotransferase is a PLP (pyridoxal 5′-phosphate) (a coenzyme form of vitamin B6)-independent aminotransferase which catalyses a reversible transamination reaction between pyridoxamine and pyruvate to form pyridoxal and L-alanine. The gene encoding the enzyme has been identified, cloned and overexpressed for the first time. The mlr6806 gene on the chromosome of a symbiotic nitrogen-fixing bacterium, Mesorhizobium loti, encoded the enzyme, which consists of 393 amino acid residues. The primary sequence was identical with those of archaeal aspartate aminotransferase and rat serine–pyruvate aminotransferase, which are PLP-dependent aminotransferases. The results of fold-type analysis and the consensus amino acid residues found around the active-site lysine residue identified in the present study showed that the enzyme could be classified into class V aminotransferases of fold type I or the AT IV subfamily of the α family of the PLP-dependent enzymes. Analyses of the absorption and CD spectra of the wild-type and point-mutated enzymes showed that Lys197 was essential for the enzyme activity, and was the active-site lysine residue that corresponded to that found in the PLP-dependent aminotransferases, as had been suggested previously [Hodsdon, Kolb, Snell and Cole (1978) Biochem. J. 169, 429–432]. The Kd value for pyridoxal determined by means of CD was 100-fold lower than the Km value for it, suggesting that Schiff base formation between pyridoxal and the active-site lysine residue is partially rate determining in the catalysis of pyridoxal. The active-site structure and evolutionary aspects of the enzyme are discussed. PMID:16545075

  2. Molecular cloning and analysis of the Catsper1 gene promoter.

    PubMed

    Mata-Rocha, Minerva; Alvarado-Cuevas, Edith; Hernández-Sánchez, Javier; Cerecedo, Doris; Felix, Ricardo; Hernández-Reyes, Adriana; Tesoro-Cruz, Emiliano; Oviedo, Norma

    2013-05-01

    CatSper channels are essential for hyperactivity of sperm flagellum, progesterone-mediated chemotaxis and oocyte fertilization. Catsper genes are exclusively expressed in the testis during spermatogenesis, but the function and regulation of the corresponding promoter regions are unknown. Here, we report the cloning and characterization of the promoter regions in the human and murine Catsper1 genes. These promoter regions were identified and isolated from genomic DNA, and transcriptional activities were tested in vitro after transfection into human embryonic kidney 293, mouse Sertoli cells 1 and GC-1spg cell lines as well as by injecting plasmids directly into mouse testes. Although the human and murine Catsper1 promoters lacked a TATA box, a well-conserved CRE site was identified. Both sequences may be considered as TATAless promoters because their transcriptional activity was not affected after deletion of TATA box-like sites. Several transcription initiation sites were revealed by RNA ligase-mediated rapid amplification of the cDNA 5'-ends. We also found that the immediate upstream region and the first exon in the human CATSPER1 gene negatively regulate transcriptional activity. In the murine Catsper1 promoter, binding sites for transcription factors SRY, SOX9 and CREB were protected by the presence of nuclear testis proteins in DNAse degradation assays. Likewise, the mouse Catsper1 promoter exhibited transcriptional activity in both orientations and displayed significant expression levels in mouse testis in vivo, whereas the suppression of transcription signals in the promoter resulted in low expression levels. This study, thus, represents the first identification of the transcriptional control regions in the genes encoding the human and murine CatSper channels.

  3. Molecular cloning and functional analysis of the goose FSHβ gene.

    PubMed

    Huang, Z; Li, X; Li, Y; Liu, R; Chen, Y; Wu, N; Wang, M; Song, Y; Yuan, X; Lan, L; Xu, Q; Chen, G; Zhao, W

    2015-01-01

    The objective of this investigation was to clone goose FSHβ-subunit cDNA and to construct a FSH fusion gene to identify the function of FSHβ mRNA during stages of the breeding cycle. The FSHβ gene was obtained by reverse transcription-PCR, and the full-length FSHβ mRNA sequence was amplified by rapid-amplification of cDNA ends. FSHβ mRNA expression was detected in reproductive tissues at different stages (pre-laying, laying period, and broody period). Additionally, the expression of 4 genes known to be involved in reproduction (FSHβ, GnRH, GH, and BMP) were evaluated in COS-7 cells expressing the fusion gene (pVITRO2-FSHαβ-CTP). The results show that the FSHβ gene consists of a 16 base pair (bp) 5'-untranslated region (UTR), 396 bp open reading frame, and alternative 3'-UTRs at 518 bp and 780 bp, respectively. qPCR analyses revealed that FSHβ mRNA is highly transcribed in reproductive tissues, including the pituitary, hypothalamus, ovaries, and oviduct. FSHβ mRNA expression increased and subsequently decreased in the pituitary, ovaries, and oviduct during the reproductive stages. Stable FSH expression was confirmed using enzyme-linked immunosorbent assays after transfection with the pVITRO2-FSHαβ-CTP plasmid. FSHβ, GnRH, and BMP expression increased significantly 36 h and 48 h after transfection with the fusion gene in COS-7 cells. The results demonstrate that the FSHβ subunit functions in the goose reproductive cycle and provides a theoretical basis for future breeding work.

  4. Molecular cloning and characterization of novel ficolins from Xenopus laevis.

    PubMed

    Kakinuma, Yuji; Endo, Yuichi; Takahashi, Minoru; Nakata, Munehiro; Matsushita, Misao; Takenoshita, Seiichi; Fujita, Teizo

    2003-04-01

    Ficolins are proteins characterized by the presence of collagen- and fibrinogen-like domains. Two of three human ficolins, L-ficolin and H-ficolin, are serum lectins and are thought to play crucial roles in host defense through opsonization and complement activation. To elucidate the evolution of ficolins and the primordial complement lectin pathway, we cloned four ficolin cDNAs from Xenopus laevis, termed Xenopus ficolin (XeFCN) 1, 2, 3 and 4. The deduced amino acid sequences of the four ficolins revealed the conserved collagen- and fibrinogen-like domains. The full sequences of the four ficolins showed a 42-56% identity to human ficolins, and 60-83% between one another. Northern blots showed that XeFCN1 was expressed mainly in liver, spleen and heart, and XeFCN2 and XeFCN4 mainly in peripheral blood leukocytes, lung and spleen. We isolated ficolin proteins from Xenopus serum by affinity chromatography on N-acetylglucosamine-agarose, followed by ion-exchange chromatography. The final eluate showed polymeric bands composed of two components of 37 and 40 kDa. The N-terminal amino acid sequences and treatment with endoglycosidase F showed that the two bands are the same XeFCN1 protein with different masses of N-linked sugar. The polymeric form of the two types of XeFCN1 specifically recognized GlcNAc and GalNAc residues. These results suggest that like human L-ficolin, XeFCN1 functions in the circulation through its lectin activity.

  5. Molecular cloning and characterization of leucine aminopeptidase from Fasciola gigantica.

    PubMed

    Changklungmoa, Narin; Chaithirayanon, Kulathida; Kueakhai, Pornanan; Meemon, Krai; Riengrojpitak, Suda; Sobhon, Prasert

    2012-07-01

    M17 leucine aminopeptidase (LAP) is one of a family of metalloexopeptidases, of which short peptide fragments are cleaved from the N-terminals. In this study, the full length of cDNA encoding Fasciola gigantica LAP (FgLAP) was cloned from adult parasites. The amino acid sequences of FgLAP showed a high degree of identity (98%) with that from Fasciola hepatica and a low degree of identities (11% and 9%) with those from cattle and human. Phylogenetic analysis revealed that the FgLAP was closely related and grouped with F. hepatica LAP (FhLAP). Northern analysis showed that FgLAP transcriptional products have 1800 base pairs. Analysis by RNA in situ hybridization indicated that LAP gene was expressed in the cecal epithelial cells of adult parasites. A polyclonal antibody to a recombinant FgLAP (rFgLAP) detected the native LAP protein in various developmental stages of the parasite. In a functional test, this rFgLAP displayed aminolytic activity using a fluorogenic Leu-MCA substrate, and was significantly inhibited by bestatin. Its maximum activity was at pH 8.0 and enhanced by Mn(2+) ions. Localization of LAP proteins by immunohistochemistry and immunofluorescence techniques indicated that the enzyme was distributed in the apical cytoplasm of cecal epithelial cells. Because of its important metabolic role and fairly exposed position, FgLAP is a potential drug target and a possible vaccine candidate against fasciolosis. Copyright © 2012 Elsevier Inc. All rights reserved.

  6. Ecdysteroid receptor from the American lobster Homarus americanus: EcR/RXR isoform cloning and ligand-binding properties.

    PubMed

    Tarrant, Ann M; Behrendt, Lars; Stegeman, John J; Verslycke, Tim

    2011-09-01

    In arthropods, ecdysteroids regulate molting by activating a heterodimer formed by the ecdysone receptor (EcR) and retinoid X receptor (RXR). While this mechanism is similar in insects and crustaceans, variation in receptor splicing, dimerization and ligand affinity adds specificity to molting processes. This study reports the EcR and RXR sequences from American lobster, a commercially and ecologically important crustacean. We cloned two EcR splice variants, both of which specifically bind ponasterone A, and two RXR variants, both of which enhance binding of ponasterone A to the EcR. Lobster EcR has high affinity for ponasterone A and muristerone and moderately high affinity for the insecticide tebufenozide. Bisphenol A, diethyl phthalate, and two polychlorinated biphenyls (PCB 29 and PCB 30), environmental chemicals shown to interfere with crustacean molting, showed little or no affinity for lobster EcR. These studies establish the molecular basis for investigation of lobster ecdysteroid signaling and signal disruption by environmental chemicals.

  7. Recent advances in the molecular pharmacology of the alpha 1-adrenergic receptors.

    PubMed

    Guarino, R D; Perez, D M; Piascik, M T

    1996-08-01

    This review is intended to discuss recent developments in the molecular pharmacology of the alpha 1-adrenergic receptor (alpha 1-AR) subtypes. After a brief historical development, we will focus on the more contemporary issues having to do with this receptor family. Emphasis will be put on recent data regarding the cloning, nomenclature, signalling mechanisms, and genomic organization of the alpha 1-AR subtypes. We will also highlight recent mutational studies that identify key amino acid residues involved in ligand binding, as well as the role of the alpha 1-AR subtypes in regulating physiologic processes.

  8. Molecular cloning and characterization of l-methionine γ-lyase from Streptomyces avermitilis.

    PubMed

    Kudou, Daizou; Yasuda, Eri; Hirai, Yoshiyuki; Tamura, Takashi; Inagaki, Kenji

    2015-10-01

    A pyridoxal 5'-phosphate-dependent methionine γ-lyase (MGL) was cloned from Streptomyces avermitilis catalyzed the degradation of methionine to α-ketobutyrate, methanethiol, and ammonia. The sav7062 gene (1,242 bp) was corresponded to 413 amino acid residues with a molecular mass of 42,994 Da. The deduced amino acid sequence showed a high degree of similarity to those of other MGL enzymes. The sav7062 gene was overexpressed in Escherichia coli. The enzyme was purified to homogeneity and exhibited the MGL catalytic activities. We cloned the enzyme that has the MGL activity in Streptomyces for the first time.

  9. Construction and characterization of an infectious molecular clone of Koala retrovirus.

    PubMed

    Shojima, Takayuki; Hoshino, Shigeki; Abe, Masumi; Yasuda, Jiro; Shogen, Hiroko; Kobayashi, Takeshi; Miyazawa, Takayuki

    2013-05-01

    Koala retrovirus (KoRV) is a gammaretrovirus that is currently endogenizing into koalas. Studies on KoRV infection have been hampered by the lack of a replication-competent molecular clone. In this study, we constructed an infectious molecular clone, termed plasmid pKoRV522, of a KoRV isolate (strain Aki) from a koala reared in a Japanese zoo. The virus KoRV522, derived from pKoRV522, grew efficiently in human embryonic kidney (HEK293T) cells, attaining 10(6) focus-forming units/ml. Several mutations in the Gag (L domain) and Env regions reported to be involved in reduction in viral infection/production in vitro are found in pKoRV522, yet KoRV522 replicated well, suggesting that any effects of these mutations are limited. Indeed, a reporter virus pseudotyped with pKoRV522 Env was found to infect human, feline, and mink cell lines efficiently. Analyses of KoRV L-domain mutants showed that an additional PPXY sequence, PPPY, in Gag plays a critical role in KoRV budding. Altogether, our results demonstrate the construction and characterization of the first infectious molecular clone of KoRV. The infectious clone reported here will be useful for elucidating the mechanism of endogenization of the virus in koalas and screening for antiretroviral drugs for KoRV-infected koalas.

  10. Expression cloning of the murine interferon gamma receptor cDNA.

    PubMed

    Munro, S; Maniatis, T

    1989-12-01

    A cDNA encoding a receptor for murine interferon gamma (IFN-gamma) was isolated from an expression library made from murine thymocytes. The clone was identified by transfecting the library into monkey COS cells and probing the transfected monolayer with radiolabeled murine IFN-gamma. Cells expressing the receptor were identified by autoradiography and plasmids encoding the receptor were directly rescued from those cells producing a positive signal. A partial cDNA so obtained was used to isolate a full-length cDNA from mouse L929 cells by conventional means. When this cDNA was expressed in COS cells it produced a specific binding site for murine IFN-gamma with an affinity constant similar to that of the receptor found on L929 cells. The predicted amino acid sequence of the murine IFN-gamma receptor shows homology to that previously reported for the human IFN-gamma receptor. However, although the two proteins are clearly related, they show less than 60% identity in both the putative extracellular domain and the intracellular domain.

  11. Cloning and functional expression of alternative spliced variants of the ρ1 γ-aminobutyrate receptor

    PubMed Central

    Martínez-Torres, Ataúlfo; Vazquez, Ana E.; Panicker, Mitradas M.; Miledi, Ricardo

    1998-01-01

    The ρ1 γ-aminobutyrate receptor (GABAρ1) is expressed predominantly in the retina and forms homomeric GABA-gated Cl− channels that are clearly different from the multisubunit GABAA receptors. In contrast to these, GABAρ1 receptors desensitize very little and are not blocked by bicuculline. In addition to GABAρ1, two new variants were identified in human retina cDNA libraries. Cloning and sequence analysis showed that both variants contain large deletions in the putative extracellular domain of the receptor. These deletions extend from a common 5′ site to different 3′ sites. The cDNA with the largest deletion, named GABAρ1Δ450, contains a complete ORF identical to that of GABAρ1 but missing 450 nt. This cDNA encodes a protein of 323 aa, identical to the GABAρ1, but has a deletion of 150 aa in the amino-terminal extracellular domain. GABAρ1Δ450 mRNA injected into Xenopus oocytes did not produce functional GABA receptors. The second GABAρ1 variant (GABAρ1Δ51) contains a 51-nt deletion. In Xenopus oocytes, GABAρ1Δ51 led to the expression of GABA receptors that had the essential GABAρ1 characteristics of low desensitization and bicuculline resistance. Therefore, alternative splicing increases the coding potential of this gene family expressed in the human retina, but the functional diversity created by the alternative spliced forms is still not understood. PMID:9520485

  12. Cloning of human genes encoding novel G protein-coupled receptors

    SciTech Connect

    Marchese, A.; Docherty, J.M.; Heiber, M.

    1994-10-01

    We report the isolation and characterization of several novel human genes encoding G protein-coupled receptors. Each of the receptors contained the familiar seven transmembrane topography and most closely resembled peptide binding receptors. Gene GPR1 encoded a receptor protein that is intronless in the coding region and that shared identity (43% in the transmembrane regions) with the opioid receptors. Northern blot analysis revealed that GPR1 transcripts were expressed in the human hippocampus, and the gene was localized to chromosome 15q21.6. Gene GPR2 encoded a protein that most closely resembled an interleukin-8 receptor (51% in the transmembrane regions), and this gene, not expressed in the six brain regions examined, was localized to chromosome 17q2.1-q21.3. A third gene, GPR3, showed identity (56% in the transmembrane regions) with a previously characterized cDNA clone from rat and was localized to chromosome 1p35-p36.1. 31 refs., 5 figs., 1 tab.

  13. Cloning and expression of a cDNA for mouse prostaglandin E receptor EP2 subtype.

    PubMed

    Honda, A; Sugimoto, Y; Namba, T; Watabe, A; Irie, A; Negishi, M; Narumiya, S; Ichikawa, A

    1993-04-15

    A functional cDNA clone encoding mouse EP2 subtype of prostaglandin (PG) E receptor was isolated from a mouse cDNA library by cross-hybridization with the mouse EP3 subtype PGE receptor cDNA. The mouse EP2 receptor consists of 513 amino acid residues with putative seven-transmembrane domains. In contrast to EP3 receptor, this receptor possesses long third intracellular loop and carboxyl-terminal tail. [3H] PGE2 specifically bound to the membrane of mammalian COS cells transfected with the cDNA. The binding to the membrane was displaced with unlabeled PG in the order of PGE2 = PGE1 > iloprost > or = PGF2 alpha > or = PGD2. The binding was also inhibited by misoprostol, an EP2 and EP3 agonist, but not by sulprostone, an EP1 and EP3 agonist, and SC-19220, an EP1 antagonist. PGE2 markedly increased cAMP level in COS cells transfected with the cDNA. These results suggest that this receptor is EP2 subtype. Northern blot analysis demonstrated that the EP2 mRNA is widely expressed in various tissues, the abundant expression being observed in ileum, thymus, and mastocytoma P-815 cells.

  14. Molecular Cloning and Characterization of Apricot Fruit Polyphenol Oxidase

    PubMed Central

    Chevalier, Tony; de Rigal, David; Mbéguié-A-Mbéguié, Didier; Gauillard, Frédéric; Richard-Forget, Florence; Fils-Lycaon, Bernard R.

    1999-01-01

    A reverse transcriptase-polymerase chain reaction experiment was done to synthesize a homologous polyphenol oxidase (PPO) probe from apricot (Prunus armeniaca var Bergeron) fruit. This probe was further used to isolate a full-length PPO cDNA, PA-PPO (accession no. AF020786), from an immature-green fruit cDNA library. PA-PPO is 2070 bp long and contains a single open reading frame encoding a PPO precursor peptide of 597 amino acids with a calculated molecular mass of 67.1 kD and an isoelectric point of 6.84. The mature protein has a predicted molecular mass of 56.2 kD and an isoelectric point of 5.84. PA-PPO belongs to a multigene family. The gene is highly expressed in young, immature-green fruit and is turned off early in the ripening process. The ratio of PPO protein to total proteins per fruit apparently remains stable regardless of the stage of development, whereas PPO specific activity peaks at the breaker stage. These results suggest that, in addition to a transcriptional control of PPO expression, other regulation factors such as translational and posttranslational controls also occur. PMID:10198084

  15. Cloning, expression and functional characterization of carp, Cyprinus carpio, estrogen receptors and their differential activations by estrogens.

    PubMed

    Katsu, Yoshinao; Lange, Anke; Miyagawa, Shinichi; Urushitani, Hiroshi; Tatarazako, Norishisa; Kawashima, Yukio; Tyler, Charles R; Iguchi, Taisen

    2013-01-01

    Sex-steroid hormones are essential for normal reproductive activity in both sexes. Estrogens are necessary for ovarian differentiation during a critical developmental stage in vertebrates and promote the growth and differentiation of the female reproductive system. Importantly, environmental estrogens can influence the reproductive system and have been shown to disrupt gametogenesis in males. To understand the molecular mechanisms of estrogen actions and to evaluate estrogen receptor ligand interactions in the carp, Cyprinus carpio, a species used widely for both field- and laboratory-based studies, we cloned all three carp estrogen receptors (ER; ERα, ERβ1 and ERβ2) and applied an estrogen-responsive (ERE)-luciferase reporter assay system to characterize the interactions of these receptors with steroidal and synthetic estrogens. DNA fragments encoding all three ERs in carp, ERα, ERβ1 and ERβ2, were obtained from the ovary using degenerate primer sets and PCR techniques, and full-length carp ER (cER) cDNAs were then obtained using RACE (rapid amplification of the cDNA end) techniques. Amino acid sequences of cERs showed overall homology of 46% (α vs β1), 49% (α vs β2) and 53% (β1 vs β2). In the transient transfection ERE-luciferase reporter assay system (using mammalian cells) the cER proteins displayed estrogen-dependent activation of transcription and cERβ2 showed a higher sensitivity to the natural steroid oestrogen, 17β-estradiol, than cERα. The assay system developed is a powerful assay for toxicology and provides a tool for future studies examining the receptor-environmental chemical interactions and estrogen-disrupting mechanisms in carp. The data presented also expand our knowledge of estrogen receptor evolution.

  16. Cloning and expression of melatonin receptors in the mudskipper Boleophthalmus pectinirostris: their role in synchronizing its semilunar spawning rhythm.

    PubMed

    Hong, Lu Yan; Hong, Wan Shu; Zhu, Wen Bo; Shi, Qiong; You, Xin Xin; Chen, Shi Xi

    2014-01-01

    The mudskipper Boleophthalmus pectinirostris, a burrow-dwelling fish inhabiting intertidal mudflats, spawns only once during the spawning season around either the first or last lunar quarters. To understand the molecular mechanisms regulating this semilunar spawning rhythm, we cloned all melatonin receptor subtypes (mtnr1a1.4, mtnr1a1.7, mtnr1b, and mtnr1c). Expression of three melatonin receptor subtypes (except mtnr1c) was found in the ovaries. In contrast, the expression of all receptor subtypes was found in the diencephalon and the pituitary. In the fully-grown follicles, only mtnr1a1.7 mRNA was detected in both the isolated follicle layers and denuded oocytes. Interestingly, the transcript levels of both mtnr1a1.4 in the diencephalon and mtnr1a1.7 in the ovary displayed two cycles within one lunar month, and peaked around the first and last lunar quarters. We used 17α,20β-dihydroxy-4-pregnen-3-one (DHP), a maturation-inducing hormone, as a biomarker to examine the involvement of melatonin receptors in the control of the spawning cycle. Melatonin significantly increased the plasma DHP level 1h post intraperitoneal injection. Melatonin also directly stimulated ovarian fragments in vitro to produce a significantly higher amount of DHP. Taken together, these results provided the first evidence that melatonin receptors were involved in the synchronization of the semilunar spawning rhythm in the female mudskipper by acting through the HPG axis and/or directly on ovarian tissues to stimulate the production of DHP.

  17. A fast and robust method to clone and functionally validate T-cell receptors.

    PubMed

    Birkholz, Katrin; Hofmann, Christian; Hoyer, Stefanie; Schulz, Birgit; Harrer, Thomas; Kämpgen, Eckhart; Schuler, Gerold; Dörrie, Jan; Schaft, Niels

    2009-07-31

    Sequencing, cloning and functional testing of T-cell-receptor (TCR) alpha- and beta-chains from T-cell clones is often required in immunotherapy and in immunological research. However, the determination of the TCR chains by a simple PCR is not possible, since, in contrast to the 3' constant domain and untranslated region (UTR), no conserved sequences are present in the 5' region. Furthermore, subsequent functional testing of cloned TCRs requires laborious cell culture experiments, often involving primary human material and time-consuming viral transduction strategies. Here we present a universal PCR-based protocol, adapted from the capswitch technology, that allows for amplification of the TCR alpha- and beta-chain mRNAs without knowledge of the TCR variable domain subtype by attaching a designed sequence to the mRNA's 5' end. Two different MelanA/HLA-A2-specific and one HIVgag/HLA-A2-specific TCR were cloned that way, and were functionally tested by a newly developed easy, fast, and low-cost method: we electroporated Jurkat T cells simultaneously with TCR-encoding RNA and an NFAT-reporter construct, and measured the activation status of the cells upon specific stimulation. The results of this assay correlated with the cytokine release, functional avidity, proliferative activity, and the ability to recognize MelanA/HLA-A2-presenting tumor cells of bulk T cells electroporated with RNA encoding the same TCR. Together these two protocols represent a rapid and low-cost tool for the identification and functional testing of TCRs of T-cell clones, which can then be applied in immunotherapy or immunological research.

  18. Cloning of the bovine 215-kDa cation-independent mannose 6-phosphate receptor.

    PubMed Central

    Lobel, P; Dahms, N M; Breitmeyer, J; Chirgwin, J M; Kornfeld, S

    1987-01-01

    Four overlapping cDNA clones encoding a partial sequence of the cation-independent 215-kDa mannose 6-phosphate receptor have been identified by screening a fetal calf liver cDNA library with oligonucleotide probes. RNA hybridization analysis showed that the length of the mRNA is approximately 9.5 kilobases. Sequence analysis demonstrated that the clones consist of 4647 contiguous nucleotides and contain an open reading frame coding for a polypeptide of 1461 amino acids, which we estimate represents greater than 75% of the primary structure of the receptor. The deduced amino acid sequence indicates that the receptor has a carboxyl-terminal cytoplasmic domain of 163 amino acids that is rich in acidic residues, a 23-amino acid transmembrane segment, and an extracellular domain containing at least eight homologous repeats of approximately 145 amino acids. One of the repeats contains an additional 43-residue segment that is similar to the type II repeat of fibronectin. Each repeat contains a highly conserved 13-amino acid unit bordered by cysteine residues that may be functionally important. Images PMID:2951738

  19. The mouse prostaglandin E receptor EP2 subtype: cloning, expression, and northern blot analysis.

    PubMed

    Katsuyama, M; Nishigaki, N; Sugimoto, Y; Morimoto, K; Negishi, M; Narumiya, S; Ichikawa, A

    1995-09-25

    A functional cDNA clone for the mouse prostaglandin (PG) E receptor EP2 subtype was isolated from a mouse cDNA library. The mouse EP2 receptor consists of 362 amino acid residues with seven putative transmembrane domains. [3H]PGE2 bound specifically to the membrane of Chinese hamster ovary cells stably expressing the cloned receptor. This binding was displaced by unlabeled prostanoids in the order of PGE2 = PGE1 > iloprost, a stable PGI2 agonist > PGF2 alpha > PGD2. Binding was also inhibited by butaprost (an EP2 agonist) and to a lesser extent by M&B 28767 (an EP3 agonist), but not by sulprostone (an EP1 and EP3 agonist) or SC-19220 (an EP1 antagonist). PGE2 and butaprost increased the cAMP level in the Chinese hamster ovary cells in a concentration-dependent manner. Northern blot analysis revealed that EP2 mRNA is expressed most abundantly in the uterus, followed by the spleen, lung, thymus, ileum, liver, and stomach.

  20. Molecular cloning and characterization of a cDNA encoding endonuclease from potato (Solanum tuberosum).

    PubMed

    Larsen, Knud

    2005-11-01

    A cDNA, StEN1, encoding a potato (Solanum tuberosum) endonuclease was cloned and sequenced. The nucleotide sequence of this clone contains an open reading frame of 906 nucleotides encoding a protein of 302 amino acids, and with a calculated molecular mass of 34.4kDa and a Pi of 5.6. The deduced StEN1 protein contains a putative signal sequence of 25 amino acid residues. The StEN1 encoded protein shows substantial homology to both plant and fungal endonucleases isolated and cloned from other sources. The highest identity (73%) was observed with AgCEL I from celery, Apium graveolens, ZEN1 from Zinnia elegans (69%) and DSA6 from daylily, Hemerocallis (68%). RT-PCR expression analysis demonstrated that the potato StEN1 gene is constitutively expressed in potato, although minor differences in expression level in different tissues were observed.

  1. Molecular cloning and characterization of protein phosphatase 2C of vomeronasal sensory epithelium of garter snakes.

    PubMed

    Wang, Dalton; Liu, Weimin; Liu, Jinming; Chen, Ping; Quan, Wei; Halpern, Mimi

    2002-12-15

    The earthworm-derived chemoattractant ES20 interacts with its G-protein-coupled receptors on the plasma membrane of vomeronasal (VN) sensory neurons of garter snakes, resulting in an increase in inositol trisphosphate [J. Biol. Chem. 269 (1994) 16867] and a rapid phosphorylation of the membrane-bound proteins, p42/44 [Biochim. Biophys. Acta 1450 (1999) 320]. The phosphorylation of p42/44 proteins are countervailingly regulated by a protein kinase and an okadaic acid-insensitive but fluoride-sensitive protein phosphatase (PPase) [J. Liu et al. (loc. cit.)]. The phosphorylation of p42/44 induced by ES20 appears to play a role in the regulation of signal transduction pathways by modulating the GTPase activity [J. Liu et al. (loc. cit.)]. A 564-bp fragment of cDNA was obtained from VN RNA of garter snakes by reverse transcription polymerase chain reaction with degenerate primers. The 564-bp fragment was amplified, cloned, and sequenced. Northern blot analysis revealed that both the VN organ (VNO) and brain contained the gene of PPase 2C. A full-length complementary 4119-bp DNA containing an open reading frame of 1146bp that encodes a protein of 382 amino acids with a molecular mass of 49,123Da was obtained from the VN cDNA library of garter snakes. The deduced amino acid sequence showed 88% amino acid identity to bovine protein phosphatase 2C alpha and 87% identity to human and rat PP2C alpha and to Mg(2+)-dependent protein phosphatase 1A of rat and rabbit. In situ hybridization revealed that the mRNA of VN protein phosphatase 2C is expressed in the vomeronasal sensory epithelium. This is the first report of the identification of a type 2C serine/threonine protein phosphatase in the VN system.

  2. Cloning of the cDNA for the human. beta. /sub 1/-adrenergic receptor

    SciTech Connect

    Frielle, T.; Collins, S.; Daniel, K.W.; Caron, M.G.; Lefkowitz, R.J.; Kobilka, B.K.

    1987-11-01

    Screening of a human placenta lambdagt11 library has led to the isolation of the cDNA for the human ..beta../sub 1/-adrenergic receptor (..beta../sub 1/AR). Used as the probe was the human genomic clone termed G-21. This clone, which contains an intronless gene for a putative receptor, was previously isolated by virtue of its cross hybridization with the human ..beta../sub 2/-adrenergic receptor (..beta../sub 2/AR). The 2.4-kilobase cDNA for the human ..beta../sub 1/AR encodes a protein of 477 amino acid residues that is 69% homologous with the avian ..beta..AR but only 54% homologous with the human ..beta../sub 2/AR. This suggests that the avian gene encoding ..beta..AR and the human gene encoding ..beta../sub 1/AR evolved from a common ancestral gene. RNA blot analysis indicates a message of 2.5 kilobases in rat tissues, with a pattern of tissue distribution consistent with ..beta../sub 1/AR binding. This pattern is quite distinct from the pattern obtained when the ..beta../sub 2/AR cDNA is used as a probe. Expression of receptor protein in Xenopus laevis oocytes conveys adenylate cyclase responsiveness to catecholamines with a typical ..beta../sub 1/AR specificity. This contrasts with the typical ..beta../sub 2/ subtype specificity observed when the human ..beta../sub 2/AR cDNA is expressed in this system. Mammalian ..beta../sub 1/AR and ..beta../sub 2/AR are thus products of distinct genes, both of which are apparently related to the putative G-21 receptor.

  3. Cloning and functional characterization of the rabbit C-C chemokine receptor 2

    PubMed Central

    Lu, Deshun; Yuan, Xiu-juan; Evans, Robert J; Pappas, Amy T; Wang, He; Su, Eric W; Hamdouchi, Chafiq; Venkataraman, Chandrasekar

    2005-01-01

    Background CC-family chemokine receptor 2 (CCR2) is implicated in the trafficking of blood-borne monocytes to sites of inflammation and is implicated in the pathogenesis of several inflammatory diseases such as rheumatoid arthritis, multiple sclerosis and atherosclerosis. The major challenge in the development of small molecule chemokine receptor antagonists is the lack of cross-species activity to the receptor in the preclinical species. Rabbit models have been widely used to study the role of various inflammatory molecules in the development of inflammatory processes. Therefore, in this study, we report the cloning and characterization of rabbit CCR2. Data regarding the activity of the CCR2 antagonist will provide valuable tools to perform toxicology and efficacy studies in the rabbit model. Results Sequence alignment indicated that rabbit CCR2 shares 80 % identity to human CCR2b. Tissue distribution indicated that rabbit CCR2 is abundantly expressed in spleen and lung. Recombinant rabbit CCR2 expressed as stable transfectants in U-937 cells binds radiolabeled 125I-mouse JE (murine MCP-1) with a calculated Kd of 0.1 nM. In competition binding assays, binding of radiolabeled mouse JE to rabbit CCR2 is differentially competed by human MCP-1, -2, -3 and -4, but not by RANTES, MIP-1α or MIP-1β. U-937/rabbit CCR2 stable transfectants undergo chemotaxis in response to both human MCP-1 and mouse JE with potencies comparable to those reported for human CCR2b. Finally, TAK-779, a dual CCR2/CCR5 antagonist effectively inhibits the binding of 125I-mouse JE (IC50 = 2.3 nM) to rabbit CCR2 and effectively blocks CCR2-mediated chemotaxis. Conclusion In this study, we report the cloning of rabbit CCR2 and demonstrate that this receptor is a functional chemotactic receptor for MCP-1. PMID:16001983

  4. Development and Characterization of an In Vivo Pathogenic Molecular Clone of Equine Infectious Anemia Virus

    PubMed Central

    Cook, R. Frank; Leroux, Caroline; Cook, Sheila J.; Berger, Sandra L.; Lichtenstein, Drew L.; Ghabrial, Nadia N.; Montelaro, Ronald C.; Issel, Charles J.

    1998-01-01

    An infectious nonpathogenic molecular clone (19-2-6A) of equine infectious anemia virus (EIAV) was modified by substitution of a 3.3-kbp fragment amplified by PCR techniques from a pathogenic variant (EIAVPV) of the cell culture-adapted strain of EIAV (EIAVPR). This substitution consisted of coding sequences for 77 amino acids at the carboxyl terminus of the integrase, the S1 (encoding the second exon of tat), S2, and S3 (encoding the second exon of rev) open reading frames, the complete env gene (including the first exon of rev), and the 3′ long terminal repeat (LTR). Modified 19-2-6A molecular clones were designated EIAVPV3.3, and infection of a single pony (678) with viruses derived from a mixture of five of these molecular clones induced clinical signs of acute equine infectious anemia (EIA) at 23 days postinfection (dpi). As a consequence of this initial study, a single molecular clone, EIAVPV3.3#3 (redesignated EIAVUK), was selected for further study and inoculated into two ponies (613 and 614) and two horses (700 and 764). Pony 614 and the two horses developed febrile responses by 12 dpi, which was accompanied by a 48 to 64% reduction in platelet number, whereas pony 613 did not develop fever (40.6°C) until 76 dpi. EIAV could be isolated from the plasma of these animals by 5 to 7 dpi, and all became seropositive for antibodies to this virus by 21 dpi. Analysis of the complete nucleotide sequence demonstrated that the 3.3-kbp 3′ fragment of EIAVUK differed from the consensus sequence of EIAVPV by just a single amino acid residue in the second exon of the rev gene. Complete homology with the EIAVPV consensus sequence was observed in the hypervariable region of the LTR. However, EIAVUK was found to contain an unusual 68-bp nucleotide insertion/duplication in a normally conserved region of the LTR sequence. These results demonstrate that substitution of a 3.3-kbp fragment from the EIAVPV strain into the infectious nonpathogenic molecular clone 19-2-6A leads

  5. Molecular Cloning and Immunochemical Characterization of a New Japanese Cedar Pollen Allergen Homologous to Plant Subtilisin-Like Serine Protease

    PubMed Central

    2010-01-01

    Protease activities in allergen sources are thought to be involved in triggering allergic inflammation through the disruption of epithelial barrier or the induction of proinflammatory cytokines. Protease allergens may also work as type 2 helper T cell (TH2) adjuvants through the cleavage of cell surface receptors. Here, we report molecular cloning and immunochemical characterization of a new Japanese cedar (Cryptomeria japonica) pollen allergen (CPA9) homologous to serine protease, which is initially found as a high IgE-binding spot on our two-dimensional (2-D) IgE immunoblotting map. The cpa9 cDNA encoded a 757 amino acid polypeptide showing a significant sequence identity with plant subtilisin-like serine protease family members including melon major allergen Cuc m 1. We found that native CPA9 purified from C. japonica pollen showed a high IgE-binding frequency and IgE cross-reactivity with melon extract. PMID:23282945

  6. cDNA cloning of the bovine low density lipoprotein receptor: feedback regulation of a receptor mRNA.

    PubMed Central

    Russell, D W; Yamamoto, T; Schneider, W J; Slaughter, C J; Brown, M S; Goldstein, J L

    1983-01-01

    The low density lipoprotein (LDL) receptor belongs to a class of migrant cell surface proteins that mediate endocytosis of macromolecular ligands. No cDNAs for this class of proteins have been isolated to date. In the current paper, we report the isolation of a cDNA clone for the LDL receptor from a bovine adrenal cDNA library. The library was constructed by the Okayama-Berg method from poly(A)+ RNA that had been enriched in receptor mRNA by immunopurification of polysomes. Mixtures of synthetic oligonucleotides encoding the amino acid sequence of two neighboring regions of a single cyanogen bromide fragment were used as hybridization probes to identify a recombinant plasmid containing the LDL receptor cDNA. This plasmid, designated pLDLR-1, contains a 2.8-kilobase (kb) insert that includes a sequence which corresponds to the known amino acid sequence of a 36-residue cyanogen bromide fragment of the receptor. pLDLR-1 hybridized to a mRNA of approximately equal to 5.5 kb in the bovine adrenal gland. This mRNA, like the receptor protein, was 9-fold more abundant in bovine adrenal than in bovine liver. pLDLR-1 cross-hybridized to a mRNA of approximately equal to 5.5 kb in cultured human epidermoid carcinoma A-431 cells. This mRNA was markedly reduced in amount when sterols were added to the culture medium, an observation that explains the previously observed feedback regulation of LDL receptor protein. Southern blot analysis of bovine genomic DNA with 32P-labeled pLDLR-1 revealed a simple pattern of hybridization, consistent with a single-copy gene containing introns. Images PMID:6143315

  7. Cloning and expression of the VHDL receptor from fat body of the corn ear worm, Helicoverpa zea.

    PubMed

    Persaud, Deryck R; Haunerland, Norbert H

    2004-01-01

    In Noctuids, storage proteins are taken up into fat body by receptor-mediated endocytosis. These include arylphorin and a second, structurally unrelated very high-density lipoprotein (VHDL). Previously, we have isolated a single storage protein receptor from the corn earworm, Helicoverpa zea, which binds both VHDL and arylphorin. The receptor protein is a basic, N-terminally blocked, approximately 80 kDa protein that is associated with fat body membranes. Microsequencing of proteolytic fragments of the isolated receptor protein revealed internal sequences that were used to clone the complete cDNA of the VHDL receptor by 3' and 5' RACE techniques. The receptor protein, when expressed in vitro via a suitable insect expression vector, reacted with antibodies against the native VHDL receptor and bound strongly to its ligand VHDL, thus confirming that the cloned cDNA represents indeed the previously purified VHDL receptor. The receptor protein and a second, similar protein also found associated with the fat body membrane show considerable homology to putative basic juvenile hormone suppressible proteins cloned previously from other Noctuid species. Sequence analysis revealed that the receptor is likely a peripheral membrane protein that may mediate the selective uptake of VHDL.

  8. CDNA CLONING OF FATHEAD MINNOW (PIMEPHALES PROMELAS) ESTROGEN AND ANDROGEN RECEPTORS FOR USE IN STEROID RECEPTOR EXTRAPOLATION STUDIES FOR ENDOCRINE DISRUPTING CHEMICALS

    EPA Science Inventory

    cDNA Cloning of Fathead minnow (Pimephales promelas) Estrogen and Androgen Receptors for Use in Steroid Receptor Extrapolation Studies for Endocrine Disrupting Chemicals.

    Wilson, V.S.1,, Korte, J.2, Hartig P. 1, Ankley, G.T.2, Gray, L.E., Jr 1, , and Welch, J.E.1. 1U.S...

  9. CDNA CLONING OF FATHEAD MINNOW (PIMEPHALES PROMELAS) ESTROGEN AND ANDROGEN RECEPTORS FOR USE IN STEROID RECEPTOR EXTRAPOLATION STUDIES FOR ENDOCRINE DISRUPTING CHEMICALS

    EPA Science Inventory

    cDNA Cloning of Fathead minnow (Pimephales promelas) Estrogen and Androgen Receptors for Use in Steroid Receptor Extrapolation Studies for Endocrine Disrupting Chemicals.

    Wilson, V.S.1,, Korte, J.2, Hartig P. 1, Ankley, G.T.2, Gray, L.E., Jr 1, , and Welch, J.E.1. 1U.S...

  10. Ca2+ channel inhibition by endomorphins via the cloned mu-opioid receptor expressed in NG108-15 cells.

    PubMed

    Mima, H; Morikawa, H; Fukuda, K; Kato, S; Shoda, T; Mori, K

    1997-12-11

    Endomorphin-1 and -2, recently isolated endogenous peptides specific for the mu-opioid receptor, inhibited Ca2+ channel currents with EC50 of 6 and 9 nM, respectively, in NG108-15 cells transformed to express the cloned rat mu-opioid receptor. On the other hand, they elicited no response in nontransfected NG108-15 cells. It is concluded that endomorphin-1 and -2 induce Ca2+ channel inhibition by selectively activating the mu-opioid receptor.

  11. Particle infectivity of HIV-1 full-length genome infectious molecular clones in a subtype C heterosexual transmission pair following high fidelity amplification and unbiased cloning

    SciTech Connect

    Deymier, Martin J.; Claiborne, Daniel T.; Ende, Zachary; Ratner, Hannah K.; Kilembe, William; Hunter, Eric

    2014-11-15

    The high genetic diversity of HIV-1 impedes high throughput, large-scale sequencing and full-length genome cloning by common restriction enzyme based methods. Applying novel methods that employ a high-fidelity polymerase for amplification and an unbiased fusion-based cloning strategy, we have generated several HIV-1 full-length genome infectious molecular clones from an epidemiologically linked transmission pair. These clones represent the transmitted/founder virus and phylogenetically diverse non-transmitted variants from the chronically infected individual's diverse quasispecies near the time of transmission. We demonstrate that, using this approach, PCR-induced mutations in full-length clones derived from their cognate single genome amplicons are rare. Furthermore, all eight non-transmitted genomes tested produced functional virus with a range of infectivities, belying the previous assumption that a majority of circulating viruses in chronic HIV-1 infection are defective. Thus, these methods provide important tools to update protocols in molecular biology that can be universally applied to the study of human viral pathogens. - Highlights: • Our novel methodology demonstrates accurate amplification and cloning of full-length HIV-1 genomes. • A majority of plasma derived HIV variants from a chronically infected individual are infectious. • The transmitted/founder was more infectious than the majority of the variants from the chronically infected donor.

  12. [Construction of standard allelic ladder of miniSTR loci by molecular cloning].

    PubMed

    Bai, Xue; Cong, Bin; Li, Shu-Jin; Hou, Zhi-Ping; Gu, Jian-Li; Liu, Ning; Li, Xia; Guo, Xia

    2009-04-01

    To investigate the genetic polymorphism of five miniSTR loci (D9S2157, D9S1122, D10S1435, D12ATA63, D2S1776) in Hebei Han population and to construct standard allelic ladders. Polymorphism of the five miniSTR loci in 120 unrelated individuals was analyzed by fluorescence PCR and ABI 310 Genetic Analyzer. Molecular cloning technique was employed to construct standard allelic ladder of the 5 loci. Of the five miniSTR loci, 8, 8, 7, 5 and 8 alleles were found, respectively. The polymorphism information component were 0.790, 0.720, 0.750, 0.630 and 0.850, respectively. The five loci have relatively abundant polymorphic information and their standard allelic ladders constructed by molecular cloning technique are useful in forensic science.

  13. Cloning and characterization of the retinoid X receptor from a primitive crustacean Daphnia magna.

    PubMed

    Wang, Ying H; Wang, Guirong; LeBlanc, Gerald A

    2007-01-15

    Terpenoid hormones function as morphogens throughout the animal kingdom and many of these activities are mediated through members of the retinoid X group of nuclear receptors (RXR; NR2B). In the present study, RXR was cloned from the water flea Daphnia magna, a primitive crustacean of the class Branchiopoda, and characterized with respect to phylogeny, developmental expression, and hormonal regulation. The full length daphnid RXR cDNA was cloned by initial PCR amplification of a cDNA fragment from the highly conserved DNA-binding domain followed by extension of the fragment using RACE PCR. The full length cDNA was 1888 base pairs in length and coded for a 400 amino acid protein that exhibited the five-domain structure of a nuclear receptor superfamily member. The RXR protein shared significant identity with other NR2B group members. Phylogenetic analyses of the ligand-binding domain of the receptor revealed that daphnid RXR clustered with RXR from decapod crustaceans on a branch of the phylogenetic tree that was distinct from RXRs known to bind retinoic acids and juvenile hormones. Daphnid RXR mRNA levels were greatest in embryos that were early in development and progressively declined through the initial five stages of embryo development. Adult females expressed higher levels of RXR mRNA than did males and exposure of females to the terpenoid mimic pyriproxyfen reduced RXR mRNA to levels approaching levels in males. RXR mRNA levels in males were refractory to pyriproxyfen. These results show that branchiopod crustaceans dynamically express RXR which should be evaluated as a candidate receptor for the terpenoid hormone methyl farnesoate which functions as a sex determinant in these organisms.

  14. Human lymphocyte Fe receptor for IgE: sequence homology of its cloned cDNA with animal lectins

    SciTech Connect

    Ikuta, K.; Takami, M.; Kim, C.W.; Honjo, T.; Miyoshi, T.; Tagaya, Y.; Kawabe, T.; Yodoi, J.

    1987-02-01

    The authors have purified the human lymphocyte Fc receptor specific for IgE (Fcepsilon receptor) and its soluble form by using the anti-Fcepsilon receptor monoclonal antibody H107. Using an oligonucleotide probe corresponding to the partial amino acid sequence of the soluble Fcepsilon receptor related to IgE binding factor, they cloned, sequenced, and expressed a cDNA for the receptor. The Fcepsilon receptor has 321 amino acid residues with no NH/sub 2/-terminal signal sequence. The receptor was separated into two domains by a putative 24-amino acid residue transmembrane region located near the NH/sub 2/-terminal end. The Fcepsilon receptor showed a marked homology with animal lectins including human and rat asialoglycoprotein receptors, chicken hepatic lectin, and rat mannose binding proteins.

  15. Molecular Cloning of the Human Genes(s) Directing the Synthesis of Nervous System Cholinesterases.

    DTIC Science & Technology

    1985-12-01

    AD-8163 229 MOLECULAR CLONING OF THE HUMAN GENES (S) DIRECTING THE 1/1 SYNTHESIS OF NERYOU.. (U) NEIZMANN INST OF SCIENCE REHOVOT (ISRAEL) DEPT OF...whether these forms are produced from discrete genes or by post-transcrip- tional and post-translational processing. In addition, the amino acid...brain cholinseee (aRE.) is =*rxm yet, Which leaves open several questions Of cosdeal 1. Are the various Ch foru produiced from discrete genes , or is

  16. Molecular mechanisms of glucocorticoid receptor signaling.

    PubMed

    Labeur, Marta; Holsboer, Florian

    2010-01-01

    This review highlights the most recent findings on the molecular mechanisms of the glucocorticoid receptor (GR). Most effects of glucocorticoids are mediated by the intracellular GR which is present in almost every tissue and controls transcriptional activation via direct and indirect mechanisms. Nevertheless the glu-cocorticoid responses are tissue -and gene- specific. GR associates selectively with corticosteroid ligands produced in the adrenal gland in response to changes of humoral homeostasis. Ligand interaction with GR promotes either GR binding to genomic glucocorticoid response elements, in turn modulating gene transcription, or interaction of GR monomers with other transcription factors activated by other signalling pathways leading to transrepression. The GR regulates a broad spectrum of physiological functions, including cell differentiation, metabolism and inflammatory responses. Thus, disruption or dysregulation of GR function will result in severe impairments in the maintenance of homeostasis and the control of adaptation to stress.

  17. A molecularly cloned, pathogenic, neutralization-resistant simian immunodeficiency virus, SIVsmE543-3.

    PubMed Central

    Hirsch, V; Adger-Johnson, D; Campbell, B; Goldstein, S; Brown, C; Elkins, W R; Montefiori, D C

    1997-01-01

    An infectious molecular clone of simian immunodeficiency virus SIVsm was derived from a biological isolate obtained late in disease from an immunodeficient rhesus macaque (E543) with SIV-induced encephalitis. The molecularly cloned virus, SIVsmE543-3, replicated well in macaque peripheral blood mononuclear cells and monocyte-derived macrophages and resisted neutralization by heterologous sera which broadly neutralized genetically diverse SIV variants in vitro. SIVsmE543-3 was infectious and induced AIDS when inoculated intravenously into pig-tailed macaques (Macaca nemestrina). Two of four infected macaques developed no measurable SIV-specific antibody and succumbed to a wasting syndrome and SIV-induced meningoencephalitis by 14 and 33 weeks postinfection. The other two macaques developed antibodies reactive in Western blot and virus neutralization assays. One macaque was sacrificed at 1 year postinoculation, and the survivor has evidence of immunodeficiency, characterized by persistently low CD4 lymphocyte subsets in the peripheral blood. Plasma samples from these latter animals neutralized SIVsmE543-3 but with much lower efficiency than neutralization of other related SIV strains, confirming the difficulty by which this molecularly cloned virus is neutralized in vitro. SIVsmE543-3 will provide a valuable reagent for studying SIV-induced encephalitis, mapping determinants of neutralization, and determining the in vivo significance of resistance to neutralization in vitro. PMID:8995688

  18. The neuronal nicotinic acetylcholine receptor {alpha}7 subunit gene: Cloning, mapping, structure, and targeting in mouse

    SciTech Connect

    Orr-Urtreger, A.; Baldini, A.; Beaudet, A.L.

    1994-09-01

    The neuronal nicotinic acetylcholine receptor {alpha}7 subunit is a member of a family of ligand-gated ion channels, and is the only subunit know to bind {alpha}-bungarotoxin in mammalian brain. {alpha}-Bungarotoxin binding sites are known to be more abundant in the hippocampus of mouse strains that are particularly sensitive to nicotine-induced seizures. The {alpha}7 receptor is highly permeable to calcium, which could suggest a role in synaptic plasticity in the nervous system. Auditory gating deficiency, an abnormal response to a second auditory stimulus, is characteristic of schizophrenia. Mouse strains that exhibit a similar gating deficit have reduced hippocampal expression of the {alpha}7 subunit. We have cloned and sequenced the full length cDNA for the mouse {alpha}7 gene (Acra-7) and characterized its gene structure. The murine {alpha}7 shares amino acid identity of 99% and 93% with the rat and human {alpha}7 subunits, respectively. Using an interspecies backcross panel, the murine gene was mapped to chromosome 7 near the p locus, a region syntenic with human chromosome 15; the human gene (CHRNA7) was confirmed to map to 15q13-q14 by FISH. To generate a mouse {alpha}7 mutant by homologous recombination, we have constructed a replacement vector which will delete transmembrane domains II-IV and the cytoplasmic domain from the gene product. Recombinant embryonic stem (ES) cell clones were selected and used to develop mouse chimeras that are currently being bred to obtain germline transmission.

  19. Cloning, structural characterization, and chromosomal localization of the gene encoding the human prostaglandin E(2) receptor EP2 subtype.

    PubMed

    Smock, S L; Pan, L C; Castleberry, T A; Lu, B; Mather, R J; Owen, T A

    1999-09-17

    Northern blot analysis of human placental RNA using a probe to the 5' end of the human prostaglandin E(2) (PGE(2)) EP2 receptor subtype coding region revealed the existence of a high abundance, low molecular weight transcript. To investigate the origin of this transcript, and its possible relationship to the human EP2 mRNA, we have cloned and characterized the gene encoding the human PGE(2) EP2 receptor subtype, identified transcriptional initiation and termination sites in two tissues (spleen and thymus), and determined its chromosomal localization. The human EP2 gene consists of two exons separated by a large intron, utilizes a common initiation site in both spleen and thymus at 1113 bp upstream of the translation initiation site, and has 3' transcript termini at 1140 bp and 1149 bp downstream of the translation stop site in spleen and thymus respectively. Southern and fluorescence in situ hybridization analysis demonstrated the human EP2 gene to be a single copy gene located in band 22 of the long arm of chromosome 14 (14q22). Though our initial interest in this gene was to investigate potential differential splicing of the human EP2 gene in placenta, this work demonstrates that the atypical transcript observed in placenta probably arises from a distinct, yet related, gene. Knowledge of the sequence, structure, and transcription events associated with the human EP2 gene will enable a broader understanding of its regulation and potential role in normal physiology and disease.

  20. Immersing Undergraduate Students in the Research Experience: A Practical Laboratory Module on Molecular Cloning of Microbial Genes

    ERIC Educational Resources Information Center

    Wang, Jack T. H.; Schembri, Mark A.; Ramakrishna, Mathitha; Sagulenko, Evgeny; Fuerst, John A.

    2012-01-01

    Molecular cloning skills are an essential component of biological research, yet students often do not receive this training during their undergraduate studies. This can be attributed to the complexities of the cloning process, which may require many weeks of progressive design and experimentation. To address this issue, we incorporated an…

  1. Immersing Undergraduate Students in the Research Experience: A Practical Laboratory Module on Molecular Cloning of Microbial Genes

    ERIC Educational Resources Information Center

    Wang, Jack T. H.; Schembri, Mark A.; Ramakrishna, Mathitha; Sagulenko, Evgeny; Fuerst, John A.

    2012-01-01

    Molecular cloning skills are an essential component of biological research, yet students often do not receive this training during their undergraduate studies. This can be attributed to the complexities of the cloning process, which may require many weeks of progressive design and experimentation. To address this issue, we incorporated an…

  2. Molecular cloning and expression of a larval immunogenic protein from the cattle tick Boophilus annulatus.

    PubMed

    Shahein, Yasser Ezzat

    2008-02-15

    A full-length cDNA of an immunogenic protein was cloned from a cDNA library of the local Egyptian cattle tick Boophilus annulatus. Antibodies raised against B. annulatus larval proteins were used to screen a cDNA expression library. A 936bp cloned fragment was sequenced and showed an open reading frame of 516bp encoding a protein of 171 amino acids. Comparison of the deduced amino acid sequence with protein data bank revealed that the sequence is related to a sequence isolated from the hard tick Haemaphysalis qinghaiensis (Hq05). Southern blot analysis of B. annulatus genomic DNA showed that the cloned cDNA hybridized to double bands per restriction digest, suggesting that the cloned cDNA is a double copy gene. Amino acid analysis of the cloned gene revealed the presence of two casein kinase II phosphorylation sites in the N-terminal domain suggesting that this molecule may be involved in the signal transduction or gene expression pathways. RT-PCR and northern blotting revealed the presence of two isoforms of the Ba05 gene in salivary glands and in the 3-day-old eggs. The cloned gene without the signal peptide, was expressed in Escherichia coli under T7 promotor of pET-30b vector, and purified under denaturation conditions. The purified protein appeared as a single band on 12% SDS-PAGE with a molecular weight around 22.8kDa including the histidine tag of the vector. Antibodies raised against the purified molecule were used to detect the B. annulatus homologue to the Hq05 gene in whole tick, larvae and gut protein extracts. Immunoblotting revealed the presence of this molecule Ba05 only in whole tick and larval protein extracts and not in the gut protein extract. Using the same antibodies, homologues to the Ba05 gene were detected in other tick species as Hyalomma dromedarii and Rhipicephalus sp. but not in Ornithodoros moubata.

  3. Immune-mediated bone marrow failure syndromes of progenitor and stem cells: molecular analysis of cytotoxic T cell clones.

    PubMed

    Maciejewski, Jaroslaw P; O'Keefe, Christine; Gondek, Lukasz; Tiu, Ramon

    2007-01-01

    The unique structure of the T cell receptor (TCR) enables molecular identification of individual T cell clones and provides an unique opportunity for the design of molecular diagnostic tests based on the structure of the rearranged TCR chain e.g., using the TCR CDR3 region. Initially, clonal T cell malignancies, including T cell large granular lymphocyte leukemia (T-LGL), mucosis fungoides and peripheral T cell lymphoma were targets for the TCR-based analytic assays such as detection of clonality by T-gamma rearrangement using y-chain-specific PCR or Southern Blotting. Study of these disorders facilitated further analytic concepts and application of rational methods of TCR analysis to investigations of polyclonal T cell-mediated diseases. In hematology, such conditions include graft versus host disease (GvHD) and immune-mediated bone marrow failure syndromes. In aplastic anemia (AA), myelodysplastic syndrome (MDS) or paroxysmal nocturnal hemoglobinuria (PNH), cytotoxic T cell responses may be directed against certain antigens located on stem or more lineage-restricted progenitor cells in single lineage cytopenias. The nature of the antigenic targets driving polyclonal CTL responses remains unclear. Novel methods of TCR repertoire analysis, include VB flow cytometry, peptide-specific tetramer staining, in vitro stimulation assays and TCR CDR3-specific PCR. Such PCR assay can be either VB family-specific or multiplexed for all VB families. Amplified products can be characterized and quantitated to facilitate detection of the most immunodominant clonotypes. Such clonotypes may serve as markers for the global polyclonal T cell response. Identification of these clonotypes can be performed in blood and tissue biopsy material by various methods. Once immunodominant clonotypes corresponding to pathogenic CTL clones are identified they can serve as surrogate markers for the activity of the pathophysiologic process or even indicate the presence of specific antigens. The

  4. Bovine viral diarrhea virus: molecular cloning of genomic RNA and its diagnostic application

    SciTech Connect

    Brock, K.V.

    1987-01-01

    Molecular cloning of a field isolate of bovine viral diarrhea virus (BVDV) strain 72 RNA was done in this study. The sensitivity and specificity of cloned cDNA sequences in hybridization assays with various BVDV strains were determined. cDNA was synthesized from polyadenylated BVDV RNA templates with oligo-dT primers, reverse transcriptase, and DNA polymerase I. The newly synthesized double-stranded BVDV cDNA was C-tailed with terminal deoxytransferase and annealed into G-tailed, Pst-1-cut pUC9 plasmid. Escherichia coli was transformed with the recombinant plasmids and a library of approximately 200 BVDV specific cDNA clones varying in length from 0.5 to 2.6 kilobases were isolated. The sensitivity and specificity of hybridization between the labelled cDNA and BVDV target sequences were determined. Cloned BVDV sequences were isolated from pUC9 plasmid DNA and labelled with /sup 32/P by nick translation. The detection limit by dot blot hybridization assay was 20 pg of purified genomic BVDV RNA. cDNA hybridization probes were specific for all strains of BVDV tested, regardless of whether they were noncytopathic and cytopathic, but did not hybridize with heterologous bovine viruses tested. Probes did not hybridize with uninfected cell culture or cellular RNA. Hybridization probes were at least as sensitive as infectivity assays in detecting homologous virus.

  5. Molecular cloning, phylogenetic analysis and developmental expression of a vitellogenin (Vg) gene from the intertidal copepod Tigriopus japonicus.

    PubMed

    Lee, Kyun-Woo; Hwang, Dae-Sik; Rhee, Jae-Sung; Ki, Jang-Seu; Park, Heum Gi; Ryu, Jae-Chun; Raisuddin, Sheikh; Lee, Jae-Seong

    2008-08-01

    Vitellogenin (Vg) is the precursor of egg yolk protein vitellins, which serve as energy resource for embryonic development. Vg measurement has been used as a biomarker of exposure to endocrine-disrupting chemicals (EDCs). Therefore, Vg gene structure has been identified from several species used in environmental monitoring of EDCs. Among the copepods, except from the salmon louse, there is no report on Vg genes or their products. By using molecular cloning, we determined the full Vg gene sequence from the intertidal copepod, Tigriopus japonicus. The full cDNA sequence was of 5692 bp containing 5529 bp of open reading frame (ORF) encoding for 1842 amino acids. The phylogenetic analysis revealed that T. japonicus Vg is distinct from the other arthropods as it formed a clade with salmon louse only. The expression of Vg transcripts was negligible in nauplii; detectable only at the copepodid stage 3. Females expressed over 270 times more Vg transcripts than males. The promoter sequence of T. japonicus Vg gene revealed an estrogen receptor (ER) half site and a metal response element (MRE). When copepods were exposed to trace metals, cadmium after 96 h exposure caused significantly higher induction of Vg transcripts. Taken together, molecular analysis of T. japonicus Vg would be helpful in understanding its role in development. Previous studies have established T. japonicus as a potential model for testing of endocrine-disrupting chemicals (EDCs). The study of T. japonicus Vg will fuel momentum in using this species in comparative molecular endocrinology and biomonitoring of EDCs.

  6. Cloning, phylogeny, and regional expression of a Y5 receptor mRNA in the brain of the sea lamprey (Petromyzon marinus).

    PubMed

    Pérez-Fernández, Juan; Megías, Manuel; Pombal, Manuel A

    2014-04-01

    The NPY receptors known as Y receptors are classified into three subfamilies, Y1, Y2, and Y5, and are involved in different physiological functions. The Y5 receptor is the only member of the Y5 subfamily, and it is present in all vertebrate groups, except for teleosts. Both molecular and pharmacological studies show that Y5 receptor is highly conserved during vertebrate evolution. Furthermore, this receptor is widely expressed in the mammalian brain, including the hypothalamus, where it is thought to take part in feeding and homeostasis regulation. Lampreys belong to the agnathan lineage, and they are thought to have branched out between the two whole-genome duplications that occurred in vertebrates. Therefore, they are in a key position for studies on the evolution of gene families in vertebrates. Here we report the cloning, phylogeny, and brain expression pattern of the sea lamprey Y5 receptor. In phylogenetic studies, the lamprey Y5 receptor clusters in a basal position, together with Y5 receptors of other vertebrates. The mRNA of this receptor is broadly expressed in the lamprey brain, being especially abundant in hypothalamic areas. Its expression pattern is roughly similar to that reported for other vertebrates and parallels the expression pattern of the Y1 receptor subtype previously described by our group, as it occurs in mammals. Altogether, these results confirm that a Y5 receptor is present in lampreys, thus being highly conserved during the evolution of vertebrates, and suggest that it is involved in many brain functions, the only known exception being teleosts.

  7. Particle infectivity of HIV-1 full-length genome infectious molecular clones in a subtype C heterosexual transmission pair following high fidelity amplification and unbiased cloning.

    PubMed

    Deymier, Martin J; Claiborne, Daniel T; Ende, Zachary; Ratner, Hannah K; Kilembe, William; Allen, Susan; Hunter, Eric

    2014-11-01

    The high genetic diversity of HIV-1 impedes high throughput, large-scale sequencing and full-length genome cloning by common restriction enzyme based methods. Applying novel methods that employ a high-fidelity polymerase for amplification and an unbiased fusion-based cloning strategy, we have generated several HIV-1 full-length genome infectious molecular clones from an epidemiologically linked transmission pair. These clones represent the transmitted/founder virus and phylogenetically diverse non-transmitted variants from the chronically infected individual׳s diverse quasispecies near the time of transmission. We demonstrate that, using this approach, PCR-induced mutations in full-length clones derived from their cognate single genome amplicons are rare. Furthermore, all eight non-transmitted genomes tested produced functional virus with a range of infectivities, belying the previous assumption that a majority of circulating viruses in chronic HIV-1 infection are defective. Thus, these methods provide important tools to update protocols in molecular biology that can be universally applied to the study of human viral pathogens.

  8. Variation of LTR size in molecular clones of the BALB/c endogenous ecotropic molecular leukemia virus

    SciTech Connect

    Boone, L.R.; Myer, F.E.; Yang, D.M.; Kiggans, J.O.; Koh, C.; Tennant, R.W.; Yang, W.K.

    1982-01-01

    Retrovirus replication involves the synthesis of a DNA intermediate which integrates into the host cell genome. Structure analysis has revealed that the viral DNA contains a nucleotide sequence on both ends, termed long terminal repeat (LTR) which is derived from terminal sequences of the genomic RNA and consists of three portions: U3, R, and U5, representing respectively the 3' end, a short terminal repeated sequence, and the 5' end of the RNA. Each species of retrovirus has a characteristic length LTR; however, within a species different strains and isolates have variations in LTR size. The evolution of viral genomes during replication as an exogenous agent may involve changes which occur in the LTR. A population of molecular clones of an endogenous ecotropic virus was analyzed to identify possible changes which occur in the LTR region. Thirteen of fifteen plaque purified isolates examined have an insert which appears to be a typical viral genome with no major rearrangement or deletion. Six of these have an insert which contains a single LTR, and seven have an insert which contains two LTRs. Approximately half of the isolates, including single and double LTR clones, are infectious. More detailed analysis of plasmid subclones all containing double LTRs, revealed that the variation resided in the U3 region rather than the left LTR/right LTR junction region. Thus, the size variation of LTR in the WN1802N MuLV recombinant DNA clones appears to be different from the situation which is responsible for LTR size variability in some molecular clones of avian retrovirus. The mechanism which generates this diversity in the U3 region is not known. Perhaps there is a selective advantage for duplication/insertions in this region of the LTR. The SV40 repeat has an important activator role and it was postulated that repeats in retrovirus LTR have a similar role. (ERB)

  9. Cloning, expression of, and evidence of positive selection for, the prolactin receptor gene in Chinese giant salamander (Andrias davidianus).

    PubMed

    Hu, Qiaomu; Meng, Yan; Tian, Haifeng; Chen, Songlin; Xiao, Hanbing

    2015-12-01

    Prolactin receptor (PRLR) is a protein associated with reproduction in mammals and with osmoregulation in fish. In this study, the complete length of Chinese giant salamander Andrias davidianus prolactin receptor (AD-prlr) was cloned. Andrias davidianus prlr expression was high in the kidney, pituitary, and ovary and low in other examined tissues. The AD-prlr levels were higher in ovary than in testis, and increased in ovaries with age from 1 to 6 years. To determine effect of exogenous androgen and aromatase inhibitor on AD-prlr expression, methyltestosterone (MT) and letrozole (LE) were injected, resulting in decreased AD-prlr in both brain and ovary, with MT repressing prlr transcription more rapidly than did LE. The molecular evolution of prlr was assessed, and found to have undergone a complex evolution process. The obranch-site test detected four positively selected sites in ancestral lineages prior to the separation of mammals and birds. Fourteen sites underwent positive selection in ancestral lineages of birds and six were positively selected in amphibians. The site model showed that 16, 7, and 30 sites underwent positive selection in extant mammals, amphibians, and birds, respectively. The positively selected sites in amphibians were located outside the transmembrane domain, with four in the extracellular and three in the intracellular domain, indicating that the transmembrane region might be conserved and essential for protein function. Our findings provide a basis for further studies of AD-prlr function and molecular evolution in Chinese giant salamander. J. Exp. Zool. (Mol. Dev. Evol.) 324B: 707-719, 2015. © 2015 Wiley Periodicals, Inc.

  10. Molecular cloning, functional expression and characterization of (E)-beta farnesene synthase from Citrus junos.

    PubMed

    Maruyama, T; Ito, M; Honda, G

    2001-10-01

    We cloned the gene of the acyclic sesquiterpene synthase, (E)-beta-farnesene synthase (CJFS) from Yuzu (Citrus junos, Rutaceae). The function of CJFS was elucidated by the preparation of recombinant protein and subsequent enzyme assay. CJFS consisted of 1867 nucleotides including 1680 bp of coding sequence encoding a protein of 560 amino acids with a molecular weight of 62 kDa. The deduced amino acid sequence possessed characteristic amino acid residues, such as the DDxxD motif, which are highly conserved among terpene synthases. This is the first report of the cloning of a terpene synthase from a Rutaceous plant. A possible reaction mechanism for terpene biosynthesis is also discussed on the basis of sequence comparison of CJFS with known sesquiterpene synthase genes.

  11. Taenia hydatigena: isolation of mitochondrial DNA, molecular cloning, and physical mitochondrial genome mapping.

    PubMed

    Yap, K W; Thompson, R C; Rood, J I; Pawlowski, I D

    1987-06-01

    Mitochondrial DNA was isolated from Taenia hydatigena, T. crassiceps, and Echinococcus granulosus using a cetyltrimethylammonium bromide precipitation technique. The technique is simple, rapid, reproducible, and does not require extensive high speed ultracentrifugation. The advantage of using mitochondrial DNA from taeniid cestodes for comparative restriction analysis was demonstrated. Mitochondrial DNA of T. hydatigena was isolated as covalently closed circular molecules. These were linearized by single digestion with BamHI and the molecular weight was estimated from the linear form of 17.6 kb. The mitochondrial DNA of T. hydatigena is therefore similar in size and structure to that of many other animal species. The entire mitochondrial genome was cloned into pBR322 in Escherichia coli and a restriction map of the recombinant molecule was constructed. The potential of using the cloned mitochondrial genome as a probe in speciation studies as well as for providing functional information on the role of the cestode mitochondrion is discussed.

  12. Molecular cloning and nucleotide sequence of rat lingual lipase cDNA.

    PubMed Central

    Docherty, A J; Bodmer, M W; Angal, S; Verger, R; Riviere, C; Lowe, P A; Lyons, A; Emtage, J S; Harris, T J

    1985-01-01

    Purified rat lingual lipase (EC3113), a glycoprotein of approximate molecular weight 52,000, was used to generate polyclonal antibodies which were able to recognise the denatured and deglycosylated enzyme. These immunoglobulins were used to screen a cDNA library prepared from mRNA isolated from the serous glands of rat tongue cloned in E. coli expression vectors. An almost full length cDNA clone was isolated and the nucleotide and predicted amino acid sequence obtained. Comparison with the N-terminal amino acid sequence of the purified enzyme confirmed the identity of the cDNA and indicated that there was a hydrophobic signal sequence of 18 residues. The amino acid sequence of mature rat lingual lipase consists of 377 residues and shares little homology with porcine pancreatic lipase apart from a short region containing a serine residue at an analogous position to the ser 152 of the porcine enzyme. Images PMID:3839077

  13. Characterization of U-97775 as a GABAA receptor ligand of dual functionality in cloned rat GABAA receptor subtypes.

    PubMed Central

    Im, H. K.; Im, W. B.; Pregenzer, J. F.; Carter, D. B.; Jacobsen, E. J.; Hamilton, B. J.

    1995-01-01

    1. U-97775 (tert-butyl 7-chloro-4,5-dihydro-5-[(1-(3,4,5-trimethyl)piperazino)carbonyl]- imidazo[1,5-a])quinoxaline-3-carboxylate) is a novel GABAA receptor ligand of dual functionality and was characterized for its interactions with cloned rat GABAA receptors expressed in human embryonic kidney cells. 2. The drug produced a bell-shaped dose-response profile in the alpha 1 beta 2 gamma 2 receptor subtype as monitored with GABA-induced Cl- currents in the whole cell patch-clamp technique. At low concentrations (< 0.5 microM), U-97775 enhanced the currents with a maximal increase of 120% as normalized to 5 microM GABA response (control). An agonist interaction of U-97775 with the benzodiazepine site is suggested, because Ro 15-1788 (an antagonist at the benzodiazepine site) abolished the current increase and [3H]-flunitrazepam binding was inhibited by U-97775 with a Ki of 1.2 nM. 3. The enhancement of GABA currents progressively disappeared as the U-97775 concentration was raised above 1 microM, and the current amplitude was reduced to 40% below the control at 10 microM U-97775. The current inhibition by U-97775 (10 microM) was not affected by Ro 15-1788. It appears that U-97775 interacts with a second site on GABA receptors, distinct from the benzodiazepine site, to reverse its agonistic activity on the benzodiazepine site and also to inhibit GABA currents. 4. U-97775 at low concentrations reduced and at high concentrations enhanced [35S]-TBPS binding. Ro 15-1788 selectively blocked the effect of U-97775 at low concentrations.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7647975

  14. Teaching molecular genetics: chapter 4—positional cloning of genetic disorders

    PubMed Central

    Puliti, Aldamaria; Caridi, Gianluca; Ravazzolo, Roberto

    2007-01-01

    Positional cloning is the approach of choice for the identification of genetic mutations underlying the pathological development of diseases with simple Mendelian inheritance. It consists of different consecutive steps, starting with recruitment of patients and DNA collection, that are critical to the overall process. A genetic analysis of the enrolled patients and their families is performed, based on genetic recombination frequencies generated by meiotic cross-overs and on genome-wide molecular studies, to define a critical DNA region of interest. This analysis culminates in a statistical estimate of the probability that disease features may segregate in the families independently or in association with specific molecular markers located in known regions. In this latter case, a marker can be defined as being linked to the disease manifestations. The genetic markers define an interval that is a function of their recombination frequencies with the disease, in which the disease gene is localised. The identification and characterisation of chromosome abnormalities as translocations, deletions and duplications by classical cytogenetic methods or by the newly developed microarray-based comparative genomic hybridisation (array CGH) technique may define extensions and borders of the genomic regions involved. The step following the definition of a critical genomic region is the identification of candidate genes that is based on the analysis of available databases from genome browsers. Positional cloning culminates in the identification of the causative gene mutation, and the definition of its functional role in the pathogenesis of the disorder, by the use of cell-based or animal-based experiments. More often, positional cloning ends with the generation of mice with homologous mutations reproducing the human clinical phenotype. Altogether, positional cloning has represented a fundamental step in the research on genetic renal disorders, leading to the definition of several

  15. Molecular cloning and expression patterns of the molt-regulating transcription factor HHR3 from Helicoverpa armigera.

    PubMed

    Zhao, X-F; Wang, J-X; Xu, X-L; Li, Z-M; Kang, C-J

    2004-08-01

    Molt-regulating transcription factors, hormone receptor 3 (HR3), play important roles in regulating expression of tissue-specific genes involved in insect molting and metamorphosis. A 1668 bp cDNA encoding a molt-regulating transcription factor (HHR3) was cloned from Helicoverpa armigera, which encodes a protein made up of 556 amino acids. This 62 kDa protein was found to have an isoelectric point (pI) of 6.52. There was no signal peptide or N-glycosylation site found in this cDNA. A DNA-binding region signature of nuclear hormone receptor was found from amino acids 107-133. A possible outside to inside transmembrane helice was found from amino acids 72-90. Northern blots of the larvae revealed five bands of HHR3 named as band 0, 1, 2, 3 and 4 with molecular masses determined as 2.1, 2.6, 3.6, 4.5 and 5.5 kb, respectively. The expression patterns of HHR3 in vivo were variable with developmental stages and tissues. Results showed that band 1-4 of HHR3 was only briefly expressed during molting, which suggested these bands are involved in the regulation of molting cascade, whereas band 0 was expressed in both molting and feeding larvae. Band 1 and 2 of HHR3 could be induced from epidermis of newly molted 6th instar larvae by non-steroidal ecdysone agonist, RH-2485.

  16. Molecular Mechanisms of Opioid Receptor-Dependent Signaling and Behavior

    PubMed Central

    Al-Hasani, Ream; Bruchas, Michael R.

    2013-01-01

    Opioid receptors have been targeted for the treatment of pain and related disorders for thousands of years, and remain the most widely used analgesics in the clinic. Mu (μ), kappa (κ), and delta (δ) opioid receptors represent the originally classified receptor subtypes, with opioid receptor like-1 (ORL1) being the least characterized. All four receptors are G-protein coupled, and activate inhibitory G-proteins. These receptors form homo- and hetereodimeric complexes, signal to kinase cascades, and scaffold a variety of proteins. In this review, we discuss classical mechanisms and developments in understanding opioid tolerance, opioid receptor signaling, and highlight advances in opioid molecular pharmacology, behavioral pharmacology, and human genetics. We put into context how opioid receptor signaling leads to the modulation of behavior with the potential for therapeutic intervention. Finally, we conclude that there is a continued need for more translational work on opioid receptors in vivo. PMID:22020140

  17. Structural and Molecular Modeling Features of P2X Receptors

    PubMed Central

    Alves, Luiz Anastacio; da Silva, João Herminio Martins; Ferreira, Dinarte Neto Moreira; Fidalgo-Neto, Antonio Augusto; Teixeira, Pedro Celso Nogueira; de Souza, Cristina Alves Magalhães; Caffarena, Ernesto Raúl; de Freitas, Mônica Santos

    2014-01-01

    Currently, adenosine 5′-triphosphate (ATP) is recognized as the extracellular messenger that acts through P2 receptors. P2 receptors are divided into two subtypes: P2Y metabotropic receptors and P2X ionotropic receptors, both of which are found in virtually all mammalian cell types studied. Due to the difficulty in studying membrane protein structures by X-ray crystallography or NMR techniques, there is little information about these structures available in the literature. Two structures of the P2X4 receptor in truncated form have been solved by crystallography. Molecular modeling has proven to be an excellent tool for studying ionotropic receptors. Recently, modeling studies carried out on P2X receptors have advanced our knowledge of the P2X receptor structure-function relationships. This review presents a brief history of ion channel structural studies and shows how modeling approaches can be used to address relevant questions about P2X receptors. PMID:24637936

  18. Cloning, expression and functional analysis of the duck Toll-like receptor 5 (TLR5) gene

    PubMed Central

    Cheng, Yuqiang; Sun, Yingjie; Wang, Hengan; Shi, Shuduan; Yan, Yaxian; Li, Jing

    2015-01-01

    Toll-like receptor 5 (TLR5) is responsible for the recognition of bacterial flagellin in vertebrates. In the present study, the first TLR5 gene in duck was cloned. The open reading frame (ORF) of duck TLR5 (dTLR5) cDNA is 2580 bp and encodes a polypeptide of 859 amino acids. We also cloned partial sequences of myeloid differentiation factor 88, 2'-5'-oligoadenylate synthetase (OAS), and myxovirus resistance (Mx) genes from duck. dTLR5 mRNA was highly expressed in the bursa of Fabricius, spleen, trachea, lung, jejunum, rectum, and skin; moderately expressed in the muscular and glandular tissues, duodenum, ileum, caecum, and pancreas; and minimally expressed in the heart, liver, kidney, and muscle. DF-1 or HeLa cells transfected with DNA constructs encoding dTLR5 can activate NF-κB leading to the activation of interleukin-6 (IL-6) promoter. When we challenged ducks with a Herts33 Newcastle disease virus (NDV), mRNA transcription of the antiviral molecules Mx, Double stranded RNA activated protein kinase (PKR), and OAS was up-regulated in the liver, lung, and spleen 1 and 2 days post-inoculation. PMID:25269719

  19. A macrophage receptor for apolipoprotein B48: Cloning, expression, and atherosclerosis

    PubMed Central

    Brown, Matthew L.; Ramprasad, M. P.; Umeda, Patrick K.; Tanaka, Akira; Kobayashi, Yasushi; Watanabe, Teruo; Shimoyamada, Hiroaki; Kuo, Wen-Lin; Li, Ran; Song, Ruiling; Bradley, William A.; Gianturco, Sandra H.

    2000-01-01

    We have cloned a human macrophage receptor that binds to apolipoprotein (apo)B48 of dietary triglyceride (TG)-rich lipoproteins. TG-rich lipoprotein uptake by the apoB48R rapidly converts macrophages and apoB48R-transfected Chinese hamster ovary cells in vitro into lipid-filled foam cells, as seen in atherosclerotic lesions. The apoB48R cDNA (3,744 bp) encodes a protein with no known homologs. Its ≈3.8-kb mRNA is expressed primarily by reticuloendothelial cells: monocytes, macrophages, and endothelial cells. Immunohistochemistry shows the apoB48R is in human atherosclerotic lesion foam cells. Normally, the apoB48R may provide essential lipids to reticuloendothelial cells. If overwhelmed, foam cell formation, endothelial dysfunction, and atherothrombogenesis may ensue, a mechanism for cardiovascular disease risk of elevated TG. PMID:10852956

  20. CD11b of Ovis canadensis and Ovis aries: molecular cloning and characterization.

    PubMed

    Lawrence, Paulraj K; Srikumaran, Subramaniam

    2007-10-15

    Leukotoxin (Lkt) is the primary virulence factor secreted by Mannheimia haemolytica which causes pneumonia in ruminants. Previously, we have shown that CD18, the beta subunit of beta(2) integrins, mediates Lkt-induced cytolysis of ruminant leukocytes. CD18 associates with four distinct alpha subunits giving rise to four beta(2) integrins, CD11a/CD18 (LFA-1), CD11b/CD18 (Mac-1), CD11c/CD18 (CR4), and CD11d/CD18. It is not known whether all the beta(2) integrins serve as a receptor for Lkt. Since PMNs are the leukocyte subset that is most susceptible to Lkt, and Mac-1 expression on PMNs exceeds that of other beta(2) integrins, it is of interest to determine whether Mac-1 serves as a receptor for Lkt which necessitates the cloning of CD11b and CD18. In this study, we cloned and sequenced the cDNA encoding CD11b of Ovis canadensis (bighorn sheep) and Ovis aries (domestic sheep). CD11b cDNA is 3455 nucleotides long encoding a polypeptide of 1152 amino acids. CD11b polypeptides from these two species exhibit 99% identity with each other, and 92% with that of cattle, and 70-80% with that of the non-ruminants analyzed.

  1. Molecular cloning and characterization of a novel glucocerebrosidase of Paenibacillus sp. TS12.

    PubMed

    Sumida, Tomomi; Sueyoshi, Noriyuki; Ito, Makoto

    2002-08-01

    We report here the molecular cloning and characterization of a glucocerebrosidase [EC 3.2.1.45] from Paenibacillus sp. TS12. The open reading frame of the glucocerebrosidase gene consisted of 2,493 bp nucleotides and encoded 831 amino acid residues. The enzyme exhibited no sequence similarity with a classical glucocerebrosidase belonging to glycoside hydrolase (GH) family 30, but rather showed significant similarity with GH family 3 beta-glucosidases from Clostridium thermocellum, Ruminococcus albus, and Aspergillus aculeateus. The recombinant enzyme, expressed in Escherichia coli BL21(DE3)pLysS, had a molecular weight of 90.7 kDa and hydrolyzed NBD-labeled glucosylceramide, but not galactosylceramide, GM1a or sphingomyelin. The enzyme was most active at pH 6.5, and its apparent Km and Vmax values for NBD-labeled glucosylceramide and p-nitrophenyl-beta-glucopyranoside were 223 microM and 1.60 micromol/min/mg of protein, and 593 microM and 112 micromol/min/mg of protein, respectively. Site-directed mutagenesis indicated that Asp-223 is an essential amino acid for the catalytic reaction and possibly functions a catalytic nucleophile, as in GH family 3 beta-glucosidases. This is the first report of the molecular cloning and characterization of a glucocerebrosidase from a procaryote.

  2. Molecular transformation, gene cloning, and gene expression systems for filamentous fungi

    USGS Publications Warehouse

    Gold, Scott E.; Duick, John W.; Redman, Regina S.; Rodriguez, Rusty J.

    2001-01-01

    This chapter discusses the molecular transformation, gene cloning, and gene expression systems for filamentous fungi. Molecular transformation involves the movement of discrete amounts of DNA into cells, the expression of genes on the transported DNA, and the sustainable replication of the transforming DNA. The ability to transform fungi is dependent on the stable replication and expression of genes located on the transforming DNA. Three phenomena observed in bacteria, that is, competence, plasmids, and restriction enzymes to facilitate cloning, were responsible for the development of molecular transformation in fungi. Initial transformation success with filamentous fungi, involving the complementation of auxotrophic mutants by exposure to sheared genomic DNA or RNA from wt isolates, occurred with low transformation efficiencies. In addition, it was difficult to retrieve complementing DNA fragments and isolate genes of interest. This prompted the development of transformation vectors and methods to increase efficiencies. The physiological studies performed with fungi indicated that the cell wall could be removed to generate protoplasts. It was evident that protoplasts could be transformed with significantly greater efficiencies than walled cells.

  3. Cloning yeast actin cDNA leads to an investigative approach for the molecular biology laboratory.

    PubMed

    Black, Michael W; Tuan, Alice; Jonasson, Erin

    2008-05-01

    The emergence of molecular tools in multiple disciplines has elevated the importance of undergraduate laboratory courses that train students in molecular biology techniques. Although it would also be desirable to provide students with opportunities to apply these techniques in an investigative manner, this is generally not possible in the classroom because of the preparation, expense, and logistics involved in independent student projects. The authors have designed a 10-week lab series that mimics the research environment by tying separate fundamental lab techniques to a common goal: to build a plasmid with yeast actin cDNA cloned in a particular orientation. In the process of completing this goal, a problem arises in that students are unable to obtain the target plasmid and instead only recover the gene cloned in the opposite orientation. To address this problem, students identify four plausible hypotheses and work in teams to address them by designing and executing experiments. This project reinforces the utility and flexibility of techniques covered earlier in the class and serves to develop their skills in experimental design and analysis. As the project is focused on one problem, the diversity of experimental approaches is limited and may be prepared in advance with little additional expense in reagents or technical support. Copyright © 2008 International Union of Biochemistry and Molecular Biology, Inc.

  4. Cloning and molecular characterization of a cubilin-related serine proteinase from the hard tick Haemaphysalis longicornis.

    PubMed

    Miyoshi, Takeharu; Tsuji, Naotoshi; Islam, M Khyrul; Kamio, Tsugihiko; Fujisaki, Kozo

    2004-08-01

    Serine proteinases are one of the largest proteolytic families of enzymes, and have diverse cellular activities in mammalian tissues. We report here the cloning and molecular characterization of a cDNA encoding the serine proteinase of the hard tick Haemaphysalis longicornis (HlSP). The HlSP cDNA is 1570 bp long and the deduced precursor protein consists of 464 amino acids with a predicted molecular mass of 50.4 kDa and a pI of 8.2. The preprotein, consisting of 443 amino acids, was predicted to include a complement C1r/C1s, Uegf, and bone morphogenic protein-1 domain, a low-density lipoprotein receptor class A domain, and a catalytic domain. HlSP sequence analysis showed high similarity to serine proteinases reported from arthropods and vertebrate animal species. Two-dimensional immunoblot analysis revealed endogenous HlSP in adult tick extracts at 50 kDa. Endogenous HlSP was also expressed in all lifecycle stages of H. longicornis. Immunohistochemical studies detected the endogenous enzyme in the midgut epithelial cells of an adult tick. The Escherichia coli-expressed recombinant HlSP was demonstrated to degrade bovine serum albumin and hydrolyze the substrate Bz-L-Arg-pNA at the rate of 30.2 micromol/min/mg protein. Further, HlSP expression was up-regulated during a blood-feeding process, indicating its involvement in the digestion of host blood components.

  5. Physical mapping and molecular cloning of mung bean yellow mosaic virus DNA.

    PubMed

    Morinaga, T; Ikegami, M; Miura, K

    1990-01-01

    Viral single-stranded DNA of mung bean yellow mosaic virus (MYMV) was converted to the double-stranded state in vitro, and physical mapping was carried out. The genome of MYMV was found to consist of two major components (designated as DNA 1 and DNA 2). In addition, some minor components were detected. Molecular cloning of the major components was carried out, using in vitro double-stranded DNA and replicative intermediate DNAs. DNA 1 is about 2.72 and DNA 2 about 2.67 kilobase pairs. No similarities were observed when the two restriction maps of DNA 1 and 2 were compared.

  6. Molecular cloning and functional analysis of a 10-epi-junenol synthase from Inula hupehensis.

    PubMed

    Gou, Jun-Bo; Li, Zhen-Qiu; Li, Chang-Fu; Chen, Fang-Fang; Lv, Shi-You; Zhang, Yan-Sheng

    2016-09-01

    Junenol based-eudesmanolides have been detected in many compositae plant species and were reported to exhibit various pharmacological activities. So far, the gene encoding junenol synthase has never been isolated. Here we report the molecular cloning and functional analysis of a 10-epi-junenol synthase from Inula hupehensis (designated IhsTPS1). IhsTPS1 converts the substrate farnesyl diphosphate into multiple sesquiterpenes with the product 10-epi-junenol being predominant. The transcript levels of IhsTPS1 correlate well with the accumulation pattern of 10-epi-junenol in I. hupehensis organs, supporting its biochemical roles in vivo.

  7. Molecular cloning of a gene involved in methotrexate uptake by DNA-mediated gene transfer.

    PubMed

    Underhill, T M; Williams, F M; Murray, R C; Flintoff, W F

    1992-07-01

    A methotrexate-resistant Chinese hamster ovary cell line deficient in methotrexate uptake has been complemented to methotrexate sensitivity by transfection with DNA isolated from a wild-type Chinese hamster ovary genomic cosmid library. Primary and secondary transfectants, which contain a limited number of cosmid sequences, have been shown to regain methotrexate sensitivity and to take up methotrexate. Furthermore, the DNA from three cosmid clones, isolated from a primary methotrexate-sensitive transfectant, after transfection rescued the methotrexate-resistant phenotype at a high frequency. Restriction endonuclease analysis of the DNA of these cosmid clones indicated that they overlapped extensively and shared two regions of Chinese hamster ovary DNA of 6.6 kb and 20.6 kb. These observations indicate that a gene involved in methotrexate uptake is contained in its entirety within one of these regions. This is the first report of the functional molecular cloning of a gene involved in methotrexate uptake. A general strategy is also described for screening large cosmid libraries from primary transfectants.

  8. Molecular cloning of a lectin cDNA from Alocasia macrorrhiza and prediction of its characteristics.

    PubMed

    Zhu, Ya-Ran; Wang, Jie; Huang, Bing-Qiu; Hou, Xue-Wen

    2006-12-01

    The cDNA of Alocasia macrorrhiza lectin (aml, GenBank accession number: DQ340864) was cloned by RACE-PCR and its characteristics were predicted by various bioinformatics tools. GSPs (Gene Specific Primers) were designed according to the conserved regions of the genes encoded for lectins and similar proteins from the same family Araceae. Total RNAs were extracted from the tubers of A macrorrhiza by Qiagen RNeasy mini kit. The 3'- and 5'-RACE-PCRs were performed with the isolated total RNAs by SMART(TM)RACE cDNA amplification kit from BD Biosciences Clontech Company, respectively. The purified PCR products were ligated with pMD 18-T vector, and the confirmed clones were sequenced. The full-length cDNA of aml was obtained by combination of 3'- and 5'-end sequences, and was then confirmed by full-length 3'-RACE-PCR. The aml cDNA is 1 124 bp long. The deduced amino acid length of AML lectin is 270 aa. Its relative molecular weight is 29.7 kD. The results of homologous analysis showed a high similarity between AML and other mannose-binding lectins and similar proteins from Araceae family. Two typical B-lectin domains and three mannose- binding motifs were found in the sequence of AML. With all these taken together, it can be concluded that this newly cloned aml cDNA encodes for a mannose-binding lectin.

  9. Molecular cloning, expression, and sequence of the pilin gene from nontypeable Haemophilus influenzae M37.

    PubMed Central

    Coleman, T; Grass, S; Munson, R

    1991-01-01

    Nontypeable Haemophilus influenzae M37 adheres to human buccal epithelial cells and exhibits mannose-resistant hemagglutination of human erythrocytes. An isogenic variant of this strain which was deficient in hemagglutination was isolated. A protein with an apparent molecular weight of 22,000 was present in the sodium dodecyl sulfate-polyacrylamide gel profile of sarcosyl-insoluble proteins from the hemagglutination-proficient strain but was absent from the profile of the isogenic hemagglutination-deficient variant. A monoclonal antibody which reacts with the hemagglutination-proficient isolate but not with the hemagglutination-deficient isolate has been characterized. This monoclonal antibody was employed in an affinity column for purification of the protein as well as to screen a genomic library for recombinant clones expressing the gene. Several clones which contained overlapping genomic fragments were identified by reaction with the monoclonal antibody. The gene for the 22-kDa protein was subcloned and sequenced. The gene for the type b pilin from H. influenzae type b strain MinnA was also cloned and sequenced. The DNA sequence of the strain MinnA gene was identical to that reported previously for two other type b strains. The DNA sequence of the strain M37 gene is 77% identical to that of the type b pilin gene, and the derived amino acid sequence is 68% identical to that of the type b pilin. Images PMID:1673447

  10. Molecular cloning and expression of rat brain endopeptidase 3.4.24.16.

    PubMed

    Dauch, P; Vincent, J P; Checler, F

    1995-11-10

    We have isolated by immunological screening of a lambda ZAPII cDNA library constructed from rat brain mRNAs a cDNA clone encoding endopeptidase 3.4.24.16. The longest open reading frame encodes a 704-amino acid protein with a theoretical molecular mass of 80,202 daltons and bears the consensus sequence of the zinc metalloprotease family. The sequence exhibits a 60.2% homology with those of another zinc metallopeptidase, endopeptidase 3.4.24.15. Northern blot analysis reveals two mRNA species of about 3 and 5 kilobases in rat brain, ileum, kidney, and testis. We have transiently transfected COS-7 cells with pcDNA3 containing the cloned cDNA and established the overexpression of a 70-75-kDa immunoreactive protein. This protein hydrolyzes QFS, a quenched fluorimetric substrate of endopeptidase 3.4.24.16, and cleaves neurotensin at a single peptide bond, leading to the formation of neurotensin (1-10) and neurotensin (11-13). QFS and neurotensin hydrolysis are potently inhibited by the selective endopeptidase 3.4.24.16 dipeptide blocker Pro-Ile and by dithiothreitol, while the enzymatic activity remains unaffected by phosphoramidon and captopril, the specific inhibitors of endopeptidase 3.4.24.11 and angiotensin-converting enzyme, respectively. Altogether, these physicochemical, biochemical, and immunological properties unambiguously identify endopeptidase 3.4.24.16 as the protein encoded by the isolated cDNA clone.

  11. Molecular cloning and expression of a new gene, GON-SJTU1 in the rat testis

    PubMed Central

    2010-01-01

    Background Spermatogenesis is a complex process involving cell development, differentiation and apoptosis. This process is governed by a series of genes whose expressions are highly regulated. Male infertility can be attributed to multiple genetic defects or alterations that are related to spermatogenesis. The discovery, cloning and further functional study of genes related to spermatogenesis is of great importance to the elucidation of the molecular mechanism of spermatogenesis. It is also physiologically and pathologically significant to the therapy of male infertility. Methods GON-SJTU1 was identified and cloned from rat testis by cDNA library screening and 3'-and 5'-RACE. The products of GON-SJTU1 were assessed by Northern and Western blotting. The expression of GON-SJTU1 was also examined by In situ hybridization and immunohistochemistry. Results Here we identified and cloned a new gene, GON-SJTU1, with the biological process of spermatogenesis. GON-SJTU1 is highly expressed in the testis from day 1 to 15 and then decreased, suggesting that GON-SJTU1 might be a time-related gene and involved in the early stage of spermatogenesis. And the expression of GON-SJTU1 in the testis occurred in some male germ cells, particularly in gonocytes and spermatogonial stem cells. Conclusion GON-SJTU1 may play a role in the biological process of spermatogenesis. PMID:20462432

  12. Molecular cloning and expression of a new gene, GON-SJTU1 in the rat testis.

    PubMed

    Yang, Zhao-juan; Sun, Ning; Wang, Shu-qin; Tian, Geng G; Wu, Ji

    2010-05-12

    Spermatogenesis is a complex process involving cell development, differentiation and apoptosis. This process is governed by a series of genes whose expressions are highly regulated. Male infertility can be attributed to multiple genetic defects or alterations that are related to spermatogenesis. The discovery, cloning and further functional study of genes related to spermatogenesis is of great importance to the elucidation of the molecular mechanism of spermatogenesis. It is also physiologically and pathologically significant to the therapy of male infertility. GON-SJTU1 was identified and cloned from rat testis by cDNA library screening and 3'-and 5'-RACE. The products of GON-SJTU1 were assessed by Northern and Western blotting. The expression of GON-SJTU1 was also examined by In situ hybridization and immunohistochemistry. Here we identified and cloned a new gene, GON-SJTU1, with the biological process of spermatogenesis. GON-SJTU1 is highly expressed in the testis from day 1 to 15 and then decreased, suggesting that GON-SJTU1 might be a time-related gene and involved in the early stage of spermatogenesis. And the expression of GON-SJTU1 in the testis occurred in some male germ cells, particularly in gonocytes and spermatogonial stem cells. GON-SJTU1 may play a role in the biological process of spermatogenesis.

  13. Molecular Structure of P2Y Receptors: Mutagenesis, Modeling, and Chemical Probes

    PubMed Central

    Jayasekara, M.P. Suresh; Costanzi, Stefano

    2012-01-01

    There are eight subtypes of P2Y receptors (P2YRs) that are activated, and in some cases inhibited, by a range of extracellular nucleotides. These nucleotides are ubiquitous, but their extracellular concentration can rise dramatically in response to hypoxia, ischemia, or mechanical stress, injury, and release through channels and from vesicles. Two subclasses of P2YRs were defined based on clustering of sequences, second messengers, and receptor sequence analysis. The numbering system for P2YR subtypes is discontinuous; i.e., P2Y1–14Rs have been defined, but six of the intermediate-numbered cloned receptor sequences (e.g., P2y3, P2y5, P2y7–10) are not functional mammalian nucleotide receptors. Of these two clusters, the P2Y12–14 subtypes couple via Gαi to inhibit adenylate cyclase, while the remaining subtypes couple through Gαq to activate phospholipase C. Collectively, the P2YRs respond to both purine and pyrimidine nucleotides, in the form of 5′-mono- and dinucleotides and nucleoside-5′-diphosphosugars. In recent years, the medicinal chemistry of P2Y receptors has advanced significantly, to provide selective agonists and antagonists for many but not all of the subtypes. Ligand design has been aided by insights from structural probing using molecular modelling and mutagenesis. Currently, the molecular modelling of the receptors is effectively based on the X-ray structure of the CXCR4 receptor, which is the closest to the P2Y receptors among all the currently crystallized receptors in terms of sequence similarity. It is now a challenge to develop novel and selective P2YR ligands for disease treatment (although antagonists of the P2Y12R are already widely used as antithrombotics). PMID:23336097

  14. Molecular evolution of GPCRs: Ghrelin/ghrelin receptors.

    PubMed

    Kaiya, Hiroyuki; Kangawa, Kenji; Miyazato, Mikiya

    2014-06-01

    After the discovery in 1996 of the GH secretagogue-receptor type-1a (GHS-R1a) as an orphan G-protein coupled receptor, many research groups attempted to identify the endogenous ligand. Finally, Kojima and colleagues successfully isolated the peptide ligand from rat stomach extracts, determined its structure, and named it ghrelin. The GHS-R1a is now accepted to be the ghrelin receptor. The existence of the ghrelin system has been demonstrated in many animal classes through biochemical and molecular biological strategies as well as through genome projects. Our work, focused on identifying the ghrelin receptor and its ligand ghrelin in laboratory animals, particularly nonmammalian vertebrates, has provided new insights into the molecular evolution of the ghrelin receptor. In mammals, it is assumed that the ghrelin receptor evolution is in line with the plate tectonics theory. In contrast, the evolution of the ghrelin receptor in nonmammalian vertebrates differs from that of mammals: multiplicity of the ghrelin receptor isoforms is observed in nonmammalian vertebrates only. This multiplicity is due to genome duplication and polyploidization events that particularly occurred in Teleostei. Furthermore, it is likely that the evolution of the ghrelin receptor is distinct from that of its ligand, ghrelin, because only one ghrelin isoform has been detected in all species examined so far. In this review, we summarize current knowledge related to the molecular evolution of the ghrelin receptor in mammalian and nonmammalian vertebrates. © 2014 Society for Endocrinology.

  15. Molecular cloning of the avian myelocytomatosis virus genome and recovery of infectious virus by transfection of chicken cells.

    PubMed Central

    Vennström, B; Moscovici, C; Goodman, H M; Bishop, J M

    1981-01-01

    The avian retrovirus myelocytomatosis virus 19 (MCV) possesses an interesting diversity of oncogenic potentials, but the virus has proven difficult to study because of its inability to replicate without the assistance of a helper virus. We have therefore isolated and amplified the genome of MCV by molecular cloning in a procaryotic vector. The topography of the cloned DNA was explored by the use of restriction endonucleases and radioactive complementary DNAs representing specific domains in avian retrovirus genomes. The cloned DNA appeared to be an authentic representation of the MCV genome: the size and genetic topography of the DNA were comparable to those of MCV, and transfection of the cloned DNA into chicken cells (in company with the DNA of a suitable helper virus) gave rise to virus with the genome and transforming potentials of MCV. The availability of cloned MCV DNA should facilitate a variety of genetic and biochemical manipulations directed at elucidating the mechanism of oncogenesis by MCV. Images PMID:6268847

  16. Cloning and mapping of the mouse {alpha}7-neuronal nicotinic acetylcholine receptor

    SciTech Connect

    Orr-Urtreger, A.; Baldini, A.; Beaudet, A.L.

    1995-03-20

    We report the isolation of cDNA clones for the mouse {alpha}7 neuronal nicotinic acetylcholine receptor subunit (gene symbol Acra7), the only nicotinic receptor subunit known to bind a-bungarotoxin in mammalian brain. This gene may have relevance to nicotine sensitivity and to some electrophysiologic findings in schizophrenia. The mouse {alpha}7 subunit gene encodes a protein of 502 amino acids with substantial identity to the rat (99.6%), human (92.8%), and chicken (87.5%) amino acid sequences. The {alpha}7 gene was mapped to mouse chromosome 7 near the p locus with the following gene order from proximal to distal: Myod1-3.5 {+-}1.7 cM-Gas2-0.9 cM {+-} 0.9 cM-D7Mit70-1.8 {+-} 1.2 cM- Acra7-4.4 {+-}1.0 cM-Hras1-ps11/Igf1r/Snrp2a. The human gene was confirmed to map to the homologous region of human chromosome 15q13-q14. 26 refs., 3 figs.

  17. P2X4: an ATP-activated ionotropic receptor cloned from rat brain.

    PubMed Central

    Soto, F; Garcia-Guzman, M; Gomez-Hernandez, J M; Hollmann, M; Karschin, C; Stühmer, W

    1996-01-01

    Extracellular ATP exerts pronounced biological actions in virtually every organ or tissue that has been studied. In the central and peripheral nervous system, ATP acts as a fast excitatory transmitter in certain synaptic pathways [Evans, R.J., Derkach, V. & Surprenant, A. (1992) Nature (London) 357, 503-505; Edwards, F.A., Gigg, A.J. & Colquhoun, D. (1992) Nature (London) 359, 144-147]. Here, we report the cloning and characterization of complementary DNA from rat brain, encoding an additional member (P2X4) of the emerging multigenic family of ligand-gated ATP channels, the P2X receptors. Expression in Xenopus oocytes gives an ATP-activated cation-selective channel that is highly permeable to Ca2+ and whose sensitivity is modulated by extracellular Zn2+. Surprisingly, the current elicited by ATP is almost insensitive to the common P2X antagonist suramin. In situ hybridization reveals the expression of P2X4 mRNA in central nervous system neurons. Northern blot and reverse transcription-PCR (RT-PCR) analysis demonstrate a wide distribution of P2X4 transcripts in various tissues, including blood vessels and leukocytes. This suggests that the P2X4 receptor might mediate not only ATP-dependent synaptic transmission in the central nervous system but also a wide repertoire of biological responses in diverse tissues. Images Fig. 3 Fig. 4 PMID:8622997

  18. Cloning and expression analysis of a muscarinic cholinergic receptor from the brain of ant, Polyrhachis vicina.

    PubMed

    Lü, Shu-Min; Zhao, Zhuo; Li, Ke; Zhang, Ya-Lin; Xi, Geng-Si

    2011-09-01

    Muscarinic acetylcholine receptors (mAchRs) are the predominant cholinergic receptors in the central and peripheral nervous systems of animals. They also have been found in various insect nervous systems. In this article, a full-length cDNA of a pupative mAchR (PmAchR) was obtained from the brains of ant Polyrhachis vicina by homology cloning in combination with rapid amplification of cDNA ends. PmAchR encodes a 599-amino acid protein that exhibits a high degree of homology with other mAchRs. Real-time quantitative RT-PCR analysis showed that PmAchR is differentially expressed in the brains of workers, males, and females. By in situ hybridization, it is revealed that PmAchR is widely expressed in different soma clusters of the brain, including the mushroom bodies, the antennal lobes, as well as the optic lobes (OL), and the most intensely staining is found in Kenyon cells. Nonetheless, there are more positive nerve fibers in the OL of males' brains than in females' and workers' brains.

  19. Cloning and tissue distribution of the chicken type 2 corticotropin-releasing hormone receptor.

    PubMed

    de Groef, Bert; Grommen, Sylvia V H; Mertens, Inge; Schoofs, Liliane; Kühn, Eduard R; Darras, Veerle M

    2004-08-01

    We report the cloning of the complete coding sequence of the putative chicken type 2 corticotropin-releasing hormone receptor (CRH-R2) by rapid amplification of cDNA ends (RACE). The chicken CRH-R2 is a 412-amino acid 7-transmembrane G protein-coupled receptor, showing 87% identity to the Xenopus laevis and Oncorhynchus keta CRH-R2s, and 78-80% to mammalian CRH-R2s. The distribution of CRH-R2 mRNA was studied by RT-PCR analysis and compared to CRH-R1 distribution. Both CRH-R1 and CRH-R2 mRNA are expressed in the main chicken brain parts. In peripheral organs, CRH-R1 mRNA shows a more restricted distribution, whereas CRH-R2 mRNA is expressed in every tissue investigated, indicating that a number of actions of CRH and/or CRH-like peptides remain to be discovered in the chicken as well as in other vertebrates.

  20. From lesions to viral clones: biological and molecular diversity amongst autochthonous Brazilian vaccinia virus.

    PubMed

    Oliveira, Graziele; Assis, Felipe; Almeida, Gabriel; Albarnaz, Jonas; Lima, Maurício; Andrade, Ana Cláudia; Calixto, Rafael; Oliveira, Cairo; Diomedes Neto, José; Trindade, Giliane; Ferreira, Paulo César; Kroon, Erna Geessien; Abrahão, Jônatas

    2015-03-16

    Vaccinia virus (VACV) has had an important role for humanity because of its use during the smallpox eradication campaign. VACV is the etiologic agent of the bovine vaccinia (BV), an emerging zoonosis that has been associated with economic, social, veterinary and public health problems, mainly in Brazil and India. Despite the current and historical VACV importance, there is little information about its circulation, prevalence, origins and maintenance in the environment, natural reservoirs and diversity. Brazilian VACV (VACV-BR) are grouped into at least two groups based on genetic and biological diversity: group 1 (G1) and group 2 (G2). In this study, we went to the field and investigated VACV clonal diversity directly from exanthemous lesions, during BV outbreaks. Our results demonstrate that the G1 VACV-BR were more frequently isolated. Furthermore, we were able to co-detect the two variants (G1 and G2) in the same sample. Molecular and biological analysis corroborated previous reports and confirmed the co-circulation of two VACV-BR lineages. The detected G2 clones presented exclusive genetic and biological markers, distinct to reference isolates, including VACV-Western Reserve. Two clones presented a mosaic profile, with both G1 and G2 features based on the molecular analysis of A56R, A26L and C23L genes. Indeed, some SNPs and INDELs in A56R nucleotide sequences were observed among clones of the same virus population, maybe as a result of an increased mutation rate in a mixed population. These results provide information about the diversity profile in VACV populations, highlighting its importance to VACV evolution and maintenance in the environment.

  1. From Lesions to Viral Clones: Biological and Molecular Diversity amongst Autochthonous Brazilian Vaccinia Virus

    PubMed Central

    Oliveira, Graziele; Assis, Felipe; Almeida, Gabriel; Albarnaz, Jonas; Lima, Maurício; Andrade, Ana Cláudia; Calixto, Rafael; Oliveira, Cairo; Neto, José Diomedes; Trindade, Giliane; Ferreira, Paulo César; Kroon, Erna Geessien; Abrahão, Jônatas

    2015-01-01

    Vaccinia virus (VACV) has had an important role for humanity because of its use during the smallpox eradication campaign. VACV is the etiologic agent of the bovine vaccinia (BV), an emerging zoonosis that has been associated with economic, social, veterinary and public health problems, mainly in Brazil and India. Despite the current and historical VACV importance, there is little information about its circulation, prevalence, origins and maintenance in the environment, natural reservoirs and diversity. Brazilian VACV (VACV-BR) are grouped into at least two groups based on genetic and biological diversity: group 1 (G1) and group 2 (G2). In this study, we went to the field and investigated VACV clonal diversity directly from exanthemous lesions, during BV outbreaks. Our results demonstrate that the G1 VACV-BR were more frequently isolated. Furthermore, we were able to co-detect the two variants (G1 and G2) in the same sample. Molecular and biological analysis corroborated previous reports and confirmed the co-circulation of two VACV-BR lineages. The detected G2 clones presented exclusive genetic and biological markers, distinct to reference isolates, including VACV-Western Reserve. Two clones presented a mosaic profile, with both G1 and G2 features based on the molecular analysis of A56R, A26L and C23L genes. Indeed, some SNPs and INDELs in A56R nucleotide sequences were observed among clones of the same virus population, maybe as a result of an increased mutation rate in a mixed population. These results provide information about the diversity profile in VACV populations, highlighting its importance to VACV evolution and maintenance in the environment. PMID:25785515

  2. Molecular cloning and expression analysis of Annexin A2 gene in sika deer antler tip.

    PubMed

    Xia, Yanling; Qu, Haomiao; Lu, Binshan; Zhang, Qiang; Li, Heping

    2017-08-16

    Molecular cloning and bioinformatics analysis of Annexin A2 gene in sika deer antler tip were conducted. The role of Annexin A2 gene in the growth and development of the antler were analyzed initially. The reverse transcriptase-polymerase chain reaction (RT-PCR) was used to clone the cDNA sequence of the Anxa2 gene from antler tip of sika deer (Cervus nippon hortulorum) and the bioinformatics tools provided on the web site were applied to analyze the amino acid sequence of Anxa2 protein. The mRNA expression levels of the Anxa2 gene in different growth stages were examined by real-time RT-PCR. The nucleotide sequence analysis revealed an ORF of 1020 bp encoding 339 amino acids long protein of calculated molecular weight 38.6 kDa and pI value 6.09. Homologous sequence alignment and phylogenetic analysis indicated that the Anxa2 mature protein of sika deer had the closest genetic distance with Cervus elaphus and Bos mutus. Real-time RT-PCR results showed that the gene had differential expression levels in different growth stages, and the expression level of the Anxa2 gene was the highest at metaphase (rapid growing period). Anxa2 gene may promote the cell proliferation, and the finding suggested Anxa2 as an important candidate for regulating the growth and development of deer antler.

  3. Melatonin receptor of a reef fish with lunar-related rhythmicity: cloning and daily variations.

    PubMed

    Park, Yong-Ju; Park, Ji-Gweon; Kim, Se-Jae; Lee, Young-Don; Saydur Rahman, Md; Takemura, Akihiro

    2006-09-01

    Melatonin receptors are expressed in neural and peripheral tissues and mediate melatonin actions on the regulation of circadian rhythms in various species. For overall understanding of 'circa' rhythms in the golden rabbitfish, Siganus guttatus, which exhibits restricted lunar-related rhythms and spawns synchronously around the first quarter moon, the aim of the present study was to clone a melatonin receptor (Mel(lb)) cDNA and examine daily variations of Mel(lb) mRNA expression in certain tissues of the rabbitfish. The full-length Mel(lb) cDNA (1808 bp) contained an open reading frame to encode a protein with a length of 354 amino acids, which was highly homologous to a protein of nonmammalian species. Northern blot analysis showed transcripts of Mel(lb) in the brain and retina. Real-time quantitative polymerase chain reaction analysis also revealed expression of Mel(lb) in all tissues tested. Significantly high expression of the gene during daytime was evident in the liver and kidney. When the expression of Mel(lb) was examined in the brain and retina under conditions of light/dark cycles or constant darkness, daily and circadian variations of gene expression with two increases during daytime and nighttime for the brain and a single increase during nighttime for the retina were recognized. Moreover, daily variations in the expression of Mel(lb) were observed in the cultured pineal gland. These results suggest that the melatonin receptor plays a role in integration of melatonin actions in various tissues and that daily variations of Mel(lb) in the neural tissues may be related to regulation of circadian clock.

  4. Molecular cloning and nucleotide sequence of a transforming gene detected by transfection of chicken B-cell lymphoma DNA

    NASA Astrophysics Data System (ADS)

    Goubin, Gerard; Goldman, Debra S.; Luce, Judith; Neiman, Paul E.; Cooper, Geoffrey M.

    1983-03-01

    A transforming gene detected by transfection of chicken B-cell lymphoma DNA has been isolated by molecular cloning. It is homologous to a conserved family of sequences present in normal chicken and human DNAs but is not related to transforming genes of acutely transforming retroviruses. The nucleotide sequence of the cloned transforming gene suggests that it encodes a protein that is partially homologous to the amino terminus of transferrin and related proteins although only about one tenth the size of transferrin.

  5. Molecular cloning of the avian erythroblastosis virus genome and recovery of oncogenic virus by transfection of chicken cells.

    PubMed Central

    Vennström, B; Fanshier, L; Moscovici, C; Bishop, J M

    1980-01-01

    Avian erythroblastosis virus (AEV) causes erythroblastosis and sarcomas in birds and transforms both erythroblasts and fibroblasts to neoplastic phenotypes in culture. The viral genetic locus required for oncogenesis by AEV is at present poorly defined; moreover, we know very little of the mechanism of tumorigenesis by the virus. To facilitate further analysis of these problems, we used molecular cloning to isolate the genome of AEV as recombinant DNA in a procaryotic vector. The identity of the isolated DNA was verified by mapping with restriction endonucleases and by tests for biological activity. The circular form of unintegrated AEV DNA was purified from synchronously infected quail cells and cloned into the EcoRI site of lambda gtWES x B. A restriction endonuclease cleavage map was established. By hybridization with complementary DNA probes representing specific parts of avian retrovirus genomes, the restriction map of the cloned AEV DNAs was correlated with a genetic map. These data show that nucleotide sequences unique to AEV comprise at least 50% of the genome and are located approximately in the middle of the AEV genome. Our data confirm and extend previous descriptions of the AEV genome obtained by other procedures. We studied in detail two recombinant clones containing AEV DNA: the topography of the viral DNA in the two clones was virtually identical, except that one clone apparently contained two copies of the terminal redundancy that occurs in linear viral DNA isolated from infected cells; the other clone probably contained only one copy of the redundant sequence. To recover infectious virus from the cloned DNA, we developed a procedure for transfection that compensated for the defectiveness of AEV in replication. We accomplished this by ligating cloned AEV DNA to the cloned DNA of a retrovirus (Rous-associated virus type 1) whose genome could complement the deficiencies of AEV. Ligation of the two viral DNAs was facilitated by using a neutral fragment

  6. Cloning and Expression of Ecdysone Receptor and Retinoid X Receptor from Procambarus clarkii: Induction by Eyestalk Ablation

    PubMed Central

    Dai, Tian-Hao; Sserwadda, Ali; Song, Kun; Zang, Ya-Nan; Shen, Huai-Shun

    2016-01-01

    Ecdysone receptor and retinoid X receptor are key regulators in molting. Here, full length ecdysone receptor (PcEcR) and retinoid X receptor (PcRXR) cDNAs from Procambarus clarkii were cloned. Full length cDNA of PcEcR has 2500 bp, encoding 576 amino acid proteins, and full length cDNA of PcRXR has 2593 bp, in which a 15 bp and a 204 bp insert/deletion splice variant regions in DNA binding domain and hinge domain were identified. The two splice variant regions in PcRXR result four isoforms: PcRXR1-4, encoding 525, 520, 457 and 452 amino acids respectively. PcEcR was highly expressed in the hepatopancreas and eyestalk and PcRXR was highly expressed in the eyestalk among eight examined tissues. Both PcEcR and PcRXR had induced expression after eyestalk ablation (ESA) in the three examined tissues. In muscle, PcEcR and PcRXR were upregulated after ESA, PcEcR reached the highest level on day 3 after ESA and increased 33.5-fold relative to day 0, and PcRXR reached highest the level on day 1 after ESA and increased 2.7-fold relative to day 0. In the hepatopancreas, PcEcR and PcRXR dEcReased continuously after ESA, and the expression levels of PcEcR and PcRXR were only 0.7% and 1.7% on day 7 after ESA relative to day 0, respectively. In the ovaries, PcEcR was upregulated after ESA, reached the highest level on day 3 after ESA, increased 3.0-fold relative to day 0, and the expression level of PcRXR changed insignificantly after ESA (p > 0.05). The different responses of PcEcR and PcRXR after ESA indicates that different tissues play different roles (and coordinates their functions) in molting. PMID:27763563

  7. Cloning and Expression of Ecdysone Receptor and Retinoid X Receptor from Procambarus clarkii: Induction by Eyestalk Ablation.

    PubMed

    Dai, Tian-Hao; Sserwadda, Ali; Song, Kun; Zang, Ya-Nan; Shen, Huai-Shun

    2016-10-18

    Ecdysone receptor and retinoid X receptor are key regulators in molting. Here, full length ecdysone receptor (PcEcR) and retinoid X receptor (PcRXR) cDNAs from Procambarus clarkii were cloned. Full length cDNA of PcEcR has 2500 bp, encoding 576 amino acid proteins, and full length cDNA of PcRXR has 2593 bp, in which a 15 bp and a 204 bp insert/deletion splice variant regions in DNA binding domain and hinge domain were identified. The two splice variant regions in PcRXR result four isoforms: PcRXR1-4, encoding 525, 520, 457 and 452 amino acids respectively. PcEcR was highly expressed in the hepatopancreas and eyestalk and PcRXR was highly expressed in the eyestalk among eight examined tissues. Both PcEcR and PcRXR had induced expression after eyestalk ablation (ESA) in the three examined tissues. In muscle, PcEcR and PcRXR were upregulated after ESA, PcEcR reached the highest level on day 3 after ESA and increased 33.5-fold relative to day 0, and PcRXR reached highest the level on day 1 after ESA and increased 2.7-fold relative to day 0. In the hepatopancreas, PcEcR and PcRXR dEcReased continuously after ESA, and the expression levels of PcEcR and PcRXR were only 0.7% and 1.7% on day 7 after ESA relative to day 0, respectively. In the ovaries, PcEcR was upregulated after ESA, reached the highest level on day 3 after ESA, increased 3.0-fold relative to day 0, and the expression level of PcRXR changed insignificantly after ESA (p > 0.05). The different responses of PcEcR and PcRXR after ESA indicates that different tissues play different roles (and coordinates their functions) in molting.

  8. Cloning of the gene coding for the outer membrane receptor protein for ferric pseudobactin, a siderophore from a plant growth-promoting Pseudomonas strain.

    PubMed

    Magazin, M D; Moores, J C; Leong, J

    1986-01-15

    Plant growth-promoting Pseudomonas B10 produces its yellow-green, fluorescent siderophore (microbial iron transport agent) pseudobactin under iron-limiting conditions. A structural gene encoding the 85,000-Da putative outer membrane receptor protein for ferric pseudobactin was identified in a gene bank from Pseudomonas B10 prepared with the broad host-range conjugative cosmid cloning vector pLAFR1. Transposon Tn5 mutagenesis of recombinant plasmid pJLM300 localized the functional gene to a region of approximately 2.4 kilobases consistent with the apparent molecular weight of the receptor protein. Mobilization of pJLM300 into Pseudomonas A124 and A225, whose growth was inhibited by Pseudomonas B10 or pseudobactin, rendered these strains no longer susceptible to iron starvation by pseudobactin because they were now able to transport ferric pseudobactin. Pseudobactin biosynthetic genes flanked this receptor gene on both sides and were on separate operons. Transposon Tn5 insertion mutants of Pseudomonas B10 lacking this receptor protein were generated by a marker exchange technique and were defective in ferric pseudobactin transport. Such mutants could be complemented in trans by pJLM300. The production of pseudobactin, the receptor protein, and four other outer membrane proteins in Pseudomonas B10 was coordinately regulated by the level of intracellular iron.

  9. Cloning of the rat ecotropic retroviral receptor and studies of its expression in intestinal tissues

    SciTech Connect

    Puppi, M.; Henning, S.J.

    1995-05-01

    A long-term goal of our laboratory is to establish a rat model to study the feasibility of using the intestinal tract as a site for somatic gene therapy. As a step toward that goal, the current study reports the cloning of the rat ecotropic retroviral receptor (EcoR) cDNA and the study of various aspects of its expression in the intestinal cDNA library with mouse EcoR cDNA. A clone of approximately 7 kb, designated MP10, was obtained. Partial sequencing of MP10 from the 5{prime} end revealed a level of similarity of 92% compared with mouse EcoR. The presence of a 5{prime} untranslated region and a 3{prime} poly(A)tract, together with the overall size of the cDNA, suggest that is very close to being a full-length cDNA for this large transcript. Northern blots with MP10 showed an RNA of approximately 7.9 kb present along the entire length of the small intestine and somewhat less abundant in the colon. Developmental studies showed high levels of EcoR in fetal rat intestine, a decline in the early postnatal period, then a gradual rise to adulthood. Caco-2 cells were used to assess the expression of EcoR in proliferating compared with differentiated intestinal epithelial cells. EcoR mRNA was found to be very much more abundant in nondifferentiated cells and declined to low levels as the cells underwent spontaneous differentiation. These patterns of EcoR expression indicate that ecotropic retroviruses should be suitable vectors with which to attempt gene transfer into the intestinal epithelium. In addition, since the endogenous role of EcoR is as the y{sup +} cationic amino acid transporter, these data have significance for understanding patterns of amino acid transport in the intestinal epithelium. 37 refs., 4 figs.

  10. Cloning of the rat ecotropic retroviral receptor and studies of its expression in intestinal tissues.

    PubMed

    Puppi, M; Henning, S J

    1995-05-01

    A long-term goal of our laboratory is to establish a rat model to study the feasibility of using the intestinal tract as a site for somatic gene therapy. As a step toward that goal, the current study reports the cloning of the rat ecotropic retroviral receptor (EcoR) cDNA and the study of various aspects of its expression in the intestinal tissues. The cDNA for rat EcoR was cloned by screening a size-selected rat intestinal cDNA library with mouse EcoR cDNA. A clone of approximately 7 kb, designated MP10, was obtained. Partial sequencing of MP10 from the 5' end revealed a level of similarity of 92% compared with mouse EcoR. The presence of a 5' untranslated region and a 3' poly(A) tract, together with the overall size of the cDNA, suggest that is very close to being a full-length cDNA for this large transcript. Northern blots with MP10 showed an RNA of approximately 7.9 kb present along the entire length of the small intestine and somewhat less abundant in the colon. Developmental studies showed high levels of EcoR in fetal rat intestine, a decline in the early postnatal period, then a gradual rise to adulthood. Caco-2 cells were used to assess the expression of EcoR in proliferating compared with differentiated intestinal epithelial cells. EcoR mRNA was found to be very much more abundant in nondifferentiated cells and declined to low levels as the cells underwent spontaneous differentiation. These patterns of EcoR expression indicate that ecotropic retroviruses should be suitable vectors with which to attempt gene transfer into the intestinal epithelium. In addition, since the endogenous role of EcoR is as the y+ cationic amino acid transporter, these data have significance for understanding patterns of amino acid transport in the intestinal epithelium.

  11. Molecular cloning, expression, and chromosome 19 localization of a human Ro/SS-A autoantigen.

    PubMed Central

    McCauliffe, D P; Lux, F A; Lieu, T S; Sanz, I; Hanke, J; Newkirk, M M; Bachinski, L L; Itoh, Y; Siciliano, M J; Reichlin, M

    1990-01-01

    Ro/SS-A antibodies are found in a number of human autoimmune disorders including Sjogren's syndrome and several systemic lupus erythematosus-related disorders. These heterogeneous autoantibodies are known to recognize several distinct cellular antigens. With synthetic oligonucleotides corresponding to amino acid sequence information we have isolated a full-length cDNA clone which encodes a human Ro ribonucleoprotein autoantigen. The 1,890-base pair clone contains an open reading frame that encodes a 417-amino acid, 48-kD polypeptide that migrates aberrantly at 60 kD by SDS-PAGE. Rabbit antibodies raised against this protein's recently described amino-terminal epitope react with a previously identified 52-kD human Ro protein and immunoprecipitate the human cytoplasmic RNAs. Ultraviolet light cross-linking studies suggest that this Ro protein binds each of the four major human cytoplasmic RNAs. The deduced amino acid sequence is 63% homologous to an Onchocerca volvulus antigen. Southern filter hybridization analysis indicates that this gene is not highly polymorphic and exists as a single copy in the human genome. Chromosomal localization studies place this gene on the short arm of chromosome 19 near the gene encoding the low density lipoprotein receptor. Images PMID:2332496

  12. Molecular cloning and gene expression of canine apoptosis inhibitor of macrophage.

    PubMed

    Tomura, Shintaro; Uchida, Mona; Yonezawa, Tomohiro; Kobayashi, Masato; Bonkobara, Makoto; Arai, Satoko; Miyazaki, Toru; Tamahara, Satoshi; Matsuki, Naoaki

    2014-12-01

    Apoptosis inhibitor of macrophage (AIM) plays roles in survival of macrophages. In this study, we cloned canine AIM cDNA and observed its transcriptional expression levels in various tissues. The coding sequence of canine AIM was 1,023 bp encoding 340 amino acid residues, which had around 65% homology with those of the human, mouse and rat. Transcriptional expression of AIM was observed in the spleen, lung, liver and lymph node, which confirmed the expression of canine AIM in tissue macrophages. Moreover, AIM was highly expressed in one of the canine histiocytic sarcoma cell lines. CD36, the receptor of AIM, was also expressed in various tissues and these cell lines. These findings are useful to reveal the actual functions of canine AIM.

  13. The relaxant 5-HT receptor in the dog coronary artery smooth muscle: pharmacological resemblance to the cloned 5-ht7 receptor subtype.

    PubMed Central

    Terrón, J. A.

    1996-01-01

    1. The relaxant effect of 5-hydroxytryptamine (5-HT) in the dog isolated coronary artery deprived of endothelium is mediated by a receptor unrelated to the 5-HT1, 5-HT2, 5-HT3 or 5-HT4 types. Based upon the pharmacological characteristics of this relaxant 5-HT receptor and those reported for the new members of the 5-HT receptor family, the present study explored the possibility that the relaxant 5-HT receptor referred to above, corresponds to the cloned 5-ht7 subtype. Thus, the relaxing and/or blocking effects of several 5-HT receptor drugs as well as some typical and atypical antipsychotic drugs with high affinity for the cloned 5-ht7 receptor in precontracted ring segments were analyzed. 2. 5-HT, 5-carboxamidotryptamine (5-CT) and 5-methoxytryptamine, but not 8-OH-DPAT or sumatriptan, produced concentration-dependent relaxations in endothelium-denuded canine coronary artery rings precontracted with prostaglandin F2a (2 microM). Clozapine (1 microM) produced in some cases a small relaxing effect and antagonized 5-HT- and 5-CT-induced relaxation suggesting a partial agonist effect. In the presence of the 5-HT1D receptor antagonist, GR127935 (100 nM), the rank order of agonist potency was 5-CT > 5-HT > clozapine > or = 5-methoxytryptamine. 8-OH-DPAT and sumatriptan remained inactive as agonists. 3. In GR127935-treated preparations, methiothepin (3 nM) and mianserin (1 microM), as well as the antipsychotics, clozapine (1 microM), pimozide (300 nM), risperidone (3 nM) and spiperone (1 microM), failed to induce a significant relaxation in prostaglandin F2x-precontracted vessels, but produced significant rightward displacements of the concentration-response curves to 5-HT and 5-CT without significantly reducing the Emax. In a final set of experiments with 5-CT, metergoline (100 nM) and mesulergine (300 nM) behaved as competitive antagonists. In contrast, lisuride (3 nM) noncompetitively antagonized 5-CT-induced relaxation. The estimated affinity (apparent pKa values) of

  14. T cell receptor genes in an alloreactive CTL clone: implications for rearrangement and germline diversity of variable gene segments.

    PubMed Central

    Chou, H S; Behlke, M A; Godambe, S A; Russell, J H; Brooks, C G; Loh, D Y

    1986-01-01

    Both cDNA and genomic clones of the T cell receptor (TCR) alpha- and beta-chain genes of the alloreactive cytotoxic T lymphocyte (CTL) clone F3 were examined. Two distinct rearrangement events, one functional and one non-functional, were found for both the alpha and beta loci. Thus only a single functional TCR alpha beta heterodimer could be defined, consistent with allelic exclusion in the TCR genes. The V alpha gene employed by F3 is part of a six-member V alpha subfamily. Genomic clones containing each member of this subfamily were isolated and the V alpha nucleotide sequences determined. Five of these six genes are functional; these genes differ from each other by 7-14% at the amino acid level. A single dominant hypervariable region was defined within this subfamily, in contrast to the pattern of variability seen between V alpha genes in general. Images Fig. 4. Fig. 5. PMID:3490968

  15. Molecular Cloning, Characterization, and Expression of the M Antigen of Histoplasma capsulatum

    PubMed Central

    Zancopé-Oliveira, Rosely M.; Reiss, Errol; Lott, Timothy J.; Mayer, Leonard W.; Deepe, George S.

    1999-01-01

    The major diagnostic antigens of Histoplasma capsulatum are the H and M antigens, pluripotent glycoproteins that elicit both humoral and T-cell-mediated immune responses. These antigens may play a role in the pathogenesis of histoplasmosis. M antigen is considered immunodominant because antibodies against it are the first precipitins to arise in acute histoplasmosis and are commonly present during all phases of infection. The biological activity of monomolecular M antigen and its ability to elicit a protective immune response to H. capsulatum are largely unknown. A molecular approach was used to identify the biological nature of M antigen, including its purification from histoplasmin, partial digestion with proteinases, and reverse-phase high-performance liquid chromatography to separate the released peptides. The amino acid sequences of the purified peptides were obtained by Edman degradation, and using degenerate oligonucleotide primers for PCR, a 321-bp fragment of the gene encoding the M antigen was amplified from genomic H. capsulatum DNA. This fragment was used to screen an H. capsulatum genomic DNA library, leading to the isolation, cloning, and sequencing of the full-length gene. The M gene consists of 2,187-bp DNA encoding a protein of 80,719 Da, which has significant homology to catalases from Aspergillus fumigatus, Aspergillus niger, and Eimericella nidulans. A cDNA was generated by reverse transcription-PCR and cloned into the expression vector pQE40. The identity of the cloned, expressed protein was confirmed by Western blotting. The recombinant fusion protein was immunoreactive with monoclonal antibodies raised against M antigen, with polyclonal mouse anti-M antiserum, and with a serum sample from a patient with histoplasmosis. The gene encoding the major immunodominant M antigen of H. capsulatum is a presumptive catalase, and the recombinant protein retains serodiagnostic activity. PMID:10085041

  16. Molecular cloning, expression and characterization of a functional GSTmu class from the cattle tick Boophilus annulatus.

    PubMed

    Shahein, Yasser Ezzat; El Sayed El-Hakim, Amr; Abouelella, Amira Mohamed Kamal; Hamed, Ragaa Reda; Allam, Shaimaa Abdul-Moez; Farid, Nevin Mahmoud

    2008-03-25

    A full-length cDNA of a glutathione S-transferase (GST) was cloned from a cDNA library of the local Egyptian cattle tick Boophilus annulatus. The 672 bp cloned fragment was sequenced and showed an open reading frame encoding a protein of 223 amino acids. Comparison of the deduced amino acid sequence with GSTs from other species revealed that the sequence is closely related to the mammalian mu-class GST. The cloned gene was expressed in E. coli under T7 promotor of pET-30b vector, and purified under native conditions. The purified enzyme appeared as a single band on 12% SDS-PAGE and has a molecular weight of 30.8 kDa including the histidine tag of the vector. The purified enzyme was assayed upon the chromogenic substrate 1-chloro-2,4-dinitrobenzene (CDNB) and the recombinant enzyme showed high level of activity even in the presence of the beta-galactosidase region on its 5' end and showed maximum activity at pH 7.5. The Km values for CDNB and GSH were 0.57 and 0.79 mM, respectively. The over expressed rBaGST showed high activity toward CDNB (121 units/mg protein) and less toward DCNB (29.3 units/mg protein). rBaGST exhibited peroxidatic activity on cumene hydroperoxide sharing this property with GSTs belonging to the GST alpha class. I50 values for cibacron blue and bromosulfophthalein were 0.22 and 8.45 microM, respectively, sharing this property with the mammalian GSTmu class. Immunoblotting revealed the presence of the GST molecule in B. annulatus protein extracts; whole tick, larvae, gut, salivary gland and ovary. Homologues to the GSTmu were also detected in other tick species as Hyalomma dromedarii and Rhipicephalus sp. while in Ornithodoros moubata, GSTmu homologue could not be detected.

  17. Molecular cloning of MIS, a myeloid inhibitory siglec, that binds protein-tyrosine phosphatases SHP-1 and SHP-2.

    PubMed

    Ulyanova, T; Shah, D D; Thomas, M L

    2001-04-27

    We describe the molecular cloning and characterization of a novel myeloid inhibitory siglec, MIS, that belongs to the family of sialic acid-binding immunoglobulin-like lectins. A full-length MIS cDNA was obtained from murine bone marrow cells. MIS is predicted to contain an extracellular region comprising three immunoglobulin-like domains (V-set amino-terminal domain followed by two C-set domains), a transmembrane domain and a cytoplasmic tail with two immunoreceptor tyrosine-based inhibitory motif (ITIM)-like sequences. The closest relative of MIS in the siglec family is human siglec 8. Extracellular regions of these two siglecs share 47% identity at the amino acid level. Southern blot analysis suggests the presence of one MIS gene. MIS is expressed in the spleen, liver, heart, kidney, lung and testis tissues. Several isoforms of MIS protein exist due to the alternative splicing. In a human promonocyte cell line, MIS was able to bind Src homology 2-containing protein-tyrosine phosphatases, SHP-1 and SHP-2. This binding was mediated by the membrane-proximal ITIM of MIS. Moreover, MIS exerted an inhibitory effect on FcgammaRI receptor-induced calcium mobilization. These data suggest that MIS can play an inhibitory role through its ITIM sequences.

  18. Molecular cloning of a membrane-associated human FK506- and rapamycin-binding protein, FKBP-13.

    PubMed Central

    Jin, Y J; Albers, M W; Lane, W S; Bierer, B E; Schreiber, S L; Burakoff, S J

    1991-01-01

    The 12-kDa FK506-binding protein (FKBP-12) is a cytosolic receptor for the immunosuppressants FK506 and rapamycin. Here we report the molecular cloning and subcellular localization of a 13-kDa FKBP (FKBP-13), which has a 21-amino acid signal peptide and appears to be membrane-associated. Although no internal hydrophobic region, and thus no transmembrane domain, is apparent within the 120 amino acids of mature FKBP-13, a potential endoplasmic reticulum retention sequence (Arg-Thr-Glu-Leu) is found at its C terminus. FKBP-13 has 51% nucleotide sequence identity and 43% amino acid sequence identity to FKBP-12; the N-terminal sequences are divergent, but the 92-amino acid C-terminal sequence of FKBP-13 has 46 identical and 20 related residues when compared with FKBP-12. The conserved residues that comprise the drug binding site and rotamase active site of FKBP-12 are completely conserved in FKBP-13. Therefore, the three-dimensional structures of FKBP-12 and the FKBP-12/FK506 complex are likely to be excellent models of the corresponding FKBP-13 structure. Images PMID:1713687

  19. Chemokine receptor CXCR3 in turbot (Scophthalmus maximus): cloning, characterization and its responses to lipopolysaccharide.

    PubMed

    Chen, Yadong; Zhou, Shuhong; Jiang, Zhiqiang; Wang, Xiuli; Liu, Yang

    2016-04-01

    Chemokine (C-X-C motif) receptor 3, a member of the G protein-coupled receptors superfamily, regulates the responses of many immune responses. In this experiment, we cloned and characterized the cDNA of CXCR3 in Scophthalmus maximus (turbot). A 5'-UTR of 216-bp, a 259-bp 3'-UTR with a poly (A) tail and a 1089-bp CDS encoding 362 amino acids form the cDNA of CXCR3, which is 1564-bp long. Phylogenetic analyses indicated that turbot CXCR3 shared a high similarity with other CXCR3s and shared more similarity with CXCR5 than the other subfamilies of chemokines. The CXCR3 protein in turbot showed the highest similarity with the CXCR3b from rainbow trout (44.5%), which indicated that this CXCR3 gene/protein may be a CXCR3b isoform. Quantitative real-time PCR analysis showed that CXCR3 transcripts were constitutively expressed in all the tissues of the non-injected turbot used in this study, with the highest expression occurring in blood. Several immune-related tissues of fish, such as the spleen, head kidney, liver and blood, tissues, which were abundant of lymphocyte, were investigated in this study. CXCR3 gene was expressed at the highest level in blood than the other tested tissues. The injection experiment suggested that the CXCR3 expression level after LPS injection was significantly up-regulated in all immune-related tissues in turbot. These results improve our understanding of the functions of CXCR3 in the turbot immune response.

  20. Cloning, Characterization, and Expression Profile of Estrogen Receptor in Common Chinese Cuttlefish, Sepiella japonica.

    PubMed

    Lü, Zhen-Ming; Liu, Wan; Liu, Li-Qin; Wang, Tian-Ming; Shi, Hui-Lai; Ping, Hong-Ling; Chi, Chang-Feng; Yang, Jing-Wen; Wu, Chang-Wen

    2016-03-01

    Sex steroid hormones are widely detected in molluscs and play important roles in sex determination, gonadal tissue maturation, and gametogenesis. Nevertheless, the signaling pathways of sex steroids in cephalopod have not yet been clearly elucidated. In the present study, a full-length sequence encoding the estrogen receptor (ER) was isolated from common Chinese cuttlefish, Sepiella japonica. The sjER cDNA clone was found to contain 1,788 nucleotides including a 1,470 bp open reading frame encoding 489 amino acid (aa) residues. The deduced ER protein consisted of six nuclear receptor characteristic domains. Based on a phylogenetic analysis, the ER DNA-binding domain and ligand-binding domain are highly conserved compared to other mollusc ERs. Highest aa identities were found for sjER with common octopus (Octopus vulgaris) ER (89%) and pacific oyster (Crassostrea gigas) ER (61%). Tissue expression analysis confirmed that sjER was widely distributed among tissues and predominantly expressed in the brain, liver, gonad (testis and ovary), and other accessory sexual gland (nidamental gland). The ER expression was temporally upregulated in the brain, liver, and ovary during the early sexual maturation period in S. japonica, which is coincident with the fluctuation of ovary estradiol content. These suggest that sjER may be involved in regulating the reproductive cycle of S. japonica. A fusion protein transient transfections assay showed that sjER was mainly located in the nucleus, suggesting a possible orthodox working mechanism of S. japonica ER in the nucleus through a ligand-dependent activation of specific gene transcription. © 2016 Wiley Periodicals, Inc.

  1. Cloning and characterization of an ionotropic glutamate receptor subunit expressed in the squid nervous system.

    PubMed

    Battaglia, A A; Nardi, G; Steinhardt, A; Novakovic, A; Gentile, S; Iaccarino Idelson, P; Gilly, W F; de Santis, A

    2003-06-01

    In this paper we describe the cloning of a putative ionotropic glutamate receptor subunit, SqGluR, and its distribution in the nervous system of the squid. A full-length cDNA was assembled from a cDNA library of the stellate ganglion/giant fibre lobe complex of Loligo opalescens. The deduced amino acid sequence of the mature SqGluR displayed 44-46% amino acid identity with mammalian GluR1-GluR4 and 53% with Lym-eGluR1 from Lymnaea stagnalis. In situ hybridizations in adult squid confirmed that the SqGluR mRNA is abundant in giant fibre lobe neurons, in large, presumptive motor neurons of the stellate ganglion proper and in the supraoesophageal and optic lobes of the central nervous system. In newborn squid, SqGluR mRNA expression was detected throughout the nervous system but not elsewhere. A synthetic peptide corresponding to the last 15 amino acids of the SqGluR C-terminus was used to generate polyclonal antibodies, which were used for immunoblot analysis to demonstrate widespread expression in the squid central and peripheral nervous systems. Injection of the synthetic peptide into the postsynaptic side of the giant synapse inhibited synaptic transmission.

  2. Cloning, expression and interaction of human T-cell receptors with the bacterial superantigen SSA.

    PubMed

    De Marzí, Mauricio C; Fernández, Marisa M; Sundberg, Eric J; Molinero, Luciana; Zwirner, Norberto W; Llera, Andrea S; Mariuzza, Roy A; Malchiodi, Emilio L

    2004-10-01

    Superantigens (SAgs) are a class of disease-causing and immunostimulatory proteins of bacterial or viral origin that activate a large number of T-cells through interaction with the Vbeta domain of T-cell receptors (TCRs). In this study, recombinant TCR beta chains were constructed with human variable domains Vbeta5.2, Vbeta1 and Vbeta2.1, expressed as inclusion bodies, refolded and purified. The Streptococcus pyogenes SAg SSA-1 was cloned and expressed as a soluble periplasmic protein. SSA-1 was obtained both as a monomer and a dimer that has an intermolecular disulfide bond. We analyzed the biological activity of the recombinant SAgs by proliferation assays. The results suggest that SSA dimerization occludes the TCR interaction site. Naturally occurring SSA dimerization was also observed in supernatants of S. pyogenes isolates. An SSA mutant [SSA(C26S)] was produced to eliminate the Cys responsible for dimerization. Affinity assays using a resonant biosensor showed that both the mutant and monomeric wild type SSA have affinity for human Vbeta5.2 and Vbeta1 with Kd of 9-11 microm with a fast kass and a moderately fast kdiss. In spite of the reported stimulation of Vbeta2.1 bearing T-cells by SSA, we observed no measurable interaction.

  3. Cloning and characterization of glutamate receptors in Californian sea lions (Zalophus californianus).

    PubMed

    Gill, Santokh; Goldstein, Tracey; Situ, Donna; Zabka, Tanja S; Gulland, Frances M D; Mueller, Rudi W

    2010-05-06

    Domoic acid produced by marine algae has been shown to cause acute and chronic neurologic sequelae in Californian sea lions following acute or low-dose exposure. Histological findings in affected animals included a degenerative cardiomyopathy that was hypothesized to be caused by over-excitation of the glutamate receptors (GluRs) speculated to be present in the sea lion heart. Thus tissues from five sea lions without lesions associated with domoic acid toxicity and one animal with domoic acid-induced chronic neurologic sequelae and degenerative cardiomyopathy were examined for the presence of GluRs. Immunohistochemistry localized mGluR 2/3, mGluR 5, GluR 2/3 and NMDAR 1 in structures of the conducting system and blood vessels. NMDAR 1 and GluR 2/3 were the most widespread as immunoreactivity was observed within sea lion conducting system structures. PCR analysis, cloning and subsequent sequencing of the seal lion GluRs showed only 80% homology to those from rats, but more than 95% homologous to those from dogs. The cellular distribution and expression of subtypes of GluRs in the sea lion hearts suggests that exposure to domoic acid may induce cardiac damage and functional disturbances.

  4. Cloning of canine Toll-like receptor 7 gene and its expression in dog tissues.

    PubMed

    Okui, Yasuhumi; Kano, Rui; Maruyama, Haruhiko; Hasegawa, Atsuhiko

    2008-01-15

    Toll-like receptor 7 (TLR7) is activated by single strand RNA and imidazoquinoline compounds, and induces interferon production. In this study, canine TLR7 cDNA was cloned and sequenced. The full-length cDNA of canine TLR7 gene was 3419bp, encoding 1032 amino acids. The similarities of canine TLR7 with human and mouse TLR7 were 84 and 80% at the nucleotide sequence level, and 86 and 79% at amino acid sequence level, respectively. Further, the expression of TLR7 mRNA was investigated in canine normal tissues by semiquantitative RT-PCR analysis. The common expression level of TLR7 mRNA in tissues from three dogs examined was in large intestine, lung, pancreas, small intestine and skin, though the expression level in each tissue was varied among these healthy dogs. In other tissues (kidney, liver, lymph node, spleen, adrenal gland, and PBMCs), the level of TLR7 mRNA expression was different in individuals.

  5. Molecular cloning and immunologic reactivity of a novel low molecular mass antigen of Mycobacterium tuberculosis.

    PubMed

    Coler, R N; Skeiky, Y A; Vedvick, T; Bement, T; Ovendale, P; Campos-Neto, A; Alderson, M R; Reed, S G

    1998-09-01

    Polypeptide Ags present in the culture filtrate of Mycobacterium tuberculosis were purified and evaluated for their ability to stimulate PBMC from purified protein derivative (PPD)-positive healthy donors. One such Ag, which elicited strong proliferation and IFN-gamma production, was further characterized. The N-terminal amino acid sequence of this polypeptide was determined and used to design oligonucleotides for screening a recombinant M. tuberculosis genomic DNA library. The gene (Mtb 8.4) corresponding to the identified polypeptide was cloned, sequenced, and expressed in Escherichia coli. The predicted m.w. of the recombinant protein without its signal peptide was 8.4 kDa. By Southern analysis, the DNA encoding this mycobacterial protein was found in the M. tuberculosis substrains H37Rv, H37Ra, Erdman, and "C" strain, as well as in certain other mycobacterial species, including Mycobacterium avium and Mycobacterium bovis BCG (bacillus Calmette-Guerin, Pasteur). The Mtb 8.4 gene appears to be absent from the environmental mycobacterial species examined thus far, including Mycobacterium smegmatis, Mycobacterium gordonae, Mycobacterium chelonae, Mycobacterium fortuitum, and Mycobacterium scrofulaceum. Recombinant Mtb 8.4 Ag induced significant proliferation as well as production of IFN-gamma, IL-10, and TNF-alpha, but not IL-5, from human PBMC isolated from PPD-positive healthy donors. Mtb 8.4 did not stimulate PBMC from PPD-negative donors. Furthermore, immunogenicity studies in mice indicate that Mtb 8.4 elicits a Th1 cytokine profile, which is considered important for protective immunity to tuberculosis. Collectively, these results demonstrate that Mtb 8.4 is an immunodominant T cell Ag of M. tuberculosis.

  6. MOLECULAR PROBES FOR EXTRACELLULAR ADENOSINE RECEPTORS

    PubMed Central

    Jacobson, Kenneth A.; Ukena, Dieter; Padgett, William; Kirk, Kenneth L.; Daly, John W.

    2012-01-01

    Derivatives of adenosine receptor agonists (N6-phenyladenosines) and antagonists (1,3-dialkyl-8-phenylxanthines) bearing functionalized chains suitable for attachment to other molecules have been reported [Jacobson et al., J. med. Chem. 28, 1334 and 1341 (1985)]. The “functionalized congener” approach has been extended to the synthesis of spectroscopic and other probes for adenosine receptors that retain high affinity (Ki ~ 10−9 −10−8 M) in A1-receptor binding. The probes have been synthesized from an antagonist xanthine amine congener (XAC) and an adenosine amine congener (ADAC). [3H]ADAC has been synthesized and found to bind highly specifically to A1-adenosine receptors of rat and calf cerebral cortical membranes with KD values of 1.4 and 0.34 nM respectively. The higher affinity in the bovine brain, seen also with many of the probes derived from ADAC and XAC, is associated with phenyl substituents. The spectroscopic probes contain a reporter group attached at a distal site of the functionalized chain. These bifunctional ligands may contain a spin label (e.g. the nitroxyl radical TEMPO) for electron spin resonance spectroscopy, or a fluorescent dye, including fluorescein and 4-nitrobenz-2-oxa-1,3-diazole (NBD), or labels for 19F nuclear magnetic resonance spectroscopy. Potential applications of the spectroscopic probes in characterization of adenosine receptors are discussed. PMID:3036153

  7. Molecular cloning, characterization, and expression of a chitinase from the entomopathogenic fungus Paecilomyces javanicus.

    PubMed

    Chen, Chien-Cheng; Kumar, H G Ashok; Kumar, Senthil; Tzean, Shean-Shong; Yeh, Kai-Wun

    2007-07-01

    Paecilomyces javanicus is an entomopathogenic fungus of coleopteran and lepidopteran insects. Here we report on cloning, characterization, and expression patterns of a chitinase from P. javanicus. A strong chitinase activity was detected in P. javanicus cultures added to chitin. The full-length cDNA, designated PjChi-1, was cloned from mycelia by using both degenerate primer/reverse transcription polymerase chain reaction (RT-PCR) amplification and 5'-/3'-RACE extension. The 1.18-kb cDNA gene contains a 1035-bp open reading frame and encodes a 345-amino acid polypeptide with a deduced molecular mass of 37 kDa. A conserved motif for chitinase activity -F82DGIDIDWE90- was present in deduced amino acid sequence. Both RT-PCR and Northern analysis revealed that the expression of the PjChi gene was constitutive at low level, but enhanced to high level when chitin was the substrate. Fungal inhibitory assay showed that PjChi-1 inhibited the growth of phytopathogenic fungi such as Sclerotium rolfsii, Colletotrichum gloeosporioides, Aspergillus nidulans, and Rhizoctonia solani.

  8. Molecular cloning and expression of the Leishmania tropica KMP-11 gene.

    PubMed

    Meriee, Mouayad; Soukkarieh, Chadi; Abbady, Abdul Qader A

    2014-08-01

    Kinetoplastid membrane protein-11 (KMP-11) is a small protein of 11 kDa present in all kinetoplastid protozoa studded so far. This protein which is highly expressed in all stages of the Leishmania life cycle is considered a potential candidate for a leishmaniasis vaccine against many leishmania species. KMP-11 has been recently described in Leishmania tropica. In the present study, the KMP-11 gene was extracted from L. tropica by PCR using two oligonucleotide primers designed to amplify the entire coding region of this gene. Then, the purified PCR products were successfully ligated into a high expression vector the pRSET-GFP. This expression vector provides the opportunity to clone the desired insert as a fusion protein with a GFP and a tag, polyhistidine region. The GFP use as a carrier to improve immune response and the polyhistidine tag facilitates detection of the expressed protein with anti-His antibodies and also purification of the protein using affinity purification. After wards KMP-11 coding region was sequenced and the recombinant protein was induced and purified from Escherichia coli cultures. The results of the present study will increase our knowledge about molecular cloning and expression of the L. tropica KMP-11 gene, and this may be used as an effective target for controlling cutenous leishmaniasis.

  9. Molecular cloning and functional characterization of the human endogenous retrovirus K113.

    PubMed

    Beimforde, Nadine; Hanke, Kirsten; Ammar, Ismahen; Kurth, Reinhard; Bannert, Norbert

    2008-02-05

    The human endogenous retrovirus-K113 (HERV-K113) is the most complete HERV known to date. It contains open reading frames for all viral proteins. Depending on ethnicity, up to 30% of the human population carries the provirus on chromosome 19. To facilitate molecular and functional studies, we have cloned the HERV-K113 sequence into a small plasmid vector and characterized its functional properties. Here we show that based on a substantial LTR-promoter activity, full length messenger RNA and spliced env-, rec- and 1.5 kb (hel)-transcripts are produced. The envelope protein of HERV-K113 is synthesized as an 85 kDa precursor that is found partially processed. The accessory Rec protein is highly expressed and accumulates in the nucleus. Expression analysis revealed synthesis of the Gag precursor and the protease. However, the cloned HERV-K113 provirus is not replication competent. It carries inactivating mutations in the reverse transcriptase gene. These mutations can be reversed to reconstitute the active enzyme, but the reversion is not sufficient to reconstitute replication capacity of the virus.

  10. Molecular cloning, overexpression, purification, and sequence analysis of the giant panda (Ailuropoda melanoleuca) ferritin light polypeptide.

    PubMed

    Fu, L; Hou, Y L; Ding, X; Du, Y J; Zhu, H Q; Zhang, N; Hou, W R

    2016-08-30

    The complementary DNA (cDNA) of the giant panda (Ailuropoda melanoleuca) ferritin light polypeptide (FTL) gene was successfully cloned using reverse transcription-polymerase chain reaction technology. We constructed a recombinant expression vector containing FTL cDNA and overexpressed it in Escherichia coli using pET28a plasmids. The expressed protein was then purified by nickel chelate affinity chromatography. The cloned cDNA fragment was 580 bp long and contained an open reading frame of 525 bp. The deduced protein sequence was composed of 175 amino acids and had an estimated molecular weight of 19.90 kDa, with an isoelectric point of 5.53. Topology prediction revealed one N-glycosylation site, two casein kinase II phosphorylation sites, one N-myristoylation site, two protein kinase C phosphorylation sites, and one cell attachment sequence. Alignment indicated that the nucleotide and deduced amino acid sequences are highly conserved across several mammals, including Homo sapiens, Cavia porcellus, Equus caballus, and Felis catus, among others. The FTL gene was readily expressed in E. coli, which gave rise to the accumulation of a polypeptide of the expected size (25.50 kDa, including an N-terminal polyhistidine tag).

  11. Molecular cloning of pepsinogens A and C from adult newt (Cynops pyrrhogaster) stomach.

    PubMed

    Inokuchi, Tomofumi; Ikuzawa, Masayuki; Yamazaki, Shin; Watanabe, Yukari; Shiota, Koushiro; Katoh, Takuma; Kobayashi, Ken-Ichiro

    2013-08-01

    The full-length cDNAs of three pepsinogens (Pgs) were cloned from the stomach of newt, Cynops pyrrhogaster, and nucleotide sequences of the full-length cDNAs were determined. Molecular phylogenetic analysis showed that two Pgs, named PgC1 and PgC2, belong to the pepsinogen C group, and one Pg, named PgA, belongs to the pepsinogen A group. The sequences contain an open reading frame (ORF) encoding 385 amino acid residues for PgC1, 383 amino acid residues for PgC2 and 377 amino acid residues for PgA. In addition, all of the three amino acid sequences conserve some unique characteristics such as six cysteine residues and putative active site two aspartic acid residues. All of the pepsinogen mRNAs were detected in the stomach by RT-PCR but not in other organs. Although a slight difference at the time of the start of expression was seen among the three pepsinogen genes, all of them were expressed in the larval stage after hatching. This is the first report on cloning of pepsinogens from urodele stomach. Copyright © 2013 Elsevier Inc. All rights reserved.

  12. Dipeptidyl peptidase III is a zinc metallo-exopeptidase. Molecular cloning and expression.

    PubMed

    Fukasawa, K; Fukasawa, K M; Kanai, M; Fujii, S; Hirose, J; Harada, M

    1998-01-15

    We have purified dipeptidyl peptidase III (EC 3.4.14.4) from human placenta. It had a pH optimum of 8.8 and readily hydrolysed Arg-Arg-beta-naphthylamide. Monoamino acid-, Gly-Phe-, Gly-Pro- and Bz-Arg-beta-naphthylamides were not hydrolysed at all. The enzyme was inhibited by p-chloromercuriphenylsulphonic acid, metal chelators and 3,4-dichloroisocoumarin and contained 1 mol of zinc per mol of enzyme. The zinc dissociation constant was 250 fM at pH 7. 4 as determined by the zinc binding study. We isolated, by immunological screening of a Uni-ZAP XR cDNA library constructed from rat liver mRNA species, a cDNA clone with 2633 bp encoding the rat enzyme. The longest open reading frame encodes a 827-residue protein with a theoretical molecular mass of 92790 Da. Escherichia coli SOLR cells were infected with the pBluescript phagemid containing the cloned cDNA and established the overexpression of a protein that hydrolysed Arg-Arg-beta-naphthylamide. The recombinant protein was purified and the amino acid sequence of the protein was confirmed. We presumed that the putative zinc-binding domain involved in catalysis was present in the recombinant enzyme. It was a novel zinc-binding motif in that one amino acid residue was inserted into the conserved HEXXH motif characteristic of the metalloproteinases.

  13. Molecular cloning and functional characterization of borneol dehydrogenase from the glandular trichomes of Lavandula x intermedia.

    PubMed

    Sarker, Lukman S; Galata, Mariana; Demissie, Zerihun A; Mahmoud, Soheil S

    2012-12-15

    Several varieties of Lavandula x intermedia (lavandins) are cultivated for their essential oils (EOs) for use in cosmetic, hygiene and personal care products. These EOs are mainly constituted of monoterpenes including camphor, which contributes an off odor reducing the olfactory appeal of the oil. We have recently constructed a cDNA library from the glandular trichomes (the sites of EO synthesis) of L. x intermedia plants. Here, we describe the cloning of a borneol dehydrogenase cDNA (LiBDH) from this library. The 780 bp open reading frame of the cDNA encoded a 259 amino acid short chain alcohol dehydrogenase with a predicted molecular mass of ca. 27.5 kDa. The recombinant LiBDH was expressed in Escherichia coli, purified by Ni-NTA agarose affinity chromatography, and functionally characterized in vitro. The bacterially produced enzyme specifically converted borneol to camphor as the only product with K(m) and k(cat) values of 53 μM and 4.0 × 10(-4) s(-1), respectively. The LiBDH transcripts were specifically expressed in glandular trichomes of mature flowers indicating that like other Lavandula monoterpene synthases the expression of this gene is regulated in a tissue-specific manner. The cloning of LiBDH has far reaching implications in improving the quality of Lavandula EOs through metabolic engineering.

  14. Molecular cloning and heterologous expression of progesterone 5beta-reductase from Digitalis lanata Ehrh.

    PubMed

    Herl, Vanessa; Fischer, Gabriele; Müller-Uri, Frieder; Kreis, Wolfgang

    2006-02-01

    A full-length cDNA clone that encodes progesterone 5beta-reductase (5beta-POR) was isolated from Digitalis lanata leaves. The reading frame of the 5beta-POR gene is 1170 nucleotides corresponding to 389 amino acids. For expression, a Sph1/Sal1 5beta-POR fragment was cloned into the pQE vector and was transformed into Escherichia coli strain M15[pREP4]. The recombinant gene was functionally expressed and the recombinant enzyme was characterized. The K(m) and v(max) values for the putative natural substrate progesterone were calculated to be 0.120 mM and 45 nkat mg(-1) protein, respectively. Only 5beta-pregnane-3,20-dione but not its alpha-isomer was formed when progesterone was used as the substrate. Kinetic constants for cortisol, cortexone, 4-androstene-3,17-dione and NADPH were also determined. The molecular organization of the 5beta-POR gene in D. lanata was determined by Southern blot analysis. The 5beta-POR is highly conserved within the genus Digitalis and the respective genes and proteins share considerable homology to putative progesterone reductases from other plant species.

  15. Molecular cloning and sequence analysis of a novel chalcone synthase cDNA from Ginkgo biloba.

    PubMed

    Pang, Yongzhen; Shen, Guo-An; Liu, Chenghong; Liu, Xiaojun; Tan, Feng; Sun, Xiaofen; Tang, Kexuan

    2004-08-01

    A chalcone synthase (CHS) gene was cloned from Ginkgo biloba for the first time and it was also the first cloned gene involved in flavonoids metabolic pathway in G. biloba. The full-length cDNA of G. biloba CHS (designated as Gbchs) was 1608bp with poly(A) tailing and it contained a 1173bp open reading frame (ORF) encoding a 391 amino acid protein. Gbchs was found to have extensive homology with those of other plant chs genes via multiple alignments. The active sites of the CoA binding, coumaroyl pocket and cyclization pocket in CHS protein of Medicago sativa were also found in GbCHS. Molecular modeling of GbCHS indicated that the three-dimensional structure of GbCHS strongly resembled that of M. sativa (MsCHS2), implying GbCHS may have similar functions with MsCHS2. Phylogenetic tree analysis revealed that GbCHS had closer relationship with CHSs from gymnosperm plants than from other plants. Gbchs is a useful tool to study the regulation of flavonoids metabolism in G. biloba.

  16. Dipeptidyl peptidase III is a zinc metallo-exopeptidase. Molecular cloning and expression.

    PubMed Central

    Fukasawa, K; Fukasawa, K M; Kanai, M; Fujii, S; Hirose, J; Harada, M

    1998-01-01

    We have purified dipeptidyl peptidase III (EC 3.4.14.4) from human placenta. It had a pH optimum of 8.8 and readily hydrolysed Arg-Arg-beta-naphthylamide. Monoamino acid-, Gly-Phe-, Gly-Pro- and Bz-Arg-beta-naphthylamides were not hydrolysed at all. The enzyme was inhibited by p-chloromercuriphenylsulphonic acid, metal chelators and 3,4-dichloroisocoumarin and contained 1 mol of zinc per mol of enzyme. The zinc dissociation constant was 250 fM at pH 7. 4 as determined by the zinc binding study. We isolated, by immunological screening of a Uni-ZAP XR cDNA library constructed from rat liver mRNA species, a cDNA clone with 2633 bp encoding the rat enzyme. The longest open reading frame encodes a 827-residue protein with a theoretical molecular mass of 92790 Da. Escherichia coli SOLR cells were infected with the pBluescript phagemid containing the cloned cDNA and established the overexpression of a protein that hydrolysed Arg-Arg-beta-naphthylamide. The recombinant protein was purified and the amino acid sequence of the protein was confirmed. We presumed that the putative zinc-binding domain involved in catalysis was present in the recombinant enzyme. It was a novel zinc-binding motif in that one amino acid residue was inserted into the conserved HEXXH motif characteristic of the metalloproteinases. PMID:9425109

  17. Cloning and molecular characterization of a putative voltage-gated sodium channel gene in the crayfish.

    PubMed

    Coskun, Cagil; Purali, Nuhan

    2016-06-01

    Voltage-gated sodium channel genes and associated proteins have been cloned and studied in many mammalian and invertebrate species. However, there is no data available about the sodium channel gene(s) in the crayfish, although the animal has frequently been used as a model to investigate various aspects of neural cellular and circuit function. In the present work, by using RNA extracts from crayfish abdominal ganglia samples, the complete open reading frame of a putative sodium channel gene has firstly been cloned and molecular properties of the associated peptide have been analyzed. The open reading frame of the gene has a length of 5793 bp that encodes for the synthesis of a peptide, with 1930 amino acids, that is 82% similar to the α-peptide of a sodium channel in a neighboring species, Cancer borealis. The transmembrane topology analysis of the crayfish peptide indicated a pattern of four folding domains with several transmembrane segments, as observed in other known voltage-gated sodium channels. Upon analysis of the obtained sequence, functional regions of the putative sodium channel responsible for the selectivity filter, inactivation gate, voltage sensor, and phosphorylation have been predicted. The expression level of the putative sodium channel gene, as defined by a qPCR method, was measured and found to be the highest in nervous tissue.

  18. Molecular cloning of rat homologues of the Drosophila melanogaster dunce cAMP phosphodiesterase: evidence for a family of genes.

    PubMed Central

    Swinnen, J V; Joseph, D R; Conti, M

    1989-01-01

    To study the structure and function of cyclic nucleotide phosphodiesterases (PDEs) involved in mammalian gametogenesis, a rat testis cDNA library was screened at low stringency with a cDNA clone coding for the Drosophila melanogaster dunce-encoded PDE as a probe. This screening resulted in the isolation of two groups of cDNA clones, differing in their nucleotide sequences (ratPDE1 and ratPDE2). In the rat testis, RNA transcripts corresponding to both groups of clones were expressed predominantly in germ cells. Additional screenings of a Sertoli cell cDNA library with a ratPDE2 clone as a probe led to the isolation of two more groups of clones (rat-PDE3 and ratPDE4). Unlike ratPDE1 and ratPDE2, these clones hybridized to transcripts present predominantly in the Sertoli cell. In the middle of the coding region, all four groups of clones were homologous to each other. The deduced amino acid sequences of part of this region were also homologous to the D. melanogaster dunce PDE and to PDEs from bovine and yeast. These data indicate that a family of genes homologous to the D. melanogaster dunce-encoded PDE is present in the rat and that these genes are differentially expressed in somatic and germ cells of the seminiferous tubule. These findings provide a molecular basis for the observed heterogeneity of cAMP PDEs. Images PMID:2546153

  19. Kainate receptor trafficking: physiological roles and molecular mechanisms.

    PubMed

    Isaac, John T R; Mellor, Jack; Hurtado, David; Roche, Katherine W

    2004-12-01

    Recently, there has been intense interest in the mechanisms regulating the trafficking and synaptic targeting of kainate receptors in neurons. This topic is still in its infancy when compared with studies of trafficking of other ionotropic glutamate receptors; however, it is already clear that mechanisms exist for subunit- and splice variant-specific trafficking of kainate receptors. There is also enormous diversity of kainate receptor targeting, with the best-studied neurons in this regard being hippocampal CA3 pyramidal neurons and CA1 GABAergic interneurons. This review summarizes the current state of knowledge on this topic, focusing on the molecular mechanisms of kainate receptor trafficking and the potential for these mechanisms to regulate neuronal kainate receptor function.

  20. Molecular cloning and functional characterization of duck nucleotide-binding oligomerization domain 1 (NOD1).

    PubMed

    Li, Huilin; Jin, Hui; Li, Yaqian; Liu, Dejian; Foda, Mohamed Frahat; Jiang, Yunbo; Luo, Rui

    2017-09-01

    Nucleotide-binding oligomerization domain 1 (NOD1) is an imperative cytoplasmic pattern recognition receptor (PRR) and considered as a key member of the NOD-like receptor (NLR) family which plays a critical role in innate immunity through sensing microbial components derived from bacterial peptidoglycan. In the current study, the full-length of duck NOD1 (duNOD1) cDNA from duck embryo fibroblasts (DEFs) was cloned. Multiple sequence alignment and phylogenetic analysis demonstrated that duNOD1 exhibited a strong evolutionary relationship with chicken and rock pigeon NOD1. Tissue-specific expression analysis showed that duNOD1 was widely distributed in various organs, with the highest expression observed in the liver. Furthermore, duNOD1 overexpression induced NF-κB activation in DEFs and the CARD domain is crucial for duNOD1-mediated NF-κB activation. In addition, silencing the duNOD1 decreased the activity of NF-κB in DEFs stimulated by iE-DAP. Overexpression of duNOD1 significantly increased the expression of TNF-α, IL-6, and RANTES in DEFs. These findings highlight the crucial role of duNOD1 as an intracellular sensor in duck innate immune system. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. Receptor-mediated signalling in plants: molecular patterns and programmes

    PubMed Central

    Tör, Mahmut; Lotze, Michael T.; Holton, Nicholas

    2009-01-01

    A highly evolved surveillance system in plants is able to detect a broad range of signals originating from pathogens, damaged tissues, or altered developmental processes, initiating sophisticated molecular mechanisms that result in defence, wound healing, and development. Microbe-associated molecular pattern molecules (MAMPs), damage-associated molecular pattern molecules (DAMPs), virulence factors, secreted proteins, and processed peptides can be recognized directly or indirectly by this surveillance system. Nucleotide binding-leucine rich repeat proteins (NB-LRR) are intracellular receptors and have been targeted by breeders for decades to elicit resistance to crop pathogens in the field. Receptor-like kinases (RLKs) or receptor like proteins (RLPs) are membrane bound signalling molecules with an extracellular receptor domain. They provide an early warning system for the presence of potential pathogens and activate protective immune signalling in plants. In addition, they act as a signal amplifier in the case of tissue damage, establishing symbiotic relationships and effecting developmental processes. The identification of several important ligands for the RLK-type receptors provided an opportunity to understand how plants differentiate, how they distinguish beneficial and detrimental stimuli, and how they co-ordinate the role of various types of receptors under varying environmental conditions. The diverse roles of extra-and intracellular plant receptors are examined here and the recent findings on how they promote defence and development is reviewed. PMID:19628572

  2. Molecular Cloning and Characterisation of Farnesyl Pyrophosphate Synthase from Tripterygium wilfordii

    PubMed Central

    Zhao, Yu-Jun; Chen, Xin; Zhang, Meng; Su, Ping; Liu, Yu-Jia; Tong, Yu-Ru; Wang, Xiu-Juan; Huang, Lu-Qi; Gao, Wei

    2015-01-01

    Farnesylpyrophosphate synthase (FPS) catalyzes the biosynthesis of farnesyl pyrophosphate (FPP), which is an important precursor of sesquiterpenoids such as artemisinin and wilfordine. In the present study, we report the molecular cloning and characterization of two full-length cDNAs encoding FPSs from Tripterygium wilfordii (TwFPSs). TwFPSs maintained their capability to synthesise FPP in vitro when purified as recombinant proteins from E. coli. Consistent with the endogenous role of FPS in FPP biosynthesis, TwFPSs were highly expressed in T. wilfordii roots, and were up-regulated upon methyl jasmonate (MeJA) treatment. The global gene expression profiles suggested that the TwFPSs might play an important regulatory role interpenoid biosynthesis in T. wilfordii, laying the groundwork for the future study of the synthetic biology of natural terpene products. PMID:25938487

  3. Construction and Rescue of a Molecular Clone of Deformed Wing Virus (DWV)

    PubMed Central

    Lamp, Benjamin; Url, Angelika; Seitz, Kerstin; Eichhorn, Jürgen; Riedel, Christiane; Sinn, Leonie Janina; Indik, Stanislav; Köglberger, Hemma; Rümenapf, Till

    2016-01-01

    European honey bees are highly important in crop pollination, increasing the value of global agricultural production by billions of dollars. Current knowledge about virulence and pathogenicity of Deformed wing virus (DWV), a major factor in honey bee colony mortality, is limited. With this study, we close the gap between field research and laboratory investigations by establishing a complete in vitro model for DWV pathogenesis. Infectious DWV was rescued from a molecular clone of a DWV-A genome that induces DWV symptoms such as crippled wings and discoloration. The expression of DWV proteins, production of infectious virus progeny, and DWV host cell tropism could be confirmed using newly generated anti-DWV monoclonal antibodies. The recombinant RNA fulfills Koch’s postulates circumventing the need of virus isolation and propagation of pure virus cultures. In conclusion, we describe the development and application of a reverse genetics system for the study of DWV pathogenesis. PMID:27828961

  4. Molecular cloning and characterization of a novel mannose-binding lectin gene from Amorphophallus konjac.

    PubMed

    Fei, Jiong; Liao, Zhihua; Chai, Yourong; Pang, Yongzhen; Yao, Jianhong; Sun, Xiaofen; Tang, Kexuan

    2003-09-01

    A new lectin gene was cloned from Amorphophallus konjac. The full-length cDNA of Amorphophallus konjac agglutinin (aka) was 736 bp and contained a 474 bp open reading frame encoding a 158 amino acid protein. Homology analysis revealed that the lectin from this Araceae species belonged to the superfamily of monocot mannose-binding proteins. Molecular modeling of AKA indicated that the three-dimensional structure of AKA strongly resembles that of the snowdrop lectin. Southern blot analysis of the genomic DNA revealed that aka belonged to a low-copy gene family. Northern blot analysis demonstrated that aka expression was tissue-specific with the strongest expression being found in root.

  5. Molecular cloning of α-2-macroglobulin from hemocytes of common periwinkle Littorina littorea.

    PubMed

    Borisova, Elena A; Gorbushin, Alexander M

    2014-08-01

    We report the sequence of the proteinase inhibitor with a wide inhibition spectrum, α-2-macroglobulin (α2M), belonging to the thioester superfamily of proteins. This is the first α2M sequence from coenogastropod prosobranch snails. The full-length cDNA was cloned by RACE method, spans 7897 bp and contains an open reading frame of 5460 bp. The ORF encodes a protein of 1819 amino acids. The deduced mature protein contains 1795 amino acids with a molecular weight of 200 kDa and isoelectric point of 5.00. Littorina littorea α2M bears 4 conserved α2M domains and one internal thioester. Phylogenetic analysis showed that the sequence forms well supported cluster with Mollusca species and other representatives of Lophotrochozoa.

  6. Construction and Rescue of a Molecular Clone of Deformed Wing Virus (DWV).

    PubMed

    Lamp, Benjamin; Url, Angelika; Seitz, Kerstin; Eichhorn, Jürgen; Riedel, Christiane; Sinn, Leonie Janina; Indik, Stanislav; Köglberger, Hemma; Rümenapf, Till

    2016-01-01

    European honey bees are highly important in crop pollination, increasing the value of global agricultural production by billions of dollars. Current knowledge about virulence and pathogenicity of Deformed wing virus (DWV), a major factor in honey bee colony mortality, is limited. With this study, we close the gap between field research and laboratory investigations by establishing a complete in vitro model for DWV pathogenesis. Infectious DWV was rescued from a molecular clone of a DWV-A genome that induces DWV symptoms such as crippled wings and discoloration. The expression of DWV proteins, production of infectious virus progeny, and DWV host cell tropism could be confirmed using newly generated anti-DWV monoclonal antibodies. The recombinant RNA fulfills Koch's postulates circumventing the need of virus isolation and propagation of pure virus cultures. In conclusion, we describe the development and application of a reverse genetics system for the study of DWV pathogenesis.

  7. Molecular cloning of an Onchocerca volvulus extracellular Cu-Zn superoxide dismutase.

    PubMed Central

    James, E R; McLean, D C; Perler, F

    1994-01-01

    Onchocerca volvulus, a human parasitic nematode, is the third leading cause of preventable blindness worldwide. This study describes the molecular cloning of a novel superoxide dismutase (SOD) from the parasite. This putative O. volvulus extracellular SOD (OvEcSOD) is 628 nucleotides (nt) long, including a 22-nt 5' spliced leader (SL1) and a portion encoding an N-terminal hydrophobic 42-amino-acid signal peptide. The remainder of the cDNA shares 71% identity with an O. volvulus cytosolic SOD sequence and is 3 nt longer. All residues involved in metal ion binding, active site formation, folding, and dimer formation in SODs are conserved. Data indicate the OvEcSOD and O. volvulus cytosolic SOD are separate gene products and that the OvEcSOD appears to possess the characteristics of a membrane-bound or secreted enzyme which may be involved in the parasite defense against phagocyte-generated reactive oxygen species. Images PMID:8300230

  8. Cloning and molecular characterization of hrpX from Xanthomonas axonopodis pv. citri.

    PubMed

    Iwamoto, M; Oku, T

    2000-01-01

    The hrpX gene of plant pathogenic Xanthomonas species is essential for pathogenicity on host plants and to cause hypersensitive reaction on non-host plants. We cloned and analyzed a hrpX homologue, designated hrpXct, of X. axonopodis pv. citri, a pathogen of citrus canker. The open reading frame of hrpXct has 1431 bp in nucleotides which has a coding capacity of 476 amino acid residues with a molecular mass of 52.4 kDa. The predicted amino acid sequence of HrpXct has 90% identity to the AraC family type transcriptional activator protein HrpXc of X. campestris pv. campestris, 95% to HrpXo of X. oryzae pv. oryzae and 97% to X. vesicatoria. These findings clearly indicate and confirm that the structure of the hrpX genes in plant pathogenic Xanthomonas species is highly conserved.

  9. Molecular cloning of canine Wilms' tumor 1 for immunohistochemical analysis in canine tissues.

    PubMed

    Sakai, Osamu; Sakurai, Masashi; Sakai, Hiroki; Kubo, Masahito; Hiraoka, Hiroko; Baba, Kenji; Okuda, Masaru; Mizuno, Takuya

    2017-07-28

    Wilms' tumor 1 (WT1) expression has been investigated in various human cancers as a target molecule for cancer immunotherapy. However, few studies have focused on WT1 expression in dogs. Firstly, cDNA of canine WT1 (cWT1) was molecularly cloned from normal canine kidney. The cross-reactivity of the anti-human WT1 monoclonal antibody (6F-H2) with cWT1 was confirmed via Western blotting using cells overexpressing cWT1. Immunohistochemical staining revealed that cWT1 expression was detected in all canine lymphoma tissues and in some normal canine tissues, including the kidney and lymph node. cWT1 is a potential immunotherapy target against canine cancers.

  10. Molecular cloning of canine interleukin-31 and its expression in various tissues.

    PubMed

    Mizuno, Takuya; Kanbayashi, Satoshi; Okawa, Takumi; Maeda, Sadatoshi; Okuda, Masaru

    2009-09-15

    The newly discovered cytokine, interleukin-31 (IL-31), belongs to the short-chain cytokine group. It was reported that transgenic expression of IL-31-induced pruritus, similar to atopic dermatitis, in mice, further, excessive amounts of IL-31 was also expressed in the skin from human patients with atopic dermatitis as compared to that from normal people. In this study, canine IL-31 was molecularly cloned from concanavalin A-stimulated canine peripheral blood mononuclear cells (PBMCs), and its nucleotide sequence was determined. Canine IL-31 contains 4 alpha-helix structures characteristic of the IL-31 family, and the amino acid identity of canine IL-31 with those of human or mouse is 54% and 28%, respectively. Furthermore, we detected low levels of canine IL-31 in the thymus, testis, spleen, and kidneys, but not in the skin of atopic dogs.

  11. Molecular Cloning and Characterisation of Farnesyl Pyrophosphate Synthase from Tripterygium wilfordii.

    PubMed

    Zhao, Yu-Jun; Chen, Xin; Zhang, Meng; Su, Ping; Liu, Yu-Jia; Tong, Yu-Ru; Wang, Xiu-Juan; Huang, Lu-Qi; Gao, Wei

    2015-01-01

    Farnesylpyrophosphate synthase (FPS) catalyzes the biosynthesis of farnesyl pyrophosphate (FPP), which is an important precursor of sesquiterpenoids such as artemisinin and wilfordine. In the present study, we report the molecular cloning and characterization of two full-length cDNAs encoding FPSs from Tripterygium wilfordii (TwFPSs). TwFPSs maintained their capability to synthesise FPP in vitro when purified as recombinant proteins from E. coli. Consistent with the endogenous role of FPS in FPP biosynthesis, TwFPSs were highly expressed in T. wilfordii roots, and were up-regulated upon methyl jasmonate (MeJA) treatment. The global gene expression profiles suggested that the TwFPSs might play an important regulatory role interpenoid biosynthesis in T. wilfordii, laying the groundwork for the future study of the synthetic biology of natural terpene products.

  12. Characterization and molecular cloning of a serine hydroxymethyltransferase 1 (OsSHM1) in rice.

    PubMed

    Wang, Dekai; Liu, Heqin; Li, Sujuan; Zhai, Guowei; Shao, Jianfeng; Tao, Yuezhi

    2015-09-01

    Serine hydroxymethyltransferase (SHMT) is important for one carbon metabolism and photorespiration in higher plants for its participation in plant growth and development, and resistance to biotic and abiotic stresses. A rice serine hydroxymethyltransferase gene, OsSHM1, an ortholog of Arabidopsis SHM1, was isolated using map-based cloning. The osshm1 mutant had chlorotic lesions and a considerably smaller, lethal phenotype under natural ambient CO2 concentrations, but could be restored to wild type with normal growth under elevated CO2 levels (0.5% CO2 ), showing a typical photorespiratory phenotype. The data from antioxidant enzymes activity measurement suggested that osshm1 was subjected to significant oxidative stress. Also, OsSHM1 was expressed in all organs tested (root, culm, leaf, and young panicle) but predominantly in leaves. OsSHM1 protein is localized to the mitochondria. Our study suggested that molecular function of the OsSHM1 gene is conserved in rice and Arabidopsis.

  13. Cloning of the. gamma. -aminobutyric acid (GABA). rho. sub 1 cDNA: A GABA receptor subunit highly expressed in the retina

    SciTech Connect

    Cutting, G.R.; Lu, Luo; Kasch, L.M.; Montrose-Rafizadeh, C.; Antonarakis, S.E.; Guggino, W.B.; Kazazian, H.H. Jr. ); O'Hara, B.F.; Donovan, D.M.; Shimada, Shoichi ); Uhl, G.R. Johns Hopkins Univ. School of Medicine, Baltimore, MD )

    1991-04-01

    Type A {gamma}-aminobutyric acid (GABA{sub A}) receptors are a family of ligand-gated chloride channels that are the major inhibitory neurotransmitter receptors in the nervous system. Molecular cloning has revealed diversity in the subunits that compose this heterooligomeric receptor, but each previously elucidated subunit displays amino acid similarity in conserved structural elements. The authors have used these highly conserved regions to identify additional members of this family by using the polymerase chain reaction (PCR). One PCR product was used to isolate a full-length cDNA from a human retina cDNA library. The mature protein predicted from this cDNA sequence is 458 amino acids long and displays between 30 and 38% amino acid similarity to the previously identified GABA{sub A} subunits. This gene is expressed primarily in the retina but transcripts are also detected in the brain, lung, and thymus. Injection of Xenopus oocytes with RNA transcribed in vitro produces a GABA-responsive chloride conductance and expression of the cDNA in COS cells yields GABA-displaceable muscimol binding. These features are consistent with our identification of a GABA subunit, GABA {rho}{sub 1}, with prominent retinal expression that increases the diversity and tissue specificity of this ligand-gated ion-channel receptor family.

  14. Cloning of the gamma-aminobutyric acid (GABA) rho 1 cDNA: a GABA receptor subunit highly expressed in the retina.

    PubMed Central

    Cutting, G R; Lu, L; O'Hara, B F; Kasch, L M; Montrose-Rafizadeh, C; Donovan, D M; Shimada, S; Antonarakis, S E; Guggino, W B; Uhl, G R

    1991-01-01

    Type A gamma-aminobutyric acid (GABAA) receptors are a family of ligand-gated chloride channels that are the major inhibitory neurotransmitter receptors in the nervous system. Molecular cloning has revealed diversity in the subunits that compose this heterooligomeric receptor, but each previously elucidated subunit displays amino acid similarity in conserved structural elements. We have used these highly conserved regions to identify additional members of this family by using the polymerase chain reaction (PCR). One PCR product was used to isolate a full-length cDNA from a human retina cDNA library. The mature protein predicted from this cDNA sequence in 458 amino acids long and displays between 30 and 38% amino acid similarity to the previously identified GABAA subunits. This gene is expressed primarily in the retina but transcripts are also detected in the brain, lung, and thymus. Injection of Xenopus oocytes with RNA transcribed in vitro produces a GABA-responsive chloride conductance and expression of the cDNA in COS cells yields GABA-displaceable muscimol binding. These features are consistent with our identification of a GABA subunit, GABA rho 1, with prominent retinal expression that increases the diversity and tissue specificity of this ligand-gated ion-channel receptor family. Images PMID:1849271

  15. Molecular characterization of thyroid hormone receptors from the leopard gecko, and their differential expression in the skin.

    PubMed

    Kanaho, Yoh-Ichiro; Endo, Daisuke; Park, Min Kyun

    2006-06-01

    Thyroid hormones (THs) play crucial roles in various developmental and physiological processes in vertebrates, including squamate reptiles. The effect of THs on shedding frequency is interesting in Squamata, since the effects on lizards are quite the reverse of those in snakes: injection of thyroxine increases shedding frequency in lizards, but decreases it in snakes. However, the mechanism underlying this differential effect remains unclear. To facilitate the investigation of the molecular mechanism of the physiological functions of THs in Squamata, their two specific receptor (TRalpha and beta) cDNAs, which are members of the nuclear hormone receptor superfamily, were cloned from a lizard, the leopard gecko, Eublepharis macularius. This is the first molecular cloning of thyroid hormone receptors (TRs) from reptiles. The deduced amino acid sequences showed high identity with those of other species, especially in the C and E/F domains, which are characteristic domains in nuclear hormone receptors. Expression analysis revealed that TRs were widely expressed in many tissues and organs, as in other animals. To analyze their role in the skin, temporal expression analysis was performed by RT-PCR, revealing that the two TRs had opposing expression patterns: TRalpha was expressed more strongly after than before skin shedding, whereas TRbeta was expressed more strongly before than after skin shedding. This provides good evidence that THs play important roles in the skin, and that the roles of their two receptor isoforms are distinct from each other.

  16. Molecular cloning and analysis of the gene encoding the thermostable penicillin G acylase from Alcaligenes faecalis.

    PubMed Central

    Verhaert, R M; Riemens, A M; van der Laan, J M; van Duin, J; Quax, W J

    1997-01-01

    Alcaligenes faecalis penicillin G acylase is more stable than the Escherichia coli enzyme. The activity of the A. faecalis enzyme was not affected by incubation at 50 degrees C for 20 min, whereas more than 50% of the E. coli enzyme was irreversibly inactivated by the same treatment. To study the molecular basis of this higher stability, the A. faecalis enzyme was isolated and its gene was cloned and sequenced. The gene encodes a polypeptide that is characteristic of periplasmic penicillin G acylase (signal peptide-alpha subunit-spacer-beta subunit). Purification, N-terminal amino acid analysis, and molecular mass determination of the penicillin G acylase showed that the alpha and beta subunits have molecular masses of 23.0 and 62.7 kDa, respectively. The length of the spacer is 37 amino acids. Amino acid sequence alignment demonstrated significant homology with the penicillin G acylase from E. coli A unique feature of the A. faecalis enzyme is the presence of two cysteines that form a disulfide bridge. The stability of the A. faecalis penicillin G acylase, but not that of the E. coli enzyme, which has no cysteines, was decreased by a reductant. Thus, the improved thermostability is attributed to the presence of the disulfide bridge. PMID:9292993

  17. Characterization of nonprimate hepacivirus and construction of a functional molecular clone

    PubMed Central

    Scheel, Troels K. H.; Kapoor, Amit; Nishiuchi, Eiko; Brock, Kenny V.; Yu, Yingpu; Andrus, Linda; Gu, Meigang; Renshaw, Randall W.; Dubovi, Edward J.; McDonough, Sean P.; Van de Walle, Gerlinde R.; Lipkin, W. Ian; Divers, Thomas J.; Tennant, Bud C.; Rice, Charles M.

    2015-01-01

    Nonprimate hepacivirus (NPHV) is the closest known relative of hepatitis C virus (HCV) and its study could enrich our understanding of HCV evolution, immunity, and pathogenesis. High seropositivity is found in horses worldwide with ∼3% viremic. NPHV natural history and molecular virology remain largely unexplored, however. Here, we show that NPHV, like HCV, can cause persistent infection for over a decade, with high titers and negative strand RNA in the liver. NPHV is a near-universal contaminant of commercial horse sera for cell culture. The complete NPHV 3′-UTR was determined and consists of interspersed homopolymer tracts and an HCV-like 3′-terminal poly(U)-X-tail. NPHV translation is stimulated by miR-122 and the 3′-UTR and, similar to HCV, the NPHV NS3-4A protease can cleave mitochondrial antiviral-signaling protein to inactivate the retinoic acid-inducible gene I pathway. Using an NPHV consensus cDNA clone, replication was not observed in primary equine fetal liver cultures or after electroporation of selectable replicons. However, intrahepatic RNA inoculation of a horse initiated infection, yielding high RNA titers in the serum and liver. Delayed seroconversion, slightly elevated circulating liver enzymes and mild hepatitis was observed, followed by viral clearance. This establishes the molecular components of a functional NPHV genome. Thus, NPHV appears to resemble HCV not only in genome structure but also in its ability to establish chronic infection with delayed seroconversion and hepatitis. This NPHV infectious clone and resulting acute phase sera will facilitate more detailed studies on the natural history, pathogenesis, and immunity of this novel hepacivirus in its natural host. PMID:25646476

  18. Molecular cloning and chromosomal localization of human holocarboxylase synthetase, a gene responsible for biotin dependency

    SciTech Connect

    Suzuki, Y.; Aoki, Y.; Ishida, Y.

    1994-09-01

    Holocarboxylase synthetase (HCS) catalyzes biotin incorporation into various carboxylases that require biotin as a prosthetic group. They are acetyl-CoA carboxylase, a rate-limiting enzyme of fatty acid synthesis; pyruvate carboxylase, a key enzyme of gluconeogenesis; propionyl-CoA carboxylase and 3-methylcrotonyl-CoA carboxylase, enzymes involved in amino acid catabolism. HCS is therefore involved in various metabolic processes and is a key enzyme for biotin utilization by mammalian cells. Deficiency of HCS in man is known to cause biotin-responsive multiple carboxylase deficiency. Isolation of cDNA clones for the enzyme is essential to understand HCS and its deficiency at the molecular level. We purified bovine liver HCS and sequenced its proteolytic peptides. Degenerative oligonucleotide primers were synthesized from the two peptide sequences and used to amplify a putative HCS cDNA fragment from human liver by PCR. Using the amplified DNA fragment as a probe, we screened {lambda}gt10 human liver cDNA library and isolated 12 positive clones. The isolated cDNAs encoded a protein of 726 amino acids with molecular mass of 80,759. The protein contained several sequences identical or similar to those of peptides derived from the bovine liver HCS. The predicted protein had a homologous region with BirA which acts as both a biotin-[acetyl-CoA-carboxylase] ligase and a biotin repressor in E. coli, suggesting a functional relationship between the two proteins. We expressed the protein using pET3 a vector in E. coli (BL21 strain) and raised antiserum against the expressed protein. The antiserum immunoprecipitated HCS activities of human lymphoblasts and bovine liver. A one-base deletion and a missense mutation were found in cells from siblings with HCS deficiency. The human HCS gene was assigned to chromosome 21, region 21q22.1 by fluorescence in situ hybridization analysis.

  19. Expression and molecular cloning of interferon stimulated genes in buffalo (Bubalus bubalis).

    PubMed

    Thakur, Nipuna; Singh, Girjesh; Paul, A; Bharati, J; Rajesh, G; Gm, Vidyalakshmi; Chouhan, V S; Bhure, S K; Maurya, V P; Singh, G; Sarkar, M

    2017-09-15

    Buffalo, the most important livestock species in tropical India, remains to be a poor breeder mainly due to embryonic mortality (65%) occurring mostly between 16 and 18 days of pregnancy. Early and accurate diagnosis of pregnancy can thus become a boon for successful herd management in buffalo. However, most of the currently available methods allow diagnosis only after 30 days post AI. Interferon tau (IFNT), the first pregnancy recognition signal in ruminants is one such molecule, which stimulates expression of various Interferon stimulated genes (ISGs) in the peripheral blood mononuclear cells (PBMC's) concomitant with IFNT signaling which occurs around maternal recognition of pregnancy (MRP). Hence, the study was planned to demonstrate the expression dynamics of ISGs (OAS1, MX1, MX2 and ISG15) in PBMCs during peri-implantation period in buffalo and also molecular cloning and expression of suitable ISG coded protein (s) in suitable host. Blood was collected from two groups of multiparous buffaloes: Group1: (n = 10) inseminated/pregnant (Experimental) and Group2: (n = 10) anestrous/non pregnant (Control). The expression profile of ISGs was then analyzed using real time qPCR. Expression profile of most ISGs was observed to increase through day 14 to day 20 post AI and declined thereafter. On the basis of differential gene expression at day 18 post AI, OAS1 and MX2 were identified as suitable ISG candidate biomarkers for accurate pregnancy diagnosis within 18 days post AI. Molecular cloning and expression of selected ISGs in a suitable prokaryotic expression vector was done thereafter. Bulk expression of the recombinant proteins was done and purified by affinity chromatography and confirmed by Western blot using Mouse Monoclonal His-probe antibodies. To conclude, as OAS1 and MX2, showed distinct differential expression at day 18 post AI, they may serve as ideal biomarkers for detection of early pregnancy in buffalo. Copyright © 2017 Elsevier Inc. All rights

  20. Supernatant from a cloned helper T cell stimulates resting B cells to express transferrin and IL-2 receptors.

    PubMed

    Diu, A; Leclercq, L; Dautry-Varsat, A; Theze, J

    1987-07-01

    We describe the properties of the supernatant from a murine cloned helper T cell (clone 52.3) which is able to polyclonally activate most resting B cells in the absence of any additional stimulus. We hypothesize that an activity which we call BCAF (B-cell-activating factor(s] exists in our supernatant which can activate resting B cells alone or in conjunction with other lymphokines. In the present report, we investigate changes in the surface antigen pattern induced on resting B cells by BCAF-containing supernatant. Analysis of the cells by flow cytometry shows that transferrin receptor and IL-2 receptor expression increase on a large fraction of B cells after 2 days of activation by the T-helper-cell clone supernatant. Monoclonal anti-transferrin receptor antibody inhibits cell division but does not affect blastogenesis, while IL-2 has no effect in our experimental system. Our present results confirm that BCAF-containing supernatants can act on most resting B cells and replace helper T cells in inducing B-cell activation and proliferation.

  1. Snake acetylcholine receptor: cloning of the domain containing the four extracellular cysteines of the alpha subunit.

    PubMed Central

    Neumann, D; Barchan, D; Horowitz, M; Kochva, E; Fuchs, S

    1989-01-01

    The acetylcholine receptor (AcChoR) at the neuromuscular junction of elapid snakes binds cholinergic ligands but unlike other muscle AcChoRs does not bind alpha-bungarotoxin. Numerous studies indicate that the ligand-binding site of the AcChoR includes cysteine residues at positions 192 and 193 of the alpha subunit. We have previously shown that a synthetic dodecapeptide corresponding to residues 185-196 of the Torpedo AcChoR alpha subunit contains the essential elements of the ligand-binding site. In an attempt to elucidate the structural basis for the precise binding properties of snake AcChoR, we sequenced a portion of the snake AcChoR alpha subunit. First, a mouse AcChoR alpha-subunit cDNA probe was used to screen a size-selected snake (Natrix tessellata) genomic library. A genomic clone was isolated and was found to contain sequences homologous to the exon including the first two cysteines (Cys-128 and -142) of AcChoR alpha subunit. The domain of the alpha subunit from Natrix and cobra AcChoR (amino acid residues 119-222), which contains the four extracellular cysteines (128, 142, 192, and 193), was amplified by reverse transcription of mRNA and the polymerase chain reaction and then sequenced. The deduced amino acid sequence showed that the snake alpha subunit contains the two tandem cysteines at positions 192 and 193, resembling all other AcChoR alpha subunits. Sequence comparison revealed that the cloned region of the snake alpha subunit is highly homologous (75-80%) to other muscle AcChoRs and not to neuronal AcChoR, which also does not bind alpha-bungarotoxin. In the presumed ligand-binding site, in the vicinity of Cys-192 and Cys-193, four major substitutions occur in the snake sequence--at positions 184 (Trp----Phe), 185 (Lys----Trp), 187 (Trp----Ser), and 194 (Pro----Leu). In addition, Asn-189 is a putative N-glycosylation site, present only in the snake. These changes, or part of them, may explain the lack of alpha-bungarotoxin-binding to snake Ac

  2. Snake acetylcholine receptor: cloning of the domain containing the four extracellular cysteines of the alpha subunit.

    PubMed

    Neumann, D; Barchan, D; Horowitz, M; Kochva, E; Fuchs, S

    1989-09-01

    The acetylcholine receptor (AcChoR) at the neuromuscular junction of elapid snakes binds cholinergic ligands but unlike other muscle AcChoRs does not bind alpha-bungarotoxin. Numerous studies indicate that the ligand-binding site of the AcChoR includes cysteine residues at positions 192 and 193 of the alpha subunit. We have previously shown that a synthetic dodecapeptide corresponding to residues 185-196 of the Torpedo AcChoR alpha subunit contains the essential elements of the ligand-binding site. In an attempt to elucidate the structural basis for the precise binding properties of snake AcChoR, we sequenced a portion of the snake AcChoR alpha subunit. First, a mouse AcChoR alpha-subunit cDNA probe was used to screen a size-selected snake (Natrix tessellata) genomic library. A genomic clone was isolated and was found to contain sequences homologous to the exon including the first two cysteines (Cys-128 and -142) of AcChoR alpha subunit. The domain of the alpha subunit from Natrix and cobra AcChoR (amino acid residues 119-222), which contains the four extracellular cysteines (128, 142, 192, and 193), was amplified by reverse transcription of mRNA and the polymerase chain reaction and then sequenced. The deduced amino acid sequence showed that the snake alpha subunit contains the two tandem cysteines at positions 192 and 193, resembling all other AcChoR alpha subunits. Sequence comparison revealed that the cloned region of the snake alpha subunit is highly homologous (75-80%) to other muscle AcChoRs and not to neuronal AcChoR, which also does not bind alpha-bungarotoxin. In the presumed ligand-binding site, in the vicinity of Cys-192 and Cys-193, four major substitutions occur in the snake sequence--at positions 184 (Trp----Phe), 185 (Lys----Trp), 187 (Trp----Ser), and 194 (Pro----Leu). In addition, Asn-189 is a putative N-glycosylation site, present only in the snake. These changes, or part of them, may explain the lack of alpha-bungarotoxin-binding to snake AcChoR.

  3. Cloning, expression, and functional analysis of human dopamine D1 receptors.

    PubMed

    Sun, Wan-chun; Jin, Lei; Cao, Yan; Wang, Li-zhen; Meng, Fan; Zhu, Xing-zu

    2005-01-01

    To construct an HEK293 cell line stably expressing human dopamine D1 receptor (D1R). cDNA was amplified by RT-PCR using total RNA from human embryo brain tissue as the template. The PCR products were subcloned into the plasmid pcDNA3 and cloned into the plasmid pcDNA3.1. The cloned D1R cDNA was sequenced and stably expressed in HEK293 cells. Expression of D1R in HEK293 cells was monitored by the [3H]SCH23390 binding assay. The function of D1R was studied by the cAMP accumulation assay, CRE-SEAP reporter gene activity assay, and intracellular calcium assay. An HEK293 cell line stably expressing human D1R was obtained. A saturation radioligand binding experiment with [3H]SCH23390 demonstrated that the Kd and Bmax values were 1.5+/-0.2 nmol/L and 2.94+/-0.15 nmol/g of protein, respectively. In the [3H]SCH23390 competition assay, D1R agonist SKF38393 displaced [3H]SCH23390 with an IC50 value of 2.0 (1.5-2.8) micromol/L. SKF38393 increased the intracellular cAMP level and CRE-SEAP activity through D1R expressed in HEK293 cells in a concentration-dependent manner with an EC50 value of 0.25 (0.12-0.53) micromol/L and 0.39 (0.27-0.57) micromol/L at 6 h/0.59 (0.22-1.58) micromol/L at 12 h, respectively. SKF38393 also increased the intracellular calcium level in a concentration-dependent manner with EC50 value of 27 (8.6-70) nmol/L. An HEK293 cell line stably expressing human D1R was obtained successfully. The study also demonstrated that the CRE-SEAP activity assay could be substituted for the cAMP accumulation assay for measuring increase in cAMP levels. Thus, both intracellular calcium measurements and the CRE-SEAP activity assay are suitable for high-throughput screening in drug research.

  4. The 5' untranslated region of a novel infectious molecular clone of the dicistrovirus cricket paralysis virus modulates infection.

    PubMed

    Kerr, Craig H; Wang, Qing S; Keatings, Kathleen; Khong, Anthony; Allan, Douglas; Yip, Calvin K; Foster, Leonard J; Jan, Eric

    2015-06-01

    Dicistroviridae are a family of RNA viruses that possesses a single-stranded positive-sense RNA genome containing two distinct open reading frames (ORFs), each preceded by an internal ribosome entry site that drives translation of the viral structural and nonstructural proteins, respectively. The type species, Cricket paralysis virus (CrPV), has served as a model for studying host-virus interactions; however, investigations into the molecular mechanisms of CrPV and other dicistroviruses have been limited as an established infectious clone was elusive. Here, we report the construction of an infectious molecular clone of CrPV. Transfection of in vitro-transcribed RNA from the CrPV clone into Drosophila Schneider line 2 (S2) cells resulted in cytopathic effects, viral RNA accumulation, detection of negative-sense viral RNA, and expression of viral proteins. Transmission electron microscopy, viral titers, and immunofluorescence-coupled transwell assays demonstrated that infectious viral particles are released from transfected cells. In contrast, mutant clones containing stop codons in either ORF decreased virus infectivity. Injection of adult Drosophila flies with virus derived from CrPV clones but not UV-inactivated clones resulted in mortality. Molecular analysis of the CrPV clone revealed a 196-nucleotide duplication within its 5' untranslated region (UTR) that stimulated translation of reporter constructs. In cells infected with the CrPV clone, the duplication inhibited viral infectivity yet did not affect viral translation or RNA accumulation, suggesting an effect on viral packaging or entry. The generation of the CrPV infectious clone provides a powerful tool for investigating the viral life cycle and pathogenesis of dicistroviruses and may further understanding of fundamental host-virus interactions in insect cells. Dicistroviridae, which are RNA viruses that infect arthropods, have served as a model to gain insights into fundamental host-virus interactions in

  5. Molecular cloning of osteoma-inducing replication-competent murine leukemia viruses from the RFB osteoma virus stock.

    PubMed Central

    Pedersen, L; Behnisch, W; Schmidt, J; Luz, A; Pedersen, F S; Erfle, V; Strauss, P G

    1992-01-01

    We report the molecular cloning of two replication-competent osteoma-inducing murine leukemia viruses from the RFB osteoma virus stock (M. P. Finkel, C. A. Reilly, Jr., B. O. Biskis, and I. L. Greco, p. 353-366, in C. H. G. Price and F. G. M. Ross, ed., Bone--Certain Aspects of Neoplasia, 1973). Like the original RFB osteoma virus stock, viruses derived from the molecular RFB clones induced multiple osteomas in mice of the CBA/Ca strain. The cloned RFB viruses were indistinguishable by restriction enzyme analysis and by nucleotide sequence analysis of their long-terminal-repeat regions and showed close relatedness to the Akv murine leukemia virus. Images PMID:1326664

  6. Molecular cloning, expression pattern, and molecular evolution of the spleen tyrosine kinase in lamprey, Lampetra japonica.

    PubMed

    Liu, Chang; Su, Peng; Li, Ranran; Zhang, Qiong; Zhu, Ting; Liu, Xin; Li, Qingwei

    2015-04-01

    Spleen tyrosine kinase (Syk), a member of Syk family of cytoplasmic non-receptor tyrosine kinases, is a key component of B cell receptor signaling and regulates multiple physiological functions of B lymphocytes in vertebrates. In the current study, a Syk homologue was identified in the lamprey Lampetra japonica (Lj-Syk). The cDNA fragment of Lj-Syk contains a 1953-bp open reading frame which encodes 651 amino acids, a 12-bp fragment of 5'-untranslated region, and a 1029-bp 3'-untranslated region. The same as vertebrate's Syks, Lj-Syk protein also contains a tyrosine kinase catalytic domain which functions as its kinase activity center and two Src homology 2 (SH2) domains which are the targets when Syk is recruited by phosphorylated immunoreceptor tyrosine-based activation motif. It is revealed by multiple sequence alignment that the tyrosine kinase catalytic domain and two SH2 domains are conserved throughout the Syk gene family in vertebrates. The evolutionary dynamics of Syks were analyzed by MEME software using conserved motifs as markers. Among 19 conserved motifs elicited from 22 Syks or Syk-like proteins, 12 motifs that locate at N-terminal, two tandem SH2, Inter SH2, and Tyrkc domains are conserved in Syks from jawless to jawed vertebrates. From the absence and existence of the other seven motifs, it can be concluded that the primary Syk gene evolved to modern functional gene through short insertion and deletion strategy in their gene sequence rather than gene duplication. The expression of lamprey Syk was examined by real-time quantitative PCR and Western blot methods in leukocyte cells, gills, supraneural myeloid bodies, kidneys, and hearts of lampreys before and after the animals were stimulated with lipopolysaccharide (LPS). The transcriptional level of lamprey Syk was upregulated in gill, kidney, heart, and leukocyte cells, and the protein expression level is upregulated in leukocyte cells and supraneural myeloid bodies after stimulated with LPS. It

  7. Solution structures and molecular interactions of selective melanocortin receptor antagonists.

    PubMed

    Lee, Chul-Jin; Yun, Ji-Hye; Lim, Sung-Kil; Lee, Weontae

    2010-12-01

    The solution structures and inter-molecular interaction of the cyclic melanocortin antagonists SHU9119, JKC363, HS014, and HS024 with receptor molecules have been determined by NMR spectroscopy and molecular modeling. While SHU9119 is known as a nonselective antagonist, JKC363, HS014, and HS024 are selective for the melanocortin subtype-4 receptor (MC4R) involved in modulation of food intake. Data from NMR and molecular dynamics suggest that the conformation of the Trp9 sidechain in the three MC4R-selective antagonists is quite different from that of SHU9119. This result strongly supports the concept that the spatial orientation of the hydrophobic aromatic residue is more important for determining selectivity than the presence of a basic, "arginine-like" moiety responsible for biological activity. We propose that the conformation of hydrophobic residues of MCR antagonists is critical for receptor-specific selectivity.

  8. Molecular cloning and characterization of two hypersensitive induced reaction genes from wheat infected by stripe rust pathogen

    USDA-ARS?s Scientific Manuscript database

    A novel gene induced during hypersensitive reaction (HIR) in wheat was identified using in silico cloning and designated as TaHIR2. The TaHIR2 gene was deduced to encode a 284-amino acid protein, whose molecular mass and isoelectric point (pI) were 31.05 kD and 5.18, respectively. Amino acid sequenc...

  9. Isolation, characterization, molecular cloning and molecular modelling of two lectins of different specificities from bluebell (Scilla campanulata) bulbs.

    PubMed Central

    Wright, L M; Van Damme, E J; Barre, A; Allen, A K; Van Leuven, F; Reynolds, C D; Rouge, P; Peumans, W J

    1999-01-01

    Two lectins have been isolated from bluebell (Scilla campanulata) bulbs. From their isolation by affinity chromatography, they are characterized as a mannose-binding lectin (SCAman) and a fetuin-binding lectin (SCAfet). SCAman preferentially binds oligosaccharides with alpha(1,3)- and alpha(1,6)-linked mannopyranosides. It is a tetramer of four identical protomers of approx. 13 kDa containing 119 amino acid residues; it is not glycosylated. The fetuin-binding lectin (SCAfet), which is not inhibited by any simple sugars, is also unglycosylated. It is a tetramer of four identical subunits of approx. 28 kDa containing 244 residues. Each 28 kDa subunit is composed of two 14 kDa domains. Both lectins have been cloned from a cDNA library and sequenced. X-ray crystallographic analysis and molecular modelling studies have demonstrated close relationships in sequence and structure between these lectins and other monocot mannose-binding lectins. A refined model of the molecular evolution of the monocot mannose-binding lectins is proposed. PMID:10229686

  10. Datura stramonium agglutinin: cloning, molecular characterization and recombinant production in Arabidopsis thaliana.

    PubMed

    Nishimoto, Keisuke; Tanaka, Kaori; Murakami, Takahiro; Nakashita, Hideo; Sakamoto, Hikaru; Oguri, Suguru

    2015-02-01

    Datura stramonium seeds contain at least three chitin-binding isolectins [termed Datura stramonium agglutinin (DSA)] as homo- or heterodimers of A and B subunits. We isolated a cDNA encoding isolectin B (DSA-B) from an immature fruit cDNA library; this contained an open reading frame encoding 279 deduced amino acids, which was confirmed by partial sequencing of the native DSA-B peptide. The sequence consisted of: (i) a cysteine (Cys)-rich carbohydrate-binding domain composed of four conserved chitin-binding domains and (ii) an extensin-like domain of 37 residues containing four SerPro4-6 motifs that was inserted between the second and third chitin-binding domains (CBDs). Although each chitin-binding domain contained eight conserved Cys residues, only the second chitin-binding domain contained an extra Cys residue, which may participate in dimerization through inter-disulfide bridge formation. Using matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry, the molecular mass of homodimeric lectin composed of two B-subunits was determined as 68,821 Da. The molecular mass of the S-pyridilethylated B-subunit were found to be 37,748 Da and that of the de-glycosylated form was 26,491 Da, which correlated with the molecular weight estimated from the deduced sequence. Transgenic Arabidopsis plants overexpressing the dsa-b demonstrated hemagglutinating activity. Recombinant DSA-B was produced as a homodimeric glycoprotein with a similar molecular mass to that of the native form. Moreover, the N-terminus of the purified recombinant DSA-B protein was identical to that of the native DSA-B, confirming that the cloned cDNA encoded DSA-B.

  11. On the Molecular Evolution of Leptin, Leptin Receptor, and Endospanin.

    PubMed

    Londraville, Richard Lyle; Prokop, Jeremy W; Duff, Robert Joel; Liu, Qin; Tuttle, Matthew

    2017-01-01

    Over a decade passed between Friedman's discovery of the mammalian leptin gene (1) and its cloning in fish (2) and amphibians (3). Since 2005, the concept of gene synteny conservation (vs. gene sequence homology) was instrumental in identifying leptin genes in dozens of species, and we now have leptin genes from all major classes of vertebrates. This database of LEP (leptin), LEPR (leptin receptor), and LEPROT (endospanin) genes has allowed protein structure modeling, stoichiometry predictions, and even functional predictions of leptin function for most vertebrate classes. Here, we apply functional genomics to model hundreds of LEP, LEPR, and LEPROT proteins from both vertebrates and invertebrates. We identify conserved structural motifs in each of the three leptin signaling proteins and demonstrate Drosophila Dome protein's conservation with vertebrate leptin receptors. We model endospanin structure for the first time and identify endospanin paralogs in invertebrate genomes. Finally, we argue that leptin is not an adipostat in fishes and discuss emerging knockout models in fishes.

  12. Molecular neuroimaging in rodents: assessing receptor expression and function.

    PubMed

    Mueggler, Thomas; Baltes, Christof; Rudin, Markus

    2009-11-01

    Multimodal non-invasive neuroimaging in rodents constitutes an attractive tool for studying neurobiological processes in vivo. At present, imaging studies of brain anatomy and function as well as the investigation of structure-function relationships belong to the standard repertoire of neuroscientists. Molecular imaging adds a new perspective. The mapping of the receptor distribution and receptor occupancy can nowadays be complemented by specific readouts of receptor function either by visualizing the activity of signaling pathways or mapping the physiological consequences of receptor stimulation. Molecular information is obtained through the use of imaging probes that combine a target-specific ligand with a reporter moiety that generates a signal that can be detected from outside the body. For imaging probes targeting the central nervous system, penetration of the intact blood-brain barrier constitutes a major hurdle. Molecular imaging generates specific information and therefore has a large potential for disease phenotyping (diagnostics), therapy development and monitoring of treatment response. Molecular imaging is still in its infancy and major developments in imaging technology, probe design and data analysis are required in order to make an impact. Rodent molecular neuroimaging will play an important role in the development of these tools.

  13. Hyperspectral molecular imaging of multiple receptors using immunolabeled plasmonic nanoparticles

    NASA Astrophysics Data System (ADS)

    Seekell, Kevin; Crow, Matthew J.; Marinakos, Stella; Ostrander, Julie; Chilkoti, Ashutosh; Wax, Adam

    2011-11-01

    This work presents simultaneous imaging and detection of three different cell receptors using three types of plasmonic nanoparticles (NPs). The size, shape, and composition-dependent scattering profiles of these NPs allow for a system of multiple distinct molecular markers using a single optical source. With this goal in mind, tags consisting of anti-epidermal growth factor receptor gold nanorods, anti-insulin-like growth factor 1-R silver nanospheres, and human epidermal growth factor receptor 2Ab gold nanospheres were developed to monitor the expression of receptors commonly overexpressed by cancer cells. These labels were chosen because they scatter strongly in distinct spectral windows. A hyperspectral darkfield microspectroscopy system was developed to record the scattering spectra of cells labeled with these molecular tags. Simultaneous monitoring of multiple tags may lead to applications such as profiling of cell line immunophenotype and investigation of receptor signaling pathways. Single, dual, and triple tag experiments were performed to analyze NP tag specificity as well as their interactions. Distinct resonance peaks were observed in these studies, showing the ability to characterize cell lines using conjugated NPs. However, interpreting shifts in these peaks due to changes in a cellular dielectric environment may be complicated by plasmon coupling between NPs bound to proximal receptors and other coupling mechanisms due to the receptors themselves.

  14. Molecular cloning of MER-2, a human chromosome-11-encoded red blood cell antigen, using linkage of cotransfected markers.

    PubMed

    Bill, J; Palmer, E; Jones, C

    1987-09-01

    We report the molecular cloning of a human gene MER-2 located on chromosome 11 that encodes a cell surface antigen which is polymorphic on red blood cells. An essential element of the cloning strategy was cotransfection-induced linkage of pSV2-neo, which encodes resistance to the antibiotic G418, to the human MER-2 gene. An important feature of the pSV2-neo construct is that the same gene (the transposon, Tn5) that encodes G418 resistance in eukaryotic cells confers neomycin resistance in bacteria. Chinese hamster ovary (CHO) cells were cotransfected with pSV2-neo and genomic DNA from a CHO X human cell hybrid containing a single human chromosome (chromosome 11). Transfectants expressing both the human MER-2 gene and G418 resistance were isolated by selection in the antibiotic G418, followed by indirect immunofluorescence using the monoclonal antibody 1D12, which recognizes the MER-2 antigen, manual enrichment, and single-cell cloning. Genomic DNA from a primary transfectant positive for MER-2 expression and G418 resistance was used to construct a cosmid library and cosmid clones able to grow in neomycin were isolated. Of 150,000 cosmid clones screened, 90 were resistant to neomycin and of these, 11 contained human repetitive sequences. Five neomycin-resistant cosmid clones containing human repetitive DNA were able to transfect CHO cells for G418 resistance and MER-2 expression.

  15. Cloning and expression of progesterone receptor isoforms A and B in bovine corpus luteum.

    PubMed

    Rekawiecki, Robert; Kowalik, Magdalena Karolina; Kotwica, Jan

    2015-09-01

    Progesterone (P4) affects a cell through its nuclear receptor (PGR), which has two main isoforms: A (PGRA) and B (PGRB). A partial section of previously unknown PGRB cDNA from cattle was cloned. Next, mRNA and protein levels for these two isoforms in corpora lutea (CL) collected during different stages of the oestrous cycle and pregnancy were determined. The PGRB mRNA level was highest on Days 2-5 of the oestrous cycle, decreased over the next few days (P<0.01) and increased again slightly on Days 17-20 (P<0.05). During pregnancy, PGRB mRNA was at its lowest level during Weeks 3-5 (P<0.01) and highest during Weeks 6-12 (P<0.01). The profile of PGRA mRNA levels was similar to that of PGRB throughout the oestrous cycle. The PGRA protein level was highest on Days 2-10 of the oestrous cycle, decreased continuously to its lowest concentration on Days 17-20 (P<0.01) and during Weeks 3-5 of pregnancy (P>0.05) and increased during Weeks 6-12 (P<0.05). PGRB protein concentration followed a similar pattern but at a markedly lower level. Both PGRA and PGRB isoforms are involved in the regulation of P4 action, especially in the newly formed CL and developed CL in the first trimester of pregnancy. These data suggest that the variable expression of these isoforms during the oestrous cycle may depend on the influence of P4.

  16. Cloning and characterization of an ovine intracellular seven transmembrane receptor for progesterone that mediates calcium mobilization.

    PubMed

    Ashley, R L; Clay, C M; Farmerie, T A; Niswender, G D; Nett, T M

    2006-09-01

    Classically, progesterone has been thought to act only through the well-known genomic pathway involving hormone binding to nuclear receptors (nPR) and subsequent modulation of gene expression. However, there is increasing evidence for rapid, nongenomic effects of progesterone in a variety of tissues in mammals, and it seems likely that a membrane PR (mPR) is causing these events. The objective of this study was to isolate and characterize an ovine mPR distinct from the nPR. A cDNA clone was isolated from ovine genomic DNA by PCR. The ovine mPR is a 350-amino acid protein that, based on computer hydrophobicity analysis, possesses seven transmembrane domains and is distinct from the nPR. Message for the ovine mPR was detected in hypothalamus, pituitary, uterus, ovary, and corpus luteum by RT-PCR. In CHO cells that overexpressed a mPR-green fluorescent protein fusion protein, the ovine mPR was localized to the endoplasmic reticulum and not the plasma membrane. Specific binding of 3H-progesterone to membrane fractions was demonstrated in CHO cells that expressed the ovine mPR but not in nontransfected cells. Furthermore, progesterone and 17 alpha-hydroxy-progesterone stimulated intracellular Ca2+ mobilization in CHO cells that expressed ovine mPR in Ca2+-free medium (P < 0.05) but not in CHO cells transfected with empty vector. This rise in intracellular Ca2+ is believed to be from the endoplasmic reticulum as intracellular Ca2+ mobilization is absent when mPR transfected cells are first treated with thapsigargin to deplete Ca2+ stores from the endoplasmic reticulum. Isolation, identification, tissue distribution, cellular localization, steroid binding, and a functional response for a unique intracellular mPR in the sheep are presented.

  17. Isolation and partial characterization of infectious molecular clones of feline immunodeficiency virus obtained directly from bone marrow DNA of a naturally infected cat.

    PubMed Central

    Siebelink, K H; Chu, I H; Rimmelzwaan, G F; Weijer, K; Osterhaus, A D; Bosch, M L

    1992-01-01

    Replication-competent molecular clones of feline immunodeficiency virus (FIV) were isolated directly from the DNA of bone marrow cells of a naturally FIV-infected cat. After transfection in a feline kidney cell line (CrFK) and subsequent cocultivation with peripheral blood mononuclear cells (PBMC), the viral progeny of the clones was infectious for PBMC but not for CrFK cells. PBMC infected with these clones showed syncytium formation, a decrease in cell viability, and gradual loss of CD4+ cells. The restriction maps of these clones differed from those obtained for previously described molecular clones of FIV derived from cats in the United States. The predicted amino acid sequence similarity of the envelope genes of the two clones was 99.3%, whereas the similarities of the sequences of the clones to those of two molecular clones from the United States, Petaluma and PPR, were 86 and 88%, respectively. Most of the differences between the amino acid sequences of the two clones and those of the clones from the United States were found in five different hypervariable (HV) regions, HV-1 through HV-5. The viral progeny of one of these clones was inoculated into two specific-pathogen-free cats. The animals seroconverted, and the virus could be reisolated from their PBMC. Images PMID:1309891

  18. Characterization and cloning of a receptor for BMP-2 and BMP-4 from NIH 3T3 cells.

    PubMed Central

    Koenig, B B; Cook, J S; Wolsing, D H; Ting, J; Tiesman, J P; Correa, P E; Olson, C A; Pecquet, A L; Ventura, F; Grant, R A

    1994-01-01

    The bone morphogenetic proteins (BMPs) are a group of transforming growth factor beta (TGF-beta)-related factors whose only receptor identified to date is the product of the daf-4 gene from Caenorhabditis elegans. Mouse embryonic NIH 3T3 fibroblasts display high-affinity 125I-BMP-4 binding sites. Binding assays are not possible with the isoform 125I-BMP-2 unless the positively charged N-terminal sequence is removed to create a modified BMP-2, 125I-DR-BMP-2. Cross-competition experiments reveal that BMP-2 and BMP-4 interact with the same binding sites. Affinity cross-linking assays show that both BMPs interact with cell surface proteins corresponding in size to the type I (57- to 62-kDa) and type II (75- to 82-kDa) receptor components for TGF-beta and activin. Using a PCR approach, we have cloned a cDNA from NIH 3T3 cells which encodes a novel member of the transmembrane serine/threonine kinase family most closely resembling the cloned type I receptors for TGF-beta and activin. Transient expression of this receptor in COS-7 cells leads to an increase in specific 125I-BMP-4 binding and the appearance of a major affinity-labeled product of approximately 64 kDa that can be labeled by either tracer. This receptor has been named BRK-1 in recognition of its ability to bind BMP-2 and BMP-4 and its receptor kinase structure. Although BRK-1 does not require cotransfection of a type II receptor in order to bind ligand in COS cells, complex formation between BRK-1 and the BMP type II receptor DAF-4 can be demonstrated when the two receptors are coexpressed, affinity labeled, and immunoprecipitated with antibodies to either receptor subunit. We conclude that BRK-1 is a putative BMP type I receptor capable of interacting with a known type II receptor for BMPs. Images PMID:8065329

  19. Applications of molecular replacement to G protein-coupled receptors

    SciTech Connect

    Kruse, Andrew C.; Manglik, Aashish; Kobilka, Brian K.; Weis, William I.

    2013-11-01

    The use of molecular replacement in solving the structures of G protein-coupled receptors is discussed, with specific examples being described in detail. G protein-coupled receptors (GPCRs) are a large class of integral membrane proteins involved in regulating virtually every aspect of human physiology. Despite their profound importance in human health and disease, structural information regarding GPCRs has been extremely limited until recently. With the advent of a variety of new biochemical and crystallographic techniques, the structural biology of GPCRs has advanced rapidly, offering key molecular insights into GPCR activation and signal transduction. To date, almost all GPCR structures have been solved using molecular-replacement techniques. Here, the unique aspects of molecular replacement as applied to individual GPCRs and to signaling complexes of these important proteins are discussed.

  20. Molecular Cloning and Characterization of Novel Glutamate-Gated Chloride Channel Subunits from Schistosoma mansoni

    PubMed Central

    Dufour, Vanessa; Beech, Robin N.; Wever, Claudia; Dent, Joseph A.; Geary, Timothy G.

    2013-01-01

    Cys-loop ligand-gated ion channels (LGICs) mediate fast ionotropic neurotransmission. They are proven drug targets in nematodes and arthropods, but are poorly characterized in flatworms. In this study, we characterized the anion-selective, non-acetylcholine-gated Cys-loop LGICs from Schistosoma mansoni. Full-length cDNAs were obtained for SmGluCl-1 (Smp_096480), SmGluCl-2 (Smp_015630) and SmGluCl-3 (Smp_104890). A partial cDNA was retrieved for SmGluCl-4 (Smp_099500/Smp_176730). Phylogenetic analyses suggest that SmGluCl-1, SmGluCl-2, SmGluCl-3 and SmGluCl-4 belong to a novel clade of flatworm glutamate-gated chloride channels (GluCl) that includes putative genes from trematodes and cestodes. The flatworm GluCl clade was distinct from the nematode-arthropod and mollusc GluCl clades, and from all GABA receptors. We found no evidence of GABA receptors in S. mansoni. SmGluCl-1, SmGluCl-2 and SmGluCl-3 subunits were characterized by two-electrode voltage clamp (TEVC) in Xenopus oocytes, and shown to encode Cl−-permeable channels gated by glutamate. SmGluCl-2 and SmGluCl-3 produced functional homomers, while SmGluCl-1 formed heteromers with SmGluCl-2. Concentration-response relationships revealed that the sensitivity of SmGluCl receptors to L-glutamate is among the highest reported for GluCl receptors, with EC50 values of 7–26 µM. Chloride selectivity was confirmed by current-voltage (I/V) relationships. SmGluCl receptors are insensitive to 1 µM ivermectin (IVM), indicating that they do not belong to the highly IVM-sensitive GluClα subtype group. SmGluCl receptors are also insensitive to 10 µM meclonazepam, a schistosomicidal benzodiazepine. These results provide the first molecular evidence showing the contribution of GluCl receptors to L-glutamate signaling in S. mansoni, an unprecedented finding in parasitic flatworms. Further work is needed to elucidate the roles of GluCl receptors in schistosomes and to explore their potential as drug targets. PMID:24009509

  1. Molecular cloning and characterization of CD4 in an aquatic mammal, the white whale Delphinapterus leucas.

    PubMed

    Romano, T A; Ridgway, S H; Felten, D L; Quaranta, V

    1999-05-01

    Given the importance of the cell surface recognition protein, CD4, in immune function, the cloning and characterization of CD4 at the molecular level from an odontocete cetacean, the white whale (Delphinapterus leucas), was carried out. Whale CD4 cDNA contains 2662 base pairs and translates into a protein containing 455 amino acids. Whale CD4 shares 64% and 51% identity with the human and mouse CD4 protein, respectively, and is organized in a similar manner. Unlike human and mouse, however, the cytoplasmic domain, which is highly conserved, contains amino acid substitutions unique to whale. Moreover, only one of the seven potential N-linked glycosylation sites present in whale is shared with human and mouse. Evolutionarily, the whale CD4 sequence is most similar to pig and structurally similar to dog and cat, in that all lack the cysteine pair in the V2 domain. These differences suggest that CD4 may have a different secondary structure in these species, which may affect binding of class II and subsequent T-cell activation, as well as binding of viral pathogens. Interestingly, as a group, species with these CD4 characteristics all have high constitutive expression of class II molecules on T lymphocytes, suggesting potential uniqueness in the interaction of CD4, class II molecules, and the immune response. Molecular characterization of CD4 in an aquatic mammal provides information on the CD4 molecule itself and may provide insight into adaptive evolutionary changes of the immune system.

  2. Molecular Modeling of Estrogen Receptor Using Molecular Operating Environment

    ERIC Educational Resources Information Center

    Roy, Urmi; Luck, Linda A.

    2007-01-01

    Molecular modeling is pervasive in the pharmaceutical industry that employs many of our students from Biology, Chemistry and the interdisciplinary majors. To expose our students to this important aspect of their education we have incorporated a set of tutorials in our Biochemistry class. The present article describes one of our tutorials where…

  3. Molecular Modeling of Estrogen Receptor Using Molecular Operating Environment

    ERIC Educational Resources Information Center

    Roy, Urmi; Luck, Linda A.

    2007-01-01

    Molecular modeling is pervasive in the pharmaceutical industry that employs many of our students from Biology, Chemistry and the interdisciplinary majors. To expose our students to this important aspect of their education we have incorporated a set of tutorials in our Biochemistry class. The present article describes one of our tutorials where…

  4. Cloning and Characterization of a 7 Transmembrane Receptor from the Adherent Cells of Chicken Peripheral Blood Mononuclear Cells

    PubMed Central

    Chen, Yu San; Wu, Hsing Chieh; Shien, Jui Hung; Chiu, Hua Hsien; Lee, Long Huw

    2014-01-01

    A cDNA encoding a 7 transmembrane (7TM) receptor gene from the adherent cells of chicken peripheral blood mononuclear cells (PBMC) was cloned and characterized. The open reading frame of the chicken-7TM (Ch-7TM) receptor gene was 1008 nucleotides long, encoding a protein of 335 amino acid residues with a molecular mass of approximately 37.1 kDa. Hydrophobic stretches indicated the presence of 7 TM domains. Moreover, the complete nucleotide sequences encoding 7TM of duck (Du-7TM) and goose (Go-7TM), corresponding to the open reading frame of Ch-7TM, were determined. Each of the Du- and Go-7TM encoding regions comprised 990 nucleotides, representing an 18-nucleotide deletion in alignment with the Ch-7TM encoding region, resulting in a 6-amino-acid deletion at the 3′-end. No signal peptides were predicted. Six phosphorylation sites were predicted and conserved for all three 7TMs. The proteins of the three 7TMs were similar, with 11 conserved cysteine residues. No glycosylation sites could be predicted. The results of the pairwise comparisons indicated that the Ch-7TM encoding region and Ch-7TM protein were the least similar to those of Du- and Go-7TMs. These results were in accordance with those of the phylogenetic analysis, which indicated that the Du- and Go-7TM encoding regions clustered, but were separated from the Ch-7TM encoding region. Monoclonal antibody B28D5 was prepared from spleens of mice immunized with the bacterially expressed N-terminal (55 amino acid residues) region of the Ch-7TM protein for further use. Double staining with B28D5 and KUL01 suggested that Ch-7TM was expressed in subsets of the adherent cells, among which a subset that was recognized with both antibodies was likely of monocyte and macrophage lineage. However, the fluorescence intensities of B28D5 and, particularly, KUL01 decreased after the adherent cells were incubated for additional 48 h. PMID:24466279

  5. Human substance P receptor (NK-1): Organization of the gene, chromosome localization, and functional expression of cDNA clones

    SciTech Connect

    Gerard, N.P.; Paquet, J.L. Children's Hospital, Boston, MA Harvard Medical School, Boston, MA ); Garraway, L.A. Harvard Medical School, Boston, MA ); Eddy, R.L. Jr.; Shows, T.B. ); Iijima, Hideya Harvard School of Public Health, Boston, MA ); Gerard, C. )

    1991-11-05

    The gene for the human substance P receptor (NK-1) was cloned using cDNA probes made by the polymerase chain reaction from primers based on the rat sequence. The gene spans 45-60 kb and is contained in five exons, with introns interrupting at sites homologous to those in the NK-2 receptor gene. Analysis of restriction digests of genomic DNA from mouse/human cell hybrids indicates the NK-1 receptor is a single-copy gene located on human chromosome 2. Polymerase chain reaction using primers based on the 5{prime} and 3{prime} ends of the coding sequence was used to generate full-length cDNAs from human lung and from IM9 lymphoblast cells. When transfected into COS-7 cells, the NK-1 receptor binds {sup 125}I-BHSP with a K{sub d} of 0.35 {plus minus} 0.07 nM and mediates substance P induced phosphatidylinositol metabolism. The receptor is selective for substance P; the relative affinity for neurokinin A and neurokinin B is 100- and 500-fold lower, respectively. Human IM9 lymphoblast cells express relatively high levels of the NK-1 receptor, and Northern blot analysis indicates modulation of mRNA levels by glucocorticoids and growth factors, suggesting that this cell line may be useful as a model for studying the control of NK-1 receptor gene expression.

  6. Molecular cloning and functional characterization of a rainbow trout liver Oatp

    SciTech Connect

    Steiner, Konstanze; Hagenbuch, Bruno; Dietrich, Daniel R.

    2014-11-01

    Cyanobacterial blooms have an impact on the aquatic ecosystem due to the production of toxins (e.g. microcystins, MCs), which constrain fish health or even cause fish death. However the toxicokinetics of the most abundant toxin, microcystin-LR (MC-LR), are not yet fully understood. To investigate the uptake mechanism, the novel Oatp1d1 in rainbow trout (rtOatp1d1) was cloned, identified and characterized. The cDNA isolated from a clone library consisted of 2772 bp containing a 2115 bp open reading frame coding for a 705 aa protein with an approximate molecular mass of 80 kDa. This fish specific transporter belongs to the OATP1 family and has most likely evolved from a common ancestor of OATP1C1. Real time PCR analysis showed that rtOatp1d1 is predominantly expressed in the liver, followed by the brain while expression in other organs was not detectable. Transient transfection in HEK293 cells was used for further characterization. Like its human homologues OATP1A1, OATP1B1 and OATP1B3, rtOatp1d1 displayed multi-specific transport including endogenous and xenobiotic substrates. Kinetic analyses revealed a K{sub m} value of 13.9 μM and 13.4 μM for estrone-3-sulfate and methotrexate, respectively and a rather low affinity for taurocholate with a K{sub m} value of 103 μM. Furthermore, it was confirmed that rtOatp1d1 is a MC-LR transporter and therefore most likely plays a key role in the susceptibility of rainbow trout to MC intoxications. - Highlights: • A new Oatp1d1 in rainbow trout (rtOatp1d1) was cloned, identified and characterized. • rtOatp1d1 is predominantly expressed in the liver. • rtOatp1d1 displays multi-specific transport of endogenous and xenobiotic substrates. • rtOatp1d1 is a homologue of the OATP1A1, OATP1B1 and OATP1B3. • rtOatp1d1 is a microcystin (MC) transporter.

  7. Molecular cloning and characterization of the human beta-like globin gene cluster.

    PubMed

    Fritsch, E F; Lawn, R M; Maniatis, T

    1980-04-01

    The genes encoding human embryonic (epsilon), fetal (G gamma, A gamma) and adult (delta, beta) beta-like globin polypeptides were isolated as a set of overlapping cloned DNA fragments from bacteriophage lambda libraries of high molecular weight (15-20 kb) chromosomal DNA. The 65 kb of DNA represented in these overlapping clones contains the genes for all five beta-like polypeptides, including the embryonic epsilon-globin gene, for which the chromosomal location was previously unknown. All five genes are transcribed from the same DNA strand and are arranged in the order 5'-epsilon-(13.3 kb)-G gamma-(3.5 kb)-A gamma-(13.9 kb)-delta-(5.4 kb)-beta-3'. Thus the genes are positioned on the chromosome in the order of their expression during development. In addition to the five known beta-like globin genes, we have detected two other beta-like globin sequences which do not correspond to known polypeptides. One of these sequences has been mapped to the A gamma-delta intergenic region while the other is located 6-9 kb 5' to the epsilon gene. Cross hybridization experiments between the intergenic sequences of the gene cluster have revealed a nonglobin repeat sequence (*) which is interspersed with the globin genes in the following manner: 5'-**epsilon-*G gamma-A gamma*-**delta-beta*-3'. Fine structure mapping of the region located 5' to the delta-globin gene revealed two repeats with a maximum size of 400 bp, which are separated by approximately 700 bp of DNA not repeated within the cluster. Preliminary experiments indicate that this repeat family is also repeated many times in the human genome.

  8. Molecular cloning and characterization of wheat calreticulin (CRT) gene involved in drought-stressed responses.

    PubMed

    Jia, Xiao-Yun; Xu, Chong-Yi; Jing, Rui-Lian; Li, Run-Zhi; Mao, Xin-Guo; Wang, Ji-Ping; Chang, Xiao-Ping

    2008-01-01

    Calreticulin (CRT) is a highly conserved and ubiquitously expressed Ca(2+)-binding protein in multicellular eukaryotes. CRT plays a crucial role in many cellular processes including Ca(2+) storage and release, protein synthesis, and molecular chaperone activity. To elucidate the function of CRTs in plant responses against drought, a main abiotic stress limiting cereal crop production worldwide, a full-length cDNA encoding calreticulin protein namely TaCRT was isolated from wheat (Triticum aestivum L.). The deduced amino acid sequence of TaCRT shares high homology with other plant CRTs. Phylogenetic analysis indicates that TaCRT cDNA clone encodes a wheat CRT3 isoform. Southern analysis suggests that the wheat genome contains three copies of TaCRT. Subcellular locations of TaCRT were the cytoplasm and nucleus, evidenced by transient expression of GFP fused with TaCRT in onion epidermal cells. Enhanced accumulation of TaCRT transcript was observed in wheat seedlings in response to PEG-induced drought stress. To investigate further whether TaCRT is involved in the drought-stress response, transgenic plants were constructed. Compared to the wild-type and GFP-expressing plants, TaCRT-overexpressing tobacco (Nicotiana benthamiana) plants grew better and exhibited less wilt under the drought stress. Moreover, TaCRT-overexpressing plants exhibited enhanced drought resistance to water deficit, as shown by their capacity to maintain higher WUE (water use efficiency), WRA (water retention ability), RWC (relative water content), and lower MDR (membrane damaging ratio) (P < or = 0.01) under water-stress conditions. In conclusion, a cDNA clone encoding wheat CRT was successfully isolated and the results suggest that TaCRT is involved in the plant response to drought stress, indicating a potential in the transgenic improvements of plant water-stress.

  9. Molecular cloning and functional characterization of a rainbow trout liver Oatp

    PubMed Central

    Steiner, Konstanze; Hagenbuch, Bruno; Dietrich, Daniel R.

    2014-01-01

    Cyanobacterial blooms have an impact on the aquatic ecosystem due to the production of toxins (e.g. microcystins, MCs), which constrains fish health or even cause fish death. However the toxicokinetics of the most abundant toxin, microcystin-LR (MC-LR), are not yet fully understood. To investigate the uptake mechanism, the novel Oatp1d1 in rainbow trout (rtOatp1d1) was cloned, identified and characterized. The cDNA isolated from a clone library consisted of 2772 bp containing a 2115 bp open reading frame coding for a 705 aa protein with an approximate molecular mass of 80 kDa. This fish specific transporter belongs to the OATP1 family and has most likely evolved from a common ancestor of OATP1C1. Real time PCR analysis showed that rtOatp1d1 is predominantly expressed in the liver, followed by the brain while expression in other organs was not detectable. Transient transfection in HEK293 cells was used for further characterization. Like its human homologs OATP1A1, OATP1B1 and OATP1B3, rtOatp1d1 displayed multi-specific transport including endogenous and xenobiotic substrates. Kinetic analyses revealed a Km value of 13.9 μM and 13.4 μM for estrone-3-sulfate and methotrexate, respectively and a rather low affinity for taurocholate with a Km value of 103 μM. Furthermore, it was confirmed that rtOatp1d1 is a MC-LR transporter and therefore most likely plays a key role in the susceptibility of rainbow trout to MC intoxications. PMID:25218291

  10. Molecular cloning and characterization of genistein 4'-O-glucoside specific glycosyltransferase from Bacopa monniera.

    PubMed

    Ruby; Santosh Kumar, R J; Vishwakarma, Rishi K; Singh, Somesh; Khan, Bashir M

    2014-07-01

    Health related benefits of isoflavones such as genistein are well known. Glycosylation of genistein yields different glycosides like genistein 7-O-glycoside (genistin) and genistein 4'-O-glycoside (sophoricoside). This is the first report on isolation, cloning and functional characterization of a glycosyltransferase specific for genistein 4'-O-glucoside from Bacopa monniera, an important Indian medicinal herb. The glycosyltransferase from B. monniera (UGT74W1) showed 49% identity at amino acid level with the glycosyltransferases from Lycium barbarum. The UGT74W1 sequence contained all the conserved motifs present in plant glycosyltransferases. UGT74W1 was cloned in pET-30b (+) expression vector and transformed into E. coli. The molecular mass of over expressed protein was found to be around 52 kDa. Functional characterization of the enzyme was performed using different substrates. Product analysis was done using LC-MS and HPLC, which confirmed its specificity for genistein 4'-O-glucoside. Immuno-localization studies of the UGT74W1 showed its localization in the vascular bundle. Spatio-temporal expression studies under normal and stressed conditions were also performed. The control B. monniera plant showed maximum expression of UGT74W1 in leaves followed by roots and stem. Salicylic acid treatment causes almost tenfold increase in UGT74W1 expression in roots, while leaves and stem showed decrease in expression. Since salicylic acid is generated at the time of injury or wound caused by pathogens, this increase in UGT74W1 expression under salicylic acid stress might point towards its role in defense mechanism.

  11. Molecular Cloning and Characterization of an Acetylcholinesterase cDNA in the Brown Planthopper, Nilaparvata lugens

    PubMed Central

    Yang, Zhifan; Chen, Jun; Chen, Yongqin; Jiang, Sijing

    2010-01-01

    A full cDNA encoding an acetylcholinesterase (AChE, EC 3.1.1.7) was cloned and characterized from the brown planthopper, Nilaparvata lugens Stål (Hemiptera: Delphacidae). The complete cDNA (2467 bp) contains a 1938-bp open reading frame encoding 646 amino acid residues. The amino acid sequence of the AChE deduced from the cDNA consists of 30 residues for a putative signal peptide and 616 residues for the mature protein with a predicted molecular weight of 69,418. The three residues (Ser242, Glu371, and His485) that putatively form the catalytic triad and the six Cys that form intra-subunit disulfide bonds are completely conserved, and 10 out of the 14 aromatic residues lining the active site gorge of the AChE are also conserved. Northern blot analysis of poly(A)+ RNA showed an approximately 2.6-kb transcript, and Southern blot analysis revealed there likely was just a single copy of this gene in N. lugens. The deduced protein sequence is most similar to AChE of Nephotettix cincticeps with 83% amino acid identity. Phylogenetic analysis constructed with 45 AChEs from 30 species showed that the deduced N. lugens AChE formed a cluster with the other 8 insect AChE2s. Additionally, the hypervariable region and amino acids specific to insect AChE2 also existed in the AChE of N. lugens. The results revealed that the AChE cDNA cloned in this work belongs to insect AChE2 subgroup, which is orthologous to Drosophila AChE. Comparison of the AChEs between the susceptible and resistant strains revealed a point mutation, Gly185Ser, is likely responsible for the insensitivity of the AChE to methamidopho in the resistant strain. PMID:20874389

  12. Molecular cloning and characterization of beta-expansin gene related to root hair formation in barley.

    PubMed

    Kwasniewski, Miroslaw; Szarejko, Iwona

    2006-07-01

    Root hairs are specialized epidermal cells that play a role in the uptake of water and nutrients from the rhizosphere and serve as a site of interaction with soil microorganisms. The process of root hair formation is well characterized in Arabidopsis (Arabidopsis thaliana); however, there is a very little information about the genetic and molecular basis of root hair development in monocots. Here, we report on isolation and cloning of the beta-expansin (EXPB) gene HvEXPB1, tightly related to root hair initiation in barley (Hordeum vulgare). Using root transcriptome differentiation in the wild-type/root-hairless mutant system, a cDNA fragment present in roots of wild-type plants only was identified. After cloning of full-length cDNA and genomic sequences flanking the identified fragment, the subsequent bioinformatics analyses revealed homology of the protein coded by the identified gene to the EXPB family. Reverse transcription-PCR showed that expression of HvEXPB1 cosegregated with the root hair phenotype in F2 progeny of the cross between the hairless mutant rhl1.a and the wild-type Karat parent variety. Expression of the HvEXPB1 gene was root specific; it was expressed in roots of wild-type forms, but not in coleoptiles, leaves, tillers, and spikes. The identified gene was active in roots of two other analyzed root hair mutants: rhp1.a developing root hair primordia only and rhs1.a with very short root hairs. Contrary to this, a complete lack of HvEXPB1 expression was observed in roots of the spontaneous root-hairless mutant bald root barley. All these observations suggest a role of the HvEXPB1 gene in the process of root hair formation in barley.

  13. Molecular cloning and characterization of two genes encoding 2-Cys peroxiredoxins from Fasciola gigantica.

    PubMed

    Chaithirayanon, Kulathida; Sobhon, Prasert

    2010-06-01

    In Fasciola species, peroxiredoxin (Prx) serves as the major antioxidant enzyme to remove hydrogen peroxide that is generated from various metabolic reactions, because the parasites lack catalase, and only express glutathione peroxidases at minimal levels. We have cloned and characterized two genes, FgPrx-1 and FgPrx-2, belonging to the 2-Cys Prx family, by immunoscreening of an expressed adult stage Fasciola gigantica cDNA library using a rabbit anti-serum against its tegumental antigens. Predicted FgPrx-1 and FgPrx-2 consisted of 218 amino acids each with predicted molecular weights at 24.63 kDa and 24.57 kDa, respectively. The two predicted F. gigantica Prx proteins exhibited 98% identity to each other, and 52% identity to Prx from oxen which is the natural host. A phylogenetic analysis revealed that FgPrx-1 and FgPrx-2 appear to be closely related to those of Fasciola hepatica. The nucleotide sequences of FgPrx-2 are 654 bp, which is similar to that cloned from newly excysted juveniles of F. hepatica. The FgPrx genes were found to be constitutively expressed in all developmental stages, and with a similar pattern. In the adult parasite, FgPrx transcripts were located in the gut epithelial cells, tegument cells, and cells of reproductive organs, including prostate gland, vitelline glands, testis and ovary. In 4-week-old juveniles, a similar distribution pattern was observed. Metacercaria and newly excysted juveniles exhibited strongest signals for mRNA transcripts in the gut epithelium, and moderately in the tegumental cells. Because of their key role in protecting the parasite and specificities, these proteins may have immunodiagnostic as well as vaccine potentials. Copyright (c) 2010 Elsevier Inc. All rights reserved.

  14. Molecular cloning, over expression and characterization of thermoalkalophilic esterases isolated from Geobacillus sp.

    PubMed

    Tekedar, Hasan Cihad; Sanlı-Mohamed, Gülşah

    2011-03-01

    Due to potential use for variety of biotechnological applications, genes encoding thermoalkalophilic esterase from three different Geobacillus strains isolated from thermal environmental samples in Balçova (Agamemnon) geothermal site were cloned and respective proteins were expressed in Escherichia coli (E.coli) and characterized in detail. Three esterases (Est1, Est2, Est3) were cloned directly by PCR amplification using consensus degenerate primers from genomic DNA of the strains Est1, Est2 and Est3 which were from mud, reinjection water and uncontrolled thermal leak, respectively. The genes contained an open reading frame (ORF) consisting of 741 bp for Est1 and Est2, which encoded 246 amino acids and ORF of Est3 was 729 bp encoded 242 amino acids. The esterase genes were expressed in E. coli and purified using His-Select HF nickel affinity gel. The molecular mass of the recombinant enzyme for each esterase was approximately 27.5 kDa. The three esterases showed high specific activity toward short chain p-NP esters. Recombinant Est1, Est2, Est3 have exhibited similar activity and the highest esterase activity of 1,100 U/mg with p-nitrophenyl acetate (pNPC(2)) as substrate was observed with Est1. All three esterase were most active around 65°C and pH 9.5-10.0. The effect of organic solvents, several metal ions, inhibitors and detergents on enzyme activity for purified Est1, Est2, Est3 were determined separately and compared.

  15. Molecular cloning and analysis of the endogenous retrovirus chemically induced from RFM/Un mouse cell cultures

    SciTech Connect

    Liou, R.S.; Boone, L.R.; Kiggans, J.O.; Yang, D.M.; Wang, T.W.; Tennant, R.W.; Yang, W.K.

    1983-04-01

    An N-tropic ecotropic retrovirus induced with iododeoxyuridine from RFM/Un mouse cell cultures was molecularly cloned and analyzed. Based on the restriction map, the RFM/Un virus appears to be indistinguishable from other induced N-tropic retroviruses. A nucleotide sequence analysis of the long terminal repeat of an infectious clone revealed structural features characteristic of murine type C retrovirus long terminal repeats. The U3 region of the RFM/Un virus long terminal repeat, however, contained no short sequence duplication or insertion found in other murine leukemia virus isolates.

  16. Production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone

    SciTech Connect

    Adachi, A.; Gendelman, H.E.; Koenig, S.; Folks, T.; Willey, R.; Rabson, A.; Martin, M.A.

    1986-08-01

    The authors considered an infectious molecular clone of acquired immunodeficiency syndrome-associated retrovirus. Upon transfection, this clone directed the production of infectious virus particles in a wide variety of cells in addition to human T4 cells. The progeny, infectious virions, were synthesized in mouse, mink, monkey, and several human non-T cell lines, indicating the absence of any intracellular obstacle to viral RNA or protein production or assembly. During the course of these studies, a human colon carcinoma cell line, exquisitely sensitive to DNA transfection, was identified.

  17. Molecular cloning of ICAM-3, a third ligand for LFA-1, constitutively expressed on resting leukocytes.

    PubMed

    Fawcett, J; Holness, C L; Needham, L A; Turley, H; Gatter, K C; Mason, D Y; Simmons, D L

    1992-12-03

    The co-ordinated function of effector and accessory cells in the immune system is assisted by adhesion molecules on the cell surface that stabilize interactions between different cell types. Leukocyte function-associated antigen 1 (LFA-1) is expressed on the surface of all white blood cells and is a receptor for intercellular adhesion molecules (ICAM) 1 and 2 (ref. 3) which are members of the immunoglobulin superfamily. The interaction of LFA-1 with ICAMs 1 and 2 provides essential accessory adhesion signals in many immune interactions, including those between T and B lymphocytes and cytotoxic T cells and their targets. In addition, both ICAMs are expressed at low levels on resting vascular endothelium; ICAM-1 is strongly upregulated by cytokine stimulation and plays a key role in the arrest of leukocytes in blood vessels at sites of inflammation and injury. Recent work has indicated that resting leukocytes express a third ligand, ICAM-3, for LFA-1 (refs 11, 12). ICAM-3 is potentially the most important ligand for LFA-1 in the initiation of the immune response because the expression of ICAM-1 on resting leukocytes is low. We report the expression cloning of a complementary DNA, pICAM-3, encoding a protein constitutively expressed on all leukocytes, which binds LFA-1. ICAM-3 is closely related to ICAM-1, consists of five immunoglobulin domains, and binds LFA-1 through its two N-terminal domains.

  18. Molecular cloning, characterization and expression of the energy homeostasis-associated gene in piglet.

    PubMed

    Wang, Sheng-ping; Gao, Yun-ling; Liu, Gang; Deng, Dun; Chen, Rong-jun; Zhang, Yu-zhe; Li, Li-li; Wen, Qing-qi; Hou, Yong-qing; Feng, Ze-meng; Guo, Zhao-hui

    2015-06-01

    The energy homeostasis-associated (Enho) gene encodes a secreted protein, adropin, which regulates the expression of hepatic lipogenic genes and adipose tissue peroxisome proliferator-activated receptor γ, a major regulator of lipogenesis. In the present study, the porcine (Sus scrofa) homologue of the Enho gene, which was named pEnho, was amplified by reverse transcriptase polymerase chain reaction (RT-PCR) using oligonucleotide primers derived from in silico sequences. The gene sequence was submitted into the GenBank of NCBI, and the access number is GQ414763. The pEnho encodes a protein of 76 amino acids which shows 75% similarity to Homo sapiens adropin. The expression profile of pEnho in tissues (liver, muscle, anterior jejunum, posterior jejunum, and ileum) was determined by quantitative real-time RT-PCR. pEnho was localized on porcine chromosome 10 and no introns were found. In conclusion, pEnho was cloned and analysed with the aim of increasing knowledge about glucose and lipid metabolism in piglets and helping to promote the health and growth of piglets through adropin regulation.

  19. Molecular cloning, characterization and expression of the energy homeostasis-associated gene in piglet*

    PubMed Central

    Wang, Sheng-ping; Gao, Yun-ling; Liu, Gang; Deng, Dun; Chen, Rong-jun; Zhang, Yu-zhe; Li, Li-li; Wen, Qing-qi; Hou, Yong-qing; Feng, Ze-meng; Guo, Zhao-hui

    2015-01-01

    The energy homeostasis-associated (Enho) gene encodes a secreted protein, adropin, which regulates the expression of hepatic lipogenic genes and adipose tissue peroxisome proliferator-activated receptor γ, a major regulator of lipogenesis. In the present study, the porcine (Sus scrofa) homologue of the Enho gene, which was named pEnho, was amplified by reverse transcriptase polymerase chain reaction (RT-PCR) using oligonucleotide primers derived from in silico sequences. The gene sequence was submitted into the GenBank of NCBI, and the access number is GQ414763. The pEnho encodes a protein of 76 amino acids which shows 75% similarity to Homo sapiens adropin. The expression profile of pEnho in tissues (liver, muscle, anterior jejunum, posterior jejunum, and ileum) was determined by quantitative real-time RT-PCR. pEnho was localized on porcine chromosome 10 and no introns were found. In conclusion, pEnho was cloned and analysed with the aim of increasing knowledge about glucose and lipid metabolism in piglets and helping to promote the health and growth of piglets through adropin regulation. PMID:26055914

  20. Comparative analysis of cloned cDNAs encoding Chinese yellow cattle and Gansu black swine integrin receptors for foot-and-mouth disease virus.

    PubMed

    Du, Ping; Shang, Youjun; Yin, Shuanghui; Zhang, Keshan; Wang, Guangxiang; Lv, Zhanlu; Yang, Shunli; Wu, Jinyan; Jin, Ye; Chen, Yan; Liu, Yongjie; Tian, Hong; Liu, Xiangtao

    2013-10-01

    To analyze foot-and-mouth disease virus tropism and host range with respect to the integrin receptor, we cloned cDNAs encoding the integrin αν, β1, β3, β6 and β8 subunits from Chinese yellow cattle and Gansu black swine and carried out comparative analysis of their molecular characteristics. The lengths of the mature proteins and the functional domains of the four integrin β subunits were the same between bovine and swine; however, the number of putative N-linked glycosylation sites and cysteine residues and their arrangement varied. Homology analysis of the nucleotide and amino acid sequences showed that FMDV integrin receptors of Chinese yellow cattle and Gansu black swine are highly conserved. Phylogenetic analysis showed that all FMDV integrin receptor subunits of cattle and swine are clustered into the Artiodactyla group; however, Chinese yellow cattle are phylogenetically closer to sheep than to Gansu black swine. We postulate that the host tropism of FMDV may, in part, be related to the divergence of integrin subunits among different species.

  1. Cloning and identification of a novel thyroid hormone receptor β isoform expressed in the pituitary gland.

    PubMed

    Zhao, Rong-Lan; Sun, Bei; Liu, Ying; Li, Jing-Hua; Xiong, Wei-Li; Liang, Dong-Chun; Guo, Gang; Zuo, Ai-Jun; Zhang, Jing-Yu

    2014-04-01

    We have previously identified a novel Trβ isoform (TrβΔ) in the rat, in which a novel exon N (108 bps) was found between exon 3 and exon 4 of TrβΔ, which represents the only difference between TrβΔ and Trβ1. In this study, we searched for an elongated Trβ2-like subtype with one additional exon N. We successfully isolated the entire mRNA/cDNA of a novel elongated Trβ2 isoform via PCR in the rat pituitary gland. The mRNA/cDNA was only 108 bps (exon N) longer than that Trβ2, and the extension of the sequence was between exon 3 and 4 of Trβ. The whole sequence of this novel Trβ isoform has been published in NCBI GenBank (HM043807.1); it is named TRbeta2Delta (Trβ2Δ). In adult rat pituitary tissue, quantitative real-time RT-PCR analysis showed that the mRNA levels of Trβ2Δ and Trβ2 were roughly equal (P > 0.05). We cloned, expressed, and purified the His-Trβ2Δ protein [recombinant TRβ2Δ (rTRβ2Δ)]. SDS-PAGE and western blotting revealed that the molecular weight of rTRβ2Δ was 58.2 kDa. Using a radioligand binding assay and an electrophoretic mobility shift assay, rTRβ2Δ-bound T3 with high affinity and recognized thyroid hormone response element (TRE) binding sites. Finally, in vitro transfection experiments further confirmed that rTRβ2Δ binding T3 significantly promotes the transcription of target genes via the TRE. Here, we have provided evidence suggesting that rTRβ2Δ is a novel functional TR isoform.

  2. Two base changes restore infectivity to a noninfectious molecular clone of Moloney murine Leukemia virus (pMLV-1)

    SciTech Connect

    Miller, A.D.; Verma, I.M.

    1984-01-01

    The complete nucleotide sequence of a molecular clone of Moloney murine leukemia virus (pMLV-1) has previously been reported. However, pMLV-1 does not generate infectious virus after transfection into cells. The lesion in pMLV-1 has been localized by determining the biological activity of recombinants containing DNA from kilobase pair region which spans the gag-pol junction of pMLV-1 with the corresponding DNA fragment from the infectious clone (pMLV-48) and pMLV-1 reveals two single base pair changes. The mutation in the pol gene does not affect the production of infectious virus but renders them XC negative, whereas the mutation in the gag gene appears to be lethal. The complete nucleotide sequence of an infectious clone of Moloney murine leukemia virus can now be deduced.

  3. Molecular cloning of the unintegrated squirrel monkey retrovirus genome: organization and distribution of related sequences in primate DNAs.

    PubMed Central

    Chiu, I M; Andersen, P R; Aaronson, S A; Tronick, S R

    1983-01-01

    The closed circular form of the endogenous squirrel monkey type D retrovirus (SMRV) was molecularly cloned in a bacteriophage vector. The restriction map of the biologically active clone was determined and found to be identical to that of the parental SMRV linear DNA except for the deletion of one long terminal repeat. Restriction enzyme analysis and Southern blotting indicated that the SMRV long terminal repeat was approximately 300 base pairs long. The SMRV restriction map was oriented to the viral RNA by using a gene-specific probe from baboon endogenous virus. Restriction enzyme digests of a variety of vertebrate DNAs were analyzed for DNA sequence homology with SMRV by using the cloned SMRV genome as a probe. Consistent with earlier studies, multiple copies of SMRV were detected in squirrel monkey DNA. Related fragments were also detected in the DNAs from other primate species, including humans. Images PMID:6312076

  4. Cell and molecular biology of epidermal growth factor receptor.

    PubMed

    Ceresa, Brian P; Peterson, Joanne L

    2014-01-01

    The epidermal growth factor receptor (EGFR) has been one of the most intensely studied cell surface receptors due to its well-established roles in developmental biology, tissue homeostasis, and cancer biology. The EGFR has been critical for creating paradigms for numerous aspects of cell biology, such as ligand binding, signal transduction, and membrane trafficking. Despite this history of discovery, there is a continual stream of evidence that only the surface has been scratched. New ways of receptor regulation continue to be identified, each of which is a potential molecular target for manipulating EGFR signaling and the resultant changes in cell and tissue biology. This chapter is an update on EGFR-mediated signaling, and describes some recent developments in the regulation of receptor biology.

  5. Construction and characterization of HIV type 1 CRF07_BC infectious molecular clone from men who have sex with men.

    PubMed

    Jiang, Yan-Ling; Bai, Wen-Wei; Qu, Fan-Wei; Ma, Hua; Jiang, Run-Sheng; Shen, Bao-Sheng

    2016-03-01

    This study aimed to investigate the biological characterization of HIV type 1 (HIV-1) CRF07_BC infection among men who have sex with men (MSM). From November 2011 to November 2013, a total of 66 blood samples were collected from MSM with acute HIV-1 infection with CRF07_BC subgroup strains. Deletion in the gag p6 region was detected by sequence alignment and comparative analysis. Peripheral blood mononuclear cells (PBMCs) of HNXX1301-1307 samples were separated by density gradient centrifugation. Nested polymerase chain reaction (nPCR) was used to amplify the viral DNA. The near full-length HIV-1 DNA products were ligated to the long terminal repeat (LTR) vector plasmid (07BCLTR) to construct a full-length HIV clone. The molecular clone was transfected into HEK-293T cells, TZM-b1 cells and patients' PBMCs. The pregenome of an infectious molecular clone of HIV-1 (pNL4-3) was amplified, and a subclone with CRF07_BC was developed to construct the full-length chimeric molecular clone pNL4-3/07BCLTR. Detection of p24 antigen and luciferase activity was used to measure the in vitro infectivity of pNL4-3/07BCLTR. Among the 66 MSM patients infected with CRF07_BC strains, deletion mutations of the Gag P6 proteins were found in 7 of 18CRF07_BC strains; deletion mutations of 2-13 amino acids in different regions were discovered in 6 strains; and the remaining 42 strains did not show deletions. Seven strains with amino acids deficiency in the P6 protein accounted for 27% of all strains and 75% of all deletion genotype strains. A total of 186 full-length molecular clones of CRF07_BC were constructed. There were 5, 9, 10 and 11 clones of HNXX1302, HNXX1304, HNXX1305 and HNXX1306 that resulted in p24-positive supernatant when transfected into HEK-293T cells. Full-length clones of HNXX1302, HNXX1304, HNXX1305 and HNXX1306 showed slight infection in the transfected TZM-b1 cells, as judged by the fluorescence values of TZM-b1 cells 48h post-transfection. However, we were unable to

  6. Human Fc. gamma. RIII: Cloning, expression, and identification of the chromosomal locus of two Fc receptors for IgG

    SciTech Connect

    Peltz, G.A.; Moore, K.W. ); Grundy, H.O.; Lebo, R.V.; Barsh, G.S. ); Yssel, H. )

    1989-02-01

    A cDNA clone encoding a human receptor for the Fc portion of IgG Fc{gamma}RIII or CD16, was isolated from a human leukocyte library by a transient expression-immunoselection procedure. This cDNA (pGP5) encodes a 46-kDa phosphatidylinositol-linked cell surface protein with CD16 determinants and affinity for human IgG. The deduced protein sequence is most homologous to the murine receptor Fc{gamma}RII{alpha}, with slightly less homology to the human receptors Fc{gamma}RII and Fc{epsilon}RI. The cDNA hybridizes to a 2.2 kilobase mRNA in human leukocytes and a cloned human natural killer cell line. Fc{gamma}RIII is mapped to chromosome 1 by spot-blot analysis of sorted human chromosomes. Hybridization of Fc{gamma}RII and Fc{gamma}RIII probes to restriction digests of human genomic DNA separated by pulsed-field gel electrophoresis demonstrates physical linkage of the two genes within a maximum distance of 200 kilobases. The results identify a locus for at least two Fc{gamma}R genes on human chromosome 1.

  7. Nuclear receptors CAR and PXR: Molecular, functional, and biomedical aspects.

    PubMed

    di Masi, Alessandra; De Marinis, Elisabetta; Ascenzi, Paolo; Marino, Maria

    2009-10-01

    Nuclear receptors (NRs) are ligand-activated transcription factors sharing a common evolutionary history and having similar sequence features at the protein level. Selective ligand(s) for some NRs is not known, therefore these NRs have been named "orphan receptors". Whenever ligands have been recognized for any of the orphan receptor, it has been categorized and grouped as "adopted" orphan receptor. This group includes the constitutive androstane receptor (CAR) and the pregnane X receptor (PXR). They function as sensors of toxic byproducts derived from endogenous metabolites and of exogenous chemicals, in order to enhance their elimination. This unique function of CAR and PXR sets them apart from the steroid hormone receptors. The broad response profile has established that CAR and PXR are xenobiotic sensors that coordinately regulate xenobiotic clearance in the liver and intestine via induction of genes involved in drug and xenobiotic metabolism. In the past few years, research has revealed new and mostly unsuspected roles for CAR and PXR in modulating hormone, lipid, and energy homeostasis as well as cancer and liver steatosis. The purpose of this review is to highlight the structural and molecular bases of CAR and PXR impact on human health, providing information on mechanisms through which diet, chemical exposure, and environment ultimately impact health and disease.

  8. Molecular cloning, sequencing and structural studies of granulocyte-macrophage colony-stimulating factor (GM-CSF) from Indian water buffalo (Bubalus bubalis).

    PubMed

    Sugumar, Thennarasu; Pugalenthi, Ganesan; Harishankar, Murugesan; Dhinakar Raj, G

    2014-02-01

    Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine that is essential for growth and development of progenitors of granulocytes and monocytes/macrophages. In this study, we report molecular cloning, sequencing and characterization of GM-CSF from Indian water buffalo, Bubalus bubalis. In addition, we performed sequence and structural analysis for buffalo GM-CSF. Buffalo GM-CSF has been compared with 17 mammalian GM-CSFs using multiple sequence alignment and phylogenetic tree. Three-dimensional model for buffalo GM-CSF and human receptor complex was built using homology modelling to study cross-reactivity between two species. Detailed analysis was performed to study GM-CSF interface and various interactions at the interface. © 2013 John Wiley & Sons Ltd.

  9. Human eosinophil cationic protein. Molecular cloning of a cytotoxin and helminthotoxin with ribonuclease activity

    PubMed Central

    1989-01-01

    We have isolated a 725-bp full-length cDNA clone for the human eosinophil cationic protein (ECP). ECP is a small, basic protein found in the matrix of the eosinophil's large specific granule that has cytotoxic, helminthotoxic, and ribonuclease activity, and is a member of the ribonuclease multigene family. The cDNA sequence shows 89% sequence identity with that reported for the related granule protein, eosinophil-derived neurotoxin (EDN). The open reading frame encodes a previously unidentified 27-amino acid leader sequence preceding a 133- residue mature ECP polypeptide with a molecular mass of 15.6 kD. The encoded amino acid sequence of ECP shows 66% identity to that of EDN and 31% identity to that of human pancreatic ribonuclease, including conservation of the essential structural cysteine and cataytic lysine and histidine residues. mRNA for ECP was detected in eosinophil- enriched peripheral granulocytes and in a subclone of the promyelocytic leukemia line, HL-60, induced toward eosinophilic differentiation with IL-5. No ECP mRNA was detected in uninduced HL-60 cells, or in HL-60 cells induced toward monocytic differentiation with vitamin D3 or toward neutrophilic differentiation with DMSO. In contrast, mRNA for EDN was detected in uninduced HL-60 cells and was upregulated in HL-60 cells induced with DMSO. Despite similarities in sequence and cellular localization, these results suggest that ECP and EDN are subject to different regulatory mechanisms. PMID:2473157

  10. Molecular cloning and evolutionary analysis of the GJA1 (connexin43) gene from bats (Chiroptera).

    PubMed

    Wang, Li; Li, Gang; Wang, Jinhong; Ye, Shaohui; Jones, Gareth; Zhang, Shuyi

    2009-04-01

    Gap junction protein connexin43 (Cx43), encoded by the GJA1 gene, is the most abundant connexin in the cardiovascular system and was reported as a crucial factor maintaining cardiac electrical conduction, as well as having a very important function in facilitating the recycling of potassium ions from hair cells in the cochlea back into the cochlear endolymph during auditory transduction processes. In mammals, bats are the only taxon possessing powered flight, placing exceptional demand on many organismal processes. To meet the demands of flying, the hearts of bats show many specialties. Moreover, ultrasonic echolocation allows bat species to orientate and often detect and locate food in darkness. In this study, we cloned the full-length coding region of GJA1 gene from 12 different species of bats and obtained orthologous sequences from other mammals. We used the maximum likelihood method to analyse the evolution of GJA1 gene in mammals and the lineage of bats. Our results showed this gene is much conserved in mammals, as well as in bats' lineage. Compared with other mammals, we found one private amino acid substitution shared by bats, which is located on the inner loop domain, as well as some species-specific amino acid substitutions. The evolution rate analyses showed the signature of purifying selection on not only different classification level lineages but also the different domains and amino acid residue sites of this gene. Also, we suggested that GJA1 gene could be used as a good molecular marker to do the phylogenetic reconstruction.

  11. Identification, purification, and molecular cloning of a putative plastidic glucose translocator.

    PubMed

    Weber, A; Servaites, J C; Geiger, D R; Kofler, H; Hille, D; Gröner, F; Hebbeker, U; Flügge, U I

    2000-05-01

    During photosynthesis, part of the fixed carbon is directed into the synthesis of transitory starch, which serves as an intermediate carbon storage facility in chloroplasts. This transitory starch is mobilized during the night. Increasing evidence indicates that the main route of starch breakdown proceeds by way of hydrolytic enzymes and results in glucose formation. This pathway requires a glucose translocator to mediate the export of glucose from the chloroplasts. We have reexamined the kinetic properties of the plastidic glucose translocator and, using a differential labeling procedure, have identified the glucose translocator as a component of the inner envelope membrane. Peptide sequence information derived from this protein was used to isolate cDNA clones encoding a putative plastidic glucose translocator from spinach, potato, tobacco, Arabidopsis, and maize. We also present the molecular characterization of a candidate for a hexose transporter of the plastid envelope membrane. This transporter, initially characterized more than 20 years ago, is closely related to the mammalian glucose transporter GLUT family and differs from all other plant hexose transporters that have been characterized to date.

  12. Purification and molecular cloning of a new galactose-specific lectin from Bauhinia variegata seeds.

    PubMed

    Pinto, Luciano S; Nagano, Celso S; Oliveira, Taianá M; Moura, Tales R; Sampaio, Alexandre H; Debray, Henri; Pinto, Vicente P; Dellagostin, Odir A; Cavada, Benildo S

    2008-09-01

    A new galactose-specific lectin was purified from seeds of a Caesalpinoideae plant, Bauhinia variegata, by affinity chromatography on lactose-agarose. Protein extracts haemagglutinated rabbit and human erythrocytes (native and treated with proteolytic enzymes), showing preference for rabbit blood treated with papain and trypsin. Among various carbohydrates tested, the lectin was best inhibited by D-galactose and its derivatives, especially lactose. SDS-PAGE showed that the lectin, named BVL, has a pattern similar to other lectins isolated from the same genus, Bauhinia purpurea agglutinin (BPA). The molecular mass of BVL subunit is 32 871 Da, determined by MALDI-TOF spectrometry. DNA extracted from B.variegata young leaves and primers designed according to the B. purpurea lectin were used to generate specific fragments which were cloned and sequenced, revealing two distinct isoforms. The bvl gene sequence comprised an open reading frame of 876 base pairs which encodes a protein of 291 amino acids. The protein carried a putative signal peptide. The mature protein was predicted to have 263 amino acid residues and 28 963 Da in size.

  13. Molecular cloning, heterologous expression, and characterization of Ornithine decarboxylase from Oenococcus oeni.

    PubMed

    Bonnin-Jusserand, Maryse; Grandvalet, Cosette; David, Vanessa; Alexandre, Hervé

    2011-08-01

    Ornithine decarboxylase (ODC) is responsible for the production of putrescine, the major biogenic amine found in wine. Oenococcus oeni is the most important lactic acid bacterium in the winemaking process and is involved in malolactic fermentation. We report here the characterization of ODC from an O. oeni strain isolated from wine. Screening of 263 strains isolated from wine and cider from all over the world revealed that the presence of the odc gene appears to be strain specific in O. oeni. After cloning, heterologous expression in Escherichia coli, and characterization, the enzyme was found to have a molecular mass of 85 kDa and a pI of 6.2 and revealed maximal activity at pH 5.5 and an optimum temperature of 35°C. Kinetic studies showed that O. oeni ODC is specific for L-ornithine with a K(m) value of 1 mM and a V(max) of 0.57 U·mg(-1). The hypothesis that cadaverine, which results from lysine decarboxylation, may be linked to putrescine production is not valid since O. oeni ODC cannot decarboxylate L-lysine. As no lysine decarboxylase was detected in any of the O. oeni genomes sequenced, cadaverine synthesis may result from another metabolic pathway. This work is the first characterization of an ODC from a lactic acid bacterium isolated from a fermented product.

  14. Molecular cloning and characterization of a cDNA encoding the Paracoccidioides brasiliensis 135 ribosomal protein.

    PubMed

    Jesuino, Rosália S A; Pereira, Maristela; Felipe, M Sueli S; Azevedo, Maristella O; Soares, Célia M A

    2004-06-01

    A 630 bp cDNA encoding an L35 ribosomal protein of Paracoccidioides brasiliensis, designated as Pbl35, was cloned from a yeast expression library. Pbl35 encodes a polypeptide of 125 amino acids, with a predicted molecular mass of 14.5 kDa and a pI of 11.0. The deduced PbL35 shows significant conservation in respect to other described ribosomal L35 proteins from eukaryotes and prokaryotes. Motifs of ribosomal proteins are present in PbL35, including a bipartite nuclear localization signal (NLS) that could be related to the protein addressing to the nucleolus for the ribosomal assembly. The mRNA for PbL35, about 700 nucleotides in length, is expressed at a high level in P. brasiliensis. The PbL35 and the deduced amino acid sequence constitute the first description of a ribosomal protein in P. brasiliensis. The cDNA was deposited in GenBank under accession number AF416509.

  15. Monofunctional catalase P of Paracoccidioides brasiliensis: identification, characterization, molecular cloning and expression analysis.

    PubMed

    Moreira, Sabrina F I; Bailão, Alexandre M; Barbosa, Mônica S; Jesuino, Rosalia S A; Felipe, M Sueli Soares; Pereira, Maristela; de Almeida Soares, Célia Maria

    2004-01-30

    Within the context of studies on genes from Paracoccidioides brasiliensis (Pb) potentially associated with fungus-host interaction, we isolated a 61 kDa protein, pI 6.2, that was reactive with sera of patients with paracoccidioidomycosis. This protein was identified as a peroxisomal catalase. A complete cDNA encoding this catalase was isolated from a Pb cDNA library and was designated PbcatP. The cDNA contained a 1509 bp ORF containing 502 amino acids, whose molecular mass was 57 kDa, with a pI of 6.5. The translated protein PbCATP revealed canonical motifs of monofunctional typical small subunit catalases and the peroxisome-PTS-1-targeting signal. The deduced and the native PbCATP demonstrated amino acid sequence homology to known monofunctional catalases and was most closely related to catalases from other fungi. The protein and mRNA were diminished in the mycelial saprobic phase compared to the yeast phase of infection. Protein synthesis and mRNA levels increased during the transition from mycelium to yeast. In addition, the catalase protein was induced when cells were exposed to hydrogen peroxide. The identification and characterization of the PbCATP and cloning and characterization of the cDNA are essential steps for investigating the role of catalase as a defence of P. brasiliensis against oxygen-dependent killing mechanisms. These results suggest that this protein exerts an influence in the virulence of P. brasiliensis.

  16. Molecular cloning and structural characterization of Ecdysis Triggering Hormone from Choristoneura fumiferana.

    PubMed

    P, Bhagath Kumar; K, Kasi Viswanath; S, Tuleshwori Devi; R, Sampath Kumar; Doucet, Daniel; Retnakaran, Arthur; Krell, Peter J; Feng, Qili; Ampasala, Dinakara Rao

    2016-07-01

    At the end of each stadium, insects undergo a precisely orchestrated process known as ecdysis which results in the replacement of the old cuticle with a new one. This physiological event is necessary to accommodate growth in arthropods since they have a rigid chitinous exoskeleton. Ecdysis is initiated by the direct action of Ecdysis Triggering Hormones on the central nervous system. Choristoneura fumiferana is a major defoliator of coniferous forests in Eastern North America. It is assumed that, studies on the ecdysis behavior of this pest might lead to the development of novel pest management strategies. Hence in this study, the cDNA of CfETH was cloned. The open reading frame of the cDNA sequence was found to encode three putative peptides viz., Pre-Ecdysis Triggering Hormone (PETH), Ecdysis Triggering Hormone (ETH), and Ecdysis Triggering Hormone Associated Peptide (ETH-AP). The CfETH transcript was detected in the epidermal tissue of larval and pupal stages, but not in eggs and adults. In order to explore the structural conformation of ETH, ab initio modelling and Molecular Dynamics (MD) Simulations were performed. Further, a library of insecticides was generated and virtual screening was performed to identify the compounds displaying high binding capacity to ETH.

  17. Characterization, molecular cloning, and expression analysis of Ecsit in the spinyhead croaker, Collichthys lucidus.

    PubMed

    Song, W; Jiang, K J; Zhang, F Y; Wang, J; Ma, L B

    2016-01-08

    Evolutionarily conserved signaling intermediate in Toll pathways (Ecsit) is reported to play an essential role in innate immunity, embryogenesis, and assembly or stability of the mitochondrial complex I. In this study, the full-length cDNA of Ecsit was cloned from the spinyhead croaker Collichthys lucidus based on the expressed sequence tags from our cDNA library constructed using the SMART technique. The cDNA was 1669 bp long, including a 5'-terminal untranslated region (UTR) of 121 bp, a 3'-terminal UTR of 183 bp, and an open reading frame of 1365 bp encoding a 454-amino acid polypeptide. The estimated molecular weight of C. lucidus Ecsit (ClEcsit) was 52.50 kDa with an isoelectric point of 6.14, and contained a typical Ecsit domain that is conserved in other Ecsits. Multiple alignment of ClEcsit with other selected Ecsits suggested that some amino acid residues were highly conserved. Phylogenetic analysis indicated that ClEcsit was more similar to its identities in Sciaenidae and grouped with Ecsits from other Perciformes. Quantitative real-time reverse transcription PCR analysis revealed broad expression of ClEcsit and the transcript was strongly expressed in the gill and weakly expressed in other tissues.

  18. Molecular cloning of cDNA encoding the Xenopus homolog of mammalian RelB.

    PubMed Central

    Suzuki, K; Yamamoto, T; Inoue, J

    1995-01-01

    We have molecularly cloned cDNA encoding a new Rel-related protein in Xenopus laevis. Nucleotide sequencing revealed that the product is most homologous to mammalian RelB in its N-terminal region. Furthermore, the putative protein kinase A phosphorylation site (RRPS), found in most of the Rel family proteins, but replaced by QRLT in mammalian RelB, is replaced by QRIT, indicating that our cDNA most likely encodes the Xenopus homolog of mammalian RelB (XrelB). As in the case of mouse RelB, XrelB alone does not bind to DNA efficiently, while XrelB/human p50 heterodimers bind to kappa B sites and activate transcription. XrelB transcripts are present at all stages of oocyte maturation and in adult tissues examined. However, in staged embryos XrelB is undetectable from neurula to stage 28 and resumes expression at stage 47, while Xrel1/XrelA, the Xenopus homolog of p65, has been demonstrated to be expressed throughout embryogenesis. These results raise the possibility that XrelB and Xrel1/XrelA play different roles in the development of X.laevis. Images PMID:8524658

  19. Molecular characterization of Staphylococcus aureus from outpatients in the Caribbean reveals the presence of pandemic clones

    PubMed Central

    Dumortier, C.; Hafer, C.; Taylor, B. S.; Sánchez E, J.; Rodriguez-Taveras, C.; Leon, P.; Rojas, R.; Olive, C.; Lowy, F. D.

    2011-01-01

    Staphylococcus aureus infections continue to pose a global public health problem. Frequently, this epidemic is driven by the successful spread of single S. aureus clones within a geographic region, but international travel has been recognized as a potential risk factor for S. aureus infections. To study the molecular epidemiology of S. aureus infections in the Caribbean, a major international tourist destination, we collected methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) isolates from community-onset infections in the Dominican Republic (n=112) and Martinique (n=143). Isolates were characterized by a combination of pulsed-field gel electrophoresis (PFGE), spa typing, and multilocus sequence typing (MLST) typing. In Martinique, MRSA infections (n=56) were mainly caused by t304-ST8 strains (n=44), whereas MSSA isolates were derived from genetically diverse backgrounds. Among MRSA strains (n=22) from the Dominican Republic, ST5, ST30, and ST72 predominated, while ST30 t665-PVL+ (30/90) accounted for a substantial number of MSSA infections. Despite epidemiological differences in sample collections from both countries, a considerable number of MSSA infections (~10%) were caused by ST5 and ST398 isolates at each site. Further phylogenetic analysis suggests the presence of lineages shared by the two countries, followed by recent genetic diversification unique to each site. Our findings also imply the frequent import and exchange of international S. aureus strains in the Caribbean. PMID:21789605

  20. Molecular cloning, expression and characterization of acylpeptide hydrolase in the silkworm, Bombyx mori.

    PubMed

    Fu, Ping; Sun, Wei; Zhang, Ze

    2016-04-10

    Acylpeptide hydrolase (APH) can catalyze the release of the N-terminal amino acid from acetylated peptides. There were many documented examples of this enzyme in various prokaryotic and eukaryotic organisms. However, knowledge about APH in insects still remains unknown. In this study, we cloned and sequenced a putative silkworm Bombyx mori APH (BmAPH) gene. The BmAPH gene encodes a protein of 710 amino acids with a predicted molecular mass of 78.5kDa. The putative BmAPH and mammal APHs share about 36% amino acid sequence identity, yet key catalytic residues are conserved (Ser566, Asp654, and His686). Expression and purification of the recombinant BmAPH in Escherichia coli showed that it has acylpeptide hydrolase activity toward the traditional substrate, Ac-Ala-pNA. Furthermore, organophosphorus (OP) insecticides, chlorpyrifos, phoxim, and malathion, significantly inhibited the activity of the APH both in vitro and in vivo. In addition, BmAPH was expressed in all tested tissues and developmental stages of the silkworm. Finally, immunohistochemistry analysis showed that BmAPH protein was localized in the basement membranes. These results suggested that BmAPH may be involved in enhancing silkworm tolerance to the OP insecticides. In a word, our results provide evidence for understanding of the biological function of APH in insects. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. Molecular cloning and functional expression of human connexin37, an endothelial cell gap junction protein.

    PubMed Central

    Reed, K E; Westphale, E M; Larson, D M; Wang, H Z; Veenstra, R D; Beyer, E C

    1993-01-01

    Gap junctions allow direct intercellular coupling between many cells including those in the blood vessel wall. They are formed by a group of related proteins called connexins, containing conserved transmembrane and extracellular domains, but unique cytoplasmic regions that may confer connexin-specific physiological properties. We used polymerase chain reaction amplification and cDNA library screening to clone DNA encoding a human gap junction protein, connexin37 (Cx37). The derived human Cx37 polypeptide contains 333 amino acids, with a predicted molecular mass of 37,238 D. RNA blots demonstrate that Cx37 is expressed in multiple organs and tissues (including heart, uterus, ovary, and blood vessel endothelium) and in primary cultures of vascular endothelial cells. Cx37 mRNA is coexpressed with connexin43 at similar levels in some endothelial cells, but at much lower levels in others. To demonstrate that Cx37 could form functional channels, we stably transfected communication-deficient Neuro2A cells with the Cx37 cDNA. The induced intercellular channels were studied by the double whole cell patch clamp technique. These channels were reversibly inhibited by the uncoupling agent, heptanol (2 mM). The expressed Cx37 channels exhibited multiple conductance levels and showed a pronounced voltage dependence. These electrophysiological characteristics are similar to, but distinct from, those of previously characterized connexins. Images PMID:7680674

  2. Molecular cloning, characterization and expression analysis of Tim-3 and Galectin-9 in the woodchuck model.

    PubMed

    Liu, Yanan; Wang, Junzhong; Wang, Lu; Wang, Baoju; Yang, Shangqing; Wang, Qin; Luo, Jinzhuo; Feng, Xuemei; Yang, Xuecheng; Lu, Yinping; Roggendorf, Michael; Lu, Mengji; Yang, Dongliang; Liu, Jia

    2017-03-01

    In recent years, a critical role for T cell immunoglobulin mucin domain 3 (Tim-3) and its ligand Galectin-9 (Gal-9) has emerged in infectious disease, autoimmunity and cancer. Manipulating this immune checkpoint may have immunotherapeutic potential and could represent an alternative approach for improving immune responses to viral infections and cancer. The woodchuck (Marmot monax) infected by woodchuck hepatitis virus (WHV) represents an informative animal model to study HBV infection and HCC. In the current study, the cDNA sequences of woodchuck Tim-3 and Gal-9 were cloned, sequenced and characterized. The extracellular domain of Tim-3 cDNA sequence consisted of 576bp coding sequence (CDS) that encoded 192 amino acids. The 1076bp full-length Gal-9 cDNA sequence consisted of 1059bp coding sequence (CDS) that encoded 352 amino acids with a molecular weight of 39.7kDa. The phylogenetic tree analysis revealed that the woodchuck Tim-3 and Gal-9 had the closest genetic relationship with Ictidomys tridecemlineatus. The result of quantification PCR analysis showed that ubiquitous expression of Gal-9 but not Tim-3 in different tissues of naive woodchucks. Elevated liver Gal-9 expression was observed in woodchucks with chronic WHV infection. Moreover, a polyclonal antibody against the extracellular domain of woodchuck Tim-3 were generated and identified by flow cytometry. Our results serve as a foundation for further insight into the role of Tim-3/Galectin-9 signaling pathway in viral hepatitis and HCC in the woodchuck model.

  3. Molecular cloning and biochemical characterization of a lipoxygenase in almond (Prunus dulcis) seed.

    PubMed

    Mita, G; Gallo, A; Greco, V; Zasiura, C; Casey, R; Zacheo, G; Santino, A

    2001-03-01

    We have characterized an almond (Prunus dulcis) lipoxygenase (LOX) that is expressed early in seed development. The presence of an active lipoxygenase was confirmed by western blot analysis and by measuring the enzymatic activity in microsomal and soluble protein samples purified from almond seeds at this stage of development. The almond lipoxygenase, which had a pH optimum around 6, was identified as a 9-LOX on the basis of the isomers of linoleic acid hydroperoxides produced in the enzymatic reaction. A genomic clone containing a complete lipoxygenase gene was isolated from an almond DNA library. The 6776-bp sequence reported includes an open reading frame of 4667 bp encoding a putative polypeptide of 862 amino acids with a calculated molecular mass of 98.0 kDa and a predicted pI of 5.61. Almond seed lipoxygenase shows 71% identity with an Arabidopsis LOX1 gene and is closely related to tomato fruit and potato tuber lipoxygenases. The sequence of the active site was consistent with the isolated gene encoding a 9-LOX.

  4. Molecular cloning, expression pattern and comparative analysis of chitin synthase gene B in Spodoptera exigua.

    PubMed

    Kumar, N Senthil; Tang, Bin; Chen, Xiaofei; Tian, Honggang; Zhang, Wenqing

    2008-03-01

    The chitin synthase (CHS) gene B (4781 bp) of Spodoptera exigua (SeCHSB) was cloned by reverse-transcription PCR (RT-PCR) and 3'/5' RACE from the midgut. SeCHSB contains an open reading frame of 4572 nucleotide