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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 and expression of the human interleukin 5 receptor

    PubMed Central

    1992-01-01

    Human interleukin 5 (IL-5) plays an important role in proliferation and differentiation of human eosinophils. We report the isolation of cDNA clones from cDNA libraries of human eosinophils by using murine IL-5 receptor alpha chain cDNA as a probe. Analysis of the predicted amino acid sequence indicated that the human IL-5 receptor has approximately 70% amino acid sequence homology with the murine IL-5 receptor and retains features common to the cytokine receptor superfamily. One cDNA clone encodes a glycoprotein of 420 amino acids (Mr 47,670) with an NH2- terminal hydrophobic region (20 amino acids), a glycosylated extracellular domain (324 amino acids), a transmembrane domain (21 amino acids), and a cytoplasmic domain (55 amino acids). Another cDNA encodes only the extracellular domain of this receptor molecule. Other cDNA clones encode molecules having diversified cytoplasmic domains. COS7 cells transfected with the cDNA expressed a approximately 60-kD protein and bound IL-5 with a single class of affinity (Kd = 250-590 pM). The Kd values were similar to that observed in normal human eosinophils. In contrast to the murine 60-kD alpha chain, which binds IL-5 with low affinity (Kd = approximately 10 nM), the human alpha chain homologue can bind IL-5 with much higher affinity by itself. RNA blot analysis of human cells demonstrated two transcripts (approximately 5.3 and 1.4 kb). Both of them were expressed in normal human eosinophils and in erythroleukemic cell line TF-1, which responds to IL-5. The human IL-5 receptor characterized in this paper is essential for signal transduction, because expression of this molecule in murine IL-3-dependent cell line FDC-P1 allowed these cells to proliferate in response to IL-5. PMID:1732409

  3. The 5-HT4 receptor: molecular cloning and pharmacological characterization of two splice variants.

    PubMed Central

    Gerald, C; Adham, N; Kao, H T; Olsen, M A; Laz, T M; Schechter, L E; Bard, J A; Vaysse, P J; Hartig, P R; Branchek, T A

    1995-01-01

    Molecular cloning efforts have provided primary amino acid sequence and signal transduction data for a large collection of serotonin receptor subtypes. These include five 5-HT1-like receptors, three 5-HT2 receptors, one 5-HT3 receptor, two 5-HT5 receptors, one 5-HT6 receptor and one 5-HT7 receptor. Molecular biological information on the 5-HT4 receptor is notably absent from this list. We now report the cloning of the pharmacologically defined 5-HT4 receptor. Using degenerate oligonucleotide primers, we identified a rat brain PCR fragment which encoded a '5-HT receptor-like' amino acid sequence. The corresponding full length cDNA was isolated from a rat brain cDNA library. Transiently expressed in COS-7 cells, this receptor stimulates adenylyl cyclase activity and is sensitive to the benzamide derivative cisapride. The response is also blocked by ICS-205930. Interestingly, we isolated two splice variants of the receptor, 5-HT4L and 5-HT4S, differing in the length and sequence of their C-termini. In rat brain, the 5-HT4S transcripts are restricted to the striatum, but the 5-HT4L transcripts are expressed throughout the brain, except in the cerebellum where it was barely detectable. In peripheral tissues, differential expression was also observed in the atrium of the heart where only the 5-HT4S isoform was detectable. Images PMID:7796807

  4. 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.

  5. 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.

  6. Molecular cloning, functional expression and pharmacological characterization of a mouse melanocortin receptor gene.

    PubMed Central

    Desarnaud, F; Labbe, O; Eggerickx, D; Vassart, G; Parmentier, M

    1994-01-01

    We describe the cloning of the mouse HGMP01A gene that encodes a melanocortin receptor functionally distinct from the adrenal cortex corticotropin (adrenocorticotrophic hormone; ACTH) receptor and the melanocyte-stimulating hormone (MSH) receptor expressed in melanoma. The gene encodes a protein of 323 amino acids with a calculated molecular mass of 35,800 Da, displaying potential sites for N-linked glycosylation and phosphorylation by protein kinase C. An RNAase protection assay detected weak expression in the brain, but not in adrenal gland, skin, or any of the other tissues tested. Stable CHO cell lines expressing over 100,000 receptors per cell were generated. The recombinant receptor binds iodinated [Nle4,D-Phe7]alpha-MSH (NDP-MSH) with an apparent Kd of 700 pM. Displacement of the ligand by a variety of pro-opiomelanocortin-derived peptides revealed a pharmacological profile distinct from that of the classical ACTH and MSH receptors. NDP-MSH was the most powerful competitor (IC50 1.4 nM), followed by gamma-MSH (IC50 7 nM). alpha-MSH, beta-MSH and ACTH-(1-39) were significantly less potent, with IC50 values of 30, 19 and 21 nM respectively. ACTH-(4-10) was poorly active (IC50 2.4 microM), while corticotropin-like intermediate lobe peptide (CLIP) and beta-endorphin were totally ineffective. The recombinant receptor was found to stimulate adenylate cyclase. The potency order of the agonists in this assay was consistent with that of the binding displacement assays. This receptor represents the orthologue of the human melanocortin 3 receptor reported recently. The growing family of melanocortin receptors constitute the molecular basis for the variety of actions of melanocortins that have been described over the years. The availability of functionally expressed receptors from the melanocortin family will allow the development of a specific pharmacology, and a better understanding of the function of the pro-opiomelanocortin-derived peptides. Images Figure 6 PMID

  7. Molecular cloning and functional characterization of murine toll‑like receptor 8.

    PubMed

    Li, Tingting; He, Xiaobing; Jia, Huaijie; Chen, Guohua; Zeng, Shuang; Fang, Yongxiang; Jin, Qiwang; Jing, Zhizhong

    2016-02-01

    Toll-like receptors (TLRs) are a large family of germ-line encoded pattern recognition receptors (PRRs) that recognize pathogen-associated molecular patterns and evoke the relevant innate immune responses. TLR8 is a member of several endosome nucleic acid-sensing TLRs; however little attention has been paid to murine TLR8 (mTLR8) compared with other endosome nucleic acid-sensing TLRs. In the present study, mTLR8 was cloned using reverse transcription-polymerase chain reaction from murine peripheral blood mononuclear cells and its function in regulating innate immune response was characterized. The open reading frame of mTLR8 consists of 3,099 bps and encodes 1,032 amino acids. It contains typical leucine-rich repeats, a transmembrane domain and a Toll/interleukin-1 receptor domain, and it shares a high level of identity with other mammalian species. The expression of mTLR8 has been widely observed in different tissues, and higher expression levels of mTLR8 have mainly been detected in the heart, spleen and lung. Overexpression of mTLR8 is required for the activation of transcription factor nuclear factor-κB and the production of tumor necrosis factor-α. However, mTLR8 is not able to activate interferon regulatory factor 3 or activator protein 1, nor can it induce interferon-α in HEK293T cells. These results indicate that mTLR8, as an important PRR, is indeed functional and is vital role in the activation of innate immune responses. This study may aid in determining the molecular basis of the interactions between mTLR8 and pathogens. PMID:26676274

  8. Molecular Cloning and Characterization of Growth Factor Receptor Bound-Protein in Clonorchis sinensis

    PubMed Central

    Bai, Xuelian; Lee, Ji-Yun; Kim, Tae Im; Dai, Fuhong; Lee, Tae-Jin; Hong, Sung-Jong

    2014-01-01

    Background Clonorchis sinensis causes clonorchiasis, a potentially serious disease. Growth factor receptor-bound protein 2 (Grb2) is a cytosolic protein conserved among animals and plays roles in cellular functions such as meiosis, organogenesis and energy metabolism. In the present study, we report first molecular characters of growth factor receptor bound-protein (CsGrb2) from C. sinensis as counter part of Grb2 from animals and its possible functions in development and organogenesis of C. sinensis. Methodology/Principal Findings A CsGrb2 cDNA clone retrieved from the C. sinensis transcriptome encoded a polypeptide with a SH3-SH2-SH3 structure. Recombinant CsGrb2 was bacterially produced and purified to homogeneity. Native CsGrb2 with estimated molecular weight was identified from C. sinensis adult extract by western blotting using a mouse immune serum to recombinant CsGrb2. CsGrb2 transcripts was more abundant in the metacercariae than in the adults. Immunohistochemical staining showed that CsGrb2 was localized to the suckers, mesenchymal tissues, sperms in seminal receptacle and ovary in the adults, and abundantly expressed in most organs of the metacercariae. Recombinant CsGrb2 was evaluated to be little useful as a serodiagnostic reagent for C. sinesis human infections. Conclusion Grb2 protein found in C. sinensis was conserved among animals and suggested to play a role in the organogenesis, energy metabolism and mitotic spermatogenesis of C. sinensis. These findings from C. sinensis provide wider understanding on diverse function of Grb2 in lower animals such as platyhelminths. PMID:24454892

  9. 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.

  10. 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. PMID:26896843

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 ...

  12. Molecular cloning and functional expression of a human intestinal lactoferrin receptor.

    PubMed

    Suzuki, Y A; Shin, K; Lönnerdal, B

    2001-12-25

    Lactoferrin (Lf), a major iron-binding protein in human milk, has been suggested to have multiple biological roles such as facilitating iron absorption, modulating the immune system, embryonic development, and cell proliferation. Our previous binding studies suggested the presence of a specific receptor for Lf (LfR) in the small intestine of newborn infants, which may facilitate iron absorption. We here report the cloning and the functional expression of the human intestinal LfR and the evidence of its involvement in iron metabolism. The entire coding region of the LfR cDNA was cloned by PCR based on amino acid sequences of the purified native LfR (nLfR). The recombinant LfR (rLfR) was then expressed in a baculovirus-insect cell system and purified by immobilized human Lf (hLf) affinity chromatography where binding of hLf to the rLfR was partially Ca(2+) dependent. The apparent molecular mass was 136 kDa under nonreducing conditions and 34 kDa under reducing conditions. 125I-hLf bound to the rLfR with an apparent K(d) of approximately 360 nM. These biochemical properties of the rLfR are similar to those of the nLfR. RT-PCR revealed that the gene was expressed at high levels in fetal small intestine and in adult heart and at lower levels in Caco-2 cells. PI-PLC treatment of Caco-2 cells indicated that the LfR is GPI anchored. In Caco-2 cells transfected with the LfR gene, 125I-hLf binding and 59Fe-hLf uptake were increased by 1.7 and 3.4 times, respectively, compared to those in mock-transfected cells. Our findings demonstrate the presence of a unique receptor-mediated mechanism for nutrient uptake by the newborn.

  13. Molecular cloning and functional expression of a brain-specific somatostatin receptor.

    PubMed Central

    Bruno, J F; Xu, Y; Song, J; Berelowitz, M

    1992-01-01

    The PCR and conventional library screening were used to clone the brain-specific somatostatin receptor rSSTR-4 from a rat genomic library. The deduced amino acid sequence encodes a protein of 384 amino acids and displays structural and sequence homologies with members of the G protein-receptor superfamily. The amino acid sequence of rSSTR-4 is 60% and 48% identical to that of somatostatin receptors SSTR-1 and SSTR-2, respectively, two recently cloned subtypes. Competition curve analysis of the binding properties of the receptor transiently expressed in COS-1 cells revealed a higher apparent affinity for somatostatin 14 than for somatostatin 28. In contrast, the somatostatin analogs SMS 201-995, IM 4-28, and MK-678 failed to displace specific binding in transfected cells. These characteristics resemble the pharmacological binding properties of the previously described brain-specific somatostatin-receptor subtype. Examination of the tissue distribution of mRNA for rSSTR-4 revealed expression limited to various brain regions with highest levels in the cortex and hippocampus. Thus, based on the pharmacology and tissue localization of this receptor, we conclude that rSSTR-4 represents a brain-specific somatostatin receptor. Images PMID:1360663

  14. 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

  15. Rat kidney thromboxane receptor: molecular cloning, signal transduction, and intrarenal expression localization.

    PubMed Central

    Abe, T; Takeuchi, K; Takahashi, N; Tsutsumi, E; Taniyama, Y; Abe, K

    1995-01-01

    Thromboxane (TX) plays important roles in control of renal hemodynamics and water and electrolyte metabolism, and is involved in the pathophysiology of many renal diseases. The aim of the present study is to isolate a rat kidney cDNA encoding functional TX receptor, and to reveal its intrarenal expression localization. A clone (rTXR2) was isolated from a rat kidney cDNA library by a homology screening approach. rTXR2 was shown to encode the amino acid sequence containing seven transmembrane spanning domains representing rat (r) TX receptor. The membrane from COS-7 cells transiently transfected with rTXR2 cDNA was shown to be specifically bound by a thromboxane receptor antagonist, SQ29548. Either in Xenopus oocyte expression or in transfected COS-7 cells, rTX receptor was shown to be linked with Ca2+ messenger system. TX receptor-mediated increase in cytosolic Ca2+ was also observed in cultured glomerular mesangial cells. In situ hybridization showed that rTX receptor mRNA was detected in renal glomeruli, smooth muscle cells in renal arterioles, and transitional cell epithelium of renal pelvis. Reverse transcription linked to PCR applied to microdissected nephron segments indicated the presence of rTX receptor mRNA exclusively in the glomerulus. In conclusion, we have cloned a functional rat kidney TX receptor, which is expressed specifically in renal glomerulus, arterial smooth muscle cells, and transitional cell epithelium of renal pelvis. The present study will provide important insights into the etiology and pathophysiology of renal diseases with relation to TX metabolism. Images PMID:7635958

  16. [Molecular cloning and characteristics of cDNA encoding pig beta6 subunit for FMDV receptor].

    PubMed

    Gao, Shan-Dian; Du, Jun-Zheng; Chang, Hui-Yun; Cong, Guo-Zheng; Shao, Jun-Jun; Shan, Yi Hua; Zhou, Jian-Hua; Xie, Qing-Ge

    2007-09-01

    In order to study the roles of integrin beta6 in Foot-and-Mouth Disease Virus infection, pig integrin beta6 was firstly molecularly cloned from RNA of the tongue and lung of recovered pig infected experimentally with foot-and-mouth-disease virus (FMDV), and was compared with the beta6 gene of other animals available in GenBank at nucleotide and amino acid leves. GeneBank association number of the beta6 gene is EF432729. Pig integrin beta6 gene (2367bp) encodes a polypeptide of 788 amino acids consisting of 9 potential N-linked glycosylation sites, 3 Glycosaminoglycan attachment sites, a cGMP-dependent protein kinase phosphorylation site, 10 Protein kinase C phosphorylation sites, 2 EGF-like domains and 2 cysteine-rich regions. Pig integrin beta6 subunit has a 26-residue putative signal peptide, a 681-residue ectodomain, a 29-residue transmembrane domain, and a 52-residue cytoplasmic domain. 11 mutant nucleotides were found in beta6 gene coding region and 9 amino acids were changed. The nucleotide sequence similarity of integrin beta6 gene between rheses monkey, mouse, Norway rat, dog, guinea pig, human, bovine, sheep is 79.5%, 84.9%, 85.4%, 85.2%, 88.7%, 90.1%, 91.9% and 91.9%, and the amino acid sequence similarity is 93.5%, 88.2%, 88.5%, 88.3%, 91.0%, 92.8%, 93.3% and 93.4% respectively. This study will lay a foundation for understanding the interactions of FMDV with receptors. PMID:18064756

  17. Molecular cloning and expression of an additional epidermal growth factor receptor-related gene.

    PubMed Central

    Plowman, G D; Whitney, G S; Neubauer, M G; Green, J M; McDonald, V L; Todaro, G J; Shoyab, M

    1990-01-01

    Epidermal growth factor (EGF), transforming growth factor alpha (TGF-alpha), and amphiregulin are structurally and functionally related growth regulatory proteins. These secreted polypeptides all bind to the 170-kDa cell-surface EGF receptor, activating its intrinsic kinase activity. However, amphiregulin exhibits different activities than EGF and TGF-alpha in a number of biological assays. Amphiregulin only partially competes with EGF for binding EGF receptor, and amphiregulin does not induce anchorage-independent growth of normal rat kidney cells (NRK) in the presence of TGF-beta. Amphiregulin also appears to abrogate the stimulatory effect of TGF-alpha on the growth of several aggressive epithelial carcinomas that overexpress EGF receptor. These findings suggest that amphiregulin may interact with a separate receptor in certain cell types. Here we report the cloning of another member of the human EGF receptor (HER) family of receptor tyrosine kinases, which we have named "HER3/ERRB3." The cDNA was isolated from a human carcinoma cell line, and its 6-kilobase transcript was identified in various human tissues. We have generated peptide-specific antisera that recognizes the 160-kDa HER3 protein when transiently expressed in COS cells. These reagents will allow us to determine whether HER3 binds amphiregulin or other growth regulatory proteins and what role HER3 protein plays in the regulation of cell growth. Images PMID:2164210

  18. Molecular cloning of estrogen receptor alpha (ERalpha; ESR1) of the Japanese giant salamander, Andrias japonicus.

    PubMed

    Katsu, Yoshinao; Kohno, Satomi; Oka, Tomohiro; Mitsui, Naoko; Tooi, Osamu; Santo, Noriaki; Urushitani, Hiroshi; Fukumoto, Yukio; Kuwabara, Kazushi; Ashikaga, Kazuhide; Minami, Shinji; Kato, Shigeaki; Ohta, Yasuhiko; Guillette, Louis J; Iguchi, Taisen

    2006-09-26

    Estrogens are essential for normal reproductive activity in females and males and for ovarian differentiation during a critical developmental stage in many vertebrates. To understand the molecular mechanisms of estrogen action and to evaluate estrogen receptor ligand interactions in the Japanese giant salamander (Andrias japonicus), we isolated cDNA encoding the estrogen receptor (ER) from the liver. A full-length Japanese giant salamander ER cDNA (jgsER) was obtained using 5' and 3' rapid amplification cDNA ends (RACE). The deduced amino acid sequence of the jgsER showed high identity to the Xenopus ERalpha (ESR1) (77.7%). We have applied both the conventional ERE-luciferase reporter assay system and the GAL4-transactivation system to characterize this receptor. In two different transient transfection assay systems using mammalian cells, the jgsER protein displayed estrogen-dependent activation of transcription. The GAL4-transactivation system showed about 10-fold greater activity of the estrogen receptor by hormone when compared to the conventional ERE-luciferase reporter assay system. Tissue distribution of ERalpha mRNA was examined and kidney, ovary and liver exhibited expression. This is the first isolation of an estrogen receptor from a salamander and also is the first functional cDNA obtained from the Japanese giant salamander, an endangered species considered a special natural monument of Japan.

  19. Molecular cloning, characterization, and expression profiles of androgen receptors in spotted scat (Scatophagus argus).

    PubMed

    Chen, H P; Deng, S P; Dai, M L; Zhu, C H; Li, G L

    2016-01-01

    Androgen plays critical roles in vertebrate reproductive systems via androgen receptors (ARs). In the present study, the full-length spotted scat (Scatophagus argus) androgen receptor (sAR) cDNA sequence was cloned from testis. The sAR cDNA measured 2448 bp in length with an open-reading frame of 2289 bp, encoding 763 amino acids. Amino acid alignment analyses showed that the sARs exhibited highly evolutionary conserved functional domains. Phylogenetically, the sARs clustered within the ARβ common vertebrate group. Real-time polymerase chain reaction (RT-PCR) revealed that sAR expression varied in level and distribution throughout the tissues of both females and males. sAR expression was detected during testicular development by quantitative RT-PCR. The results showed that the highest transcription of sARs was observed in the mid-testicular stage, and remained at a high expression level until the late-testicular stage. In addition, the effects of 17α-methyltestosterone (MT) and estrogen (E2) on the expression of sARs in ovaries were determined using quantitative RT-PCR. sAR expression increased at 12 and 24 h post-MT treatment and decreased with E2 treatment. The present study provides preliminary evidence indicating gonadal plasticity of spotted scat under exogenous steroidal hormone treatments. It also provides a theoretical basis for sex reversal and production of artificial pseudo-males for female monosex breeding. PMID:27173207

  20. Molecular cloning and characterisation of the griffon vulture (Gyps fulvus) toll-like receptor 1.

    PubMed

    de la Lastra, José Manuel Pérez; de la Fuente, José

    2007-01-01

    The toll-like receptor (TLR) family is an ancient pattern recognition receptor family, conserved from insects to mammals. Members of the TLR family are vital to immune function through the sensing of pathogenic agents and initiation of an appropriate immune response. In this study, we cloned a cDNA encoding for a griffon vulture (Gyps fulvus) orthologue of mammalian TLR1 (CD281). The predicted 650 amino acid sequence comprised an extracellular domain with five leucine-rich repeats (LRR) and an LRR-C-terminal (LRR-CT) motif, followed by a 23 amino acid transmembrane segment, and a 190 amino acid intracytoplasmic region containing the Toll/IL-1R (TIR) domain. Vulture TLR1 and TIR domain showed 64% and 86% amino acid sequence similarity with chicken sequences. The tissue and cell expression pattern of vulture TLR1 were analysed by real time-PCR (RT-PCR) and correlated with the ability to respond to various pathogenic challenges. Despite the similarities in the overall structure and expression pattern of vulture TLR1 with other vertebrate TLRs, the length of the vulture TLR ectodomain, number and position of LRRs and N-glycosylation sites suggest structural differences that may have functional implications.

  1. Molecular cloning and expression analysis of mannose receptor C type 1 in grass carp (Ctenopharyngodon idella).

    PubMed

    Wang, Li; Liu, Lichun; Zhou, Yang; Zhao, Xiaoheng; Xi, Mingjun; Wei, Shun; Fang, Rui; Ji, Wei; Chen, Nan; Gu, Zemao; Liu, Xueqin; Wang, Weimin; Asim, Muhammad; Liu, Xiaoling; Lin, Li

    2014-03-01

    Mannose receptor C type 1 (MRC1) is a pattern-recognition receptor (PRR) which plays a significant role in immune responses. Much work on MRC1 has been done in mammals and birds while little in fish. In this study, we cloned and characterized MRC1 in grass carp (gcMR). The full-length gcMR contained 5291bp encoding a putative protein of 1432 amino acids. The predicted amino acid sequences showed that gcMR contained a signal peptide, a cysteine-rich (CR) domain, a fibronectin type II (FN II) domain, eight C-type lectin-like domains (CTLDs), a transmembrane domain and a short cytoplasmic domain. gcMR were constitutively expressed in different organs with the higher expression in spleen and head kidney. During embryonic development, gcMR transcript levels were highest at cleavage stage. The up-regulation expression of gcMR, IL-1β and TNF-α in liver, spleen, head kidney and intestine after Aeromonas hydrophila infection indicating it involved in innate immune regulation during bacterial infections. PMID:24184700

  2. Molecular cloning, characterization, and expression profiles of androgen receptors in spotted scat (Scatophagus argus).

    PubMed

    Chen, H P; Deng, S P; Dai, M L; Zhu, C H; Li, G L

    2016-01-01

    Androgen plays critical roles in vertebrate reproductive systems via androgen receptors (ARs). In the present study, the full-length spotted scat (Scatophagus argus) androgen receptor (sAR) cDNA sequence was cloned from testis. The sAR cDNA measured 2448 bp in length with an open-reading frame of 2289 bp, encoding 763 amino acids. Amino acid alignment analyses showed that the sARs exhibited highly evolutionary conserved functional domains. Phylogenetically, the sARs clustered within the ARβ common vertebrate group. Real-time polymerase chain reaction (RT-PCR) revealed that sAR expression varied in level and distribution throughout the tissues of both females and males. sAR expression was detected during testicular development by quantitative RT-PCR. The results showed that the highest transcription of sARs was observed in the mid-testicular stage, and remained at a high expression level until the late-testicular stage. In addition, the effects of 17α-methyltestosterone (MT) and estrogen (E2) on the expression of sARs in ovaries were determined using quantitative RT-PCR. sAR expression increased at 12 and 24 h post-MT treatment and decreased with E2 treatment. The present study provides preliminary evidence indicating gonadal plasticity of spotted scat under exogenous steroidal hormone treatments. It also provides a theoretical basis for sex reversal and production of artificial pseudo-males for female monosex breeding.

  3. 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

  4. 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. PMID:25174962

  5. 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.

  6. Molecular cloning, characterization, subcellular localization and dynamics of p23, the mammalian KDEL receptor

    PubMed Central

    1993-01-01

    We have isolated a cDNA clone (mERD2) for the mammalian (bovine) homologue of the yeast ERD2 gene, which codes for the yeast HDEL receptor. The deduced amino acid sequence bears extensive homology to its yeast counterpart and is almost identical to a previously described human sequence. The sequence predicts a very hydrophobic protein with multiple membrane spanning domains, as confirmed by analysis of the in vitro translation product. The protein encoded by mERD2 (p23) has widespread occurrence, being present in all the cell types examined. p23 was localized to the cis-side of the Golgi apparatus and to a spotty intermediate compartment which mediates ER to Golgi transport. A majority of the intracellular staining could be accumulated in the intermediate compartment by a low temperature (15 degrees C) or brefeldin A. During recovery from these treatments, the spotty intermediate compartment staining of p23 was shifted to the perinuclear staining of the Golgi apparatus and tubular structures marked by p23 were observed. These tubular structures may serve to mediate transport between the intermediate compartment and the Golgi apparatus. PMID:8380600

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

    PubMed Central

    Jiang, Hongbo; Wei, Zhaojun; Nachman, Ronald J.; Park, Yoonseong

    2013-01-01

    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 its ligand specificities in a heterologous reporter system. HzDHr was expressed in Chinese Hamster Ovary (CHO) cells, which were co-transfected with the aequorin reporter, and was used to measure the ligand activities. A total of 68 chemicals, including natural DH analogs and structurally similar peptide mimetics, were tested for agonistic and antagonistic activities. Several peptide mimetics with a 2-amino-7-bromofluorene-succinoyl (2Abf-Suc) N-terminal modification showed strong agonistic activities; these mimetics included 2Abf-Suc-F[dA]PRLamide, 2Abf-Suc-F[dR]PRLamide, 2Abf-Suc-FKPRLamide and 2Abf-Suc-FGPRLamide. Antagonistic activity was found in the ecdysis triggering hormone in Drosophila melanogaster (FFLKITKNVPRLamide). Interestingly, HzDHr does not discriminate between DH (WFGPRLamide C-terminal motif) and another closely related endogenous peptide, pyrokinin 1 (FXPRXamide; a C-terminal motif that is separate from WFGPRLamide). We provide large-scale in vitro data that serve as a reference for the development of agonists and antagonists to disrupt the DH signaling pathway. PMID:24257143

  8. 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.

  9. Molecular cloning of a somatostatin-28 receptor and comparison of its expression pattern with that of a somatostatin-14 receptor in rat brain.

    PubMed Central

    Meyerhof, W; Wulfsen, I; Schönrock, C; Fehr, S; Richter, D

    1992-01-01

    The tetradecapeptide somatotropin-release inhibiting factor somatostatin-14 regulates the release of peptide hormones and also functions as neurotransmitter. The octacosapeptide somatostatin-28, the N-terminally extended form of somatostatin-14, shows similar biological activities yet with different potencies. Both peptides most likely function through distinct receptors. Here we report on the molecular and functional characterization of a somatostatin-28 receptor (SSR-28) cloned from a rat brain cDNA library. The nucleotide sequence contains an open reading frame for a protein of 428 amino acid residues with a predicted molecular mass of 47 kDa. Binding assays using radiolabeled somatostatin-14 and membranes from COS cells transfected with the cloned cDNA show that this receptor, SSR-28, has a higher binding affinity for somatostatin-28 (IC50 = 0.24 nM) than for somatostatin-14 (IC50 = 0.89 nM). RNA blot analysis reveals a 4.4-kilobase mRNA in rat cerebellum and at significantly lower abundance in other brain regions. In situ hybridization indicates that SSR-28 mRNA is present in the granular and Purkinje cell layers of the cerebellum and in the large cells of the hypoglossal nucleus of the brain stem. Signals for SSR-28 mRNA do not overlap with those of a previously cloned rat receptor that preferentially binds somatostatin-14 (SSR-14). SSR-14 mRNA is found in the medial cerebellar nucleus, horizontal limb of the diagonal band, various hypothalamic nuclei, and in layers IV and V of the cortex. In the rat cerebellum, SSR-14 and SSR-28 mRNAs are developmentally regulated; the levels of the former are highest around birth and levels of the latter are highest at the adult stage. Images PMID:1279674

  10. Molecular characterization and expression of cloned human galanin receptors GALR2 and GALR3.

    PubMed

    Kolakowski, L F; O'Neill, G P; Howard, A D; Broussard, S R; Sullivan, K A; Feighner, S D; Sawzdargo, M; Nguyen, T; Kargman, S; Shiao, L L; Hreniuk, D L; Tan, C P; Evans, J; Abramovitz, M; Chateauneuf, A; Coulombe, N; Ng, G; Johnson, M P; Tharian, A; Khoshbouei, H; George, S R; Smith, R G; O'Dowd, B F

    1998-12-01

    Galanin is a 29- or 30-amino acid peptide with wide-ranging effects on hormone release, feeding behavior, smooth muscle contractility, and somatosensory neuronal function. Three distinct galanin receptor (GALR) subtypes, designated GALR1, 2, and 3, have been cloned from the rat. We report here the cloning of the human GALR2 and GALR3 genes, an initial characterization of their pharmacology with respect to radioligand binding and signal transduction pathways, and a profile of their expression in brain and peripheral tissues. Human GALR2 and GALR3 show, respectively, 92 and 89% amino acid sequence identity with their rat homologues. Radioligand binding studies with 125I-galanin show that recombinant human GALR2 binds with high affinity to human galanin (K(D) = 0.3 nM). Human GALR3 binds galanin with less affinity (IC50 of 12 nM for porcine galanin and 75 nM for human galanin). Human GALR2 was shown to couple to phospholipase C and elevation of intracellular calcium levels as assessed by aequorin luminescence in HEK-293 cells and by Xenopus melanophore pigment aggregation and dispersion assays, in contrast to human GALR1 and human GALR3, which signal predominantly through inhibition of adenylate cyclase. GALR2 mRNA shows a wide distribution in the brain (mammillary nuclei, dentate gyrus, cingulate gyrus, and posterior hypothalamic, supraoptic, and arcuate nuclei), and restricted peripheral tissue distribution with highest mRNA levels detected in human small intestine. In comparison, whereas GALR3 mRNA was expressed in many areas of the rat brain, there was abundant expression in the primary olfactory cortex, olfactory tubercle, the islands of Calleja, the hippocampal CA regions of Ammon's horn, and the dentate gyrus. GALR3 mRNA was highly expressed in human testis and was detectable in adrenal gland and pancreas. The genes for human GALR2 and 3 were localized to chromosomes 17q25 and 22q12.2-13.1, respectively.

  11. 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.

  12. 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

  13. 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.

  14. 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

  15. Molecular cloning, tissue distribution, and pharmacological characterization of melanocortin-4 receptor in spotted scat, Scatophagus argus.

    PubMed

    Li, Jian-Tao; Yang, Zhao; Chen, Hua-Pu; Zhu, Chun-Hua; Deng, Si-Ping; Li, Guang-Li; Tao, Ya-Xiong

    2016-05-01

    Melanocortin-4 receptor (MC4R) plays an important role in the regulation of food intake and energy expenditure in mammals. The functions of the MC4R in fish have not been investigated extensively. We herein reported on the cloning, tissue distribution, and pharmacological characterization of spotted scat (Scatophagus argus) MC4R (SAMC4R). It consisted of a 984bp open reading frame predicted to encode a protein of 327 amino acids. Sequence analysis revealed that SAMC4R was highly homologous (>80%) at amino acid levels to several teleost MC4Rs. Phylogenetic analyses showed that SAMC4R was closely related to piscine MC4R. Using RT-PCR, we showed that in addition to brain, pituitary, and gonads, mc4r mRNA was also widely expressed in peripheral tissues of spotted scat in sexually divergent pattern. With human MC4R (hMC4R) as a control, several agonists including α-melanocyte stimulating hormone (α-MSH), [Nle(4), D-Phe(7)]-α-MSH (NDP-MSH), adrenocorticotropic hormone (ACTH) and THIQ (N-[(3R)-1,2,3,4-tetrahydroisoquinolinium3-ylcarbonyl]-(1R)-1-(4-chlorobenzyl)-2-[4-cyclohexyl-4-(1H-1,2,4-triazol-1-ylmethyl)piperidin-1-yl]-2-oxoethylamine), were used to investigate the binding and signaling properties of SAMC4R. The results showed that SAMC4R bound NDP-MSH with the highest affinity followed by ACTH (1-24) and α-MSH. Similar ranking was also found for hMC4R, although SAMC4R had two to five-fold higher affinities for these ligands. THIQ did not displace NDP-MSH from SAMC4R, different from hMC4R. α-MSH, NDP-MSH, and ACTH (1-24) were identified as potent agonists to stimulate cAMP generation followed by THIQ in SAMC4R. The availability of SAMC4R and its pharmacological characteristics will facilitate the investigation of its function in regulating diverse physiological processes in spotted scat.

  16. [Molecular cloning, tissue distribution and expression in engineered cells of human orphan receptor GPR81].

    PubMed

    Wu, Fang-Ming; Huang, Huo-Gao; Hu, Ming; Gao, Yue; Liu, Yong-Xue

    2006-05-01

    The gpr81 was amplified by polymerase chain reaction (PCR) using human fetus kidney cDNA and whole blood genome DNA as template, respectively. The expression profile of gpr81 in human fetus was analyzed by RT-PCR and the result indicated GPR81 mRNA was most abundant in fetus liver and heart. In addition, the deduced amino acid of GPR81 was compared with other related molecules by Clustal w/x software, and a molecular phylogenetic tree was constructed with Treeview software. It was showed that GPR81 had the highest homology with nicotinic acid receptor in amino acids. After sequence identification, gpr81 was inserted into the plasmid pcDNA3. 1 (-)/his-mycA and then transfected into Chinese hamster ovary cell (CHO-K1). With the selection of G418, an engineered cell line which could stably express gpr81 was obtained by the indication of RT-PCR and Western-blot detection. The establishment of the cell line will serve as means for further study of GPR81.

  17. 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.

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

    PubMed

    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

  19. 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

  20. 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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  2. 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

  3. Molecular cloning and characterization of a Toll receptor gene from Macrobrachium rosenbergii.

    PubMed

    Srisuk, Chutima; Longyant, Siwaporn; Senapin, Saengchan; Sithigorngul, Paisarn; Chaivisuthangkura, Parin

    2014-02-01

    Toll receptors are cell surface molecules acting as pattern recognition receptors (PRRs) that have been implicated in the signaling pathway of innate immune responses. In this study, the full-length cDNA of a Toll receptor gene of Macrobrachium rosenbergii, designated MrToll, was successfully isolated using designed degenerate primers and the rapid amplification of cDNA ends (RACE). The MrToll gene sequence contained an open reading frame (ORF) of 2799 nucleotides encoding a protein of 932 amino acid residues. The protein contained distinct structural motifs of the Toll-like receptor (TLR) family, including an extracellular domain containing 15 leucine-rich repeats (LRRs), a transmembrane segment of 23 amino acids, and a cytoplasmic Toll/interleukin-1R (TIR) domain of 139 residues. Phylogenetic analysis revealed that MrToll and Toll receptor of Marsupenaeus japonicus (MjToll) evolved closely. However, the MrToll ORF demonstrated only 48-49% identity with shrimp Toll1, suggesting that MrToll isolated from a palaemonid shrimp might belong to a novel class of Toll receptors in shrimp. The transcripts of the MrToll gene were constitutively expressed in various tissues, with high levels in hemocytes, the stomach and muscle. A reverse transcriptase PCR assay demonstrated that the expression patterns of MrToll were distinctly modulated after Aeromonas caviae stimulation, with significant enhancement at 3-12 h post-challenge and a decline to basal levels at 24 h post-challenge. In addition, when MrToll-silenced shrimp were challenged with A. caviae, there was a significant increase in mortality and bacterial CFU counts. These results suggest that MrToll might be involved in host innate defense, especially against the pathogen A. caviae.

  4. Molecular cloning and characterization of a Toll receptor gene from Macrobrachium rosenbergii.

    PubMed

    Srisuk, Chutima; Longyant, Siwaporn; Senapin, Saengchan; Sithigorngul, Paisarn; Chaivisuthangkura, Parin

    2014-02-01

    Toll receptors are cell surface molecules acting as pattern recognition receptors (PRRs) that have been implicated in the signaling pathway of innate immune responses. In this study, the full-length cDNA of a Toll receptor gene of Macrobrachium rosenbergii, designated MrToll, was successfully isolated using designed degenerate primers and the rapid amplification of cDNA ends (RACE). The MrToll gene sequence contained an open reading frame (ORF) of 2799 nucleotides encoding a protein of 932 amino acid residues. The protein contained distinct structural motifs of the Toll-like receptor (TLR) family, including an extracellular domain containing 15 leucine-rich repeats (LRRs), a transmembrane segment of 23 amino acids, and a cytoplasmic Toll/interleukin-1R (TIR) domain of 139 residues. Phylogenetic analysis revealed that MrToll and Toll receptor of Marsupenaeus japonicus (MjToll) evolved closely. However, the MrToll ORF demonstrated only 48-49% identity with shrimp Toll1, suggesting that MrToll isolated from a palaemonid shrimp might belong to a novel class of Toll receptors in shrimp. The transcripts of the MrToll gene were constitutively expressed in various tissues, with high levels in hemocytes, the stomach and muscle. A reverse transcriptase PCR assay demonstrated that the expression patterns of MrToll were distinctly modulated after Aeromonas caviae stimulation, with significant enhancement at 3-12 h post-challenge and a decline to basal levels at 24 h post-challenge. In addition, when MrToll-silenced shrimp were challenged with A. caviae, there was a significant increase in mortality and bacterial CFU counts. These results suggest that MrToll might be involved in host innate defense, especially against the pathogen A. caviae. PMID:24398262

  5. Molecular cloning and expression analysis of GABA(A) receptor-associated protein (GABARAP) from small abalone, Haliotis diversicolor.

    PubMed

    Bai, Rongyao; You, Weiwei; Chen, Jun; Huang, Heqing; Ke, Caihuan

    2012-10-01

    GABA(A) receptor-associated protein (GABARAP), a multifunctional protein participating in autophagy process, is evolutionarily conserved and involves in innate immunity in eukaryotic cells, but currently there is no research on the relationship between GABARAP and innate immunity in mollusc. In the present study, the GABARAP full-length cDNA and its genomic DNA were firstly cloned from small abalone (Haliotis diversicolor), which was named as saGABARAP. Its full-length cDNA is 963 bp with a 354 bp open reading frame encoding a protein of 117 aa, a 276 bp 5'-UTR, and a 333 bp 3'-UTR including a poly(A) tail, two typical polyadenylation signals (AATAA) and two RNA instability motifs (ATTTA). The deduced protein has an estimated molecular weight of 13.9 kDa and a predicted PI of 8.73. Its genomic DNA comprises 4352 bp, containing three exons and two introns. Quantitative real-time PCR analysis revealed that saGABARAP was constitutively expressed in all examined tissues, with the highest expression level in hepatopancreas, and was upregulated in hepatopancreas and hemocytes after bacterial challenge. In addition, saGABARAP was ubiquitously expressed at all examined embryonic and larval development stages. These results suggested that saGABARAP could respond to bacteria challenge and may play a vital role in the adult innate immune system against pathogens and the development process of abalone embryo and larvae.

  6. 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. PMID:27252725

  7. 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.

  8. 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

  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. PMID:23467070

  10. Molecular cloning of the gene encoding the mouse parathyroid hormone/parathyroid hormone-related peptide receptor.

    PubMed Central

    McCuaig, K A; Clarke, J C; White, J H

    1994-01-01

    The parathyroid hormone/parathyroid hormone-related peptide receptor (PTHR) is a G-protein-coupled receptor containing seven predicted transmembrane domains. We have isolated and characterized recombinant bacteriophage lambda EMBL3 genomic clones containing the mouse PTHR gene, including 10 kilobases of the promoter region. The gene spans > 32 kilobases and is divided into 15 exons, 8 of which contain the transmembrane domains. The PTHR exons containing the predicted membrane-spanning domains are heterogeneous in length and three of the exon-intron boundaries fall within putative transmembrane sequences, suggesting that the exons did not arise from duplication events. This arrangement is closely related to that of the growth hormone releasing factor receptor gene, particularly in the transmembrane region, providing strong evidence that the two genes evolved from a common precursor. Transcription is initiated principally at a series of sites over a 15-base-pair region. The proximal promoter region is highly (G+C)-rich and lacks an apparent TATA box or initiator element homologies but does contain CCGCCC motifs. The presumptive amino acid sequence of the encoded receptor is 99%, 91%, and 76% identical to those of the rat, human, and opossum receptors, respectively. There is no consensus polyadenylation signal in the 3' untranslated region. The poly(A) tail of the PTHR transcript begins 32 bases downstream of a 35-base-long A-rich sequence, suggesting that this region directs polyadenylylation. Images PMID:8197183

  11. Molecular cloning and expression of the porcine trigeminal ganglion cDNA encoding a 5-ht(1F) receptor.

    PubMed

    Bhalla, Pankaj; Sharma, Hari S; Wurch, Thierry; Pauwels, Petrus J; Saxena, Pramod R

    2002-02-01

    Using a combination of reverse transcription polymerase chain reaction (RT-PCR) and inverse-PCR techniques, we amplified, cloned and sequenced a full-length porcine 5-hydroxytryptamine 1F (5-ht(1F)) receptor complementary DNA (cDNA) derived from porcine trigeminal ganglion. Sequence analysis revealed 1101 base pairs (bp) encoding an open reading frame of 366 amino acids showing a high similarity (>90%) with the 5-ht(1F) receptor sequences from other species, including human. The recombinant porcine 5-ht(1F) receptor was expressed in African green monkey kidney cell lines (COS-7 cells) and its ligand binding profile was determined using [3H]5-HT. The affinities of several agonists (LY334370 (5-(4-fluorobenzoyl)amino-3-(1-methylpiperidin-4-yl)-1H-indole fumarate)>CP122638 (N-methyl-3 [pyrrolidin 2(R)-yl methyl]-1H-indol-5-ylmethyl sulphonamide)=naratriptan =5HT>eletriptan>sumatriptan>frovatriptan =avitriptan>dihydroergotamine>zolmitriptan>5-carboxamidotryptamine>rizatriptan>alniditan=donitriptan>L694247 (2-[5-[3-(4-methylsulphonylamino)benzyl-1,2,4-oxadiazol-5-yl]-1H-indole-3-yl] ethylamine) and putative antagonists (methiothepin>GR127935 (N-[4-methoxy-3-(4-methyl-1-piperazinyl) phenyl]-2'-methyl 4'-(5-methyl-1,2,4-oxadiazol-3-yl) [1,1-biphenyl]-4-carboxamide hydrochloride)>ritanserin>SB224289 (2,3,6,7-tetrahydro-1'-methyl-5-[2'-methyl-4'(5-methyl-1,2,4-oxadiazol-3-yl) biphenyl-4-carbonyl] furo [2,3-f] indole-3-spiro-4'-piperidine hydrochloride)>BRL155572 ([1-(3-chlorophenyl)-4-[3,3-diphenyl (2-(S,R) hydroxypropanyl)piperazine] hydrochloride)>ketanserin=pindolol) correlated highly with those described for the recombinant human 5-ht(1F) receptor (Spearman correlation coefficient; r(s)=0.942). Nevertheless, as compared to the human homologue, some triptans (i.e. sumatriptan, zolmitriptan and rizatriptan) displayed a 10- to 15-fold lower affinity for the porcine 5-ht(1F) receptor. Using RT-PCR technique, the expression of porcine 5-ht(1F) receptor mRNA was observed in

  12. The receptor of Bacillus sphaericus binary toxin in Culex pipiens (Diptera: Culicidae) midgut: molecular cloning and expression.

    PubMed

    Darboux, I; Nielsen-LeRoux, C; Charles, J F; Pauron, D

    2001-09-01

    Culex pipiens larval midgut is the primary target of the binary toxin (Bin) present in parasporal inclusions of Bacillus sphaericus. Cpm1, a 60-kDa protein purified from brush border membranes, has been proposed as the receptor of the Bin toxin in the midgut epithelial cells of mosquitoes. We have cloned and characterized the corresponding cDNA from midgut of Culex pipiens larvae. The open reading frame predicted a 580 amino-acid protein with a putative signal peptide at the N-terminus and a putative GPI-anchoring signal at the C-terminus. The amino acid sequence of the cloned Cpm1 exhibited 39-43% identities with insect maltases (alpha-glucosidases and alpha-amylases). Recombinant Cpm1 expressed in E. coli specifically bound to the Bin toxin and had a significant alpha-glucosidase activity but no alpha-amylase activity. These results support the view that Cpm1 is an alpha-glucosidase expressed in Culex midgut where it constitutes the receptor for the Bin toxin. To date, this is the first component involved in the mosquitocidal activity of the Bacillus sphaericus Bin toxin to be characterized. Its identification provides a key step to elucidate the mode of action of the Bin toxin and the mechanisms of resistance developed against it by some mosquito strains. PMID:11483434

  13. Molecular cloning and tissue-specific expression of a gonadotropin-releasing hormone receptor in the Japanese eel.

    PubMed

    Okubo, K; Suetake, H; Usami, T; Aida, K

    2000-08-01

    Gonadotropin-releasing hormone (GnRH) is a key regulatory neuropeptide involved in the control of reproduction in vertebrates. In the Japanese eel, one of the most primitive teleost species, two molecular forms of GnRH, mammalian-type GnRH and chicken-II-type GnRH (cGnRH-II), have been identified. This study has isolated a full-length cDNA for a GnRH receptor from the pituitary of the eel. The 3233-bp cDNA encodes a 380-amino acid protein which contains seven hydrophobic transmembrane domains and N- and C-terminal regions. The exon/intron organization of the open reading frame of the eel GnRH receptor gene was also determined. The open reading frame consists of three exons and two introns. The exon-intron splice site is similar to that of the GnRH receptor genes of mammals reported so far. Expression of the eel GnRH receptor was detected in various parts of the brain, pituitary, eye, olfactory epithelium, and testis. This result suggests that GnRH has local functions in these tissues in addition to its actions on gonadotropin synthesis and release in the pituitary. This tissue-specific expression pattern is similar to that of the eel cGnRH-II. Furthermore, the present eel receptor shows very high amino acid identity with the catfish and goldfish GnRH receptors, which are highly selective for the cGnRH-II. These results suggest that the cGnRH-II acts through binding to the present receptor in the eel.

  14. 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

    2015-01-01

    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

  15. 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

  16. Molecular cloning and characterization of estrogen, androgen, and progesterone nuclear receptors from a freshwater turtle (Pseudemys nelsoni).

    PubMed

    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 (ERalpha), an androgen receptor (AR), and a progesterone receptor (PR). The full-length red-belly slider turtle (t)ERalpha, 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 (tERalpha, 90%; tAR, 71%; tPR, 71%). Using transient transfection assays of mammalian cells, tERalpha 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 tERalpha, 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

  17. Molecular cloning and expression of toll-like receptor 4 (tlr4) in the blunt snout bream (Megalobrama amblycephala).

    PubMed

    Lai, Ruifang; Liu, Han; Jakovlić, Ivan; Zhan, Fanbin; Wei, Jin; Yang, Pinhong; Wang, Weimin

    2016-06-01

    Toll-like receptors (TLRs) play a pivotal role in teleost innate immune system. In this study, Megalobrama amblycephala (ma) tlr4 gene was cloned, its putative polypeptide product characterized, and expression analysed. Matlr4 cDNA is 2862 bp long, with an open reading frame of 2364 bp encoding 787 amino acids. MaTlr4 is a typical TLR protein, including the extracellular part with nine leucine-rich repeat motifs, a transmembrane region and a cytoplasmic Toll/interleukin-1 receptor domain. MaTlr4 has the highest level of identity (94%) and similarity (97%) with the grass carp Tlr4.2 homolog. This was also corroborated by the phylogenetic analysis, which placed MaTlr4 in a cluster with other cyprinid homologs. Matlr4 mRNA was ubiquitously expressed in all examined tissues and during all sampled developmental stages. The observed peak in matlr4 mRNA expression during gastrula and somite stages is in good agreement with its proposed role in the development of the neural system. Temporal expression patterns of matlr4 and maMyD88 mRNAs and proteins were analyzed in liver, spleen, head kidney, trunk kidney and intestine after Aeromonas hydrophila infection. And mRNA expression varied between different time-points. Both MaTlr4 and MaMyD88 protein expressions at 12 hpi were significantly enhanced in head kidney and intestine. These results indicate that matlr4 is involved in the immune response in M. amblycephala, and that it is indeed a functional homologue of tlr4s described in other animal species. PMID:26802439

  18. Molecular cloning and characterization of gilthead sea bream (Sparus aurata) growth hormone receptor (GHR). Assessment of alternative splicing.

    PubMed

    Calduch-Giner, Josep A; Mingarro, Mónica; Vega-Rubín de Celis, Silvia; Boujard, Daniel; Pérez-Sánchez, Jaume

    2003-09-01

    The full-length growth hormone receptor (GHR) of gilthead sea bream (Sparus aurata) was cloned and sequenced by RT-PCR and rapid amplification of 5'and 3'ends. The open reading frame codes for a mature 609 amino acid protein with a hydrophobic transmembrane region and all the characteristic motifs of GHRs. Sequence analysis revealed a 96 and 76% of amino acid identity with black sea bream (Acanthopagrus schlegeli) and turbot (Scophthalmus maximus) GHRs, respectively, but this amino acid identity decreases up to 52% for goldfish (Carassius auratus) GHR. By means of real-time PCR assays, concurrent changes in the hepatic expression of GHRs and insulin-like growth factor-I (IGF-I) was evidenced. Moreover, their regulation occurred in conjunction with the summer spurt of growth rates and circulating levels of GH and IGF-I. Search of alternative splicing was carried out exhaustively for gilthead sea bream GHR, but Northern blot and 3' RACE failed to demonstrate the occurrence of short alternative messengers. Besides, RT-PCR screening did not reveal deletions or insertions that could lead to alternative reading frames. In agreement with this, cross-linking assays only evidenced two protein bands that match well with the size of glycosylated and non-glycosylated forms of the full-length GHR. If so, it appears that alternative splicing at the 3'end does not occur in gilthead sea bream, although different messengers for truncated or longer GHR variants already exist in turbot and black sea bream, respectively. The physiological relevance of this finding remains unclear, but perhaps it points out large inter-species differences in the heterogeneity of the GHR population.

  19. Molecular cloning and functional characterization of the oxytocin receptor from a rat pancreatic cell line (RINm5F).

    PubMed

    Jeng, Y J; Lolait, S J; Strakova, Z; Chen, C; Copland, J A; Mellman, D; Hellmich, M R; Soloff, M S

    1996-12-01

    Oxytocin (OT) and vasopressin (AVP) stimulate insulin and glucagon release from the pancreas, and evoke insulin secretion from the rat insulinoma cell line, RINm5F. To determine which AVP/OT receptor subtype is expressed in RINm5F cells, we used PCR with degenerate primers to two transmembrane domains of the AVP (V1a, V1b (or V3), V2) and OT receptors (OTRs). The single PCR fragment identified was used to obtain a full length cDNA from a RINm5F cDNA library. Comparison of the deduced amino acid sequence of this clone with uterine OTR sequences from several species (human, sheep, bovine) and to the pig kidney epithelial cell (LLC-PK1) OTR reveals a very high degree of homology. After the RIN cell OTR cDNA was stably transfected into CHO cells (CHO-OTR), the cell membranes bound iodinated oxytocin antagonist with an apparent Kd comparable to that of RIN cell membranes and those from other OT target cells. Comparison of the ligand specificities of CHO-OTR and RIN cells membranes showed that the relative Ki values of a series of OT analogues were approximately equivalent in both preparations. The rank order of apparent Ki values also corresponded to published values for the rat myometrium, where OT elicits intracellular calcium transients, and increases inositol phosphate production. In uterin endometrium and amnion cells, OT stimulates prostaglandin release. Stimulation of CHO-OTR cells with OT caused an increase in cytosolic calcium concentration originating from both intracellular and extracellular sources, and a dose-dependent increase in inositol phosphate levels. Arachidonic acid release and PGE2 synthesis were also stimulated by OT. These findings (amino acid sequence homology, binding specificity, and signal transduction/second messenger production) suggest that OTRs from RINm5F cells are indistinguishable from OTRs that have been described in other tissues. The expression of OTR in pancreatic cells implies that OT plays a role in pancreatic function.

  20. 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.

  1. Cannabinoid receptors are widely expressed in goldfish: molecular cloning of a CB2-like receptor and evaluation of CB1 and CB2 mRNA expression profiles in different organs

    PubMed Central

    Cottone, Erika; Pomatto, Valentina; Cerri, Fulvio; Campantico, Ezio; Mackie, Ken; Delpero, Massimiliano; Guastalla, Alda; Dati, Claudio; Bovolin, Patrizia; Franzoni, Maria Fosca

    2013-01-01

    Cannabinoids, the bioactive constituents of Cannabis sativa, and endocannabinoids, among which the most important are anandamide and 2-arachidonoylglycerol, control various biological processes by binding to specific G protein-coupled receptors, namely CB1 and CB2 cannabinoid receptors. While a vast amount of information on the mammalian endocannabinoid system does exist, few data have been reported on bony fish. In the goldfish, Carassius auratus, the CB1 receptor has been cloned and its distribution has been analyzed in the retina, brain and gonads, while CB2 had not yet been isolated. In the present paper we cloned the goldfish CB2 receptor and show that it presents a quite high degree of amino acid identity with zebrafish Danio rerio CB2A and CB2B receptors, while the percentage of identity is lower with the pufferfish Fugu rubripes CB2, as also confirmed by the phylogenetic analysis. The sequence identity becomes much lower when comparing the goldfish and the mammalian CB2 sequences; as for other species, goldfish CB2 and CB1 amino acid sequences share moderate levels of identity. Western-blotting analysis shows the CB2 receptor as two major bands of about 53 kDa and 40 kDa, and other faint bands with apparent molecular masses around 70 kDa, 57 kDa and 55 kDa. Since the distribution of a receptor could give information on its physiological role, we evaluated and compared CB1 and CB2 mRNA expression in different goldfish organs by means of quantitative Real-Time PCR. Our results show that both CB1 and CB2 receptors are widely expressed in the goldfish, displaying some tissue specificities, thus opening the way for further functional studies on bony fish and other non-mammalian vertebrates. PMID:23504102

  2. Cannabinoid receptors are widely expressed in goldfish: molecular cloning of a CB2-like receptor and evaluation of CB1 and CB2 mRNA expression profiles in different organs.

    PubMed

    Cottone, Erika; Pomatto, Valentina; Cerri, Fulvio; Campantico, Ezio; Mackie, Ken; Delpero, Massimiliano; Guastalla, Alda; Dati, Claudio; Bovolin, Patrizia; Franzoni, Maria Fosca

    2013-10-01

    Cannabinoids, the bioactive constituents of Cannabis sativa, and endocannabinoids, among which the most important are anandamide and 2-arachidonoylglycerol, control various biological processes by binding to specific G protein-coupled receptors, namely CB1 and CB2 cannabinoid receptors. While a vast amount of information on the mammalian endocannabinoid system does exist, few data have been reported on bony fish. In the goldfish, Carassius auratus, the CB1 receptor has been cloned and its distribution has been analyzed in the retina, brain and gonads, while CB2 had not yet been isolated. In the present paper, we cloned the goldfish CB2 receptor and show that it presents a quite high degree of amino acid identity with zebrafish Danio rerio CB2A and CB2B receptors, while the percentage of identity is lower with the puffer fish Fugu rubripes CB2, as also confirmed by the phylogenetic analysis. The sequence identity becomes much lower when comparing the goldfish and the mammalian CB2 sequences; as for other species, goldfish CB2 and CB1 amino acid sequences share moderate levels of identity. Western-blotting analysis shows the CB2 receptor as two major bands of about 53 and 40 kDa and other faint bands with apparent molecular masses around 70, 57 and 55 kDa. Since the distribution of a receptor could give information on its physiological role, we evaluated and compared CB1 and CB2 mRNA expression in different goldfish organs by means of qReal-Time PCR. Our results show that both CB1 and CB2 receptors are widely expressed in the goldfish, displaying some tissue specificities, thus opening the way for further functional studies on bony fish and other nonmammalian vertebrates.

  3. 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.

  4. 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.

  5. 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. PMID:24333435

  6. Cloning and molecular analysis of genes affecting expression of binding substance, the recipient-encoded receptor(s) mediating mating aggregate formation in Enterococcus faecalis.

    PubMed

    Bensing, B A; Dunny, G M

    1993-11-01

    Transfer of the conjugative plasmid pCF10 in Enterococcus faecalis strains involves production of a plasmid-encoded aggregation substance on the surface of donor cells in response to stimulation by a pheromone secreted by recipient cells. Aggregation substance then facilitates attachment to recipient cells via a chromosomally encoded receptor, termed binding substance (BS). A BS mutant, strain INY3000, generated by random Tn916 insertions, was previously found to carry copies of the transposon at four unique sites (K. M. Trotter and G. M. Dunny, Plasmid 24:57-67, 1990). In the present study, DNA flanking the Tn916 insertions was used to complement the BS mutation of INY3000 following Tn916 excision from cloned chromosomal fragments. Complementation results showed that three of the four regions mutated in INY3000 play some role in BS expression. Tn5 mutagenesis and DNA sequence analysis of the complementing fragment from one of these regions indicated the presence of three genes (ebsA, ebsB, and ebsC) that affect BS expression. The ebsA and ebsB genes encode peptides likely to function in cell wall metabolism, whereas ebsC may encode a product that suppresses the function or expression of EbsB.

  7. Cloning and molecular analysis of genes affecting expression of binding substance, the recipient-encoded receptor(s) mediating mating aggregate formation in Enterococcus faecalis.

    PubMed Central

    Bensing, B A; Dunny, G M

    1993-01-01

    Transfer of the conjugative plasmid pCF10 in Enterococcus faecalis strains involves production of a plasmid-encoded aggregation substance on the surface of donor cells in response to stimulation by a pheromone secreted by recipient cells. Aggregation substance then facilitates attachment to recipient cells via a chromosomally encoded receptor, termed binding substance (BS). A BS mutant, strain INY3000, generated by random Tn916 insertions, was previously found to carry copies of the transposon at four unique sites (K. M. Trotter and G. M. Dunny, Plasmid 24:57-67, 1990). In the present study, DNA flanking the Tn916 insertions was used to complement the BS mutation of INY3000 following Tn916 excision from cloned chromosomal fragments. Complementation results showed that three of the four regions mutated in INY3000 play some role in BS expression. Tn5 mutagenesis and DNA sequence analysis of the complementing fragment from one of these regions indicated the presence of three genes (ebsA, ebsB, and ebsC) that affect BS expression. The ebsA and ebsB genes encode peptides likely to function in cell wall metabolism, whereas ebsC may encode a product that suppresses the function or expression of EbsB. Images PMID:8226689

  8. 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.

  9. Cloning, Expression Analysis, and Molecular Modeling of the Gamma-Aminobutyric Acid Receptor Alpha2 Subunit Gene from the Common Cutworm, Spodoptera litura

    PubMed Central

    Zuo, Hongliang; Gao, Lu; Hu, Zhen; Liu, Haiyuan; Zhong, Guohua

    2013-01-01

    Intensive research on the molecule structures of the gamma-nminobutyric acid (GABA) receptor in agricultural pests has great significance to the mechanism investigation, resistance prevention, and molecular design of novel pesticides. The GABA receptor a2 (SlGABARα2) subunit gene in Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae) was cloned using the technologies of reverse transcription PCR and rapid amplification of cDNA ends. The gemonic DNA sequence of SlGABARα2 has 5164 bp with 8 exons and 7 introns that were in accordance with the GT-AG splicing formula. The complete mRNA sequence of SlGABARα2 was 1965 bp, with an open reading frame of 1500 bp encoding a protein of 499 amino acids. The GABA receptor is highly conserved among insects. The conserved regions include several N-glycosylation, Oglycosylation, and phosphorylation sites, as well as 4 transmembrane domains. The identities that SlGABARα2 shared with the GABA receptor a2 subunit of Spodoptera exigua, Heliothis virescens, Chilo suppressalis, Plutella xylostella, Bombyx mori ranged from 99.2% to 87.2% at the amino acid level. The comparative 3-dimensional model of SlGABARα2 showed that its tertiary structure was composed of 4 major α-helixes located at the 4 putative transmembrane domains on one side, with some β-sheets and 1 small α-helix on the other side. SlGABARα2 may be attached to the membrane by 4 α-helixes that bind ions in other conserved domains to transport them through the membrane. The results of quantitative real time PCR demonstrated that SlGABARα2 was expressed in all developmental stages of S. litura. The relative expression level of SlGABARα2 was the lowest in eggs and increased with larval growth, while it declined slightly in pupae and reached the peak in adults. The expressions of SlGABARα2 in larvae varied among different tissues; it was extremely high in the brain but was low in the midgut, epicuticle, Malpighian tube, and fat body. PMID:23909412

  10. Cloning, expression analysis, and molecular modeling of the gamma-aminobutyric acid receptor alpha2 subunit gene from the common cutworm, Spodoptera litura.

    PubMed

    Zuo, Hongliang; Gao, Lu; Hu, Zhen; Liu, Haiyuan; Zhong, Guohua

    2013-01-01

    Intensive research on the molecule structures of the gamma-nminobutyric acid (GABA) receptor in agricultural pests has great significance to the mechanism investigation, resistance prevention, and molecular design of novel pesticides. The GABA receptor a2 (SlGABARα2) subunit gene in Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae) was cloned using the technologies of reverse transcription PCR and rapid amplification of cDNA ends. The gemonic DNA sequence of SlGABARα2 has 5164 bp with 8 exons and 7 introns that were in accordance with the GT-AG splicing formula. The complete mRNA sequence of SlGABARα2 was 1965 bp, with an open reading frame of 1500 bp encoding a protein of 499 amino acids. The GABA receptor is highly conserved among insects. The conserved regions include several N-glycosylation, Oglycosylation, and phosphorylation sites, as well as 4 transmembrane domains. The identities that SlGABARα2 shared with the GABA receptor a2 subunit of Spodoptera exigua, Heliothis virescens, Chilo suppressalis, Plutella xylostella, Bombyx mori ranged from 99.2% to 87.2% at the amino acid level. The comparative 3-dimensional model of SlGABARα2 showed that its tertiary structure was composed of 4 major α-helixes located at the 4 putative transmembrane domains on one side, with some β-sheets and 1 small α-helix on the other side. SlGABARα2 may be attached to the membrane by 4 α-helixes that bind ions in other conserved domains to transport them through the membrane. The results of quantitative real time PCR demonstrated that SlGABARα2 was expressed in all developmental stages of S. litura. The relative expression level of SlGABARα2 was the lowest in eggs and increased with larval growth, while it declined slightly in pupae and reached the peak in adults. The expressions of SlGABARα2 in larvae varied among different tissues; it was extremely high in the brain but was low in the midgut, epicuticle, Malpighian tube, and fat body. PMID:23909412

  11. 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.

  12. Molecular cloning and functional expression of two monocyte chemoattractant protein 1 receptors reveals alternative splicing of the carboxyl-terminal tails.

    PubMed Central

    Charo, I F; Myers, S J; Herman, A; Franci, C; Connolly, A J; Coughlin, S R

    1994-01-01

    Monocyte chemoattractant protein 1 (MCP-1) is a member of the chemokine family of cytokines that mediate leukocyte chemotaxis. The potent and specific activation of monocytes by MCP-1 may mediate the monocytic infiltration of tissues in atherosclerosis and other inflammatory diseases. We have isolated cDNAs that encode two MCP-1-specific receptors with alternatively spliced carboxyl tails. Expression of the receptors in Xenopus oocytes conferred robust mobilization of intracellular calcium in response to nanomolar concentrations of MCP-1 but not to related chemokines. The MCP-1 receptors are most closely related to the receptor for the chemokines macrophage inflammatory protein 1 alpha and RANTES (regulated on activation, normal T expressed and secreted). The identification of the MCP-1 receptor and cloning of two distinct isoforms provide powerful tools for understanding the specificity and signaling mechanisms of this important chemokine. Images PMID:8146186

  13. 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.

  14. Corticotropin releasing factor receptor type 1: molecular cloning and investigation of alternative splicing in the hamster skin.

    PubMed

    Pisarchik, Alexander; Slominski, Andrzej

    2002-06-01

    The coding region of the hamster corticotropin releasing factor receptor type 1 was sequenced. Hamster gene appeared to be similar to mouse, rat, and human sequences with 95%, 94%, and 91% homology, respectively. Protein substitutions were generally found in the corticotropin releasing factor-binding domain. Thus, this domain can be more prone to mutations leading to changes in amino acid sequence. Hamster pituitary, eye, spleen, heart, skin, and four melanoma lines differentially expressed nine corticotropin releasing factor-R1 isoforms. These included the corticotropin releasing factor-R1alpha and corticotropin releasing factor-R1d homologs of human isoforms as well as e, f, h, j, k, m, and n isoforms. Corticotropin releasing factor-R1e mRNA had deletion of exons 3 and 4, CRF-R1j of exon 5, CRF-R1f of exon 11, CRF-R1k of exon 10, CRF-R1m of exons 11 and 12, and CRF-R1n of exons 10, 11, and 12. Corticotropin releasing factor-R1h had an insertion of a cryptic exon between exons 4 and 5. Reading frames of isoforms e, f, j, k, m, and h contained frameshifts, expected to produce truncated proteins. Corticotropin releasing factor-R1n isoform preserved the reading frame, but the transmembrane domains 6, 7, and one-third of the fifth were deleted. The AbC1 hamster melanoma cell line changed the pattern of alternative splicing after irradiation with ultraviolet light or induction of melanogenesis; this suggests that corticotropin releasing factor receptor alternative splicing may be regulated by common stressors, through modifications of activity and/or availability of splicing factors.

  15. Molecular cloning and expression analysis of interleukin (IL)-15 and IL-15 receptor α from rock bream, Oplegnathus fasciatus.

    PubMed

    Bae, Jin-Sol; Shim, Sang Hee; Hwang, Seong Don; Kim, Ju-Won; Park, Dae-Won; Park, Chan-Il

    2013-10-01

    Mammalian interleukin (IL)-15 plays an important role in the activation of CD8(+) T cells and natural killer (NK) cells along with its receptor α (IL-15Rα). To understand the potential roles of IL-15 and IL-15Rα in fish, we identified IL-15 and IL-15Rα cDNA from rock bream (Oplegnathus fasciatus) and investigated their gene expression profiles after bacterial and viral infection. Coding regions of rock bream (Rb) IL-15 and RbIL-15Rα cDNAs were 534 and 402 bp encoding 177 and 133 amino acid residues, respectively. The sushi domain of IL-15Rα was highly conserved between rock bream and other species. Unlike other IL-15Rαs, RbIL-15Rα does not have a transmembrane region. Gene expression of RbIL-15 and RbIL-15Rα was widely expressed in different tissues of healthy fish, especially immune-related tissues. RbIL-15 and RbIL-15Rα were highly induced in the kidney and spleen after infection with Edwardsiella tarda, Streptococcus iniae and red seabream iridovirus. Gene expression patterns of RbIL-15 and RbIL-15Rα were similar in the kidney and spleen after pathogen infection. However, these genes were differentially induced in the liver after pathogen infection. These results suggest that the different responses of RbIL-15 and RbIL-15Rα to pathogen infection may be induced by different tissues or cell types. PMID:23911652

  16. 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.

  17. 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. PMID:27546552

  18. 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. PMID:27417233

  19. 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.

  20. 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. PMID:27212643

  1. 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

  2. Molecular cloning, characterization and expression analysis of receptor for activated C kinase 1 (RACK1) from pearl oyster (Pinctada martensii) challenged with bacteria and exposed to cadmium.

    PubMed

    Chen, Jinhui; Liu, Jifang; Xiao, Shu; Yu, Ziniu

    2011-12-01

    Receptor for activated C kinase 1 (RACK1) is involved in superoxide anion generation and play an important role in the immune response. In the study, we cloned the full-length sequence of pearl oyster, Pinctada martensii, RACK1 (designated as PmRACK1) by a combination of expression sequence tag (EST) analysis and rapid amplification of cDNA ends (RACE). The full-length cDNA of PmRACK1 is 1176 bp in length, containing a 5' UTR of 83 bp, a 3' UTR of 139, and an open reading frame (ORF) of 954 bp encoding 317 amino acids. Analysis of protein domain features showed that the deduced polypeptide contain seven WD domains characteristic of RACK1 protein family. The tissue distribution of PmRACK1 in unchallenged pearl oysters and temporal expression pattern of PmRACK1 in pearl oysters challenged with bacteria and exposed to 0.1 ppm cadmium were analyzed by quantitative real-time PCR (qRT-PCR). The transcript was detected in all tissues tested, and the expression level was highest in hepatopancreas and lowest in adductor muscle. After challenge with bacteria, expression level of PmRACK1 in haemocytes was gradually decreased until 6 h post challenge, and then up-regulated over time. After exposure to cadmium, its expression level in gill decreased on 1 d post exposure, and then increased as time elapsed, and its expression level in hepatopancreas gradually decreased until 2 d post exposure, and then increased over time. These results suggested that PmRACK1 was involved in oxidative stress response caused by bacteria and cadmium and was a useful biomarker for cadmium exposure. The expression pattern of PmRACK1 in response to bacterial challenge also has a potential link with organism's immune response. PMID:21782956

  3. Molecular cloning, characterization and expression analysis of receptor for activated C kinase 1 (RACK1) from pearl oyster (Pinctada martensii) challenged with bacteria and exposed to cadmium.

    PubMed

    Chen, Jinhui; Liu, Jifang; Xiao, Shu; Yu, Ziniu

    2011-12-01

    Receptor for activated C kinase 1 (RACK1) is involved in superoxide anion generation and play an important role in the immune response. In the study, we cloned the full-length sequence of pearl oyster, Pinctada martensii, RACK1 (designated as PmRACK1) by a combination of expression sequence tag (EST) analysis and rapid amplification of cDNA ends (RACE). The full-length cDNA of PmRACK1 is 1176 bp in length, containing a 5' UTR of 83 bp, a 3' UTR of 139, and an open reading frame (ORF) of 954 bp encoding 317 amino acids. Analysis of protein domain features showed that the deduced polypeptide contain seven WD domains characteristic of RACK1 protein family. The tissue distribution of PmRACK1 in unchallenged pearl oysters and temporal expression pattern of PmRACK1 in pearl oysters challenged with bacteria and exposed to 0.1 ppm cadmium were analyzed by quantitative real-time PCR (qRT-PCR). The transcript was detected in all tissues tested, and the expression level was highest in hepatopancreas and lowest in adductor muscle. After challenge with bacteria, expression level of PmRACK1 in haemocytes was gradually decreased until 6 h post challenge, and then up-regulated over time. After exposure to cadmium, its expression level in gill decreased on 1 d post exposure, and then increased as time elapsed, and its expression level in hepatopancreas gradually decreased until 2 d post exposure, and then increased over time. These results suggested that PmRACK1 was involved in oxidative stress response caused by bacteria and cadmium and was a useful biomarker for cadmium exposure. The expression pattern of PmRACK1 in response to bacterial challenge also has a potential link with organism's immune response.

  4. 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

  5. Goat liver X receptor α, molecular cloning, functional characterization and regulating fatty acid synthesis in epithelial cells of goat mammary glands.

    PubMed

    Wang, Wei; Luo, Jun; Zhong, Yu; Lin, Xian-Zi; Shi, Heng-Bo; Zhu, Jiang-Jiang; Li, Jun; Sun, Yu-Ting; Zhao, Wang-Sheng

    2012-08-15

    The liver X receptor α (LXRα) is a nuclear receptor of the transcription factor and is known to play a crucial role in lipid metabolism processes such as bile acid and fatty acid synthesis in humans and rodents. However, very little information is available on the role of LXRα in the regulation of fatty acid synthesis in the goat mammary gland. In this investigation, a cDNA was isolated from the mammary gland of Xinong Saanen dairy goats and designated as goat LXRα. RT-PCR and RACE gave rise to the full-length cDNA of LXRα, which was comprised of 1654 bp and characterized by an ORF of 1344 bp and 5'- and 3'-UTR regions of 150 and 160 bp, respectively. The deduced amino acid sequence encodes 477 amino acids with a predicted molecular weight (MW) of 50.4kDa and a theoretical isoelectric point (pI) of 6.3. Additionally, homology search and sequence multi-alignment indicated that the putative goat LXRα amino acid sequence is very similar to those of cattle, mice, rats, swine, and humans. Bioinformatic predictions demonstrated that the LXRα protein is located in the nucleus, containing characteristic signatures of a nuclear receptor with DNA-binding domain (DBD) and ligand-binding domain (LBD). Real-time quantitative PCR suggested that LXRα was predominantly expressed in the small intestine, liver, spleen and mammary gland. Treatment of goat mammary gland epithelial cells (GMEC) with different concentrations (i.e., 0.01, 0.1, 1 μM) of T0901317, a synthetic agonist of LXRα, resulted in elevated sterol regulatory element binding protein 1 (SREBP1) and fatty acid synthase (FASN) mRNA levels in response to LXRα activation. The association between different T0901317 concentrations and fatty acid composition in GMEC also was examined using gas chromatography (GC). The results showed that activation of LXRα significantly increased GMEC C18:1 and C18:2 contents, but did not affect levels of saturated fatty acids (SFA). These discoveries are consistent with the

  6. 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. PMID:26159320

  7. 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.

  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. Molecular cloning of protein-based polymers.

    PubMed

    Mi, Lixin

    2006-07-01

    Protein-based biopolymers have become a promising class of materials for both biomedical and pharmaceutical applications, as they have well-defined molecular weights, monomer compositions, as well as tunable chemical, biological, and mechanical properties. Using standard molecular biology tools, it is possible to design and construct genes encoding artificial proteins or protein-based polymers containing multiple repeats of amino acid sequences. This article reviews some of the traditional methods used for constructing DNA duplexes encoding these repeat-containing genes, including monomer generation, concatemerization, iterative oligomerization, and seamless cloning. A facile and versatile method, called modules of degenerate codons (MDC), which uses PCR and codon degeneracy to overcome some of the disadvantages of traditional methods, is introduced. Re-engineering of the random coil spacer domain of a bioactive protein, WPT2-3R, is used to demonstrate the utility of the MDC method. MDC re-constructed coding sequences facilitate further manipulations, such as insertion, deletion, and swapping of various sequence modules. A summary of some promising emerging techniques for synthesizing repetitive sequence-containing artificial proteins is also provided. PMID:16827576

  10. Cloning and pharmacological characterization of a rat kappa opioid receptor.

    PubMed Central

    Meng, F; Xie, G X; Thompson, R C; Mansour, A; Goldstein, A; Watson, S J; Akil, H

    1993-01-01

    A full-length cDNA was isolated from a rat striatal library by using low-stringency screening with two PCR fragments, one spanning transmembrane domains 3-6 of the mouse delta opioid receptor and the other unidentified but homologous to the mouse delta receptor from rat brain. The novel cDNA had a long open reading frame encoding a protein of 380 residues with 59% identity to the mouse delta receptor and topography consistent with a seven-helix guanine nucleotide-binding protein-coupled receptor. COS-1 cells transfected with the coding region of this clone showed high-affinity binding to kappa opioid receptor-selective ligands such as dynorphin A and U-50,488 and also nonselective opioid ligands such as bremazocine, ethylketocyclazocine, and naloxone. Not bound at all (or bound with low affinity) were dynorphin A-(2-13), enantiomers of naloxone and levophanol [i.e., (+)-naloxone and dextrorphan], and selective mu and delta opioid receptor ligands. Activation of the expressed receptor by kappa receptor agonists led to inhibition of cAMP. Finally, in situ hybridization revealed a mRNA distribution in rat brain that corresponded well to the distribution of binding sites labeled with kappa-selective ligands. These observations indicate that we have cloned a cDNA encoding a rat kappa receptor of the kappa 1 subtype. Images Fig. 3 PMID:8234341

  11. Molecular cloning and comparative responses of Toll-like receptor 22 following ligands stimulation and parasitic infection in yellowtail (Seriola lalandi).

    PubMed

    Reyes-Becerril, Martha; Ascencio-Valle, Felipe; Alamillo, Erika; Hirono, Ikuo; Kondo, Hidehiro; Jirapongpairoj, Walissara; Angulo, Carlos

    2015-10-01

    TLR22 is exclusively present in teleosts and amphibians and is expected to play the distinctive role in innate immunity. In this study, we cloned the full-length cDNA sequence of yellowtail (Seriola lalandi) TLR22 (SlTLR22). The complete cDNA sequence of SlTLR22 was 4208 bp and encodes a polypeptide of 961 amino acids. Analysis of the deduced amino acid sequence indicated that SlTLR22 has typical structural features of proteins belonging to the TLR family. These included 17 LRR domains (residues 91-633) and one C-terminal LRR domain (LRR-CT, residues 693-744) in the extracellular region, and a TIR domain (residues 800-943) in the cytoplasmic region. Comparison with homologous proteins showed that the deduced SlTLR22 has the highest sequence identity to turbot TLR22 (76%). Quantitative real-time PCR (qPCR) analysis demonstrated the constitutive expression of SlTLR22 mRNA in all examined tissues with higher levels in the head kidney, intestine, skin and spleen. Further, SlTLR22 expression was significantly up-regulated following TLR ligands injection with lipopolysaccharide (LPS), CpG ODN2006 and polyinosinic: polycytidylic acid (poly I:C) in spleen and liver. Amyloodinium ocellatum infection also induced a high expression of SlTLR22 in spleen, intestine, muscle, skin and gill, with maximum increases ranging from 1000 to 100 fold upon different ligands and organs. Finally, histological examination in gill tissue confirmed infection by the parasite and histopathological lesion was observed also in spleen and skin. These findings suggest a possible role of SlTLR22 in the immune responses to the infections of a broad range of pathogens that include DNA and RNA viruses and parasites. PMID:26102460

  12. Molecular cloning and characterization of estrogen receptor gene in the scallop Chlamys farreri: expression profiles in response to endocrine disrupting chemicals.

    PubMed

    Zhang, Hui; Pan, Luqing; Zhang, Lin

    2012-06-01

    In order to gain insights into the mechanism of sex steroid signaling in molluscs, the full-length cDNA of estrogen receptor (ER) was isolated and characterized from Chlamys farreri for the first time. The positions of cysteine residues and other residues around them that constitute the two zinc finger motifs and the P-box are conserved. Phylogenetic analysis revealed that the CfER is an ortholog of the other mollusk ERs. Tissue distribution analysis of the CfER mRNA revealed that the expression of ER mRNA was observed in various tissues, and highest in the gonad of males and females. C. farreri were exposed for 10 days to endocrine disrupting chemicals including Benzo(a)pyrene (B(a)p) and polybrominated diphenyl ethers (BDE-47). B(a)p exposure at 0.4 and 2 μg/L caused significant increase in mRNA expression of ER and VTG, but B(a)p at 10 μg/L down-regulated CfER and VTG mRNA expression compared to control. Varying increase of ER and VTG mRNA transcripts was resulted in by BDE-47 at 0.1, 1 and 10 μg/L. These results elucidate potential roles of CfER induced by xenobiotics in C. farreri and can be helpful for investigating the mechanism of sex steroid signaling in bivalve mollusks. PMID:22507668

  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. 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.

  15. 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

  16. Molecular cloning, cDNA analysis, and localization of a monomer of the N-acetylglucosamine-specific receptor of the thyroid, NAGR1, to chromosome 19p13. 3-13. 2

    SciTech Connect

    Blanck, O.; Perrin, C.; Mziaut, H.; Miquelis, R. ); Darbon, H. ); Mattei, M.G. )

    1994-05-01

    The authors have proposed that the GlcNAc thyroid receptor triggers selective recycling of immature GlcNAc-bearing thyroglobulin molecules through the Golgi back to the apical membrane for further processing until maturation is achieved. This process, which is called [open quotes]receptor-mediated exocytosis,[close quotes] prevents lysosomal degradation of thyroid prohormones. In the present study, the authors report cloning of the cDNA encoding the (or one of the) monomer(s) constituting the human GlcNAc thyroid receptor. This novel gene, called NAGR1, was assigned by in situ hybridization to subbands p13.3-p13.2 of chromosome 19. Northern blot analysis showed that the mRNA encoding NAGR1 was present as a single transcript of 2.1 kb in the thyroid, but not in the heart, brain, placenta, lung, liver, skeletal muscle, kidney, and pancreas. The deduced amino acid sequence comprised a 51-kDa type I membrane protein with a single spanning region and a short intracytoplasmic domain. Sequence analysis showed that NAGR1 is a glycine-, tryptophan-, and methionine-rich protein with no cysteine residues or glycosylation site. No sequence homology with any known cDNA or protein was noted. The extracellular domain is composed of 420 amino acids and contains a region of 204 residues showing 15 repeats of 4 amino acids, each 1 having an acidic amino acid presumably involved in calcium coordination. The intracellular domain contained what appeared to be a tyrosine internalization signal. The usefulness of this clone in glycobiology, cell biology, and thyroid pathology studies is discussed. 53 refs., 6 figs.

  17. Cloning and expression of the rabbit prostaglandin EP2 receptor

    PubMed Central

    Guan, Youfei; Stillman, Brett A; Zhang, Yahua; Schneider, André; Saito, Osamu; Davis, Linda S; Redha, Reyadh; Breyer, Richard M; Breyer, Matthew D

    2002-01-01

    Background Prostaglandin E2 (PGE2) has multiple physiologic roles mediated by G protein coupled receptors designated E-prostanoid, or "EP" receptors. Evidence supports an important role for the EP2 receptor in regulating fertility, vascular tone and renal function. Results The full-length rabbit EP2 receptor cDNA was cloned. The encoded polypeptide contains 361 amino acid residues with seven hydrophobic domains. COS-1 cells expressing the cloned rabbit EP2 exhibited specific [3H]PGE2 binding with a Kd of 19.1± 1.7 nM. [3H]PGE2 was displaced by unlabeled ligands in the following order: PGE2>>PGD2=PGF2α=iloprost. Binding of [3H]PGE2 was also displaced by EP receptor subtype selective agonists with a rank order of affinity consistent with the EP2 receptor (butaprost>AH13205>misoprostol>sulprostone). Butaprost free acid produced a concentration-dependent increase in cAMP accumulation in rabbit EP2 transfected COS-1 cells with a half-maximal effective concentration of 480 nM. RNase protection assay revealed high expression in the ileum, spleen, and liver with lower expression in the kidney, lung, heart, uterus, adrenal gland and skeletal muscle. In situ hybridization localized EP2 mRNA to the uterine endometrium, but showed no distinct localization in the kidney. EP2 mRNA expression along the nephron was determined by RT-PCR and its expression was present in glomeruli, MCD, tDL and CCD. In cultured cells EP2 receptor was not detected in collecting ducts but was detected in renal interstitial cells and vascular smooth muscle cells. EP2 mRNA was also detected in arteries, veins, and preglomerular vessels of the kidney. Conclusion EP2 expression pattern is consistent with the known functional roles for cAMP coupled PGE2 effects in reproductive and vascular tissues and renal interstitial cells. It remains uncertain whether it is also expressed in renal tubules. PMID:12097143

  18. Cloning and localization of two multigene receptor families in goldfish olfactory epithelium

    PubMed Central

    Cao, Yanxiang; Oh, Bryan C.; Stryer, Lubert

    1998-01-01

    Goldfish reproduction is coordinated by pheromones that are released by ovulating females and detected by males. Two highly potent pheromones, a dihydroxyprogesterone and a prostaglandin, previously have been identified, and their effects on goldfish behavior have been studied in depth. We have cloned goldfish olfactory epithelium cDNAs belonging to two multigene G-protein coupled receptor families as a step toward elucidating the molecular basis of pheromone recognition. One gene family (GFA) consists of homologs of putative odorant receptors (≈320 residues) found in the olfactory epithelium of other fish and mammals. The other family (GFB) consists of homologs of putative pheromone receptors found in the vomeronasal organ (VNO) of mammals and also in the nose of pufferfish. GFB receptors (≈840 residues) are akin to the V2R family of VNO receptors, which possess a large extracellular N-terminal domain and are homologs of calcium-sensing and metabotropic glutamate receptors. In situ hybridization showed that the two families of goldfish receptors are differentially expressed in the olfactory epithelium. GFB mRNA is abundant in rather compact cells whose nuclei are near the apical surface. In contrast, GFA mRNA is found in elongated cells whose nuclei are positioned deeper in the epithelium. Our findings support the hypothesis that the separate olfactory organ and VNO of terrestrial vertebrates arose in evolution by the segregation of distinct classes of neurons that were differentially positioned in the olfactory epithelium of a precursor aquatic vertebrate. PMID:9751777

  19. 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

  20. 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

  1. 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.

  2. 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.

  3. Construction of chimeric vaccinia viruses by molecular cloning and packaging.

    PubMed Central

    Scheiflinger, F; Dorner, F; Falkner, F G

    1992-01-01

    Foreign DNA was inserted into unique restriction endonuclease cleavage sites (Sma I or Not I) of the 200,000-base-pair vaccinia virus genome by direct molecular cloning. The modified vaccinia virus DNA was packaged in fowlpox virus-infected avian cells, and chimeric vaccinia virus was isolated from mammalian cells not supporting the growth of the fowlpox helper virus. In contrast to the classical "in vivo" recombination technique, chimeric viruses with inserts in both possible orientations and families of chimeras with multiple inserts were obtained. The different genomic configurations of chimeric viruses provide a broader basis for screening of optimal viruses. In addition to packaging in avian cells, a second packaging procedure for vaccinia DNA, based on the abortive infection of mammalian cells with the fowlpox helper virus, was developed. This procedure permits simultaneous packaging and host-range selection for the packaged virus. The cloning/packaging procedure allows the direct insertion of foreign DNA without the need for plasmids having flanking regions homologous to viral nonessential regions and is independent of inefficient in vivo recombination events. By direct cloning and packaging, about 5-10% of the total vaccinia virus yield consisted of chimeras. The procedure is, therefore, a useful tool in molecular virology. Images PMID:1438247

  4. 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.

  5. 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.

  6. Characterization of three non-peptide endothelin receptor ligands using human cloned ETA and ETB receptors.

    PubMed Central

    Buchan, K. W.; Alldus, C.; Christodoulou, C.; Clark, K. L.; Dykes, C. W.; Sumner, M. J.; Wallace, D. M.; White, D. G.; Watts, I. S.

    1994-01-01

    1. A number of putative endothelin (ET) receptor ligands were synthesized with a view to assessing their relative affinity for human recombinant ET receptors. 2. Human (h) and endothelin ETA and ETB receptor open reading frames were cloned by reverse transcription-polymerase chain reaction into the mammalian expression vector pcDNA1 and stable cell lines were created by transfection of Chinese hamster ovary cells. 3. Scatchard analyses of saturation isotherms for the specific binding of [125I]-endothelin-1 ([125I]-ET-1) to membranes, prepared from Chinese hamster ovary cells transfected with hETA or hETB receptors, yielded values for equilibrium dissociation constants (Kd) of 20.5 +/- 1.8 pM and 25.5 +/- 5.5 pM, respectively. Hill coefficients did not differ significantly from unity, suggesting binding to homogeneous, non-interacting receptor populations. 4. Pharmacological characterization of the transfected hETA and hETB receptors was undertaken by measuring the relative abilities of ETA and ETB receptor-selective peptide ligands to inhibit binding of [125I]ET-1. For interaction with hETA receptors, the relative order of potency was ET-1 > ET-3 = FR139317 = BQ123 >[Ala1,3,11,15]-ET-1 = sarafotoxin S6c (S6c). In contrast, the relative order of potency, at hETB receptors, was ET-1 = ET-3 = [Ala1,3,11,15]-ET-1 = S6c >> FR139317 = BQ123. 5. The novel non-peptide ligands, Ro 46-2005, SB 209670 and BMS 182874, were found to inhibit [125I]-ET-1 binding to human recombinant ETA and ETB receptors.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7952888

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

    PubMed Central

    Pääbo, S

    1989-01-01

    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. Images PMID:2928314

  8. Molecular and pharmacological characteristics of the gerbil α(1a)-adrenergic receptor.

    PubMed

    Witt, Kelly M; Bockman, Charles S; Dang, Herbert K; Gruber, Daniel D; Wangemann, Philine; Scofield, Margaret A

    2012-01-01

    The spiral modiolar artery supplies blood and essential nutrients to the cochlea. Our previous functional study indicates the α(1A)-adrenergic receptor subtype mediates vasoconstriction of the gerbil spiral modiolar artery. Although the gerbil cochlea is often used as a model in hearing research, the molecular and pharmacological characteristics of the cloned gerbil α(1a)-adrenergic receptor have not been determined. Thus we cloned, expressed and characterized the gerbil α(1a)-adrenergic receptor and then compared its molecular and pharmacological properties to those of other mammalian α(1a)-adrenergic receptors. The cDNA clone contained 1404 nucleotides, which encoded a 467 amino acid peptide with a deduced sequence having 96.8, 96.4 and 91.6% identity to rat, mouse and human α(1a)-receptors, respectively. We transiently transfected the α(1a)-adrenergic receptor into COS-1 cells and determined its pharmacological characteristics by [(3)H]prazosin binding. Unlabeled prazosin had a K(i) of 0.89±0.1nM. The α(1A)-adrenergic receptor-selective antagonists, 5-methylurapidil and WB-4101, bound with high affinity and had K(i) values of 4.9±1 and 1.0±0.1nM, respectively. BMY-7378, an α(1D)-adrenergic receptor-selective antagonist, bound with low affinity (260±60nM). The 91.6% amino acid sequence identity and K(i)s of the cloned gerbil α(1a)-adrenergic receptor are similar to those of the human α(1a)-adrenergic receptor clone. These results show that the gerbil α(1a)-adrenergic receptor is representative of the human α(1a)-adrenergic receptor, lending validity to the use of the gerbil spiral modiolar artery as a model in studies of vascular disorders of the cochlea.

  9. Molecular characterization of a dual endothelin-1/Angiotensin II receptor.

    PubMed Central

    Ruiz-Opazo, N.; Hirayama, K.; Akimoto, K.; Herrera, V. L.

    1998-01-01

    BACKGROUND: The molecular recognition theory (MRT) provides a conceptual framework that could explain the evolution of intermolecular and intramolecular interaction of peptides and proteins. As such, it predicts that binding sites of peptide hormones, and its receptor binding sites were originally encoded by and evolved from complementary strands of genomic DNA. MATERIALS AND METHODS: On the basis of principles underlying the MRT, we screened a rat brain complementary DNA library using an AngII followed by an endothelin-1 (ET-1) antisense oligonucleotide probe, expecting to isolate potential cognate receptors. RESULTS: An identical cDNA clone was isolated independently from both the AngII and ET-1 oligonucleotide screenings. Structural analysis revealed a receptor polypeptide containing a single predicted transmembrane region with distinct ET-1 and AngII putative binding domains. Functional analysis demonstrated ET-1- and AngII-specific binding as well as ET-1- and AngII-induced coupling to a Ca2+ mobilizing transduction system. Amino acid substitutions within the predicted ET-1 binding domain obliterate ET-1 binding while preserving AngII binding, thus defining the structural determinants of ET-1 binding within the dual ET-1/AngII receptor, as well as corroborating the dual nature of the receptor. CONCLUSIONS: Elucidation of the dual ET-1/AngII receptor provides further molecular genetic evidence in support of the molecular recognition theory and identifies for the first time a molecular link between the ET-1 and AngII hormonal systems that could underlie observed similar physiological responses elicited by ET-1 and AngII in different organ systems. The prominent expression of the ET-1/AngII receptor mRNA in brain and heart tissues suggests an important role in cardiovascular function in normal and pathophysiological states. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:9508787

  10. 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'.

  11. Molecular properties of muscarinic acetylcholine receptors

    PubMed Central

    HAGA, Tatsuya

    2013-01-01

    Muscarinic acetylcholine receptors, which comprise five subtypes (M1-M5 receptors), are expressed in both the CNS and PNS (particularly the target organs of parasympathetic neurons). M1-M5 receptors are integral membrane proteins with seven transmembrane segments, bind with acetylcholine (ACh) in the extracellular phase, and thereafter interact with and activate GTP-binding regulatory proteins (G proteins) in the intracellular phase: M1, M3, and M5 receptors interact with Gq-type G proteins, and M2 and M4 receptors with Gi/Go-type G proteins. Activated G proteins initiate a number of intracellular signal transduction systems. Agonist-bound muscarinic receptors are phosphorylated by G protein-coupled receptor kinases, which initiate their desensitization through uncoupling from G proteins, receptor internalization, and receptor breakdown (down regulation). Recently the crystal structures of M2 and M3 receptors were determined and are expected to contribute to the development of drugs targeted to muscarinic receptors. This paper summarizes the molecular properties of muscarinic receptors with reference to the historical background and bias to studies performed in our laboratories. PMID:23759942

  12. 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. PMID:23512843

  13. 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.

  14. 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.

  15. Molecular cloning of the extracellular endodextranase of Streptococcus salivarius.

    PubMed Central

    Lawman, P; Bleiweis, A S

    1991-01-01

    We report the cloning in Escherichia coli of the gene encoding an extracellular endodextranase (alpha-1,6-glucanhydrolase, EC 3.2.1.11) from Streptococcus salivarius PC-1. Recombinants from a S. salivarius PC-1-Lambda ZAP II genomic library specifying dextranase activity were identified as plaques surrounded by zones of clearing on blue dextran agar. One such clone, PD1, had a 6.3-kb EcoRI fragment insert which encoded a 190-kDa protein with dextranase activity. The recombinant strain also produced two lower-molecular-mass polypeptides (90 and 70 kDa) that had dextranase activity. Native dextranase was recovered from concentrated culture fluids of S. salivarius as a single 110-kDa polypeptide. PD1 phage lysate and PC-1 culture supernatant fluid extract were used to measure substrate specificity of the recombinant and native forms of dextranase, respectively. Analysis of these reaction products by thin-layer chromatography revealed the expected isomaltosaccharide products yielded by the recombinant-specified enzyme but was unable to resolve the larger polysaccharide products of the native enzyme. Furthermore, S. salivarius utilized neither the substrates nor the products of dextran hydrolysis for growth. Images FIG. 1 FIG. 2 FIG. 3 FIG. 4 PMID:1938938

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

    PubMed

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

    2014-08-01

    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. PMID:25106573

  17. Molecular mechanisms of prolactin and its receptor.

    PubMed

    Brooks, Charles L

    2012-08-01

    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.

  18. 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 a chance…

  19. 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.

  20. Purification, characterization and molecular cloning of prophenoloxidases from Sarcophaga bullata.

    PubMed

    Chase, M R; Raina, K; Bruno, J; Sugumaran, M

    2000-10-01

    Prophenoloxidase (PPO) is a key enzyme associated with both melanin biosynthesis and sclerotization in insects. This enzyme is involved in three physiologically important processes viz., cuticular hardening, defense reactions and wound healing in insects. It was isolated from the larval hemolymph of Sarcophaga bullata and purified by employing ammonium sulfate precipitation, Phenyl Sepharose chromatography, DEAE-Sepharose chromatography, and Sephacryl S-200 column chromatography. The purified enzyme exhibited two closely moving bands on 7.5% SDS-PAGE under denaturing conditions. From the estimates of molecular weight on Sephacryl S-100, TSK-3000 HPLC column and SDS-PAGE, which ranged from 90,000 to 100,000, it was inferred that the enzyme is made up of a single polypeptide chain. Activation of PPO (K(a)=40 microM) was achieved by the cationic detergent, cetyl pyridinium chloride below its critical micellar concentration (0.8 mM) indicating that the detergent molecules are binding specifically to the PPO and causing the activation. Neither anionic, nor nonionic (or zwitterionic) detergents activated the PPO. The active enzyme exhibited wide substrate specificity and marked thermal unstability. Using primers designed to conserved amino acid sequences from known PPOs, we PCR amplified and cloned two PPO genes from the sarcophagid larvae. The clones encoded polypeptides of 685 and 691 amino acids. They contained two distinct copper binding regions and lacked the signal peptide sequence. They showed a high degree of homology to dipteran PPOs. Both contained putative thiol ester site, two proteolytic activation sites and a conserved C-terminal region common to all known PPOs.

  1. Cloning and retinal expression of melatonin receptors in the European sea bass, Dicentrarchus labrax.

    PubMed

    Sauzet, Sandrine; Besseau, Laurence; Herrera Perez, Patricia; Covès, Denis; Chatain, Béatrice; Peyric, Elodie; Boeuf, Gilles; Muñoz-Cueto, José Antonio; Falcón, Jack

    2008-06-01

    Melatonin contributes to synchronizing behaviors and physiological functions to daily and seasonal rhythm in fish. However, no coherent vision emerges because the effects vary with the species, sex, age, moment of the year or sexual cycle. And, scarce information is available concerning the melatonin receptors, which is crucial to our understanding of the role melatonin plays. We report here the full length cloning of three different melatonin receptor subtypes in the sea bass Dicentrarchus labrax, belonging, respectively, to the MT1, MT2 and Mel1c subtypes. MT1, the most abundantly expressed, was detected in the central nervous system, retina, and gills. MT2 was detected in the pituitary gland, blood cells and, to a lesser extend, in the optic tectum, diencephalon, liver and retina. Mel1c was mainly expressed in the skin; traces were found in the retina. The cellular sites of MT1 and MT2 expressions were investigated by in situ hybridization in the retina of pigmented and albino fish. The strongest signals were obtained with the MT1 riboprobes. Expression was seen in cells also known to express the enzymes of the melatonin biosynthesis, i.e., in the photoreceptor, inner nuclear and ganglion cell layers. MT1 receptor mRNAs were also abundant in the retinal pigment epithelium. The results are consistent with the idea that melatonin is an autocrine (neural retina) and paracrine (retinal pigment epithelium) regulator of retinal function. The molecular tools provided here will be of valuable interest to further investigate the targets and role of melatonin in nervous and peripheral tissues of fish.

  2. 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

  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

    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. PMID:27572664

  5. Molecular cloning and expression analysis of TRAF3 in chicken.

    PubMed

    Yang, H L; Feng, Z Q; Zeng, S Q; Li, S M; Zhu, Q; Liu, Y P

    2015-01-01

    Tumor necrosis factor receptor-associated factor 3 (TRAF3) is a crucial regulator that suppresses c-Jun N-terminal kinase and non-canonical nuclear factor-kB signaling, but facilitates type I interferon production. To determine TRAF3 function in innate immune responses among birds, particularly chicken, we cloned and characterized the chicken TRAF3 gene (chTRAF3) and detected its tissue expression profile in chicken. We also detected the differential expression of chTRAF3 and its downstream gene interferon-β (IFN-β) upon different stimuli in primary chicken embryo fibroblast cells. Two chTRAF3 gene products, chTRAF3-1 and chTRAF3-2, can be produced by alternative splicing. The full-length coding sequence of chTRAF3 (chTRAF3-1) was 1704 base pairs and encoded a protein of 567 amino acids with high identity to TRAF3 homologs from mammals and other birds. The deduced amino acid sequence showed typical characteristics of TRAFs, with a RING finger domain, 2 zf-TRAF motifs, and a MATH domain. Quantitative real-time polymerase chain reaction analysis revealed broad expression of chTRAF3 in all detected tissues, with abundant expression in the spleen, thymus, lung, and small intestine. Expression of chTRAF3 was significantly upregulated in a time- and concentration-dependent manner in chicken embryo fibroblast cells challenged with poly I:C or poly dA-dT. Furthermore, chTRAF3 and IFN-β mRNA expression from chicken embryo fibroblast cells challenged with Newcastle disease virus F48E9 suffered intense suppression compared with Newcastle disease virus Mukteswar infection. Our results indicate that chTRAF3 plays important roles in defending against both RNA and DNA virus infection. PMID:25966214

  6. Cloning

    MedlinePlus

    ... copies of whole animals Therapeutic cloning, which creates embryonic stem cells. Researchers hope to use these cells to grow healthy tissue to replace injured or diseased tissues in the human body. NIH: National Human Genome Research Institute

  7. Development of a mutant strain of Escherichia coli for molecular cloning of highly methylated DNA

    SciTech Connect

    Bishr, M.A.

    1991-01-01

    A mutant strain of Escherichia coli designated as GR219 that allows efficient molecular cloning of highly methylated bean DNA has been developed by UV light mutation of the parent LE392 str[sup r] strain. This mutant strain, like the parent, is streptomycin resistant and is biologically contained, because it requires thymidine for growth. Both the wild type and the mutant strain have lambda phage receptors so both can be utilized for construction of genomic libraries using the phase as a vector. The efficiency of transformation of the parent and the mutant strain with a recombinant plasmid containing bean DNA was compared to the efficiency of transformation of the PLK-F[prime] strain, which has a deletion of mcrA and mcrB genes and, therefore, allows transformation with methylated bean DNA. It has been found that the GR219 strain has the highest efficiency of transformation, while the PLK-F[prime] strain shows less, and the parent LE392 str[sup r] strain the least efficiency of transformation. These results indicate that strains of E. coli with mcrA and mcrB genes can recognize and degrade highly methylated DNA. However, other undefined factors affected by the altered gene(s) in the GR219 strain are also involved in the recognition and degradation of any cloned foreign DNA.

  8. 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.

  9. Applications of the Restriction Free (RF) cloning procedure for molecular manipulations and protein expression.

    PubMed

    Unger, Tamar; Jacobovitch, Yossi; Dantes, Ada; Bernheim, Reut; Peleg, Yoav

    2010-10-01

    Molecular manipulations, including DNA cloning and mutagenesis are basic tools used on a routine basis in all life-science disciplines. Over the last decade new methodologies have emerged that facilitated and expanded the applications for DNA cloning and mutagenesis. Ligation-Independent Cloning (LIC) techniques were developed and replaced the classical Ligation Dependent Cloning (LDC) platform. Restriction Free (RF) cloning was originally developed for introduction of foreign DNA into a plasmid at any predetermined position. RF cloning is based on PCR amplification of a DNA fragment, which serves as a mega-primer for the linear amplification of the vector and insert. Here we present several novel applications of the Restriction Free (RF) cloning platform for DNA cloning and mutagenesis. The new applications include simultaneous cloning of several DNA fragments into distinct positions within an expression vector, simultaneous multi-component assembly, and parallel cloning of the same PCR product into a series of different vectors. In addition, we have expanded the application of the RF cloning platform for multiple alterations of the target DNA, including simultaneous multiple-site mutagenesis and simultaneous introduction of deletions and insertions at different positions. We further demonstrate the robustness of the new applications for facilitating recombinant protein expression in the Escherichia coli system. PMID:20600952

  10. Chicken epidermal growth factor (EGF) receptor: cDNA cloning, expression in mouse cells, and differential binding of EGF and transforming growth factor alpha.

    PubMed Central

    Lax, I; Johnson, A; Howk, R; Sap, J; Bellot, F; Winkler, M; Ullrich, A; Vennstrom, B; Schlessinger, J; Givol, D

    1988-01-01

    The primary structure of the chicken epidermal growth factor (EGF) receptor was deduced from the sequence of a cDNA clone containing the complete coding sequence and shown to be highly homologous to the human EGF receptor. NIH-3T3 cells devoid of endogenous EGF receptor were transfected with the appropriate cDNA constructs and shown to express either chicken or human EGF receptors. Like the human EGF receptor, the chicken EGF receptor is a glycoprotein with an apparent molecular weight of 170,000. Murine EGF bound to the chicken receptor with approximately 100-fold lower affinity than to the human receptor molecule. Surprisingly, human transforming growth factor alpha (TGF-alpha) bound equally well or even better to the chicken EGF receptor than to the human EGF receptor. Moreover, TGF-alpha stimulated DNA synthesis 100-fold better than did EGF in NIH 3T3 cells that expressed the chicken EGF receptor. The differential binding and potency of mammalian EGF and TGF-alpha by the avian EGF receptor contrasts with the similar affinities of the mammalian receptor for the two growth factors. Images PMID:3260329

  11. Molecular cloning and analysis of lymphokines. Volume 13

    SciTech Connect

    Webb, D.R.; Goeddel, D.V.

    1987-01-01

    These proceedings collect papers on the subject of lymphokines. Topics include: DNA-cloning of mouse and human lymphokine genes, inteferons, interleukins, gene expression, tumor necrosis factors, and recombinant DNA.

  12. 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,…

  13. 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.

  14. Characterization of two cloned human CB1 cannabinoid receptor isoforms.

    PubMed

    Rinaldi-Carmona, M; Calandra, B; Shire, D; Bouaboula, M; Oustric, D; Barth, F; Casellas, P; Ferrara, P; Le Fur, G

    1996-08-01

    We have investigated the pharmacology of two central human cannabinoid receptor isoforms, designated CB1 and CB1A, stably expressed in Chinese hamster ovary cell lines, designated as CHO-CB1 and CHO-CB1A, respectively. In direct binding assays on isolated membranes the agonist [3H]CP 55,940 bound in a saturable and highly specific manner to both cannabinoid receptor isoforms. Competition binding experiments performed with other commonly used receptor agonists showed the following rank order of potency: CP 55,940 > tetrahydrocannabinol > WIN 55212-2 > anandamide. Except for the endogenous ligand anandamide (CB1, Ki = 359.6 nM vs. CB1A, Ki = 298 nM), these agonists bound to CB1A (CP 55,940, WIN 55212-2 and delta 9-THC, Ki = 7.24,345 and 26.7 nM, respectively) with about 3-fold less affinity than to CB1 (CP 55,940, WIN 55212-2 and delta 9-THC, Ki = 2.26, 93 and 7.1 nM, respectively). The cannabinoid receptor antagonist SR 141716A also bound to CB1A (Ki = 43.3 nM) with slightly less affinity than to CB1 (Ki = 4.9 nM). Cannabinoid receptor-linked second messenger system studies performed in the CHO-CB1 and CHO-CB1A cells showed that both receptors mediated their action through the agonist-induced inhibition of forskolin-stimulated cAMP accumulation. This activity was totally blocked by pretreatment with PTX. Additionally, both isoforms activated mitogen-activated protein kinase. The selective antagonist SR 141716A was able to selectively block these responses in both cell lines, to an extent that reflected its binding characteristics. Our results show that the amino-truncated and -modified CB1 isoform CB1A exhibits all the properties of CB1 to a slightly attenuated extent.

  15. 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.

  16. Microorganisms in the gut of beetles: evidence from molecular cloning.

    PubMed

    Zhang, Ning; Suh, Sung-Oui; Blackwell, Meredith

    2003-11-01

    We have regularly cultured yeasts from the gut of certain beetles in our ongoing research. In this study cloned PCR products amplified from the gut contents of certain mushroom-feeding and wood-ingesting beetles in four families (Erotylidae, Tenebrionidae, Ciidae, and Passalidae) were sequenced and compared with culture results. Cultural techniques detected some yeasts present in the gut of the beetles, including a Pichia stipitis-like yeast associated with wood-ingesting passalid beetles. Clone sequences similar to several ascomycete yeasts and Malassezia restricta, a fastidious basidiomycetous yeast requiring special growth media, however, were not detected by culturing. Unexpectedly, phylogenetic analysis of additional clone sequences discovered from passalid beetles showed similarity to members of the Parabasalia, protists known from other wood-ingesting insects, termites, and wood roaches. Examination of all gut regions of living passalids, however, failed to reveal parabasalids, and it is possible that they were parasites in the gut tissue present in low numbers.

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

    PubMed Central

    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. PMID:22110522

  18. T-cell receptor heterogeneity of gamma delta T-cell clones from human female reproductive tissues.

    PubMed

    Christmas, S E; Brew, R; Deniz, G; Taylor, J J

    1993-03-01

    gamma delta T cells were isolated from human decidua parietalis, decidua basalis and cervix and cloned in the presence of interleukin-2 (IL-2). T-cell receptor (TcR) expression was then analysed and compared with that of a panel of gamma delta T-cell clones from peripheral blood. Only 17/40 (42.5%) clones from decidua parietalis were V gamma 9+/V delta 2+ as compared to 68/94 (72%) of peripheral blood clones (P < 0.005). Conversely, 50% of clones from decidua parietalis but only 15% of clones from peripheral blood were V delta 1+ (P < 0.001). At least seven distinct TcR types were identified among the panel of clones from decidua parietalis and at least six different types were expressed by the panel of 17 clones from cervix. This receptor heterogeneity was not a result of interdonor variation as in all instances where more than one clone was obtained from a single sample, individual clones having between two and five receptor types were identified. However, 23/24 (95.8%) of clones from decidua basalis were V gamma 9+/V delta 2+. Most clones from decidua parietalis and cervix, whether V gamma 9+/V delta 2+ or V delta 1+, were positive for the mucosal lymphocyte marker, HML-1, but expression was often heterogeneous within a single clone. In contrast, almost all gamma delta T-cell clones from peripheral blood were HML-1-. Thus, unlike the mouse, gamma delta T cells within these human female reproductive tissues have a diverse TcR repertoire which, in decidua parietalis, is distinct from that of peripheral blood.

  19. 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…

  20. 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.

  1. GABAA receptor beta subunit heterogeneity: functional expression of cloned cDNAs.

    PubMed Central

    Ymer, S; Schofield, P R; Draguhn, A; Werner, P; Köhler, M; Seeburg, P H

    1989-01-01

    Cloned cDNAs encoding two new beta subunits of the rat and bovine GABAA receptor have been isolated using a degenerate oligonucleotide probe based on a highly conserved peptide sequence in the second transmembrane domain of GABAA receptor subunits. The beta 2 and beta 3 subunits share approximately 72% sequence identity with the previously characterized beta 1 polypeptide. Northern analysis showed that both beta 2 and beta 3 mRNAs are more abundant in the brain than beta 1 mRNA. All three beta subunit encoding cDNAs were also identified in a library constructed from adrenal medulla RNA. Each beta subunit, when co-expressed in Xenopus oocytes with an alpha subunit, forms functional GABAA receptors. These results, together with the known alpha subunit heterogeneity, suggest that a variety of related but functionally distinct GABAA receptor subtypes are generated by different subunit combinations. Images PMID:2548852

  2. Identification of a novel V1-type AVP receptor based on the molecular recognition theory.

    PubMed Central

    Herrera, V. L.; Ruiz-Opazo, N.

    2001-01-01

    BACKGROUND: The molecular recognition theory predicts that binding domains of peptide hormones and their corresponding receptor binding domains evolved from complementary strands of genomic DNA, and that a process of selective evolutionary mutational events within these primordial domains gave rise to the high affinity and high specificity of peptide hormone-receptor interactions observed today in different peptide hormone-receptor systems. Moreover, this theory has been broadened as a general hypothesis that could explain the evolution of intermolecular protein-protein and intramolecular peptide interactions. MATERIALS AND METHODS: Applying a molecular cloning strategy based on the molecular recognition theory, we screened a rat kidney cDNA library with a vasopressin (AVP) antisense oligonucleotide probe, expecting to isolate potential AVP receptors. RESULTS: We isolated a rat kidney cDNA encoding a functional V1-type vasopressin receptor. Structural analysis identified a 135 amino acid-long polypeptide with a single transmembrane domain, quite distinct from the rhodopsin-based G protein-coupled receptor superfamily. Functional analysis of the expressed V1-type receptor in Cos-1 cells revealed AVP-specific binding, AVP-specific coupling to Ca2+ mobilizing transduction system, and characteristic V1-type antagonist inhibition. CONCLUSIONS: This is the second AVP receptor cDNA isolated using AVP antipeptide-based oligonucleotide screening, thus providing compelling evidence in support of the molecular recognition theory as the basis of the evolution of this peptide hormone-receptor system, as well as adds molecular complexity and diversity to AVP receptor systems. PMID:11683375

  3. Molecular cloning and characterization of an inorganic pyrophosphatase from barley.

    PubMed

    Visser, K; Heimovaara-Dijkstra, S; Kijne, J W; Wang, M

    1998-05-01

    A cDNA clone with sequence homology to soluble inorganic pyrophosphatase (IPPase) was isolated from a library of developing barley grains. The protein encoded by this clone was produced in transgenic Escherichia coli, and showed IPPase activity. In nondormant barley grains, the gene appeared to be expressed in metabolically active tissue such as root, shoot, embryo and aleurone. During inhibition, a continuous increase of the steady state mRNA level of IPPase was observed in embryos of non-dormant grains. In the embryos of dormant grains its production declined, after an initial increase. With isolated dormant and nondormant embryos, addition of recombinant IPPase, produced by E. coli, enhanced the germination rate. On the other hand, addition of pyrophosphate (PPi), substrate for this enzyme, appeared to reduce the germination rate. A role for this IPPase in germination is discussed.

  4. 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

  5. Mannose receptor contribution to Candida albicans phagocytosis by murine E-clone J774 macrophages.

    PubMed

    Porcaro, Isabelle; Vidal, Michel; Jouvert, Sylvie; Stahl, Philip D; Giaimis, Jean

    2003-08-01

    Mannoproteins, as the main constituents of the outer layer of yeast cell walls, are able to interact with phagocytic cells in an opsonin-independent manner through the mannose receptor (MR) and to induce yeast ingestion by the professional phagocytes. Moreover, the MR also mediates endocytosis of soluble ligands through clathrin-coated pits. Here, we studied some aspects of the interaction between the MR and Candida albicans using murine E-clone macrophages and the consequences on MR trafficking. Using a pull-down assay involving mixture E-clone macrophage detergent lysate with mannosylated Sepharose beads and glutaraldehyde-fixed, heat-killed (HK) C. albicans, we found that binding of solubilized MR to mannosylated particles occurred with characteristics similar to the receptor's cell-surface mannose-binding activity. We then demonstrated that MR expressed on E-clone macrophages contributed to phagocytosis of unopsonized, HK C. albicans and that yeast phagocytosis induced a decrease in MR endocytic activity without concomitant degradation of the receptor in the time lapse studied. PMID:12885937

  6. 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.

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

    PubMed Central

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

    1996-01-01

    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. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8755605

  8. 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.

  9. Molecular cloning, phylogenetic analysis and expression of beluga whale (Delphinapterus leucas) interleukin 6.

    PubMed

    St-Laurent, G; Archambault, D

    2000-01-31

    Interleukin 6 (IL-6) is a cytokine produced primarily by the monocytes/macrophages with regulatory effects in hematopoiesis, acute phase response, and multiple aspects of the immune response. IL-6 exerts its activity through its binding to specific high affinity receptors at the surface of target cells. As yet, no molecular data have been reported for the beluga whale IL-6. In this study, we cloned and determined the entire beluga whale IL-6-encoding cDNA sequence by reverse transcription-polymerase chain reaction (RT-PCR) sequencing, and analysed its genetic relationship with those from several mammalian species including human, rodent, ruminant, carnivore and other marine species. The identity levels of beluga whale IL-6 nucleic and deduced amino acid sequences with those from these mammalian species ranged from 62.3 to 97.3%, and 42.9 to 95.6%, respectively. Phylogenetic analysis based on amino acid sequences showed that the beluga whale IL-6 was most closely related to that of the killer whale. Thereafter, beluga whale IL-6-encoding sequence was successfully expressed in Escherichia coli by using the pTHIOHisA expression vector for the production of a recombinant fusion protein. The immunogenicity of the recombinant fusion protein was then confirmed as determined by the production of a beluga whale IL-6-specific rabbit antiserum.

  10. Molecular cloning of the gene for human anti-haemophilic factor IX.

    PubMed

    Choo, K H; Gould, K G; Rees, D J; Brownlee, G G

    1982-09-01

    A functional deficiency of factor IX, one of the coagulation factors involved in blood clotting, leads to the bleeding disorder known as Christmas disease, or haemophilia B. Both this disease and haemophilia A (factor VIII (C) deficiency) are X chromosome-linked and together occur at a frequency of approximately 1 in 10,000 males. The molecular basis for the functional alteration of factor IX in Christmas disease is not clearly understood. As a first step towards the elucidation of the molecular events involved, we have attempted molecular cloning of the factor IX gene. We used a bovine factor IX cDNA clone, isolated using synthetic oligonucleotides as probes, to screen a cloned human gene library. Here we report the isolation and partial characterization of a lambda recombinant phage containing the human factor IX gene.

  11. Cloning and expression of a rat brain. alpha. sub 2B -adrenergic receptor

    SciTech Connect

    Flordellis, C.S.; Handy, D.E.; Bresnahan, M.R.; Zannis, V.I.; Gavras, H. )

    1991-02-01

    The authors isolated a cDNA clone (RB{alpha}{sub 2B}) and its homologous gene (GR{alpha}{sub 2B}) encoding an {alpha}{sub 2B}-adrenergic receptor subtype by screening a rat brain cDNA and a rat genomic library. Nucleotide sequence analysis showed that both clones code for a protein of 458 amino acids, which is 87% homologous to the human kidney glycosylated adrenergic receptor ({alpha}{sub 2}-C4) and divergent from the rat kidney nonglycosylated {alpha}{sub 2B} subtype (RNG{alpha}{sub 2}). Transient expression of RB{alpha}{sub 2B} in COS-7 cells resulted in high-affinity saturable binding for ({sup 3}H)rauwolscine and a high receptor number in the membranes of transfected COS-7 cells. Pharmacological analysis demonstrated that the expressed receptor bound adrenergic ligands with the following order of potency: rauwolscine {gt} yohimbine {gt} prazosin {gt} oxymetazoline, with a prazosin-to-oxymetazoline K{sub i} ratio of 0.34. This profile is characteristic of the {alpha}{sub 2B}-adrenergic receptor subtype. Blotting analysis of rat brain mRNA gave one major and two minor mRNA species, and hybridization with strand-specific probes showed that both DNA strands of GR{alpha}{sub 2B} may be transcriptionally active. These findings show that rat brain expresses an {alpha}{sub 2B}-adrenergic receptor subtype that is structurally different from the rat kidney nonglycosylated {alpha}{sub 2B} subtype. Thus the rat expresses at least two divergent {alpha}{sub 2B}-adrenergic receptors.

  12. Semiautomated clone verification by real-time PCR using molecular beacons.

    PubMed

    van Schie, R C; Marras, S A; Conroy, J M; Nowak, N J; Catanese, J J; de Jong, P J

    2000-12-01

    Conventional, high-throughput PCR analysis of common elements utilizing numerous primer sets and template DNA requires multiple rounds of PCR to ensure optimal conditions. Laborious gel electrophoresis and staining is then necessary to visualize amplification products. We propose novel multicolor molecular beacons, to establish a high-throughput, PCR-based sequence tagged site (STS) detection system that swiftly and accurately confirms marker content in template containing common repeat elements. A simple, one-tube, real-time PCR assay system was developed to specifically detect regions containing CA and GATA repeats. Ninety-six samples can be confirmed for marker content in a closed-tube format in 3 h, eliminating product confirmation on agarose gels and avoiding crossover contamination. Multiple STSs can be detected simultaneously in the same reaction tube by utilizing molecular beacons labeled with multicolor fluorophores. Template DNA from 260 RPCI-11 bacterial artificial chromosome (BAC) clones was examined for the presence of CA and/or GATA repeats using molecular beacon PCR and compared with conventional PCR results of the same clones. Of the 205 clones containing CA and GATA repeats, we were able to identify 129 clones (CA, n = 99; GATA, n = 30) by using molecular beacons and only 121 clones (CA, n = 92; GATA, n = 29) by conventional PCR amplification. As anticipated, 55 clones that contained sequences other than CA or GATA failed molecular beacon detection. Molecular beacon PCR, employing beacons specific for tandem repeat elements, provides a fast, accurate, and sensitive multiplex detection assay that will expedite verification of marker content in a multitude of template containing these repeats.

  13. Molecular Cloning of MAPK Gene Family Using Synthetic Oligonucleotide Probe.

    PubMed

    Zhou, Song; Wang, Qin; Chen, Jing; Chen, Jiang-Ye

    1999-01-01

    MAPK(mitogen activated protein kinase) is a kind of Ser/Thr protein kinase. The MAPKs play an important role in several different signal transduction pathways. The MAPKs may also have a role in morphorgenesis of Candida albicans. An oligonucleotide probe was used to screen novel MAPKs in C. albicans. All MAPKs shared high homogeneity in their eleven kinase subdomains, especially subdoman VII and VIII. In subdomain VII, nearly all MAPKs have the same KIDFGLAR sequence, and the two known MAPKs in C. albicans CEK1 and MKC1 have only one different nucleotide in that DNA sequence. This probe was hybridized with C. albicans genomic DNA. Under stringent conditions, the probe could only hybridize with CEK1 and MKC1 gene fragment. But when hybridized at 40 degrees in non-SDS solution, two novel bands appeared. This condition was used to screen SC5314 DNA library, and many positive clones with different hybridization density were obtained. The strongest hybridization clones were identified to contain CEK1 and MKC1 gene. From the stronger positive hybridization clones, two novel genes were identified. The first gene, named CRK1(CDC2-related protein kinase 1), shared high homogeneity to MAPKs, but was not of them. It is closest to SGV1 from S. cerevisiae (with homology 47%) and PITALRE from human (with homology 41%), both of which are CDC2-related protein kinases. The second gene called CEK2(Candida albicans extracelluar signal-regulated kinase 2) is a novel MAPK of Candida albicans, which shares the highest identity with CEK1 and its S. cerevisiae homologs, FUS3 and KSS1, two redundant MAPKs in yeast pheromone response and morphogenesis. PMID:12114967

  14. Molecular cloning of human ornithine aminotransferase mRNA

    SciTech Connect

    Inana, G.; Totsuka, S.; Redmond, M.; Dougherty, T.; Nagle, J.; Shiono, T.; Ohura, T. Kominami, E.; Katunuma, N.

    1986-03-01

    The isolation and characterization of a cDNA clone for the mRNA of human ornithine aminotransferase (OATase; ornithine-oxo-acid aminotransferase; L-ornithine:2-oxo-acid aminotransferase, EC 2.6.1.13), a nonabundant mitochondrial matrix enzyme that is severely deficient in a hereditary chorioretinal degenerative disease (gyrate atrophy), is described. Human liver, retina, and retinoblastoma (Y79) mRNAs were prepared and tested for the OATase mRNA content by in vitro translation, immunoprecipitation, and NaDodSO/sub 4//PAGE. The retinoblastoma cells were found to be expressing this enzyme at a relatively high level. The primary translation product of the OATase mRNA is larger than the pure OATase protein on NaDodSO/sub 4//PAGE. lambdagt11 cDNA libraries were prepared from the human mRNAs, and the recombinant clones were immunoscreened as plaques with two different preparations of rabbit anti-human OATase antibodies. The amino acid sequences of seven tryptic peptides (115 amino acid residues) of the pure human OATase were obtained by microsequencing. When the tryptic peptide and cDNA-derived amino acid sequences were compared, homologies in 111 of 115 residues, including a match of 20 consecutive residues, were observed. An RNA blot hybridization of /sup 32/P-labeled OATase cDNA to normal human retina and retinoblastoma mRNAs demonstrated an OATase mRNA species of approx. = 2.2 kilobases.

  15. 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.

  16. 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.

  17. 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.

  18. Molecular cloning and characterization of chitinase genes from Candida albicans.

    PubMed Central

    McCreath, K J; Specht, C A; Robbins, P W

    1995-01-01

    Chitinase (EC 3.2.1.14) is an important enzyme for the remodeling of chitin in the cell wall of fungi. We have cloned three chitinase genes (CHT1, CHT2, and CHT3) from the dimorphic human pathogen Candida albicans. CHT2 and CHT3 have been sequenced in full and their primary structures have been analyzed: CHT2 encodes a protein of 583 aa with a predicted size of 60.8 kDa; CHT3 encodes a protein of 567 aa with a predicted size of 60 kDa. All three genes show striking similarity to other chitinase genes in the literature, especially in the proposed catalytic domain. Transcription of CHT2 and CHT3 was greater when C. albicans was grown in a yeast phase as compared to a mycelial phase. A transcript of CHT1 could not be detected in either growth condition. Images Fig. 2 Fig. 5 PMID:7708682

  19. 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.

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

    PubMed Central

    Munro, S; Maniatis, T

    1989-01-01

    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. Images PMID:2531896

  1. 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...

  2. [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.

  3. (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.

  4. Molecular basis of essential fructosuria: molecular cloning and mutational analysis of human ketohexokinase (fructokinase).

    PubMed

    Bonthron, D T; Brady, N; Donaldson, I A; Steinmann, B

    1994-09-01

    Essential fructosuria is one of the oldest known inborn errors of metabolism. It is a benign condition which is believed to result from deficiency of hepatic fructokinase (ketohexokinase, KHK, E.C.2.7.1.3). This enzyme catalyses the first step of metabolism of dietary fructose, conversion of fructose to fructose-1-phosphate. Despite the early recognition of this disorder, the primary structure of human KHK and the molecular basis of essential fructosuria have not been previously defined. In this report, the isolation and sequencing of full-length cDNA clones encoding human ketohexokinase are described. Alternative mRNA species and alternative KHK isozymes are produced by alternative polyadenylation and splicing of the KHK gene. The KHK proteins show a high level of sequence conservation relative to rat KHK. Direct evidence that mutation of the KHK structural gene is the cause of essential fructosuria was also obtained. In a well-characterized family, in which three of eight siblings have fructosuria, all affected individuals are compound heterozygotes for two mutations Gly40Arg and Ala43Thr. Both mutations result from G-->A transitions, and each alters the same conserved region of the KHK protein. Neither mutation was seen in a sample of 52 unrelated control individuals. An additional conservative amino acid change (Val49IIe) was present on the KHK allele bearing Ala43Thr.

  5. 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

  6. 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. PMID:19427315

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 ...

  8. 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.

  9. Molecular cloning of ADP-glucose pyrophosphorylase from cyanobacteria

    SciTech Connect

    Kakefuda, G.; Yeeyung Charng; Iglesias, A.; McIntosh, L. )

    1991-05-01

    Bacterial and higher plant ADP-glucose pyrophosphorylase differ in structure (homotetramer vs heterotetramer respectively) and allosteric activator and inhibitor. However, highly conserved regions can be identified when sequence comparisons are made between ADP-glucose pyrophosphorylases from diverse species. The fructose 1,6 bisphosphate binding site (activator site) in E. coli is highly conserved in all species for which ADP-glucose pyrophosphorylase has been sequenced. A second conserved region, which is labeled by 8-azido-ATP, is also highly conserved in bacteria and higher plants. In previously cloned ADP-glucose pyrophosphorylases the two conserved regions are separated by approximately 80 amino acids. The authors have used these conserved amino acid sequences to design degenerate oligonucleotide primers for polymerase chain reaction amplification (PCR) of part of the ADP-glucose pyrophosphorylase geae. A predicted 240 bp fragment is amplified in PCR reactions using Anabaena sp. PCC 7120 and Synechocystis sp. PCC 6803 genomic DNA as template. The deduced amino acid sequence from the 240 bp Anabaena fragment shares 75 and 76% identity to that of the rice endosperm and spinach leaf ADP-glucose pyrophosphorylases respectively. The Anabaena amino acid sequence shares 42% identity in amino acid sequence to the E. coli enzyme. At the nucleotide level there is 66% identity of the Anabaena sequence to rice endosperm ADP-glucose pyrophosphorylase and 54% to the E. coli gene. The PCR amplified fragments are being used to screen respective Anabaena and Synechocystis genomic gene libraries.

  10. Molecular Cloning of Tomato Plasma Membrane H+-ATPase 1

    PubMed Central

    Ewing, Nicholas N.; Wimmers, Larry E.; Meyer, David J.; Chetelat, Roger T.; Bennett, Alan B.

    1990-01-01

    Two cDNA clones (LHA1 and LHA2) from tomato (Lycopersicon esculentum) which likely encode isoforms of the plasma membrane H+-ATPase were isolated. The longest cDNA (3229 base pairs), LHA1, comprises an open reading frame that encodes a 956 amino acid, 105 kilodalton polypeptide with several potential transmembrane domains. In vitro transcription and translation of LHA1 yields a major translation product of approximately 100 kilodaltons that is immunoprecipitable with antiserum to the corn root plasma membrane H+-ATPase. LHA2 encodes a portion of a coding sequence that is 96% identical to LHA1, suggesting that LHA2 encodes an isoform of the H+-ATPase. Genomic DNA gel blot analysis indicates that both LHA1 and LHA2 hybridize to a common set of six to eight restriction fragments at moderate stringency and to single distinct fragments at high stringency. LHA1 and LHA2 map to distinct sites on chromosomes three and six, respectively. RNA gel blot analysis indicates that both LHA1 and LHA2 hybridize to 3.4 kilobase pair transcripts present in both leaves and roots, although the LHA2 transcript is relatively more abundant in leaves than in roots. These results indicate that in tomato as many as six to eight genes may encode the plasma membrane H+-ATPase, two of which are expressed at the level of mRNA in both roots and leaves. Images Figure 3 Figure 4 Figure 5 Figure 7 PMID:16667929

  11. Molecular cloning and expression analysis of pig CD138.

    PubMed

    Bae, Joonbeom; Jeong, Seonah; Lee, Ju Yeon; Lee, Hyun-Jeong; Choi, Bong-Hwan; Kim, Ji-Eun; Choi, Inho; Chun, Taehoon

    2013-12-01

    CD138 (syndecan-1) interacts with various components of the extracellular matrix and associates with the actin cytoskeleton. In this study, we cloned pig CD138 cDNA and determined its complete cDNA sequence. Pig CD138 cDNA contained an open reading frame (930 bp) encoding 309 amino acids with five well conserved putative glycosaminoglycan attachment sites, a putative cleavage site for matrix metalloproteinases, and conserved motifs involved in signal transduction among mammalian species. Pig CD138 mRNA was detected in various tissues, including lymphoid and non-lymphoid organs, indicating the multicellular functions of CD138 in pigs. Western blot and flow cytometry analyses detected an approximate 35 kDa pig CD138 protein expressed on the cell surface. Further immunohistochemistry analysis revealed that CD138 expression was mainly observed in submucosa and lamina propria of the pig small intestine. Further study will be necessary to define the functional importance of CD138 during specific infectious diseases in pigs. PMID:24128845

  12. Molecular Characterization of Infectious Clones of the Minute Virus of Canines Reveals Unique Features of Bocaviruses▿

    PubMed Central

    Sun, Yuning; Chen, Aaron Yun; Cheng, Fang; Guan, Wuxiang; Johnson, F. Brent; Qiu, Jianming

    2009-01-01

    Minute virus of canines (MVC) is a member of the genus Bocavirus in the family Parvoviridae. We have molecularly cloned and sequenced the 5′- and 3′-terminal palindromes of MVC. The MVC genome, 5,404 nucleotides (nt) in length, shared an identity of 52.6% and 52.1% with that of human bocavirus and bovine parvovirus, respectively. It had distinct palindromic hairpins of 183 nt and 198 nt at the left-end and right-end termini of the genome, respectively. The left-end terminus was also found in two alternative orientations (flip or flop). Both termini shared extensive similarities with those of bovine parvovirus. Four full-length molecular clones constructed with different orientations of the left-end terminus proved to be infectious in Walter Reed canine cell/3873D (WRD) canine cells. Both MVC infection and transfection of the infectious clone in WRD cells revealed an identical RNA transcription profile that was similar to that of bovine parvovirus. Mutagenesis of the infectious clone demonstrated that the middle open reading frame encodes the NP1 protein. This protein, unique to the genus Bocavirus, was essential for MVC DNA replication. Moreover, the phospholipase A2 motif in the VP1 unique region was also critical for MVC infection. Thus, our studies revealed important information about the genus Bocavirus that may eventually help us to clone the human bocavirus and study its pathogenesis. PMID:19211770

  13. Differential Cell Tropism of Feline Immunodeficiency Virus Molecular Clones In Vivo

    PubMed Central

    Dean, Gregg A.; Himathongkham, Sunee; Sparger, Ellen E.

    1999-01-01

    Independent studies have demonstrated different cell tropisms for molecular clones of feline immunodeficiency virus (FIV). In this report, we examined three clones, FIV-pF34, FIV-14, and FIV-pPPR, for replication in Crandell feline kidney (CrFK) cells, feline peripheral blood mononuclear cells (PBMC), and feline macrophage cultures. Importantly, cell tropism for these three clones was also examined in vivo. FIV-pF34 replication was efficient in CrFK cells but severely restricted in PBMC, whereas replication of FIV-pPPR was vigorous in PBMC but severely restricted in CrFK cells. FIV-14 replication was productive in both CrFK cells and PBMC. Interestingly, all three molecular clones replicated with similar efficiencies in primary feline monocyte-derived macrophages. In vivo, FIV-pF34 proved least efficient for establishing persistent infection, and proviral DNA when detectable, was localized predominately to nonlymphoid cell populations (macrophages). FIV-pPPR proved most efficient for induction of a persistent viremia in vivo, and proviral DNA was localized predominately in CD4+ and CD8+ lymphocyte subsets. FIV-14 inoculation of cats resulted in an infection characterized by seroconversion and localization of proviral DNA in CD4+ lymphocytes only. Results of this study on diverse FIV molecular clones revealed that in vitro replication efficiency of an FIV isolate in PBMC directly correlated with replication efficiency in vivo, whereas proficiency for replication in macrophages in vitro was not predictive for replication potential in vivo. Also, infection of both CD4+ and CD8+ lymphocyte subsets was associated with higher virus load in vivo. Results of the studies on these three FIV clones, which exhibited differential cell tropism, indicated a correlation between in vitro and in vivo cell tropism and virus replication. PMID:10074104

  14. Molecular cloning and expression analysis of mulberry MAPK gene family.

    PubMed

    Wei, Congjin; Liu, Xueqin; Long, Dingpei; Guo, Qing; Fang, Yuan; Bian, Chenkai; Zhang, Dayan; Zeng, Qiwei; Xiang, Zhonghuai; Zhao, Aichun

    2014-04-01

    Mitogen-activated protein kinase (MAPK) cascades play an important role in regulating various biotic and abiotic stresses in plants. Although MAPKs have been identified and characterized in a few model plants, there is little information available for mulberry Morus sp. L., one of the most ecologically and economically important perennial trees. This study identified 47 mulberry Morus notabilis MAPK (MnMAPK) family genes: 32 MnMAPKKK, five MnMAPKK and ten MnMAPK genes, and cloned ten MnMAPK cDNA genes based on a genome-wide analysis of the morus genome database. Comparative analysis with MAPK gene families from other plants suggested that MnMAPKs could be divided into five subfamilies (groups A, B, C, D and E) and they could have similar functions in response to abiotic and biotic stresses. MnMAPK gene expression analysis of different stresses (high/low temperature, salt and drought) and signal molecules (ABA, SA, H2O2 and methyl jasmonate (MeJA)) revealed that all ten MnMAPK genes responded to high/low temperature, salt and drought stresses, and that nine of the ten MnMAPKs (MnMAPK7 excepted) could be induced by ABA, SA, H2O2 and MeJA, which suggested that MnMAPKs may play pivotal roles in signal transduction pathways. Our results indicated that almost all of the MnMAPKs may be involved in environmental stress and defense responses, which provides the basis for further characterization of the physiological functions of MnMAPKs.

  15. 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

  16. 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.

  17. The complete primary structure of the T-cell receptor genes from an alloreactive cytotoxic human T-lymphocyte clone.

    PubMed

    Leiden, J M; Fraser, J D; Strominger, J L

    1986-01-01

    The complete primary structure of the cDNAs encoding the alpha and beta chains of the T-lymphocyte receptor for antigen from a human alloreactive, cytotoxic T-cell clone, L17, is presented. Sequence analysis of these genes reveals that both are related to immunoglobulins and are composed of variable, diversity (at least in the case of the Ti beta clone), joining, and constant region sequences. Comparison of the sequence of the alpha-chain cDNA to that of previously sequenced mouse and human alpha cDNAs suggests the presence of human T-cell receptor alpha D-region sequences. Southern blot analysis confirms the finding that these cDNAs represent the functional receptor genes expressed by the L17 cytotoxic T-cell clone. The availability of these full-length T-cell receptor cDNA clones from a human T-lymphocyte clone of known antigen specificity should allow an analysis of the relationship between T-cell receptor structure and function. PMID:2426193

  18. Molecular biology of somatostatin receptor subtypes.

    PubMed

    Patel, Y C; Greenwood, M; Panetta, R; Hukovic, N; Grigorakis, S; Robertson, L A; Srikant, C B

    1996-08-01

    Somatostatin (SRIF) receptors (ssts) comprise a family of heptahelical membrane proteins encoded by five related genes that map to separate chromosomes and which, with the exception of sst1, are intronless. The ssts1-4 display weak selectivity for SRIF-14 binding, whereas sst5 is SRIF-28-selective. Based on structural similarity and reactivity for octapeptide and hexapeptide sst analogs, ssts2,3 and sst5 belong to a similar sst subclass; ssts1-4 react poorly with these analogs and belong to a separate subclass. All five ssts are functionally coupled to inhibition of adenylyl cyclase via pertussis toxin-sensitive guanosine triphosphate (GTP)-binding proteins. mRNA for ssts1-5 is widely expressed in brain and peripheral organs and displays an overlapping but characteristic pattern that is subtype-selective and tissue- and species-specific. All pituitary cell subsets express sst2 and sst5, with sst5 being more abundant. Individual pituitary cells coexpress multiple sst subtypes. The binding pocket for SRIF-14 ligand lies deep within the membrane in transmembrane domains (TMDs) 3 to 7. Except for extracellular loop 2, it does not involve the other exofacial structures. Human (h)sst2A and hsst5 undergo agonist-mediated desensitization, associated with receptor internalization. The C-tail segment of hsst5 displays positive molecular internalization signals. The ssts inhibit the growth of tumor cells directly, through blockade of mitogenic signaling leading to growth arrest and through induction of apoptosis. This process is associated with translocation of phosphotyrosine phosphatase (PTP) 1C from the cytosol to the membrane.

  19. 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.

  20. 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.

  1. Mole ghrelin: cDNA cloning, gene expression, and diverse molecular forms in Mogera imaizumii.

    PubMed

    Satou, Motoyasu; Kaiya, Hiroyuki; Nishi, Yoshihiro; Shinohara, Akio; Kawada, Shin-Ichiro; Miyazato, Mikiya; Kangawa, Kenji; Sugimoto, Hiroyuki

    2016-06-01

    Here, we describe cDNA cloning and purification of the ghrelin gene sequences and ghrelin peptides from the Japanese true mole, Mogera imaizumii. The gene spans >2.9kbp, has four exons and three introns, and shares structural similarity with those of terrestrial animals. Mature mole ghrelin peptide was predicted to be 28 amino acids long (GSSFLSPEHQKVQQRKESKKPPSKPQPR) and processed from a prepropeptide of 116 amino acids. To further elucidate molecular characteristics, we purified ghrelin peptides from mole stomach. By mass spectrometry, we found that the mole ghrelin peptides had higher ratios of the odd-number fatty acids (C9 and C11 as much as C8) attached to the third serine residue than other vertebrate ghrelin. Truncated forms of ghrelins such as [1-27], [1-19], [1-16] and [1-15], and that lacked the 14th glutamine residue (des-Gln14 ghrelin) were produced in the stomach. Marked expression of ghrelin mRNA in lung was observed as in stomach and brain. Phylogenetic analysis indicated that the branch of M. imaizumii has slightly higher dN/dS ratios (the nucleotide substitution rates at non-synonymous and synonymous sites) than did other eulipotyphlans. Peptide length was positively correlated with human ghrelin receptor activation, whereas the length of fatty-acyl chains showed no obvious functional correlation. The basal higher luciferase activities of the 5'-proximal promoter region of mole ghrelin were detected in ghrelin-negative C2C12 cells and hypoxic culture conditions impaired transcriptional activity. These results indicated that moles have acquired diverse species of ghrelin probably through distinctive fatty acid metabolism because of their food preferences. The results provide a gateway to understanding ghrelin metabolism in fossorial animals. PMID:27102942

  2. 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

  3. Mapping, cDNA cloning and tissue expression of the porcine thyrotropin-releasing hormone receptor gene.

    PubMed

    Jiang, Xiaoling; Cai, Zhaowei; Zhao, Xiaofeng; Zhang, Lifan; Chen, Zhe; Wang, Ying; Guo, Xiaoling; Xu, Ningying

    2011-01-01

    Thyrotropin-releasing hormone receptor (TRHR) is a G-protein-coupled receptor that plays a crucial role in regulating the hypothalamic-pituitary-thyroid axis by conveying the action of the hypothalamic tripeptide TRH, which is the primary central activator of this hormonal cascade. In the present study, the porcine TRHR (pTRHR) gene was localized to chromosome 4 by Radiation hybrid mapping. Quantitative trait loci affecting average backfat thickness, daily gain, and carcass and meat quality traits have been mapped to the region containing this gene. Further, the full-length cDNA of pTRHR was cloned and sequenced. pTRHR contains an open reading frame encoding 398 amino acids and shares 96.2% amino acid identity to human TRHR. Real-time quantitative RT-PCR showed that the mRNA of pTRHR is expressed in a variety of tissues, with high expression in the brain, hypothalamus, pituitary, testis, and fat tissue. The considerable expression level of TRHR mRNA found in fat tissue indicates potential direct action of TRH on lipocyte might exist. Additionally, two alternative spliced transcript variants of pTRHR were also isolated in this study. Our data provided basic molecular information which will be useful for further investigation on pTRHR gene.

  4. 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.

  5. 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. PMID:16323278

  6. Molecular cloning and sequencing of zeta-crystallin/quinone reductase cDNA from human liver.

    PubMed

    Gonzalez, P; Rao, P V; Zigler, J S

    1993-03-31

    Zeta-crystallin is an enzyme-crystallin highly expressed in the lens of some hystricomorph rodents and camels. It has been shown to have a novel NADPH: quinone oxidoreductase activity and is present at enzymatic levels in a variety of tissues from various mammals. We report here the cDNA cloning of zeta-crystallin from a human liver library. One clone with the complete open reading frame was obtained. Ten nucleotides of the 5' and 796 of the 3' nontranslated regions are present in the clone including two possible polyadenylation signals. The deduced amino acid sequence is 328 residues long with a calculated molecular mass of 34910 daltons and isoelectric point of 8.73. It shows 84% identity with the guinea pig protein.

  7. Molecular pharmacology of human NMDA receptors

    PubMed Central

    Hedegaard, Maiken K.; Hansen, Kasper B.; Andersen, Karen T.; Bräuner-Osborne, Hans; Traynelis, Stephen F.

    2012-01-01

    N-methyl-D-aspartate (NMDA) receptors are ionotropic glutamate receptors that mediate excitatory neurotransmission. NMDA receptors are also important drug targets that are implicated in a number of pathophysiological conditions. To facilitate the transition from lead compounds in pre-clinical animal models to drug candidates for human use, it is important to establish whether NMDA receptor ligands have similar properties at rodent and human NMDA receptors. Here, we compare amino acid sequences for human and rat NMDA receptor subunits and discuss inter-species variation in the context of our current knowledge of the relationship between NMDA receptor structure and function. We summarize studies on the biophysical properties of human NMDA receptors and compare these properties to those of rat orthologs. Finally, we provide a comprehensive pharmacological characterization that allows side-by-side comparison of agonists, un-competitive antagonists, GluN2B-selective non-competitive antagonists, and GluN2C/D-selective modulators at recombinant human and rat NMDA receptors. The evaluation of biophysical properties and pharmacological probes acting at different sites on the receptor suggest that the binding sites and conformational changes leading to channel gating in response to agonist binding are highly conserved between human and rat NMDA receptors. In summary, the results of this study suggest that no major detectable differences exist in the pharmacological and functional properties of human and rat NMDA receptors. PMID:22197913

  8. Development and characterization of an in vivo pathogenic molecular clone of equine infectious anemia virus.

    PubMed

    Cook, R F; Leroux, C; Cook, S J; Berger, S L; Lichtenstein, D L; Ghabrial, N N; Montelaro, R C; Issel, C J

    1998-02-01

    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 (EIAV(PV)) of the cell culture-adapted strain of EIAV (EIAV(PR)). 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 EIAV(PV3.3), 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, EIAV(PV3.3#3) (redesignated EIAV(UK)), 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 degrees 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 EIAV(UK) differed from the consensus sequence of EIAV(PV) by just a single amino acid residue in the second exon of the rev gene. Complete homology with the EIAV(PV) consensus sequence was observed in the hypervariable region of the LTR. However, EIAV(UK) 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 EIAV(PV) strain into the infectious nonpathogenic

  9. Cloning, ligand-binding, and temporal expression of ecdysteroid receptors in the diamondback moth, Plutella xylostella

    PubMed Central

    2012-01-01

    Background The diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae), is a devastating pest of cruciferous crops worldwide, and has developed resistance to a wide range of insecticides, including diacylhydrazine-based ecdysone agonists, a highly selective group of molt-accelerating biopesticides targeting the ecdysone receptors. Result In this study, we cloned and characterized the ecdysone receptors from P. xylostella, including the two isoforms of EcR and a USP. Sequence comparison and phylogenetic analysis showed striking conservations among insect ecdysone receptors, especially between P. xylostella and other lepidopterans. The binding affinity of ecdysteroids to in vitro-translated receptor proteins indicated that PxEcRB isoform bound specifically to ponasterone A, and the binding affinity was enhanced by co-incubation with PxUSP (Kd =3.0±1.7 nM). In contrast, PxEcRA did not bind to ponasterone A, even in the presence of PxUSP. The expression of PxEcRB were consistently higher than that of PxEcRA across each and every developmental stage, while the pattern of PxUSP expression is more or less ubiquitous. Conclusions Target site insensitivity, in which the altered binding of insecticides (ecdysone agonists) to their targets (ecdysone receptors) leads to an adaptive response (resistance), is one of the underlying mechanisms of diacylhydrazine resistance. Given the distinct differences at expression level and the ligand-binding capacity, we hypothesis that PxEcRB is the ecdysone receptor that controls the remodeling events during metamorphosis. More importantly, PxEcRB is the potential target site which is modified in the ecdysone agonist-resistant P. xylostella. PMID:23078528

  10. Cloning and characterization of additional members of the G protein-coupled receptor family.

    PubMed

    Lee, D K; Lynch, K R; Nguyen, T; Im, D S; Cheng, R; Saldivia, V R; Liu, Y; Liu, I S; Heng, H H; Seeman, P; George, S R; O'Dowd, B F; Marchese, A

    2000-02-29

    A search of the expressed sequence tag (EST) database retrieved a human cDNA sequence which partially encoded a novel G protein-coupled receptor (GPCR) GPR26. A human genomic DNA fragment encoding a partial open reading frame (ORF) and a rat cDNA encoding the full length ORF of GPR26 were obtained by library screening. The rat GPR26 cDNA encoded a protein of 317 amino acids, most similar (albeit distantly related) to the serotonin 5-HT(5A) and gastrin releasing hormone BB2 receptors. GPR26 mRNA expression analysis revealed signals in the striatum, pons, cerebellum and cortex. HEK293 and Rh7777 cells transfected with GPR26 cDNA displayed high basal cAMP levels, slow growth rate of clonal populations and derangements of normal cell shape. We also used a sequence reported only in the patent literature encoding GPR57 (a.k.a. HNHCI32) to PCR amplify a DNA fragment which was used to screen a human genomic library. This resulted in the cloning of a genomic fragment containing a pseudogene, psiGPR57, with a 99.6% nucleotide identity to GPR57. Based on shared sequence identities, the receptor encoded by GPR57 was predicted to belong to a novel subfamily of GPCRs together with GPR58 (a.k.a. phBL5, reported only in the patent literature), putative neurotransmitter receptor (PNR) and a 5-HT(4) pseudogene. Analysis of this subfamily revealed greatest identities (approximately 56%) between the receptors encoded by GPR57 and GPR58, each with shared identities of approximately 40% with PNR. Furthermore, psiGPR57, GPR58, PNR and the 5-HT(4) pseudogene were mapped in a cluster localized to chromosome 6q22-24. PNR and GPR58 were expressed in COS cells, however no specific binding was observed for various serotonin receptor-specific ligands.

  11. 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.

  12. [Cloning alphavirus and flavivirus sequences for use as positive controls in molecular diagnostics].

    PubMed

    Camacho, Daría; Reyes, Jesús; Franco, Leticia; Comach, Guillermo; Ferrer, Elizabeth

    2016-06-01

    The purpose of the study was to obtain a positive control to validate molecular techniques (reverse transcription- polymerase chain reaction [RT-PCR]) used in the diagnosis and research of viral infections. From strains of Chikungunya virus (CHIKV), Zika virus, and Dengue virus (DENV-1, DENV-2, DENV- 3, and DENV-4) viral RNAs were extracted to obtain complementary DNA using RT-PCR from the nsP4 (CHIKV), NS5 (Zika virus), C/prM-M, and 5'UTR-C (DENV-1, DENV-2, DENV-3, DENV-4) sequences, which were cloned into pGEM®-T Easy. Cloning was confirmed through colony PCR, from which plasmid DNA was extracted for fragment cloning verification. Cloning of cDNA corresponding to nsP4, NS5, C/prM-M, and 5'UTR-C of the different viral agents was achieved. In conclusion, recombinant plasmids were obtained with each of the sequences specified for further assessment as positive controls in molecular techniques in an effort to avoid the use of cell cultures, which can be costly, time-consuming, and potentially dangerous. PMID:27656926

  13. [Cloning alphavirus and flavivirus sequences for use as positive controls in molecular diagnostics].

    PubMed

    Camacho, Daría; Reyes, Jesús; Franco, Leticia; Comach, Guillermo; Ferrer, Elizabeth

    2016-06-01

    The purpose of the study was to obtain a positive control to validate molecular techniques (reverse transcription- polymerase chain reaction [RT-PCR]) used in the diagnosis and research of viral infections. From strains of Chikungunya virus (CHIKV), Zika virus, and Dengue virus (DENV-1, DENV-2, DENV- 3, and DENV-4) viral RNAs were extracted to obtain complementary DNA using RT-PCR from the nsP4 (CHIKV), NS5 (Zika virus), C/prM-M, and 5'UTR-C (DENV-1, DENV-2, DENV-3, DENV-4) sequences, which were cloned into pGEM®-T Easy. Cloning was confirmed through colony PCR, from which plasmid DNA was extracted for fragment cloning verification. Cloning of cDNA corresponding to nsP4, NS5, C/prM-M, and 5'UTR-C of the different viral agents was achieved. In conclusion, recombinant plasmids were obtained with each of the sequences specified for further assessment as positive controls in molecular techniques in an effort to avoid the use of cell cultures, which can be costly, time-consuming, and potentially dangerous.

  14. Molecular Cloning and Characterization of a Paramyosin from Clonorchis sinensis

    PubMed Central

    Park, Tae-Joon; Kang, Jung-Mi; Na, Byoung-Kuk

    2009-01-01

    Paramyosin is a myofibrillar protein present in helminth parasites and plays multifunctional roles in host-parasite interactions. In this study, we identified the gene encoding paramyosin of Clonorchis sinensis (CsPmy) and characterized biochemical and immunological properties of its recombinant protein. CsPmy showed a high level of sequence identity with paramyosin from other helminth parasites. Recombinant CsPmy (rCsPmy) expressed in bacteria had an approximate molecular weight of 100 kDa and bound both human collagen and complement 9. The protein was constitutively expressed in various developmental stages of the parasite. Imunofluorescence analysis revealed that CsPmy was mainly localized in the tegument, subtegumental muscles, and the muscle layer surrounding the intestine of the parasite. The rCsPmy showed high levels of positive reactions (74.6%, 56/75) against sera from patients with clonorchiasis. Immunization of experimental rats with rCsPmy evoked high levels of IgG production. These results collectively suggest that CsPmy is a multifunctional protein that not only contributes to the muscle layer structure but also to non-muscular functions in host-parasite interactions. Successful induction of host IgG production also suggests that CsPmy can be applied as a diagnostic antigen and/or vaccine candidate for clonorchiasis. PMID:19967083

  15. Cloning and olfactory expression of progestin receptors in the Chinese black sleeper Bostrichthys sinensis.

    PubMed

    Zhang, Yu Ting; Liu, Dong Teng; Zhu, Yong; Chen, Shi Xi; Hong, Wan Shu

    2016-05-01

    Our previous studies suggested that 17α,20β-dihydroxy-4-pregnen-3-one (DHP), an oocyte maturation inducing progestin, also acts as a sex pheromone in Chinese black sleeper Bostrichthys sinensis, a fish species that inhabits intertidal zones and mates and spawns inside a muddy burrow. The electro-olfactogram response to DHP increased during the breeding season. In the present study, we cloned the cDNAs of the nine progestin receptors (pgr, paqr5, 6, 7(a, b), 8, 9, pgrmc1, 2) from B. sinensis, analyzed their tissue distribution, and determined the expression in the olfactory rosette during the reproductive cycle in female and male fish. The deduced amino acid sequences of the nine progestin receptors share high sequence identities with those of other fish species and relatively lower homology with their mammalian counterparts, and phylogenetic analyses classified the nine B. sinensis progestin receptors into their respective progestin receptor groups. Tissue distribution of B. sinensis progestin receptors showed differential expression patterns, but all these nine genes were expressed in the olfactory rosette. Interestingly, paqr5 mRNA was found in the intermediate and basal parts of the olfactory epithelium but not in the central core using in situ hybridization, and its expression level was the highest in the olfactory rosette among the tissues examined. These results suggested Paqr5 may have an important role for transmitting progestin signaling in the olfactory system. The expression levels of paqr7a and paqr7b, pgr and pgrmc2 mRNA peaked around the mid meiotic stage, and that of paqr8 peaked at late meiotic stage in the olfactory rosette in males, while the olfactory expression of paqr5 decreased gradually as spermatogenesis progressed. In contrast, the expression of the progestin receptors did not change significantly during the development of the ovary in the olfactory rosette in females, except that of pgr. Interestingly, the changes of paqr8 expression in

  16. 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. PMID:24353285

  17. Molecular and functional properties of P2X receptors--recent progress and persisting challenges.

    PubMed

    Kaczmarek-Hájek, Karina; Lörinczi, Eva; Hausmann, Ralf; Nicke, Annette

    2012-09-01

    ATP-gated P2X receptors are trimeric ion channels that assemble as homo- or heteromers from seven cloned subunits. Transcripts and/or proteins of P2X subunits have been found in most, if not all, mammalian tissues and are being discovered in an increasing number of non-vertebrates. Both the first crystal structure of a P2X receptor and the generation of knockout (KO) mice for five of the seven cloned subtypes greatly advanced our understanding of their molecular and physiological function and their validation as drug targets. This review summarizes the current understanding of the structure and function of P2X receptors and gives an update on recent developments in the search for P2X subtype-selective ligands. It also provides an overview about the current knowledge of the regulation and modulation of P2X receptors on the cellular level and finally on their physiological roles as inferred from studies on KO mice.

  18. Molecular characterization of prostaglandin F receptor (FP) and E receptor subtype 3 (EP3) in chickens.

    PubMed

    Kwok, Amy H Y; Wang, Yajun; Leung, Frederick C

    2012-10-01

    Prostaglandin E and F regulate diverse physiological functions including gastrointestinal motility, fever induction and reproduction. This multitude of biological effects is mediated via their four E receptor subtypes (EP(1), EP(2), EP(3) and EP(4)) and F receptor (FP), respectively. Majority of these studies was performed in mammalian species, while investigations on their roles were impeded by inadequate information on their receptors in avian species. In present study, full-length cDNAs of chicken EP(3) (cEP(3)) and two isoforms of FP - cFPa and cFPb - were cloned from adult hen ovary. The putative cEP(3) and cFPa share high amino acid sequence identity with their respective orthologs, while the predicted cFPb is a novel middle-truncated splice variant which lacks 107 amino acids between transmembrane domains 4 and 6. RT-PCR showed that cEP(3), cFPa and cFPb are widely expressed in adult tissues examined, including ovary and oviduct. Using a pGL3-CRE luciferase reporter system, cEP(3)-expressing DF1 cells inhibited forskolin-induced luciferase activity (EC(50): <1.9 pM) upon PGE(2) treatment, suggesting that cEP(3) may functionally couple to Gi protein. Upon PGF(2α) addition, cFPa was shown to potentially couple to intracellular Ca(2+)-signaling pathway by pGL3-NFAT-RE reporter assay (EC(50): 2.9 nM), while cFPb showed no response. Using a pGL4-SRE reporter system, both cEP(3) and cFPa exhibited potential MAPK activation by PGE(2) and PGF(2α) at EC(50) 0.34 and 13 nM, respectively. Molecular characterization of these receptors paved the road to the better understanding of PGE(2) and PGF(2α) roles in avian physiology and comparative endocrinology studies. PMID:22885557

  19. Molecular Insights into Metabotropic Glutamate Receptor Allosteric Modulation

    PubMed Central

    Gregory, Karen J.

    2015-01-01

    The metabotropic glutamate (mGlu) receptors are a group of eight family C G protein–coupled receptors that are expressed throughout the central nervous system (CNS) and periphery. Within the CNS the different subtypes are found in neurons, both pre- and/or postsynaptically, where they mediate modulatory roles and in glial cells. The mGlu receptor family provides attractive targets for numerous psychiatric and neurologic disorders, with the majority of discovery programs focused on targeting allosteric sites, with allosteric ligands now available for all mGlu receptor subtypes. However, the development of allosteric ligands remains challenging. Biased modulation, probe dependence, and molecular switches all contribute to the complex molecular pharmacology exhibited by mGlu receptor allosteric ligands. In recent years we have made significant progress in our understanding of this molecular complexity coupled with an increased understanding of the structural basis of mGlu allosteric modulation. PMID:25808929

  20. 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.

  1. Molecular cloning and expression of the O4 polysaccharide gene cluster from Escherichia coli.

    PubMed

    Haraguchi, G E; Hull, R A; Krallmann-Wenzel, U; Hull, S I

    1989-02-01

    The Escherichia coli O4 serotype is common among isolates from urinary tract infections. The genes responsible for the biosynthesis of the O4 polysaccharide in a human uropathogenic E. coli were cloned and expressed in E. coli K-12. The recombinant plasmid pGH60, which conferred the O4 phenotype, encoded eight proteins with apparent molecular weights of 39, 36.5, 35, 32.8, 26, 24, 20.7 and 13 kDa.

  2. Cloning of a putative G-protein-coupled receptor from Arabidopsis thaliana.

    PubMed

    Josefsson, L G; Rask, L

    1997-10-15

    We have cloned and characterized a cDNA from Arabidopsis thaliana that most likely encodes a novel member of the vast superfamily of G-protein-coupled receptor proteins (GPCRs). By taking advantage of amino acid sequence similarities between plant expressed sequence tags (ESTs) and established G-protein-coupled receptor sequences, a probe was obtained which was used for the screening of an Arabidopsis cDNA library. The cDNA which was found is very infrequently represented in the cDNA library, suggesting a low and/or spatially restricted expression. A region of the translated sequence of the cDNA shows the highest similarity to cAMP receptors from the slime mold Dictyostelium discoideum. The same region is also similar to that in members of the animal calcitonin family of receptors. Another region of the putative receptor, however, is similar to sequences of serotonin receptors and other receptors of the so-called rhodopsin family of GPCRs. The rhodopsin family has numerous members in higher vertebrate species. Alignments and phylogenetic analyses of the regions of similarity yielded results in accordance with other evolutionary considerations. Our cDNA thus occurred on a distinct major branch in relation to the rest of the rhodopsin family. In relation to the calcitonin family, our cDNA and cAMP receptors occurred together on a distinct major branch but appear to have diverged from each other shortly after their divergence from the rest of the calcitonin family. Other features further argue for a tentative identification of it as a GPCR. It displays seven discrete and strongly predicted transmembrane domains when analyzed in hydropathy plots. The preferred orientation is with the amino terminus towards the outside. It has one Cys residue in extracellular loop 1 and another in extracellular loop 2. Cys residues in these loops are known to form disulfide bridges in many other GPCRs. Finally, it has several fully conserved amino acids that belong to the most conserved

  3. Molecular and biochemical pharmacology of the histamine H4 receptor

    PubMed Central

    Leurs, Rob; Chazot, Paul L; Shenton, Fiona C; Lim, Herman D; de Esch, Iwan JP

    2009-01-01

    The elucidation of the human genome has had a major impact on histamine receptor research. The identification of the human H4 receptor by several groups has been instrumental for a new appreciation of the role of histamine in the modulation of immune function. In this review, we summarize the historical developments and the molecular and biochemical pharmacology of the H4 receptor. PMID:19413568

  4. 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…

  5. 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

  6. Cloning, pharmacological characterization and expression analysis of Atlantic cod (Gadus morhua, L.) nuclear progesterone receptor.

    PubMed

    Chen, Shi X; Almeida, Fernanda F L; Andersson, Eva; Taranger, Geir Lasse; Schmidt, Ruben; Schulz, Rüdiger W; Bogerd, Jan

    2012-10-01

    To better understand the role(s) of progesterone in fish spermatogenesis, we cloned the nuclear progesterone receptor (Pgr) of Atlantic cod. The open-reading frame of the cod pgr consists of 2076 bp, coding for a 691-amino acids-long protein that shows the highest similarity with other piscine Pgr proteins. Functional characterization of the receptor expressed in mammalian cells revealed that the cod Pgr exhibited progesterone-specific, dose-dependent induction of reporter gene expression, with 17α,20β-dihydroxy-4-pregnen-3-one (DHP), a typical piscine progesterone, showing the highest potency in activating the receptor. During ontogenesis, the pgr mRNA was undetectable in embryo's 24 h after fertilization, but became detectable 4 days after fertilization. During the larval stage, the expression levels increased steadily with the development of the larvae. In adult fish, pgr was predominantly expressed in gonads of both sexes. During the onset of puberty, testicular pgr transcript levels started to increase during rapid spermatogonial proliferation, and peaked when spermiation started. In situ hybridization studies using testis tissue during the rapid growth phase containing all germ cell stages indicated that in cod, pgr mRNA is predominantly located in Sertoli cells that are in contact with proliferating spermatogonia. Taken together, our data suggests that the Pgr is involved in mediating progestagen stimulation of the mitotic expansion of spermatogonia, and in processes associated with the spermiation/spawning period in Atlantic cod. PMID:22885560

  7. Cloning and expression profile of ionotropic receptors in the parasitoid wasp Microplitis mediator (Hymenoptera: Braconidae).

    PubMed

    Wang, Shan-Ning; Peng, Yong; Lu, Zi-Yun; Dhiloo, Khalid Hussain; Zheng, Yao; Shan, Shuang; Li, Rui-Jun; Zhang, Yong-Jun; Guo, Yu-Yuan

    2016-07-01

    Ionotropic receptors (IRs) mainly detect the acids and amines having great importance in many insect species, representing an ancient olfactory receptor family in insects. In the present work, we performed RNAseq of Microplitis mediator antennae and identified seventeen IRs. Full-length MmedIRs were cloned and sequenced. Phylogenetic analysis of the Hymenoptera IRs revealed that ten MmedIR genes encoded "antennal IRs" and seven encoded "divergent IRs". Among the IR25a orthologous groups, two genes, MmedIR25a.1 and MmedIR25a.2, were found in M. mediator. Gene structure analysis of MmedIR25a revealed a tandem duplication of IR25a in M. mediator. The tissue distribution and development specific expression of the MmedIR genes suggested that these genes showed a broad expression profile. Quantitative gene expression analysis showed that most of the genes are highly enriched in adult antennae, indicating the candidate chemosensory function of this family in parasitic wasps. Using immunocytochemistry, we confirmed that one co-receptor, MmedIR8a, was expressed in the olfactory sensory neurons. Our data will supply fundamental information for functional analysis of the IRs in parasitoid wasp chemoreception. PMID:27208597

  8. Cloning and expression of a human kidney cDNA for an /alpha//sub 2/-adrenergic receptor subtype

    SciTech Connect

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

    1988-09-01

    An /alpha//sub 2/-adrenergic receptor subtype has been cloned from a human kidney cDNA library using the gene for the human platelet /alpha//sub 2/-adrenergic receptor as a probe. The deduced amino acid sequence resembles the human platelet /alpha//sub 2/-adrenergic receptor and is consistent with the structure of other members of he family of guanine nucleotide-binding protein-coupled receptors. The cDNA was expressed in a mammalian cell line (COS-7), and the /alpha//sub 2/-adrenergic ligand (/sup 3/H)rauwolscine was bound. Competition curve analysis with a variety of adrenergic ligands suggests that this cDNA clone represents the /alpha//sub 2/B-adrenergic receptor. The gene for this receptor is on human chromosome 4, whereas the gene for the human platelet /alpha//sub 2/-adrenergic receptor (/alpha//sub 2/A) lies on chromosome 10. This ability to express the receptor in mammalian cells, free of other adrenergic receptor subtypes, should help in developing more selective /alpha/-adrenergic ligands.

  9. Effects of receptor correlations on molecular information transmission.

    PubMed

    Singh, Vijay; Tchernookov, Martin; Nemenman, Ilya

    2016-08-01

    Cells measure concentrations of external ligands by capturing ligand molecules with cell surface receptors. The numbers of molecules captured by different receptors co-vary because they depend on the same extrinsic ligand fluctuations. However, these numbers also counter-vary due to the intrinsic stochasticity of chemical processes because a single molecule randomly captured by a receptor cannot be captured by another. Such structure of receptor correlations is generally believed to lead to an increase in information about the external signal compared to the case of independent receptors. We analyze a solvable model of two molecular receptors and show that, contrary to this widespread expectation, the correlations have a small and negative effect on the information about the ligand concentration. Further, we show that measurements that average over multiple receptors are almost as informative as those that track the states of every individual one. PMID:27627350

  10. Effects of receptor correlations on molecular information transmission

    NASA Astrophysics Data System (ADS)

    Singh, Vijay; Tchernookov, Martin; Nemenman, Ilya

    2016-08-01

    Cells measure concentrations of external ligands by capturing ligand molecules with cell surface receptors. The numbers of molecules captured by different receptors co-vary because they depend on the same extrinsic ligand fluctuations. However, these numbers also counter-vary due to the intrinsic stochasticity of chemical processes because a single molecule randomly captured by a receptor cannot be captured by another. Such structure of receptor correlations is generally believed to lead to an increase in information about the external signal compared to the case of independent receptors. We analyze a solvable model of two molecular receptors and show that, contrary to this widespread expectation, the correlations have a small and negative effect on the information about the ligand concentration. Further, we show that measurements that average over multiple receptors are almost as informative as those that track the states of every individual one.

  11. Molecular cloning and characterization of a group II chaperonin delta-subunit from soybean.

    PubMed

    Nong, Van Hai; Arahira, Masaomi; Phan, Van Chi; Kim, Chan-Shick; Zhang, Deyu; Udaka, Kyoko; Fukazawa, Chikafusa

    2002-08-01

    Molecular characterization of plant group II chaperonin (CCT, c-cpn, or TriC) still remains elusive. By PCR-based cloning techniques using soybeans, we have made a successful attempt to clone a delta-subunit homologue of CCT (CCTdelta). This subunit is responsible for the binding of an in vivo substrate, alpha-actin, by assisting the correct folding of the cytoskeletal protein in mouse, and the occurrence of the subunit homologue in plant CCT was unclear. As the cloning strategy, a putative amino acid segment, NH(2)-Gly-Gly-Gly-Ala-Pro-Glu-COOH, which is tightly conserved in all known animal and yeast CCTdelta subunits, was chosen for designing a degenerate primer of the PCR-cloning. The resultant 1881-bp cDNA was found to have an open-reading frame of 533 amino acids with a calculated molecular mass of 57,677 Da and to share about 58-65% identity overall at the amino acid level with the corresponding subunits known to date. Using antibodies raised against Escherichia coli-produced soybean insoluble CCTdelta as a monitoring tool, we purified soybean CCT from the extract of its immature seeds. STEM images demonstrated that the molecular shape of soybean CCT is a double eight-membered ring, which resembles the known group II chaperonins. The CCT also reactivated a denatured firefly luciferase with a significant, but limited level of the native enzymic activity in an in vitro system. Northern blot analysis showed that soybean CCTdelta gene, which is intronless and composed of a small family, was only expressed at a very early stage of seed development of soybean.

  12. Virtual Screening of Receptor Sites for Molecularly Imprinted Polymers.

    PubMed

    Bates, Ferdia; Cela-Pérez, María Concepción; Karim, Kal; Piletsky, Sergey; López-Vilariño, José Manuel

    2016-08-01

    Molecularly Imprinted Polymers (MIPs) are highly advantageous in the field of analytical chemistry. However, interference from secondary molecules can also impede capture of a target by a MIP receptor. This greatly complicates the design process and often requires extensive laboratory screening which is time consuming, costly, and creates substantial waste products. Herein, is presented a new technique for screening of "virtually imprinted receptors" for rebinding of the molecular template as well as secondary structures, correlating the virtual predictions with experimentally acquired data in three case studies. This novel technique is particularly applicable to the evaluation and prediction of MIP receptor specificity and efficiency in complex aqueous systems. PMID:27076379

  13. Molecular cloning of the white locus region of Drosophila melanogaster using a large transposable element

    PubMed Central

    Goldberg, M.L.; Paro, R.; Gehring, W.J.

    1982-01-01

    We report the molecular cloning of a chromosome segment including the white locus of Drosophila melanogaster. This region was isolated using a deficiency extending from the previously cloned heat-shock puff sequences at 87A7 to a large transposable element containing the loci white and roughest.FB-NOF, a 7.5 kb element with partial homology to a family of inverted repeat sequences (Potter et al., 1980), is found very near the deficiency breakpoint, and is followed by DNA originating from the white locus region. Sequences totalling ˜60 kb surrounding this initial entry point were obtained by the cloning of successively overlapping fragments from a wild-type strain. Several rearrangement breakpoints have been mapped relative to the cloned DNA; these define the limits of the white locus and further differentiate the “white proximal region”, thought to function in gene regulation, from the remainder of the locus. Insertion of the dispersed repetitive element copia into the white locus is observed in strains carrying the white-apricot allele. Analysis of several white-apricot revertants suggests that copia insertion is responsible for the apricot eye color phenotype. ImagesFig. 2.Fig. 4.Fig. 5.Fig. 6. PMID:16453411

  14. Molecular cloning, functional expression and characterisation of RCC reductase involved in chlorophyll catabolism.

    PubMed

    Wüthrich, K L; Bovet, L; Hunziker, P E; Donnison, I S; Hörtensteiner, S

    2000-01-01

    Red chlorophyll catabolite (RCC) reductase (RCCR) and pheophorbide (Pheide) a oxygenase (PaO) catalyse the key reaction of chlorophyll catabolism, porphyrin macrocycle cleavage of Pheide a to a primary fluorescent catabolite (pFCC). RCCR was purified from barley and a partial gene sequence was cloned (pHvRCCR). The gene was expressed at all stages of leaf development and in roots. By comparison with different databases, genomic sequences and expressed sequence tags similar to RCCR were found in phylogenetically diverse species, and activity of RCCR was demonstrated in two of them, Arabidopsis thaliana and Marchantia polymorpha. The gene of A. thaliana (AtRCCR) was employed for molecular cloning, heterologous expression and the production of polyclonal antibodies. With recombinant RCCR, the major product of RCC reduction was pFCC-1, but small quantities of its C1 epimer, pFCC-2, also accumulated. The reaction required reduced ferredoxin and was sensitive to oxygen. AtRCCR encoded a 35 kDa protein which was used for chloroplast import experiments. Upon transport, it was processed to a mature form of 31 kDa. The significance of cloning of RCCR is discussed in respect to the evolution of chlorophyll catabolism and to the cloning of PaO.

  15. CD8+ T-cell clones deficient in the expression of the CD45 protein tyrosine phosphatase have impaired responses to T-cell receptor stimuli.

    PubMed Central

    Weaver, C T; Pingel, J T; Nelson, J O; Thomas, M L

    1991-01-01

    CD45 is a high-molecular-weight transmembrane protein tyrosine phosphatase expressed only by nucleated cells of hematopoietic origin. To examine function, mouse CD8+ cytolytic T-cell clones were derived that had a specific defect in the expression of CD45. Northern (RNA) blot analysis indicates that the CD45 deficiency is due to either a transcriptional defect or mRNA instability. The CD45-deficient cells were greatly diminished in their ability to respond to antigen. All functional parameters of T-cell receptor signalling analyzed (cytolysis of targets, proliferation, and cytokine production) were markedly diminished. A CD45+ revertant was isolated, and the ability to respond to antigen was restored. These results support a central and immediate role for this transmembrane protein tyrosine phosphatase in T-cell receptor signalling. Images PMID:1652055

  16. A massively parallel pipeline to clone DNA variants and examine molecular phenotypes of human disease mutations.

    PubMed

    Wei, Xiaomu; Das, Jishnu; Fragoza, Robert; Liang, Jin; Bastos de Oliveira, Francisco M; Lee, Hao Ran; Wang, Xiujuan; Mort, Matthew; Stenson, Peter D; Cooper, David N; Lipkin, Steven M; Smolka, Marcus B; Yu, Haiyuan

    2014-12-01

    Understanding the functional relevance of DNA variants is essential for all exome and genome sequencing projects. However, current mutagenesis cloning protocols require Sanger sequencing, and thus are prohibitively costly and labor-intensive. We describe a massively-parallel site-directed mutagenesis approach, "Clone-seq", leveraging next-generation sequencing to rapidly and cost-effectively generate a large number of mutant alleles. Using Clone-seq, we further develop a comparative interactome-scanning pipeline integrating high-throughput GFP, yeast two-hybrid (Y2H), and mass spectrometry assays to systematically evaluate the functional impact of mutations on protein stability and interactions. We use this pipeline to show that disease mutations on protein-protein interaction interfaces are significantly more likely than those away from interfaces to disrupt corresponding interactions. We also find that mutation pairs with similar molecular phenotypes in terms of both protein stability and interactions are significantly more likely to cause the same disease than those with different molecular phenotypes, validating the in vivo biological relevance of our high-throughput GFP and Y2H assays, and indicating that both assays can be used to determine candidate disease mutations in the future. The general scheme of our experimental pipeline can be readily expanded to other types of interactome-mapping methods to comprehensively evaluate the functional relevance of all DNA variants, including those in non-coding regions.

  17. A dedicated database program for cataloging recombinant clones and other laboratory products of molecular biology technology.

    PubMed

    Jenson, H B

    1989-06-01

    A novel computer database program dedicated to storing, cataloging, and accessing information about recombinant clones and libraries has been developed for the IBM (or compatible) personal computer. This program, named CLONES, also stores information about bacterial strains and plasmid and bacteriophage vectors used in molecular biology. The advantages of this method are improved organization of data, fast and easy assimilation of new data, automatic association of new data with existing data, and rapid retrieval of desired records using search criteria specified by the user. Individual records are indexed in the database using B-trees, which automatically index new entries and expedite later access. The use of multiple windows, pull-down menus, scrolling pick-lists, and field-input techniques make the program intuitive to understand and easy to use. Daughter databases can be created to include all records of a particular type, or only those records matching user-specified search criteria. Separate databases can also be merged into a larger database. This computer program provides an easy-to-use and accurate means to organize, maintain, access, and share information about recombinant clones and other laboratory products of molecular biology technology. PMID:2631777

  18. Molecular cloning, chromosomal localization, and functional characterization of a human liver Na+/bile acid cotransporter.

    PubMed Central

    Hagenbuch, B; Meier, P J

    1994-01-01

    We have used a cDNA probe from a cloned rat liver Na+/taurocholate cotransporting polypeptide (Ntcp) to screen a human liver cDNA library. A 1,599-bp cDNA clone that encodes a human Na+/taurocholate cotransporting polypeptide (NTCP) was isolated. The human NTCP consists of 349 amino acids (calculated molecular mass of 38 kD) and exhibits 77% amino acid homology with the rat Ntcp. In vitro translation experiments indicate that the protein is glycosylated and has a molecular weight similar to the rat Ntcp. Injection of in vitro transcribed cRNA into Xenopus laevis oocytes resulted in the expression of Na(+)-dependent taurocholate uptake. Saturation kinetics indicated that the human NTCP has a higher affinity for taurocholate (apparent Km = 6 microM) than the previously cloned rat protein (apparent Km = 25 microM). NTCP-mediated taurocholate uptake into oocytes was inhibited by all major bile acid derivatives (100 microM), bumetanide (500 microM), and bromosulphophthalein (100 microM). Southern blot analysis of genomic DNA from a panel of human/hamster somatic cell hybrids mapped the human NTCP gene to chromosome 14. PMID:8132774

  19. Molecular cloning of Mu d(bla lacZ) transcriptional and translational fusions.

    PubMed Central

    Wanner, B L

    1987-01-01

    The vector pBW2 was made to selectively clone chimeric plasmids with chromosomal Mu d(bla lacZ) transcriptional or translational fusions. It was tetracycline resistant and had the carboxyl-terminal end of bla distal to its PstI site. Because ligation of PstI-digested chromosomal DNA of a Mu d(bla lacZ) insertion with pBW2 restored bla, ampicillin-resistant chimeric plasmids were selectable. These plasmids had the Mu d bla amino terminus and simultaneously acquired other Mu d sequences including lacZ, the chromosomal fusion joint, and the DNA adjacent to the nearest chromosomal PstI site. The plasmid pBW2 was useful in the molecular cloning of several psi and pho::lacZ(Mu d) fusions, as well as chromosomal genes located near Mu d insertions. PMID:3032905

  20. Stable expression of cloned rat GABAA receptor subunits in a human kidney cell line.

    PubMed

    Hamilton, B J; Lennon, D J; Im, H K; Im, W B; Seeburg, P H; Carter, D B

    1993-04-30

    A predominant form of the GABAA/benzodiazepine receptor-Cl- channel complex is believed to consist of three different 48-55 kDa subunits (alpha, beta, gamma) with unknown stoichiometry. Plasmids containing the rat GABAA receptor cDNAs coding for alpha 1, beta 2, and gamma 2 were co-transfected, along with a plasmid encoding G418 resistance, into human embryonic kidney cells previously transformed with Adenovirus 5 (HEK-293) [J. Gen. Virol., 36 (1977) 59-72]. Four percent of the G418 resistant colonies were found to express mRNA for all three of the GABAA subunits constitutively. A single cell clone derived from one of the alpha 1 beta 2 gamma 2 expressors has demonstrated stable electrophysiological characteristics over 25 passages. The GABA-activated Cl- current in this cell line is blocked by picrotoxin and bicuculline, and is modulated by a variety of agonist and inverse agonist ligands including diazepam, Ro 154513, zolpidem, and beta-CCE. The cell line has been used successfully over a 12-month period as a screen for novel drugs modulating GABA-mediated polarization of neuronal cells. PMID:7687050

  1. 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.

  2. Cloning of thyrotropin-releasing hormone precursor and receptor in rat thymus, adrenal gland, and testis.

    PubMed

    Montagne, J J; Ladram, A; Nicolas, P; Bulant, M

    1999-03-01

    TRH is a hypophysiotropic peptide that acts mainly via the hypothalamic-pituitary-thyroid axis, but TRH immunoreactivity is also detected in several peripheral tissues. PCR with two pairs of primers enabling amplification of three fragments of TRH complementary DNA (cDNA) was used to demonstrate local production of TRH. Products of the expected size were detected in the testis, adrenal gland, lymphoid organs, thymus, and spleen. The amplified cDNA fragments were cloned and sequenced to show that the TRH gene is expressed in the thymus, spleen, and adrenal gland. Competitive RT-PCR showed that the TRH messenger RNA content of the testis was about one third that of the hypothalamus, whereas the adrenal gland contained 2% and the thymus 6%. HPLC analysis of thymus and spleen extracts showed small amounts of TRH, with a particular processing pattern of pro-TRH in lymphoid organs. The expression of the TRH receptor gene in peripheral organs was investigated to determine whether TRH had an autocrine or a paracrine action. cDNA fragments that encompassed the coding region of the receptor were identified in the testis, adrenal gland and thymus. No signal was detected in the spleen. These findings indicate that TRH may have a biological activity in extrapituitary organs and may act locally in the testis, adrenal gland, and thymus.

  3. 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

  4. 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

  5. Receptor Activity-modifying Proteins 2 and 3 Generate Adrenomedullin Receptor Subtypes with Distinct Molecular Properties*

    PubMed Central

    Watkins, Harriet A.; Chakravarthy, Madhuri; Abhayawardana, Rekhati S.; Gingell, Joseph J.; Garelja, Michael; Pardamwar, Meenakshi; McElhinney, James M. W. R.; Lathbridge, Alex; Constantine, Arran; Harris, Paul W. R.; Yuen, Tsz-Ying; Brimble, Margaret A.; Barwell, James; Poyner, David R.; Woolley, Michael J.; Conner, Alex C.; Pioszak, Augen A.; Reynolds, Christopher A.

    2016-01-01

    Adrenomedullin (AM) is a peptide hormone with numerous effects in the vascular systems. AM signals through the AM1 and AM2 receptors formed by the obligate heterodimerization of a G protein-coupled receptor, the calcitonin receptor-like receptor (CLR), and receptor activity-modifying proteins 2 and 3 (RAMP2 and RAMP3), respectively. These different CLR-RAMP interactions yield discrete receptor pharmacology and physiological effects. The effective design of therapeutics that target the individual AM receptors is dependent on understanding the molecular details of the effects of RAMPs on CLR. To understand the role of RAMP2 and -3 on the activation and conformation of the CLR subunit of AM receptors, we mutated 68 individual amino acids in the juxtamembrane region of CLR, a key region for activation of AM receptors, and determined the effects on cAMP signaling. Sixteen CLR mutations had differential effects between the AM1 and AM2 receptors. Accompanying this, independent molecular modeling of the full-length AM-bound AM1 and AM2 receptors predicted differences in the binding pocket and differences in the electrostatic potential of the two AM receptors. Druggability analysis indicated unique features that could be used to develop selective small molecule ligands for each receptor. The interaction of RAMP2 or RAMP3 with CLR induces conformational variation in the juxtamembrane region, yielding distinct binding pockets, probably via an allosteric mechanism. These subtype-specific differences have implications for the design of therapeutics aimed at specific AM receptors and for understanding the mechanisms by which accessory proteins affect G protein-coupled receptor function. PMID:27013657

  6. Cloning and characterization of a binding subunit of the interleukin 13 receptor that is also a component of the interleukin 4 receptor.

    PubMed Central

    Hilton, D J; Zhang, J G; Metcalf, D; Alexander, W S; Nicola, N A; Willson, T A

    1996-01-01

    Interleukins 4 (IL-4) and 13 (IL-13) have been found previously to share receptor components on some cells, as revealed by receptor cross-competition studies. In the present study, the cloning is described of murine NR4, a previously unrecognized receptor identified on the basis of sequence similarity with members of the hemopoietin receptor family. mRNA encoding NR4 was found in a wide range of murine cells and tissues. By using transient expression in COS-7 cells, NR4 was found to encode the IL-13 receptor alpha chain, a low-affinity receptor capable of binding IL-13 but not IL-4 or interleukins 2, -7, -9, or -15. Stable expression of the IL-13 receptor alpha chain (NR4) in CTLL-2 cells resulted in the generation of high-affinity IL-13 receptors capable of transducing a proliferative signal in response to IL-13 and, moreover, led to competitive cross-reactivity in the binding of IL-4 and IL-13. These results suggest that the IL-13 receptor alpha chain (NR4) is the primary binding subunit of the IL-13 receptor and may also be a component of IL-4 receptors. Images Fig. 2 PMID:8552669

  7. 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.

  8. 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.

  9. Molecular cloning and characterization of ADP-glucose pyrophosphorylase cDNA clones isolated from pea cotyledons.

    PubMed

    Burgess, D; Penton, A; Dunsmuir, P; Dooner, H

    1997-02-01

    Three ADP-glucose pyrophosphorylase (ADPG-PPase) cDNA clones have been isolated and characterized from a pea cotyledon cDNA library. Two of these clones (Psagps1 and Psagps2) encode the small subunit of ADPG-PPase. The deduced amino acid sequences for these two clones are 95% identical. Expression of these two genes differs in that the Psagps2 gene shows comparatively higher expression in seeds relative to its expression in other tissues. Psagps2 expression also peaks midway through seed development at a time in which Psagps1 transcripts are still accumulating. The third cDNA isolated (Psagp11) encodes the large subunit of ADPG-PPase. It shows greater selectivity in expression than either of the small subunit clones. It is highly expressed in sink organs (seed, pod, and seed coat) and undetectable in leaves.

  10. Molecular cloning and sequencing of the cDNA of cop1 gene from Pisum sativum.

    PubMed

    Zhao, L; Wang, C; Zhu, Y; Zhao, J; Wu, X

    1998-02-11

    Cop1 protein plays an important role in seedling development of higher plants. The cDNA of cop1 gene from pea (Pisum sativum) was cloned and sequenced. Cop1 protein of pea is predicted to have 672 amino acids and a molecular mass of 76 kDa. Sequence comparison between Cop1 proteins of pea and Arabidopsis thaliana revealed that the two Cop1 proteins were highly homologous in the regions with functional domains and at the C-terminus. Immunoblotting performed with polyclonal antibodies against recombinant Cop1 of pea showed that Cop1 protein was present in seedlings germinated both in light and darkness.

  11. Molecular cloning of the mitogenic mannose/maltose-specific rhizome lectin from Calystegia sepium.

    PubMed

    Van Damme, E J; Barre, A; Verhaert, P; Rougé, P; Peumans, W J

    1996-11-18

    cDNA clones encoding the mitogenic mannose/maltose-specific lectin from the rhizomes of hedge bindweed (Calystegia sepium) have been isolated and sequenced. Comparison of the deduced amino acid sequence and the molecular weight of the lectin subunit as determined by mass spectrometry indicated that the mature protein comprises the entire open reading frame of the cDNA, which implies that the primary translation product contains no signal peptide and is not proteolytically processed. Searches in the databases revealed sequence homology with the previously described lectins from the taxonomically unrelated Moraceae species Artocarpus integrifolia and Maclura pomifera.

  12. 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. PMID:27231873

  13. Molecular architecture of the vanilloid receptor. Insights for drug design.

    PubMed

    Ferrer-Montiel, Antonio; García-Martínez, Carolina; Morenilla-Palao, Cruz; García-Sanz, Nuria; Fernández-Carvajal, Asia; Fernández-Ballester, Gregorio; Planells-Cases, Rosa

    2004-05-01

    The transient receptor potential channel vanilloid receptor subunit 1 (TRPV1) is a molecular integrator of physical and chemical stimuli in the peripheral nociceptor terminals. TRPV1 is an ionotropic channel that plays a critical role in both thermal nociception and inflammatory hyperalgesia. Structure-function relationships are providing fundamental insights of the modular architecture of this neuronal receptor. As a result, the molecular determinants that endow TRPV1 with its physiological properties, namely activation by heat, potentiation by extracellular acidic pH, and interaction with vanilloid-like compounds, as well as its permeation properties are being unveiled. This information can now be used to build up molecular models for the protein which, upon experimental validation, could be used as tools to thrust the target-oriented design of druggable TRPV1 ligands.

  14. 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

  15. Cloning, expression, and ligand-binding characterization of two neuropeptide Y receptor subtypes in orange-spotted grouper, Epinephelus coioides.

    PubMed

    Wang, Fei; Chen, Weimin; Lin, Haoran; Li, Wensheng

    2014-12-01

    As one of the most important multifunctional peptides, neuropeptide Y (NPY) performs its physiological functions through different subtype receptors. In this study, full-length cDNAs of two NPY receptors (YRs) in orange-spotted grouper (Epinephelus coioides) were cloned and named npy8br (y8b) and npy2r (y2). Phylogenetic analysis indicated that the Y8b receptor is an ortholog of the teleostean Y8b receptor, which belongs to the Y1 subfamily, and the Y2 receptor is an ortholog of the teleostean Y2 receptor, which belongs to the Y2 subfamily. Both of the YRs have G protein-coupled receptor family profiles. Multiple alignments demonstrate that the extracellular loop regions of YRs have distinctive residues of each species. Expression profile analysis revealed that the grouper Y8b receptor mRNA is primarily expressed in the brain, stomach and intestine, while the grouper Y2 receptor mRNA is primarily expressed in the brain, ovary, liver and heart. Double immunofluorescence analysis determined that the grouper YRs interact with the grouper NPY around the human embryonic kidney 293T cell surface. Furthermore, site-directed mutagenesis in a phage display system revealed that Asp(6.59) might be a common NPY-binding site, while Asp(2.68) of the Y8b receptor and Glu(5.24) of the Y2 receptor could be likely involved in subtype-specific binding. Combining the expression profile and ligand-binding feature, the grouper Y8b receptor could be involved in regulating food intake via the brain-gut axis and the grouper Y2 receptor might play a role in balancing the regulatory activity of the Y8b receptor and participate in metabolism in the liver and ovary.

  16. Photoaffinity labeling of the somatostatin receptor: identification of molecular subtypes.

    PubMed

    Srikant, C B; Murthy, K K; Escher, E E; Patel, Y C

    1992-05-01

    Pharmacological studies have suggested that the somatostatin (SS) receptor is heterogeneous and may exhibit subtypes selective for SS-14 and SS-28. Whether this heterogeneity can be explained by separate molecular forms of the receptor protein is unclear. In the present study, we have developed a novel photosensitive azido derivative of the octapeptide SS analog Tyr3 SMS (EE 581) and used it as a photoaffinity probe to characterize the molecular components of the SS receptor in five receptor positive tissues (normal rat brain, pituitary, pancreas, and adrenal cortex, and mouse AtT-20 pituitary tumor cells). [125I]EE-581 labeled specific high affinity binding sites in all these tissues (Kd range 1.3-1.67 nM). Photoaffinity labeled membrane SS receptors were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography. Three specifically labeled SS receptor proteins of 80 kilodaltons (kDa), 58 kDa, and 32 kDa were identified and exhibited a tissue-specific distribution. The 58 kDa species was the exclusive form in pancreas, adrenal cortex, and AtT-20 cells and the dominant form in brain. The 32 kDa receptor protein was expressed as a minor form (ratio of 58 kDa:32 kDa 3:1), exclusively in brain. The 80 kDa receptor was found only in the pituitary where it occurred as the sole SS receptor species. Competition experiments showed that the 58 kDa and 32 kDa receptor proteins in brain reacted with SS-14 greater than SS-28; in contrast, the 58 kDa protein in AtT-20 cells bound SS-28 greater than SS-14 suggesting the existence of distinct subtypes of the 58 kDa receptor in these two tissues. These data represent the first systematic evaluation of the molecular forms of SS receptor proteins by photoaffinity labeling in different target tissues and provide direct evidence for molecular heterogeneity and SS-14/SS-28 selectivity; a major 58 kDa protein present in most tissues, an additional 32 kDa protein uniquely expressed in brain, and an

  17. Molecular cloning and bacterial expression of cDNA encoding a plant cysteine synthase.

    PubMed Central

    Saito, K; Miura, N; Yamazaki, M; Hirano, H; Murakoshi, I

    1992-01-01

    Cysteine synthase (CSase) [O-acetyl-L-serine acetate-lyase (adding hydrogen sulfide), EC 4.2.99.8] catalyzes the formation of L-cysteine, the key step in sulfur assimilation in plants, from O-acetyl-L-serine and hydrogen sulfide. We report here the isolation and characterization of cDNA clones encoding cysteine synthase from spinach (Spinacia oleracea L.). Internal peptide sequences were obtained from V8 protease-digested fragments of purified CSase. A lambda gt10 cDNA library was constructed from poly(A)+ RNA of young green leaves of spinach. Screening with two synthetic mixed nucleotides encoding the partial peptide sequences revealed 19 positively hybridized clones among 2 x 10(5) clones. Nucleotide sequence analysis of two independent cDNA clones revealed a continuous open reading frame encoding a polypeptide of 325 amino acids with a calculated molecular mass of 34,185 Da. Sequence comparison of the deduced amino acids revealed 53% identity with CSases of Escherichia coli and Salmonella typhimurium. Sequence homology was also observed with other metabolic enzymes for amino acids in bacteria and yeast and with rat hemoprotein H-450. A bacterial expression vector was constructed and could genetically complement an E. coli auxotroph that lacks CSases. The accumulation of functionally active spinach CSase in E. coli was also demonstrated by immunoblotting and assaying enzymatic activity. Southern hybridization analysis showed the presence of two to three copies of the cDNA sequence in the genome of spinach. RNA blot hybridization suggested constitutive expression in leaves and roots of spinach. Images PMID:1518833

  18. Molecular pharmacology of G protein-coupled receptors.

    PubMed

    Summers, R J

    2016-10-01

    This themed issue of the British Journal of Pharmacology stems from the eighth in the series of meetings on the Molecular Pharmacology of G protein coupled receptors (MPGPCR) held as part of a joint meeting with the Australasian Society of Clinical and Experimental Pharmacologists and Toxicologists (ASCEPT) in Melbourne Australia from 7 to 11 December 2014. Linked Articles This article is part of a themed section on Molecular Pharmacology of G Protein-Coupled Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v173.20/issuetoc. PMID:27682321

  19. Molecular pharmacology of G protein-coupled receptors.

    PubMed

    Summers, R J

    2016-10-01

    This themed issue of the British Journal of Pharmacology stems from the eighth in the series of meetings on the Molecular Pharmacology of G protein coupled receptors (MPGPCR) held as part of a joint meeting with the Australasian Society of Clinical and Experimental Pharmacologists and Toxicologists (ASCEPT) in Melbourne Australia from 7 to 11 December 2014. Linked Articles This article is part of a themed section on Molecular Pharmacology of G Protein-Coupled Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v173.20/issuetoc.

  20. 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.

  1. Insect insulin receptors: insights from sequence and caste expression analyses of two cloned hymenopteran insulin receptor cDNAs from the fire ant.

    PubMed

    Lu, H-L; Pietrantonio, Patricia V

    2011-10-01

    The insulin and insulin-like growth factor (IGF) signalling (IIS) pathway in the honey bee (Apis mellifera) is linked to reproductive division of labour and foraging behaviour. Two insulin receptor genes are present in the released genomes of other social hymenopterans. Limited information is available on the IIS pathway role in ants. The predicted insulin receptor sequences from the recently released draft genome of the fire ant Solenopsis invicta (Hymenoptera: Formicidae) are incomplete and biologically significant data are also lacking. To elucidate the role of the IIS pathway in the fire ant, two putative insulin receptors (SiInR-1 and SiInR-2) were cloned; the first InR cDNAs cloned from social insects. Analyses of putative post-translational modification sites in SiInRs revealed the potential for differential regulation. We investigated the transcriptional expression of both receptors at different developmental stages, castes and queen tissues. In last instar larvae and pharate pupae of workers and reproductive, transcriptional abundance of both receptors was negatively correlated with body size and nutritional status. The expression level of both receptors in different queen tissues appears to correlate with requirements for queen reproductive physiology and behaviours. This study contributes new information to the understanding of social insects because in fire ants juvenile hormone acts as a gonadotropin and workers are fully sterile, contrary to honey bees.

  2. Cloning and expression of the estrogen receptor-alpha (Esr1) from the Harderian gland of the sea turtle (Lepidochelys olivacea).

    PubMed

    Chávez, Bertha; Ramos, Luis; Merchant-Larios, Horacio; Vilchis, Felipe

    2009-06-01

    The effects of estradiol on the Harderian gland (HG) are believed to be partially regulated by the transcriptional regulation of the estrogen-related genes via estrogen receptor (ER). In reptiles, however, it has not been well established whether the HG contains or expresses steroid nuclear receptors. As a first step toward investigating the molecular mechanisms of estrogen signalling in the HG, we isolated the cDNA for ERalpha in the sea turtle Lepidochelys olivacea. ERalpha was cloned using RT-PCR coupled with 5' and 3' RACE procedures. The cDNA contains a complete open reading frame encoding 588 amino acid residues. Comparative analysis of this amino acid sequence showed moderate to strong conservation of the ERalpha (Esr1) gene within divergent vertebrate groups. In transfection studies, the cloned ER displayed high affinity K(d)=0.25nM and high specificity for 17beta-estradiol. Binding assays using sucrose density gradients demonstrated a specific 7-7.5 S binding component in the HG cytosolic fractions. RT-qPCR analysis showed significant ERalpha mRNA expression in the liver, HG, lung and brain. Altogether, these results provide evidence for the expression of intracellular ERs in the HG of the sea turtle and suggest that ERalpha may be an important modulator of the estrogen-mediated response in the HG of reptiles.

  3. 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. PMID:26585996

  4. [Cloning and regulation of pig estrogen related receptor β gene (ESRRB) promoter].

    PubMed

    Yang, Yang; Wang, Yaxian; Du, Lixia; Wang, Huayan

    2015-04-01

    The estrogen related receptor family member Esrrb (Estrogen related receptor β) is a gene that expresses in the early stage of embryo and plays an important role in the core pluripotent network. Its function has been analyzed in human and mouse, although no report so far related to pig. Therefore, to explore its mechanism of transcriptional regulation and expression pattern, we cloned a 3.3 kb pig ESRRB promoter by PCR and constructed the green fluorescence protein (GFP) reporter vector pE3.3. We used these vectors to study the ESRRB expression pattern in 293T, Hela and C2C12. Sequence was analyzed for regulatory elements that share homology to known transcription factor binding sites by TFSEARCH and JASPER program. Some pluripotency related genes such as SMAD, STAT3, MYC, KLF4 and ESRRB have been found within the 3.3 kb sequence by co-transfected pig ESRRB promoter and these potential regulators. We found that ESRRB only expressed in 293T and SMAD could activate ESRRB expression obviously. To determine the core promoter region, a series of ESRRB promoter fragments with gradually truncated 5'-end were produced by PCR and inserted into pGL3-Basic vector. After transient transfection into 293T, dual luciferase assay was used to measure these promoter activities. The result suggested that the core promoter of pig ESRRB located within -25 bp to -269 bp region. These results suggest that these transcription factor binding sites and the core promoter region may be essential for transcriptional regulation of pig ESRRB gene. PMID:26380406

  5. 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. PMID:27076436

  6. 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.

  7. 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

  8. 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.

  9. 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.

  10. Molecular cloning and characterization of a unique type of human papillomavirus from an immune deficient patient.

    PubMed

    Ostrow, R S; Zachow, K R; Thompson, O; Faras, A J

    1984-04-01

    Several papillomas from a single patient who exhibited an unusual immune deficiency syndrome were analyzed for the presence of specific human papillomavirus (HPV) types. Preliminary analysis indicated that the HPV DNA species present in each of these tissues was quite unlike any of the previously characterized HPV types. In order to more rigorously analyze the HPV from this patient we have isolated the HPV DNA by molecularly cloning it into a bacteriophage lambda vector and have constructed a detailed restriction endonuclease map. Comparative hybridization studies using S1 nuclease analyses showed 6% or less nucleotide sequence homology of this viral DNA with HPV types 1, 2, 3, 4, 5, 6, or an HPV-11, molecularly cloned in this laboratory. Moreover, Southern blot analyses under stringent hybridization conditions revealed little, if any, hybridization to HPV types 1, 2, 4, 5, 7, 8, 10, 11, HPV-EV isolated from a patient with epidermodysplasia verruciformis (EV), or 2 previously described HPVs (HPV-P and HPV-PW) related to HPV-3. There was, however, a very weak sequence homology detected with HPV-6 and an extremely weak homology to HPV-3. No filter hybridization was observed with the recently characterized HPVs 9 or -12 to -24. These data accumulatively indicate that the HPV species from this immunosuppressed patient represents a new, hitherto unidentified HPV type. PMID:6323588

  11. Molecular cloning and characterization of five opsin genes from the marine flatfish Atlantic halibut (Hippoglossus hippoglossus).

    PubMed

    Helvik, J V; Drivenes, O; Naess, T H; Fjose, A; Seo, H C

    2001-01-01

    Most molecular studies on the visual system in fish have been performed on freshwater teleosts such as goldfish and zebrafish where cones and rods appear simultaneously. Many marine fishes have long larval phase in the upper pelagic zone before transformation into a juvenile and a benthic life style. The retina at the larval stages consists of only single cone cells; later during metamorphosis double cones and rods develop. The flatfish Atlantic halibut (Hippoglossus hippoglossus) is a typical example of a marine species with such a two-step retina development. In this study, we have cloned five different opsins from Atlantic halibut larvae and juvenile retinas. Sequence comparisons with other opsins and phylogenetic analysis show that the five genes belong to the opsins of long-wavelength sensitive (L); middle-wavelength sensitive, M(Cone) and M(Rod); and short-wavelength sensitive, S(Blue) and S(Ultraviolet), respectively. In situ hybridization analysis reveals expression in double cone (L and M(Cone)), single cone (S(Blue) and S(Ultraviolet)), and rod (M(Rod)) types of photoreceptor cells in juvenile halibut retina. The visual system in Atlantic halibut seems therefore to have all four types of cone photoreceptors in addition to rod photoreceptors. This work shows for the first time molecular isolation of a complete set of retinal visual pigment genes from a marine teleost and describes the first cloning of an ultraviolet-sensitive opsin type from a marine teleost.

  12. Molecular Cloning and Gene Expression of Canine Apoptosis Inhibitor of Macrophage

    PubMed Central

    TOMURA, Shintaro; UCHIDA, Mona; YONEZAWA, Tomohiro; KOBAYASHI, Masato; BONKOBARA, Makoto; ARAI, Satoko; MIYAZAKI, Toru; TAMAHARA, Satoshi; MATSUKI, Naoaki

    2014-01-01

    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. PMID:25649949

  13. 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…

  14. 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

  15. Molecular and Physiological Mechanisms of Membrane Receptor Systems Functioning

    PubMed Central

    Severin, E.S.; Savvateeva, M.V.

    2011-01-01

    Molecular physiology is a new interdisciplinary field of knowledge that looks into how complicated biological systems function. The living cell is a relatively simple, but at the same time very sophisticated biological system. After the sequencing of the human genome, molecular physiology has endeavored to investigate the systems of cellular interactions at a completely new level based on knowledge of the spatial organization and functions of receptors, their ligands, and protein-protein interactions. In recent years, the achievements in molecular physiology have centered on the study of sensor reception mechanisms and intercellular data transfer, as well as the immune system physiology, amongst other processes. PMID:22649671

  16. Neuromedin B and Its Receptor: Gene Cloning, Tissue Distribution and Expression Levels of the Reproductive Axis in Pigs.

    PubMed

    Ma, Zhiyu; Su, Juan; Guo, Tingting; Jin, Mengmeng; Li, Xiang; Lei, Zhihai; Hou, Yuanlong; Li, Xiaoliang; Jia, Cuicui; Zhang, Zheng; Ahmed, Ejlal

    2016-01-01

    Neuromedin B is one member of a family of bombesin-like peptides, which performs a variety of physiological functions via their receptor (NMBR) in most mammals. However, the genes encoding NMB and NMBR and their functions especially reproduction of the pigs are currently not fully understood. To research the physiological functions of NMB, we cloned and analyzed the NMB and NMBR genes, and systematically investigated the expression levels of NMB and NMBR mRNA using relative real-time PCR and the distribution of NMBR by immunohistochemistry (IHC). Experimental results show that the sequences of the amino acid and gene of NMB and NMBR were highly conservative and homology in many species, Significantly, the relative RT-PCR results revealed that NMB was mainly expressed in the central nervous system (CNS), whereas NMBR is highly expressed in peripheral tissues and organs, such as endocrine tissues, glands and reproductive organs. The IHC results show that NMBR positive cells were widely distributed in the body, such as respiratory and circulatory system, digestive system, urogenital system, in lymphatic organs and in the endocrine system. We also systematically investigated expression levels of NMB and NMBR in the reproductive axis using relative real-time PCR. In sow estrous cycle, the hypothalamic levels of both NMB and NMBR mRAN were similar, but the expression levels of the pituitary were negatively correlated. Expression levels in the ovarian system are lowest in metestrus phases and highest in proestrus and estrus phases. In boar post-natal development stages, the hypothalamic, pituitary and testicular levels of NMB and NMBR mRNAs showed developmental changes on postnatal day 30, 60, 90 and 120. Taken together, this study provided molecular and morphological data necessary for further research of physiological function of NMB/NMBR system in the pigs.

  17. Neuromedin B and Its Receptor: Gene Cloning, Tissue Distribution and Expression Levels of the Reproductive Axis in Pigs.

    PubMed

    Ma, Zhiyu; Su, Juan; Guo, Tingting; Jin, Mengmeng; Li, Xiang; Lei, Zhihai; Hou, Yuanlong; Li, Xiaoliang; Jia, Cuicui; Zhang, Zheng; Ahmed, Ejlal

    2016-01-01

    Neuromedin B is one member of a family of bombesin-like peptides, which performs a variety of physiological functions via their receptor (NMBR) in most mammals. However, the genes encoding NMB and NMBR and their functions especially reproduction of the pigs are currently not fully understood. To research the physiological functions of NMB, we cloned and analyzed the NMB and NMBR genes, and systematically investigated the expression levels of NMB and NMBR mRNA using relative real-time PCR and the distribution of NMBR by immunohistochemistry (IHC). Experimental results show that the sequences of the amino acid and gene of NMB and NMBR were highly conservative and homology in many species, Significantly, the relative RT-PCR results revealed that NMB was mainly expressed in the central nervous system (CNS), whereas NMBR is highly expressed in peripheral tissues and organs, such as endocrine tissues, glands and reproductive organs. The IHC results show that NMBR positive cells were widely distributed in the body, such as respiratory and circulatory system, digestive system, urogenital system, in lymphatic organs and in the endocrine system. We also systematically investigated expression levels of NMB and NMBR in the reproductive axis using relative real-time PCR. In sow estrous cycle, the hypothalamic levels of both NMB and NMBR mRAN were similar, but the expression levels of the pituitary were negatively correlated. Expression levels in the ovarian system are lowest in metestrus phases and highest in proestrus and estrus phases. In boar post-natal development stages, the hypothalamic, pituitary and testicular levels of NMB and NMBR mRNAs showed developmental changes on postnatal day 30, 60, 90 and 120. Taken together, this study provided molecular and morphological data necessary for further research of physiological function of NMB/NMBR system in the pigs. PMID:27010315

  18. Neuromedin B and Its Receptor: Gene Cloning, Tissue Distribution and Expression Levels of the Reproductive Axis in Pigs

    PubMed Central

    Ma, Zhiyu; Su, Juan; Guo, Tingting; Jin, Mengmeng; Li, Xiang; Lei, Zhihai; Hou, Yuanlong; Li, Xiaoliang; Jia, Cuicui; Zhang, Zheng; Ahmed, Ejlal

    2016-01-01

    Neuromedin B is one member of a family of bombesin-like peptides, which performs a variety of physiological functions via their receptor (NMBR) in most mammals. However, the genes encoding NMB and NMBR and their functions especially reproduction of the pigs are currently not fully understood. To research the physiological functions of NMB, we cloned and analyzed the NMB and NMBR genes, and systematically investigated the expression levels of NMB and NMBR mRNA using relative real-time PCR and the distribution of NMBR by immunohistochemistry (IHC). Experimental results show that the sequences of the amino acid and gene of NMB and NMBR were highly conservative and homology in many species, Significantly, the relative RT-PCR results revealed that NMB was mainly expressed in the central nervous system (CNS), whereas NMBR is highly expressed in peripheral tissues and organs, such as endocrine tissues, glands and reproductive organs. The IHC results show that NMBR positive cells were widely distributed in the body, such as respiratory and circulatory system, digestive system, urogenital system, in lymphatic organs and in the endocrine system. We also systematically investigated expression levels of NMB and NMBR in the reproductive axis using relative real-time PCR. In sow estrous cycle, the hypothalamic levels of both NMB and NMBR mRAN were similar, but the expression levels of the pituitary were negatively correlated. Expression levels in the ovarian system are lowest in metestrus phases and highest in proestrus and estrus phases. In boar post-natal development stages, the hypothalamic, pituitary and testicular levels of NMB and NMBR mRNAs showed developmental changes on postnatal day 30, 60, 90 and 120. Taken together, this study provided molecular and morphological data necessary for further research of physiological function of NMB/NMBR system in the pigs. PMID:27010315

  19. An infectious molecular clone of an unusual macrophage-tropic and highly cytopathic strain of human immunodeficiency virus type 1.

    PubMed Central

    Collman, R; Balliet, J W; Gregory, S A; Friedman, H; Kolson, D L; Nathanson, N; Srinivasan, A

    1992-01-01

    We isolated and molecularly cloned a human immunodeficiency virus type 1 (HIV-1) strain (89.6) which is unusual because it is both macrophage-tropic and extremely cytopathic in lymphocytes. Moreover, this is the first well-characterized infectious molecularly cloned macrophage-tropic HIV-1 strain derived from peripheral blood. HIV-1 89.6 differs markedly from other macrophage-tropic isolates within the envelope V3 region, which is important in determining cell tropism and cytopathicity. HIV-1 89.6 may thus represent a transitional isolate between noncytopathic macrophage-tropic viruses and cytopathic lymphocyte-tropic viruses. Images PMID:1433527

  20. Pyrrolic tripodal receptors for the molecular recognition of carbohydrates: ditopic receptors for dimannosides.

    PubMed

    Francesconi, Oscar; Nativi, Cristina; Gabrielli, Gabriele; Gentili, Matteo; Palchetti, Marco; Bonora, Beatrice; Roelens, Stefano

    2013-08-26

    Synthetic ditopic receptors, designed for the molecular recognition of dimannosides, have been prepared by bridging two monotopic units effectively recognizing mannosides with linkers of the appropriate size and flexibility, endowed with hydrogen-bonding groups. Affinities toward the α and β glycosides of the biologically relevant Manα(1-2)Man disaccharide were measured by NMR spectroscopy and isothermal titration calorimetry (ITC) in polar organic media (30-40 % DMF in chloroform). Significant selectivities and affinities in the micromolar range were observed in most cases, with two newly designed receptors being the most effective receptors of the set, together with a distinct preference of the dimannosides for the (S) enantiomer of the receptor in all cases. A 3D view of the recognition mode was elucidated by a combined NMR spectroscopic/molecular modeling approach, showing the dimannoside included in the cleft of the receptor. Compared to the monotopic precursors, the ditopic receptors showed markedly improved recognition properties, proving the efficacy of the modular receptor design for the recognition of disaccharides.

  1. A murine platelet-activating factor receptor gene: cloning, chromosomal localization and up-regulation of expression by lipopolysaccharide in peritoneal resident macrophages.

    PubMed Central

    Ishii, S; Matsuda, Y; Nakamura, M; Waga, I; Kume, K; Izumi, T; Shimizu, T

    1996-01-01

    A murine gene encoding a platelet-activating factor receptor (PAFR) was cloned. The gene was mapped to a region of the D2.2 band of chromosome 4 both by fluorescence in situ hybridization and by molecular linkage analysis. Northern blot analysis showed a high expression of the PAFR message in peritoneal macrophages. When C3H/HeN macrophages were treated with bacterial lipopolysaccharide (LPS) or synthetic lipid A, the PAFR gene expression was induced. Bacterial LPS, but not lipid A, induced the level of PAFR mRNA in LPS unresponsive C3H/HeJ macrophages. These induction patterns were parallel to those of tumor necrosis factor-alpha mRNA. Thus the PAFR in macrophages is important in LPS-induced pathologies. PMID:8670084

  2. T cell receptor junctional regions of V gamma 9+/V delta 2+ T cell clones in relation to non-MHC restricted cytotoxic activity.

    PubMed

    Flanagan, B F; Wheatcroft, N J; Thornton, S M; Christmas, S E

    1993-05-01

    Human gamma delta T cell clones having V gamma 9JP and V delta 2DJ1 T cell receptor (TCR) gene rearrangements were isolated form an individual donor and tested for non-MHC restricted cytotoxicity against the B lymphoblastoid cell line, BSM. Most clones were highly cytotoxic but 3/9 clones had very low activity, comparable to that of CD4+ alpha beta T cell clones. Although there was a tendency for clones with low cytotoxic function to produce high levels of interferon-gamma and tumor necrosis factor-alpha, this correlation was not complete. TCR gamma and delta junctional sequences were obtained and were found to be different for all clones. There were no consistent structural differences between gamma delta TCRs of cytotoxic and non-cytotoxic clones, but gamma or delta junctional regions of all three non-cytotoxic clones had unusual features. One clone had a particularly short gamma chain junctional sequence, one had a short delta chain junctional sequence and the third clone was the only one of the panel which failed to utilise the D delta 3 segment. If the gamma delta TCR is involved in target cell recognition in this model of non-MHC restricted killing, such variations in receptor structure may be sufficient to inhibit recognition and thereby reduce the cytotoxic capacity of a minority of V gamma 9+/V delta 2+ clones. Also, a panel of gamma delta T cell clones expressing V gamma 8/V delta 3 isolated from a different donor, were all highly cytotoxic against BSM, indicating that these target cells can be recognised by effector cells expressing a TCR other than the V gamma 9/V delta 2 receptor. The possible influence of other cell surface molecules on non-MHC restricted cytotoxic function is discussed.

  3. 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

  4. 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. PMID:27032955

  5. Cloning and molecular modelling of pectin degrading glycosyl hydrolase of family 28 from soil metagenomic library.

    PubMed

    Sathya, T A; Jacob, Ani Methew; Khan, Mahejibin

    2014-01-01

    Western Ghats of India is recognized as one of the 12 mega diversity regions of the world and is the hot spot for unrevealed microbial diversity. To explore the diversity of polysaccharide degrading enzymes in that region, metagenomic library was constructed from forest soil of Southern Western Ghats region. Nine pectinolytic clones with the ability to degrade citrus pectin were isolated based on function based screening of the library. Sequence analysis of pg_4 clone containing revealed that it contained GH family 28 domain (pfam00295) belonging to polygalacturonase superfamily (PLN03003). Its amino acid sequence analysis showed 25-55 % identity to the other well-characterized polygalacturonases. Molecular modeling of pg_4 revealed that it comprised of three right handed-parallel β sheets, one anti-parallel β sheet and one α helix with three conserved catalytic residue D 2263, D 284-85 and H 312 at the C terminal end. The enzyme characterized was able to hydrolyze both apple and citrus pectin with K m values of 1.685 and 1.542 mg ml(-1) and retained more that 80 % of activity at pH 5-9 and temperature 20-60 °C.

  6. Molecular cloning, structure, and reactivity of the second bromoperoxidase from Ascophyllum nodosum.

    PubMed

    Wischang, Diana; Radlow, Madlen; Schulz, Heiko; Vilter, Hans; Viehweger, Lutz; Altmeyer, Matthias O; Kegler, Carsten; Herrmann, Jennifer; Müller, Rolf; Gaillard, Fanny; Delage, Ludovic; Leblanc, Catherine; Hartung, Jens

    2012-10-01

    The sequence of bromoperoxidase II from the brown alga Ascophyllum nodosum was determined from a full length cloned cDNA, obtained from a tandem mass spectrometry RT-PCR-approach. The clone encodes a protein composed of 641 amino-acids, which provides a mature 67.4 kDa-bromoperoxidase II-protein (620 amino-acids). Based on 43% sequence homology with the previously characterized bromoperoxidase I from A. nodosum, a tertiary structure was modeled for the bromoperoxidase II. The structural model was refined on the basis of results from gel filtration and vanadate-binding studies, showing that the bromoperoxidase II is a hexameric metalloprotein, which binds 0.5 equivalents of vanadate as cofactor per 67.4 kDa-subunit, for catalyzing oxidation of bromide by hydrogen peroxide in a bi-bi-ping-pong mechanism (k(cat) = 153 s(-1), 22 °C, pH 5.9). Bromide thereby is converted into a bromoelectrophile of reactivity similar to molecular bromine, based on competition kinetic data on phenol bromination and correlation analysis. Reactivity provided by the bromoperoxidase II mimics biosynthesis of methyl 4-bromopyrrole-2-carboxylate, a natural product isolated from the marine sponge Axinella tenuidigitata. PMID:22884431

  7. 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. PMID:25597146

  8. Molecular cloning and mRNA expression of peroxiredoxin gene in black tiger shrimp (Penaeus monodon).

    PubMed

    Qiu, Lihua; Ma, Zhuojun; Jiang, Shigui; Wang, Weifang; Zhou, Falin; Huang, Jianhua; Li, Jianzhu; Yang, Qibin

    2010-07-01

    The techniques of homology cloning and anchored PCR were used to clone the peroxiredoxin (Prx) gene from black tiger shrimp (Penaeus monodon). The full length cDNA of black tiger shrimp Prx (PmPrx) contained a 5' untranslated region (UTR) of 51 bp, an ORF (open reading frame) of 582 bp encoding a polypeptide of 193 amino acids with an estimated molecular mass of 22.15 kDa and a 3' UTR of 948 bp. Sequence comparison showed that PmPrx shared higher identities with Prx IVs than that with other isoforms of Prx, indicating PmPrx was a member of the Prx IV family. A quantitative reverse transcriptase Real-Time PCR (qRT-PCR) assay was developed to assess the mRNA expression of PmPrx in different tissues and the temporal expression of PmPrx in the hepatopancreas challenged by lipopolyssacharide (LPS). Higher-level mRNA expression of PmPrx was detected in the tissues of hepatopancreas, gonad and heart. The expression of PmPrx in the hepatopancreas was up regulated after stimulated by LPS. The results indicated that PmPrx was a constitutive and inducible expressed protein and could be induced by LPS.

  9. Molecular Physiology of Membrane Guanylyl Cyclase Receptors.

    PubMed

    Kuhn, Michaela

    2016-04-01

    cGMP controls many cellular functions ranging from growth, viability, and differentiation to contractility, secretion, and ion transport. The mammalian genome encodes seven transmembrane guanylyl cyclases (GCs), GC-A to GC-G, which mainly modulate submembrane cGMP microdomains. These GCs share a unique topology comprising an extracellular domain, a short transmembrane region, and an intracellular COOH-terminal catalytic (cGMP synthesizing) region. GC-A mediates the endocrine effects of atrial and B-type natriuretic peptides regulating arterial blood pressure/volume and energy balance. GC-B is activated by C-type natriuretic peptide, stimulating endochondral ossification in autocrine way. GC-C mediates the paracrine effects of guanylins on intestinal ion transport and epithelial turnover. GC-E and GC-F are expressed in photoreceptor cells of the retina, and their activation by intracellular Ca(2+)-regulated proteins is essential for vision. Finally, in the rodent system two olfactorial GCs, GC-D and GC-G, are activated by low concentrations of CO2and by peptidergic (guanylins) and nonpeptidergic odorants as well as by coolness, which has implications for social behaviors. In the past years advances in human and mouse genetics as well as the development of sensitive biosensors monitoring the spatiotemporal dynamics of cGMP in living cells have provided novel relevant information about this receptor family. This increased our understanding of the mechanisms of signal transduction, regulation, and (dys)function of the membrane GCs, clarified their relevance for genetic and acquired diseases and, importantly, has revealed novel targets for therapies. The present review aims to illustrate these different features of membrane GCs and the main open questions in this field. PMID:27030537

  10. Cloning and expression of the Erwinia carotovora subsp. carotovora gene encoding the low-molecular-weight bacteriocin carocin S1.

    PubMed

    Chuang, Duen-yau; Chien, Yung-chei; Wu, Huang-Pin

    2007-01-01

    The purpose of this study was to clone the carocin S1 gene and express it in a non-carocin-producing strain of Erwinia carotovora. A mutant, TH22-10, which produced a high-molecular-weight bacteriocin but not a low-molecular-weight bacteriocin, was obtained by Tn5 insertional mutagenesis using H-rif-8-2 (a spontaneous rifampin-resistant mutant of Erwinia carotovora subsp. carotovora 89-H-4). Using thermal asymmetric interlaced PCR, the DNA sequence from the Tn5 insertion site and the DNA sequence of the contiguous 2,280-bp region were determined. Two complete open reading frames (ORF), designated ORF2 and ORF3, were identified within the sequence fragment. ORF2 and ORF3 were identified with the carocin S1 genes, caroS1K (ORF2) and caroS1I (ORF3), which, respectively, encode a killing protein (CaroS1K) and an immunity protein (CaroS1I). These genes were homologous to the pyocin S3 gene and the pyocin AP41 gene. Carocin S1 was expressed in E. carotovora subsp. carotovora Ea1068 and replicated in TH22-10 but could not be expressed in Escherichia coli (JM101) because a consensus sequence resembling an SOS box was absent. A putative sequence similar to the consensus sequence for the E. coli cyclic AMP receptor protein binding site (-312 bp) was found upstream of the start codon. Production of this bacteriocin was also induced by glucose and lactose. The homology search results indicated that the carocin S1 gene (between bp 1078 and bp 1704) was homologous to the pyocin S3 and pyocin AP41 genes in Pseudomonas aeruginosa. These genes encode proteins with nuclease activity (domain 4). This study found that carocin S1 also has nuclease activity.

  11. Molecular signatures of G-protein-coupled receptors.

    PubMed

    Venkatakrishnan, A J; Deupi, Xavier; Lebon, Guillaume; Tate, Christopher G; Schertler, Gebhard F; Babu, M Madan

    2013-02-14

    G-protein-coupled receptors (GPCRs) are physiologically important membrane proteins that sense signalling molecules such as hormones and neurotransmitters, and are the targets of several prescribed drugs. Recent exciting developments are providing unprecedented insights into the structure and function of several medically important GPCRs. Here, through a systematic analysis of high-resolution GPCR structures, we uncover a conserved network of non-covalent contacts that defines the GPCR fold. Furthermore, our comparative analysis reveals characteristic features of ligand binding and conformational changes during receptor activation. A holistic understanding that integrates molecular and systems biology of GPCRs holds promise for new therapeutics and personalized medicine. PMID:23407534

  12. 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

  13. 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. PMID:24830774

  14. 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

  15. Molecular cloning and expression of the human delta7-sterol reductase.

    PubMed

    Moebius, F F; Fitzky, B U; Lee, J N; Paik, Y K; Glossmann, H

    1998-02-17

    Inhibitors of the last steps of cholesterol biosynthesis such as AY9944 and BM15766 severely impair brain development. Their molecular target is the Delta7-sterol reductase (EC 1.3.1.21), suspected to be defective in the Smith-Lemli-Opitz syndrome, a frequent inborn disorder of sterol metabolism. Molecular cloning of the cDNA revealed that the human enzyme is a membrane-bound protein with a predicted molecular mass of 55 kDa and six to nine putative transmembrane segments. The protein is structurally related to plant and yeast sterol reductases. In adults the ubiquitously transcribed mRNA is most abundant in adrenal gland, liver, testis, and brain. The Delta7-sterol reductase is the ultimate enzyme of cholesterol biosynthesis in vertebrates and is absent from yeast. Microsomes from Saccharomyces cerevisiae strains heterologously expressing the human cDNA remove the C7-8 double bond in 7-dehydrocholesterol. The conversion to cholesterol depends on NADPH and is potently inhibited by AY9944 (IC50 0.013 microM), BM15766 (IC50 1.2 microM), and triparanol (IC50 14 microM). Our work paves the way to clarify whether a defect in the delta7-sterol reductase gene underlies the Smith-Lemli-Opitz syndrome. PMID:9465114

  16. Molecular cloning and expression of the human Δ7-sterol reductase

    PubMed Central

    Moebius, Fabian F.; Fitzky, Barbara U.; Lee, Joon No; Paik, Young-Ki; Glossmann, Hartmut

    1998-01-01

    Inhibitors of the last steps of cholesterol biosynthesis such as AY9944 and BM15766 severely impair brain development. Their molecular target is the Δ7-sterol reductase (EC 1.3.1.21), suspected to be defective in the Smith–Lemli–Opitz syndrome, a frequent inborn disorder of sterol metabolism. Molecular cloning of the cDNA revealed that the human enzyme is a membrane-bound protein with a predicted molecular mass of 55 kDa and six to nine putative transmembrane segments. The protein is structurally related to plant and yeast sterol reductases. In adults the ubiquitously transcribed mRNA is most abundant in adrenal gland, liver, testis, and brain. The Δ7-sterol reductase is the ultimate enzyme of cholesterol biosynthesis in vertebrates and is absent from yeast. Microsomes from Saccharomyces cerevisiae strains heterologously expressing the human cDNA remove the C7–8 double bond in 7-dehydrocholesterol. The conversion to cholesterol depends on NADPH and is potently inhibited by AY9944 (IC50 0.013 μM), BM15766 (IC50 1.2 μM), and triparanol (IC50 14 μM). Our work paves the way to clarify whether a defect in the Δ7-sterol reductase gene underlies the Smith–Lemli–Opitz syndrome. PMID:9465114

  17. Molecular cloning of the alpha-globin transcription factor CP2.

    PubMed Central

    Lim, L C; Swendeman, S L; Sheffery, M

    1992-01-01

    CP2, a transcription factor that binds the murine alpha-globin promoter, was purified and subjected to amino acid sequence analysis. Oligonucleotide primers derived from the sequence were used to obtain murine and human cDNA clones for the factor. The murine cDNA spans approximately 4 kb and contains two coextensive open reading frames (ORFs) which encode deduced polypeptides of 529 (ORF-1; molecular weight, 59,802) and 502 (ORF-2; molecular weight, 56,957) amino acids, slightly smaller than the purified factor as estimated from its mobility in sodium dodecyl sulfate-polyacrylamide gels (64,000 to 66,000). The human cDNA contains a single ORF of 501 amino acids that is nearly contiguous with murine ORF-2. Indeed, comparison of deduced human and murine amino acid sequences shows that the two polypeptides are 96.4% identical. A strictly conserved region is rich in serine and threonine (17.5%) and in proline (11%) residues (S-T-P domain). This S-T-P domain is immediately amino terminal to a string of 10 glutamines (in the human sequence) or a tract of alternating glutamine and proline residues (in the mouse sequence). Bacterial expression of the full-length (502-amino-acid) murine factor or of a core region comprising amino acids 133 to 395 generated polypeptides with the DNA binding specificity of CP2. These results confirmed the cloning of CP2 and delimited the region sufficient for specific DNA sequence recognition. Antisera produced against the core region recognized polypeptide species with Mrs of 64,000 and 66,000 in immune blots of nuclear extracts prepared from both murine and human cell lines, consistent with the size of the purified factor. Lastly, a data base search revealed that amino acids 63 to 270 of the murine factor are distantly related to a domain in the Drosophila gene regulatory factor Elf-1. Images PMID:1732747

  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. 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

  20. Mechanism of action of somatostatin: an overview of receptor function and studies of the molecular characterization and purification of somatostatin receptor proteins.

    PubMed

    Patel, Y C; Murthy, K K; Escher, E E; Banville, D; Spiess, J; Srikant, C B

    1990-09-01

    To determine whether somatostatin receptor subtypes arise from molecular heterogeneity of the receptor protein, we have cross-linked the putative receptor in normal rat tissues and in AtT-20 and GH3 cells, both chemically with SS-14, SS-28 and Tyr3 SMS ligands, as well as by photoaffinity labeling with an azido derivative of Tyr3 SMS (EE 581). Three prominent somatostatin receptor proteins of 58-kDa, 32-kDa, and 27-kDa size have been identified. These proteins exhibit a tissue-specific distribution, ligand selectivity, and relative preference for SS-14 and SS-28 binding, and thus qualify as somatostatin receptor subtypes. Using EE 581 as a photoaffinity probe, the 58-kDa and 32-kDa proteins have been purified to homogeneity from brain and AtT-20 cells by successive SDS-PAGE. The 58-kDa form has been trypsinized and amino acid sequence data obtained from four tryptic fragments. With the help of synthetic oligonucleotides derived from these sequences, work is currently in progress to clone the 58-kDa protein to elucidate its complete sequence, its expression, and its functional relationship to the somatostatin receptor and its pharmacological subtypes.

  1. Agonist Derived Molecular Probes for A2A Adenosine Receptors

    PubMed Central

    Jacobson, Kenneth A.; Pannell, Lewis K.; Ji, Xiao-duo; Jarvis, Michael F.; Williams, Michael; Hutchison, Alan J.; Barrington, William W.; Stiles, Gary L.

    2011-01-01

    The adenosine agonist 2-(4-(2-carboxyethyl)phenylethylamino)-5′-N-ethylcarboxamidoadenosine (CGS21680) was recently reported to be selective for the A2A adenosine receptor subtype, which mediates its hypotensive action. To investigate structurelactivity relationships at a distal site, CGS21680 was derivatized using a functionalized congener approach. The carboxylic group of CGS21680 has been esterified to form a methyl ester, which was then treated with ethylenediamine to produce an amine congener. The amine congener was an intermediate for acylation reactions, in which the reactive acyl species contained a reported group, or the precursor for such. For radioiodination, derivatives of p-hydroxyphenylpropionic, 2-thiophenylacetic, and p-aminophenylacetic acids were prepared. The latter derivative (PAPA-APEC) was iodinated electrophilically using [125I]iodide resulting in a radioligand which was used for studies of competition of binding to striatal A, adenosine receptors in bovine brain. A biotin conjugate and an aryl sulfonate were at least 350-fold selective for A, receptors. For spectroscopic detection, a derivative of the stable free radical tetramethyl-1-piperidinyloxy (TEMPO) was prepared. For irreversible inhibition of receptors, meta- and para-phenylenediisothiocyanate groups were incorporated in the analogs. We have demonstrated that binding at A2A receptors is relatively insensitive to distal structural changes at the 2-position, and we report high affinity molecular probes for receptor characterization by radioactive, spectroscopic and affinity labelling methodology. PMID:2561548

  2. 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

  3. 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

  4. Molecular cloning of five individual stage- and tissue-specific mRNA sequences from sea urchin pluteus embryos.

    PubMed

    Fregien, N; Dolecki, G J; Mandel, M; Humphreys, T

    1983-06-01

    Five developmentally regulated sea urchin mRNA sequences which increase in abundance between the blastula and pluteus stages of development were isolated by molecular cloning of cDNA. The regulated sequences all appeared in moderately abundant mRNA molecules of pluteus cells and represented 4% of the clones tested. There were no regulated sequences detected in the 40% of the clones which hybridized to the most abundant mRNA, and the screening procedures were inadequate to detect possible regulation in the 20 to 30% of the clones presumably derived from rare-class mRNA. The reaction of 32P[cDNA] from blastula and pluteus mRNA to dots of the cloned DNAs on nitrocellulose filters indicated that the mRNAs complementary to the different cloned pluteus-specific sequences were between 3- and 47-fold more prevalent at the pluteus stage than at the blastula stage. Polyadenylated RNA from different developmental stages was transferred from electrophoretic gels to nitrocellulose filters and reacted to the different cloned sequences. The regulated mRNAs were undetectable in the RNA of 3-h embryos, became evident at the hatching blastula stage, and reached a maximum in abundance by the gastrula or pluteus stage. Certain of the clones reacted to two sizes of mRNA which did not vary coordinately with development. Transfers of RNA isolated from each of the three cell layers of pluteus embryos that were reacted to the cloned sequences revealed that two of the sequences were found in the mRNA of all three layers, two were ectoderm specific, and one was endoderm specific. Four of the regulated sequences were complementary to one or two major bands and one to at least 50 bands on Southern transfers of restriction endonuclease-digested total sea urchin DNA. PMID:6688291

  5. The 5′ Untranslated Region of a Novel Infectious Molecular Clone of the Dicistrovirus Cricket Paralysis Virus Modulates Infection

    PubMed Central

    Kerr, Craig H.; Wang, Qing S.; Keatings, Kathleen; Khong, Anthony; Allan, Douglas; Yip, Calvin K.

    2015-01-01

    ABSTRACT 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. IMPORTANCE Dicistroviridae, which are RNA viruses that infect arthropods, have served as a model to gain insights into fundamental host

  6. 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

  7. 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. PMID:25246348

  8. T-cell receptor beta gene rearrangements in clones derived from human CD4-8- cells expressing natural killer cell activity.

    PubMed

    Christmas, S E; Moore, M

    1988-12-01

    Clones derived from highly purified human peripheral blood Leu 19+ cells in the presence of phytohaemagglutinin (PHA) and interleukin-2 (IL-2) expressed cytotoxic activity against natural killer (NK)-resistant as well as NK-sensitive targets. All 66 clones analysed had a germ line configuration of T-cell receptor (TCR) beta genes and 38/40 also had unrearranged TCR gamma genes. The two exceptions were both CD3+ clones, but these did not have a cytotoxic repertoire noticeably different from CD3- clones without TCR gamma gene rearrangements. Clones were also obtained from highly purified CD4-8- cells, most of which were also cytotoxic for NK-resistant and NK-sensitive targets. About 90% of these clones were CD3+ but only around 50% remained negative for CD4 and CD8 while a significant number (12.7%) were positive for both CD4 and CD8. All clones analysed had rearranged TCR gamma genes and most had also rearranged TCR beta genes, including 20/25 of the clones which were CD3+4-8-. Many of the clones showed two rearrangements of TCR beta genes, and 3/4 CD3- clones had rearranged TCR beta as well as TCR gamma genes. There was no correlation between cytotoxic activity and TCR gene status or phenotype of these CD4-8- derived clones, except that clones which were Leu 19+ tended to have higher cytotoxic activity against NK-sensitive and NK-resistant targets than Leu 19-clones. The results strongly indicate that TCR beta and gamma gene products are not involved in the cytotoxicity mediated by these clones. They also suggest that some CD4-8- cells may be capable of limited differentiation in vitro.

  9. Cloning of neuromedin B and its receptor in the rabbit and generating a polyclonal antibody to the neuromedin B protein.

    PubMed

    Guo, Ting-Ting; Su, Juan; Ma, Zhi-Yu; Ma, Jun-Xiao; Jin, Meng-Meng; Li, Xiang; Lei, Zhi-Hai

    2015-06-10

    Neuromedin B (NMB) is a highly conserved bombesin-related neuropeptide found in mammals. Neuromedin B (NMB) executes its effect by binding to the cell surface receptor, neuromedin B receptor (NMBR). In this study, we cloned the rabbit NMB and NMBR genes. The similarity and phylogenetic analyses of NMB and NMBR gene sequences were performed. The expression of NMB and NMBR mRNA in the rabbit was investigated using real-time RT-PCR. Our bioinformatic analysis demonstrated that the cloned rabbit NMB precursor cDNA encoded Gly-His-Phe-Met-NH2 amino acids at the C-terminus, and that its receptor possessed typical transmembrane features. The NMB mRNA was highly expressed in the CNS, while the NMBR mRNA was widely expressed in many tissues, with the highest expression in the gastrointestinal tract. The studies on the NMB distribution and function are limited by the lack of a specific antibody to this neuropeptide. In this paper, polyclonal NMB antibody was generated in mice. Western blotting analysis revealed that the prepared antibody could specifically recognize the recombinant and the endogenous NMB proteins. Immunohistochemistry analysis indicated that the NMB protein was localized in the cytoplasm of the pituitary cells. The existence of NMB protein in the hypothalamic-pituitary-gonadal axis suggests that NMB might function in rabbit reproduction.

  10. Molecular cloning and characterization of a Candida tsukubaensis alpha-glucosidase gene in the yeast Saccharomyces cerevisiae.

    PubMed

    Kinsella, B T; Larkin, A; Bolton, M; Cantwell, B A

    1991-07-01

    The molecular cloning of an alpha-glucosidase gene isolated from a Candida tsukubaensis (CBS 6389) genomic library in Saccharomyces cervisiae is reported. The cloned gene is contained within a 6.2 kb Sau3A DNA fragment and directs the synthesis and secretion of an amylolytic enzyme into the extracellular medium of the recombinant host, S. cerevisiae. The cloned enzyme was found to have an unusually broad substrate specificity and is capable of hydrolysing alpha-1,2, alpha-1,3, alpha-1,4 and alpha-1,6 linked, as well as aryl and alkyl, D-glucosides. On the basis of its substrate specificity profile, the cloned enzyme was classified as an alpha-glucosidase (E.C. 3.2.1.20). It has a pH optimum in the range 4.2-4.6, a temperature optimum of 58 degrees C and is readily inactivated at pasteurization temperature (60 degrees C). Southern blot analysis failed to reveal any homology between the cloned gene and genomic DNA isolated from other well characterized amylolytic yeasts. A rapid plate-assay, based on the utilization of a chromogenic substrate X-alpha-D-glucoside to detect the expression of the cloned alpha-glucosidase in S. cerevisiae transformants, was developed. PMID:1934116

  11. Molecular epidemiology of methicillin-resistant Staphylococcus aureus in Colombian hospitals: dominance of a single unique multidrug-resistant clone.

    PubMed

    Gomes, A R; Sanches, I S; Aires de Sousa, M; Castañeda, E; de Lencastre, H

    2001-01-01

    The first study on the molecular characterization of methicillin-resistant Staphylococcus aureus (MRSA) isolates from Colombia was performed as part of a global surveillance established by the CEM/NET Initiative, under Project RESIST. Seventy-six MRSA isolates recovered from five hospitals during 1996-1998 were analyzed by the hybridization of ClaI restriction digests with mecA- and Tn554-specific probes, and by pulsed-field gel electrophoresis (PFGE) of chromosomal SmaI digests. All MRSA isolates, with one exception, belonged to a single clonal type II::NH::D. This clone, which was previously described among MRSA isolates recovered in the early 1990s in European and New York and South American hospitals, showed resistance to beta-lactam antibiotics only and appeared to be associated almost exclusively with pediatric infections ("Pediatric clone" of MRSA). While sharing identical molecular typing properties with the Pediatric clone, the Colombian isolates differed by extensive multidrug resistance and were recovered from patients of all ages. It is also noteworthy that the Brazilian clone of MRSA (XI::B::B), another multidrug-resistant international clone currently widely spread in Brazil, Argentina, Uruguay, Chile, and also in several European countries, was completely absent from this set of isolates from Colombia.

  12. Molecular cloning and functional characterization of a putative sulfite oxidase (SO) ortholog from Nicotiana benthamiana.

    PubMed

    Xia, Zongliang; Su, Xinhong; Wu, Jianyu; Wu, Ke; Zhang, Hua

    2012-03-01

    Sulfite oxidase (SO) catalyzes the oxidation of sulfite to sulfate and thus has important roles in diverse metabolic processes. However, systematic molecular and functional investigations on the putative SO from tobacco (Nicotiana benthamiana) have hitherto not been reported. In this work, a full-length cDNA encoding putative sulfite oxidase from N. benthamiana (NbSO) was isolated. The deduced NbSO protein shares high homology and typical structural features with other species SOs. Phylogenetic analysis indicates that NbSO cDNA clone encodes a tobacco SO isoform. Southern blot analysis suggests that NbSO is a single-copy gene in the N. benthamiana genome. The NbSO transcript levels were higher in aerial tissues and were up-regulated in N. benthamiana during sulfite stress. Reducing the SO expression levels through virus-induced gene silencing caused a substantial accumulation in sulfite content and less sulfate accumulation in N. benthamiana leaves when exposed to sulfite stress, and thus resulted in decreased tolerance to sulfite stress. Taken together, this study improves our understanding on the molecular and functional properties of plant SO and provides genetic evidence on the involvement of SO in sulfite detoxification in a sulfite-oxidizing manner in N. benthamiana plants. PMID:21667106

  13. Cloning, in Vitro expression, and novel phylogenetic classification of a channel catfish estrogen receptor

    USGS Publications Warehouse

    Xia, Z.; Patino, R.; Gale, W.L.; Maule, A.G.; Densmore, L.D.

    1999-01-01

    We obtained two channel catfish estrogen receptor (ccER) cDNA from liver of female fish using RT–PCR. The two fragments were identical in sequence except that the smaller one had an out-of-frame deletion in the E domain, suggesting the existence of ccER splice variants. The larger fragment was used to screen a cDNA library from liver of a prepubescent female. A cDNA was obtained that encoded a 581-amino-acid ER with a deduced molecular weight of 63.8 kDa. Extracts of COS-7 cells transfected with ccER cDNA bound estrogen with high affinity (Kd = 4.7 nM) and specificity. Maximum parsimony and Neighbor Joining analyses were used to generate a phylogenetic classification of ccER on the basis of 18 full-length ER sequences. The tree suggested the existence of two major ER branches. One branch contained two clearly divergent clades which included all piscine ER (except Japanese eel ER) and all tetrapod ERα, respectively. The second major branch contained the eel ER and the mammalian ERβ. The high degree of divergence between the eel ER and mammalian ERβ suggested that they also represent distinct piscine and tetrapod ER. These data suggest that ERα and ERβ are present throughout vertebrates and that these two major ER types evolved by duplication of an ancestral ER gene. Sequence alignments with other members of the nuclear hormone receptor superfamily indicated the presence of 8 amino acids in the E domain that align exclusively among ER. Four of these amino acids have not received prior research attention and their function is unknown. The novel finding of putative ER splice variants in a nonmammalian vertebrate and the novel phylogenetic classification of ER offer new perspectives in understanding the diversification and function of ER.

  14. MOLECULAR TARGETS AND MECHANISMS FOR ETHANOL ACTION IN GLYCINE RECEPTORS

    PubMed Central

    Perkins, Daya I.; Trudell, James R.; Crawford, Daniel K.; Alkana, Ronald L.; Davies, Daryl L.

    2010-01-01

    Glycine receptors (GlyRs) are recognized as the primary mediators of neuronal inhibition in the spinal cord, brain stem and higher brain regions known to be sensitive to ethanol. Building evidence supports the notion that ethanol acting on GlyRs causes at least a subset of its behavioral effects and may be involved in modulating ethanol intake. For over two decades, GlyRs have been studied at the molecular level as targets for ethanol action. Despite the advances in understanding the effects of ethanol in vivo and in vitro, the precise molecular sites and mechanisms of action for ethanol in ligand-gated ion channels in general, and in GlyRs specifically, are just now starting to become understood. The present review focuses on advances in our knowledge produced by using molecular biology, pressure antagonism, electrophysiology and molecular modeling strategies over the last two decades to probe, identify and model the initial molecular sites and mechanisms of ethanol action in GlyRs. The molecular targets on the GlyR are covered on a global perspective, which includes the intracellular, transmembrane and extracellular domains. The latter has received increasing attention in recent years. Recent molecular models of the sites of ethanol action in GlyRs and their implications to our understanding of possible mechanism of ethanol action and novel targets for drug development in GlyRs are discussed. PMID:20399807

  15. Cloning and characterization of a P2X receptor expressed in the central nervous system of Lymnaea stagnalis.

    PubMed

    Bavan, Selvan; Straub, Volko A; Webb, Tania E; Ennion, Steven J

    2012-01-01

    P2X receptors are membrane ion channels gated by extracellular ATP. Mammals possess seven distinct P2X subtypes (P2X1-7) that have important functions in a wide array of physiological processes including roles in the central nervous system (CNS) where they have been linked to modulation of neurotransmitter release. We report here the cloning and functional characterization of a P2X receptor from the mollusc Lymnaea stagnalis. This model organism has a relatively simple CNS consisting of large readily identifiable neurones, a feature which together with a well characterized neuronal circuitry for important physiological processes such as feeding and respiration makes it an attractive potential model to examine P2X function. Using CODEHOP PCR we identified a single P2X receptor (LymP2X) in Lymnaea CNS which was subsequently cloned by RT-PCR. When heterologously expressed in Xenopus oocytes, LymP2X exhibited ATP evoked inward currents (EC(50) 6.2 µM) which decayed during the continued presence of agonist. UTP and ADP did not activate the receptor whereas αβmeATP was a weak agonist. BzATP was a partial agonist with an EC(50) of 2.4 µM and a maximal response 33% smaller than that of ATP. The general P2 receptor antagonists PPADS and suramin both inhibited LymP2X currents with IC(50) values of 8.1 and 27.4 µM respectively. LymP2X is inhibited by acidic pH whereas Zn(2+) and Cu(2+) ions exhibited a biphasic effect, potentiating currents up to 100 µM and inhibiting at higher concentrations. Quantitative RT-PCR and in situ hybridization detected expression of LymP2X mRNA in neurones of all CNS ganglia suggesting this ion channel may have widespread roles in Lymnaea CNS function. PMID:23209755

  16. Molecular cloning and functional characterization of a rainbow trout liver Oatp.

    PubMed

    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 2772bp containing a 2115bp open reading frame coding for a 705 aa protein with an approximate molecular mass of 80kDa. 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 Km value of 13.9μM and 13.4μM for estrone-3-sulfate and methotrexate, respectively and a rather low affinity for taurocholate with a Km value of 103μM. 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

  17. 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

  18. Molecular Cloning, Expression and Purification of Truncated hpd Fragment of Haemophilus influenzae in Escherichia coli

    PubMed Central

    Behrouzi, Ava; Bouzari, Saeid; Siadat, Seyed Davar; Jafari, Anis; Irani, Shiva

    2015-01-01

    Background: Nontypeable Haemophilus influenzae (NTHi) is a significant pathogen in children, causing otitis media, sinusitis, conjunctivitis, pneumonia, and occasionally invasive infections. Protein D (PD) belongs to the minor outer-membrane proteins of H. influenza. Moreover, it has been shown that this protein is one of the most potent vaccine candidates against the NTHi strain. Objectives: In the present study, a new truncated form of PD was designed based on conserved areas, and recombinant truncated PD was expressed. Materials and Methods: Truncated PD was designed using bioinformatics tools, and a 345 bp fragment of the truncated hpd gene was amplified by polymerase chain reaction (PCR) from H. influenzae and subsequently cloned into the prokaryotic expression vector pBAD-gIIIA. In addition, for the expression of the recombinant protein, the pBAD-truncated PD plasmid was transformed into competent TOP10 cells. The recombinant protein was expressed with Arabinose. The expressed protein was purified by affinity chromatography using Ni-NTA resin. Results: The cloning of PD was confirmed by colony-PCR and enzymatic digestion. Arabinose 0.2% was able to efficiently induce protein expression. The SDS-PAGE analysis showed that our constructed pBAD-PD-TOP10 efficiently produced a target recombinant protein with a molecular weight of 16 kDa. A high concentration of the recombinant protein was obtained via the purification process by affinity chromatography. The recombinant PD was reacted with peroxidase-conjugated rabbit anti-mouse immunoglobulins. Conclusions: Our results showed that the recombinant protein produced by the pBAD vector in the Escherichia coli system was very efficient. PMID:26464772

  19. 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.

  20. 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

  1. Purification, characterization and molecular cloning of the major chitinase from Tenebrio molitor larval midgut.

    PubMed

    Genta, Fernando A; Blanes, Lucas; Cristofoletti, Plínio T; do Lago, Claudimir L; Terra, Walter R; Ferreira, Clélia

    2006-10-01

    Insect chitinases are involved in degradation of chitin from the exoskeleton cuticle or from midgut peritrophic membrane during molts. cDNAs coding for insect cuticular and gut chitinases were cloned, but only chitinases from moulting fluid were purified and characterized. In this study the major digestive chitinase from T. molitor midgut (TmChi) was purified to homogeneity, characterized and sequenced after cDNA cloning. TmChi is secreted by midgut epithelial cells, has a molecular weight of 44 kDa and is unstable in the presence of midgut proteinases. TmChi shows strong substrate inhibition when acting on umbelliferyl-derivatives of chitobio- and chitotriosaccharides, but has normal Michaelis kinetics with the N-acetylglucosamine derivative as substrate. TmChi has very low activity against colloidal chitin, but effectively converts oligosaccharides to shorter fragments. The best substrate for TmChi is chitopentaose, with highest k(cat)/K(M) value. Sequence analysis and chemical modification experiments showed that the TmChi active site contains carboxylic groups and a tryptophane, which are known to be important for catalysis in family 18 chitinases. Modification with p-hidroximercuribenzoate of a cysteine residue, which is exposed after substrate binding, leads to complete inactivation of the enzyme. TmChi mRNA encodes a signal peptide plus a protein with 37 kDa and high similarity with other insect chitinases from family 18. Surprisingly, this gene does not encode the C-terminal Ser-Thr-rich connector and chitin-binding domain normally present in chitinases. The special features of TmChi probably result from its adaptation to digest chitin-rich food without damaging the peritrophic membrane.

  2. Molecular cloning and characterization of a heat shock protein 70 gene in swimming crab (Portunus trituberculatus).

    PubMed

    Cui, Zhaoxia; Liu, Yuan; Luan, Weisha; Li, Qianqian; Wu, Danhua; Wang, Shuangyan

    2010-01-01

    Hsp70 can stimulate cells of the innate immune system directly by acting as "danger"-signaling molecules. To understand the immune defense mechanisms of swimming crab Portunus trituberculatus (Decapoda: Brachyura: Portunidae), the cDNA of Hsp70 (designated PtHsp70) was cloned by the combination of homology cloning and rapid amplification of cDNA ends (RACE). The full-length PtHsp70 cDNA was 2195 bp, including an open reading frame (ORF) of 1950 bp encoding a polypeptide of 650 amino acids with estimated molecular mass of 71.1514 kDa and theoretical isoelectric point of 5.38. FASTA and BLAST analysis indicated that PtHsp70 should be an inducible cytosolic member of the Hsp70 family. The coding region of PtHsp70 was uninterrupted and four SNPs with 1133C/T, 1311C/T, 1551C/T and 1809 A/G were detected by direct sequencing of 20 genomic samples. Using fluorescent real-time quantitative PCR, the transcriptional expression of PtHsp70 showed a clear time-dependent response after challenge by Vibrio alginolyticus, the main causative agent of emulsification disease causing large mortality in P. trituberculatus. This is the first report on the expression of Hsp70 induced by pathogen stimulation in Brachyura. Phylogenetic analysis revealed that the inducible Hsp70s were divided into two groups in crab and PtHsp70 was clustered into the Hsp/Hsc group (Clade I) by maximum-likelihood (ML) and Bayesian inference (BI) methods. GAP repeat and GGMP motif of inducible Hsp70 gene in the crab species were only found in Clade I. PMID:19815077

  3. Molecular cloning and characterization of a type 3 iodothyronine deiodinase in the pine snake Pituophis deppei.

    PubMed

    Villalobos, Patricia; Orozco, Aurea; Valverde-R, Carlos

    2010-11-01

    The three distinct but related isotypes of the iodothyronine deiodinase family: D1, D2, and D3, have been amply studied in vertebrate homeotherms and to a lesser extent in ectotherms, particularly in reptiles. Here, we report the molecular and kinetic characteristics of both the native and the recombinant hepatic D3 from the pine snake Pituophis deppei (PdD3). The complete PdD3 cDNA (1680 bp) encodes a protein of 287 amino acids (aa), which is the longest type 3 deiodinase so far cloned. PdD3 shares 78% identity with chicken and 71% with its other orthologs. Interestingly, the hinge domain in D3s, including PdD3, is rich in proline. This structural feature is shared with D1s, the other inner-ring deiodinases, and deserves further study. The kinetic characteristics of both native and recombinant PdD3 were similar to those reported for D3 in other vertebrates. True K(m) values for T(3) IRD were 9 and 11 nM for native and recombinant PdD3, respectively. Both exhibited a requirement for a high concentration of cofactor (40 mM DTT), insensitivity to inhibition by PTU (>2 mM), and bisubstrate, sequential-type reaction kinetics. In summary, the present data demonstrate that the liver of the adult pine snake P. deppei expresses D3. Furthermore, this is the first report of the cloning and expression of a reptilian D3 cDNA. The finding of hepatic D3 expression in the adult pine snake P. deppei is consistent with results in adult piscine species in which the dietary T(3) content seems to regulate liver deiodinase expression. Thus, our present results support the proposal that hepatic D3 in adult vertebrates plays a sentinel role in avoiding an inappropriate overload of exogenous T(3) secondary to feeding in those species that devour the whole prey.

  4. 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.

  5. 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. PMID:24664316

  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, 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

  8. Molecular cloning and characterization of orange-spotted grouper (Epinephelus coioides) CXC chemokine ligand 12.

    PubMed

    Wu, Chen-Shiou; Wang, Ting-Yu; Liu, Chin-Feng; Lin, Hao-Ping; Chen, Young-Mao; Chen, Tzong-Yueh

    2015-12-01

    Chemokines are a family of soluble peptides that can recruit a wide range of immune cells to sites of infection and disease. The CXCL12 is a chemokine that binds to its cognate receptor CXCR4 and thus involved in multiple physiological and pathophysiological processes. In this study, we cloned and characterized CXCL12 from Epinephelus coioides (osgCXCL12). We found that the open reading frame of osgCXCL12 consists of 98 amino acid residues with the small cytokine C-X-C domain located between residues 29 and 87. Higher expression levels for osgCXCL12 were detected at the kitting stage, compared with the prolarva and larva shape stages. The expression patterns revealed that osgCXCL12 may play a key role in early grouper development. We detected mRNA transcripts for osgCXCL12 in healthy tissues and found the highest osgCXCL12 expression in the head kidney. Furthermore, a time-course analysis revealed significantly increased osgCXCL12 and osgCXCR4 expression levels after the nervous necrosis virus (NNV) challenge. In addition, expression of osgCXCL12 was affected by injection with microbial mimics [LPS and poly(I:C)]. These results suggest that osgCXCL12 is associated with inflammatory and developmental processes in the grouper.

  9. Molecular cloning and function characterization of a new macrophage-activating protein from Tremella fuciformis.

    PubMed

    Hung, Chih-Liang; Chang, An-Ju; Kuo, Xhao-Kai; Sheu, Fuu

    2014-02-19

    Silver ear mushroom ( Tremella fuciformis ) is an edible fungus with health benefits. In this study, we purified a new T. fuciformis protein (TFP) and demonstrated its ability to activate primary murine macrophages. The isolation procedure involved ammonium sulfate fractionation and ion exchange chromatography. TFP naturally formed a 24 kDa homodimeric protein and did not contain glycan residues. The TFP gene was cloned using the rapid amplification of cDNA ends method, and the cDNA sequence of TFP was composed of 408 nucleotides with a 336 nucleotide open reading frame encoding a 112 amino acid protein. TFP was capable of stimulating TNF-α, IL-1β, IL-1ra, and IL-12 production in addition to CD86/MHC class II expression, mRNA expression of M1-type chemokines, and nuclear NF-κB accumulation in murine peritoneal macrophage cells. Furthermore, TFP failed to stimulate TLR4-neutralized and TLR4-knockout macrophages, suggesting that TLR4 is a required receptor for TFP signaling on macrophages. Taken together, these results indicate that TFP may be an important bioactive compound from T. fuciformis that induces M1-polarized activation through a TLR4-dependent NF-κB signaling pathway. PMID:24400969

  10. Partial cloning and localization of leptin and leptin receptor in the mammary gland of the Egyptian water buffalo.

    PubMed

    Sayed-Ahmed, A; Elmorsy, S Elm; Rudas, P; Bartha, T

    2003-10-01

    Originally an overall metabolic control was attributed to the leptin hormone, which is produced mainly by the adipose tissue. Recently, leptin gene expression was demonstrated in several additional peripheral tissues. Furthermore, several isoforms of leptin receptor were found both in the central nervous system and in the peripheral tissues. Using reverse transcription and polymerase chain reaction analysis we demonstrate that leptin is expressed both in the adipose tissue and in the lactating mammary gland tissue of Egyptian water buffalo. Our results show that, short and long isoforms of leptin receptor are expressed in buffalo mammary gland tissue. We have partially cloned the buffalo leptin and its short and long isoforms of receptor, which show a high sequence homology to previously published sequences of other mammalian species especially to that of other ruminants. Localization of leptin and its receptor mRNA transcripts, as determined by in situ hybridization procedure, revealed that leptin and its receptor transcripts are expressed specifically in the alveolar epithelial cells of the mammary gland. These morphological data support that leptin could also act as an autocrine and paracrine mediator for mammary gland metabolism and as a facilitator of alveolar epithelial cell activity during lactation.

  11. The High Molecular Weight Stress Proteins: Identification, Cloning, and Utilization in Cancer Immunotherapy*

    PubMed Central

    Wang, Xiang-Yang; Subjeck, John R.

    2013-01-01

    Although the large stress/heat shock proteins (HSPs), i.e., Hsp110 and Grp170, were identified over 30 years ago, these abundant and highly conserved molecules have received much less attention compared to other conventional HSPs. Large stress proteins act as molecular chaperones with exceptional protein-holding capability and prevent the aggregation of proteins induced by thermal stress. The chaperoning properties of Hsp110 and Grp170 are integral to the ability of these molecules to modulate immune functions and are essential for developing large chaperone complex vaccines for cancer immunotherapy. The potent antitumor activity of the Hsp110/Grp170-tumor protein antigen complexes, demonstrated in preclinical studies, has led to a phase I clinical trial through the National Cancer Institute's RAID Program that is presently underway. Here we review aspects of the structure and function of these large stress proteins, their roles as molecular chaperones in the biology of cell stress, and prospects for their use in immune regulation and cancer immunotherapy. Lastly, we will discuss the recently revealed immunosuppressive activity of scavenger receptor A that binds to Hsp110 and Grp170, as well as the feasibility of targeting this receptor to promote T-cell activation and antitumor immunity induced by large HSP vaccines and other immunotherapies. PMID:23829534

  12. Molecular cloning of a cDNA encoding a novel fatty acid-binding protein from rat skin.

    PubMed

    Watanabe, R; Fujii, H; Odani, S; Sakakibara, J; Yamamoto, A; Ito, M; Ono, T

    1994-04-15

    A novel skin-type fatty acid-binding protein, termed cutaneous(C)-FABP, has been purified from rat skin and a cDNA clone for this protein has been identified. The purified protein had the ability to bind long chain fatty acids like other rat FABPs. The deduced amino acid sequence of the cDNA clone comprises residues yielding a molecular mass for the polypeptide of 15.1 kDa and exhibits around 50% identity to myelin P2 protein, adipocyte FABP and heart FABP. Our results propose that C-FABP is a new member of the FABP family.

  13. Molecular cloning of the recA gene and construction of a recA strain of Francisella novicida.

    PubMed Central

    Berg, J M; Mdluli, K E; Nano, F E

    1992-01-01

    A gene locus that is functionally analogous to the recA gene of Escherichia coli was molecularly cloned from Francisella novicida. The cloned gene was found to suppress the sensitivity of an E. coli strain to DNA-damaging agents and to support genetic recombination in E. coli. After transposon mutagenesis, the recA-like gene locus was returned to F. novicida and a UV-sensitive F. novicida strain was isolated. In contrast to the wild-type strain, this UV-sensitive strain could not be transformed with chromosomal DNA. Images PMID:1309722

  14. Human CD4-8- -derived clones. Phenotypic and functional characteristics and variation between donors in patterns of T-cell receptor gamma gene rearrangements.

    PubMed

    Christmas, S E

    1989-06-01

    Clones were derived from highly purified human CD4-8- lymphocytes from three different donors and maintained in the presence of interleukin 2 and phytohaemagglutinin. Considerable variation was noted between donors in the phenotype and T-cell receptor (TCR) gamma gene rearrangements of CD4-8- -derived clones. In one donor, most clones remained CD4-8- and all were CD3+WT31- and therefore expressed gamma/delta heterodimers. TCR gamma gene rearrangements almost all involved C gamma 1. In contrast, most clones from a second donor were CD3+WT31+, and therefore expressed alpha/beta heterodimers, and many were positive for CD4 or CD8. Most clones from a third donor were CD3+WT31- with a high proportion of TCR gamma gene rearrangements involving C gamma 2. The V gamma 9JP rearrangement was exclusively confined to CD3+WT31- clones and was present in the majority of clones. Almost all CD3+WT31- clones showed TCR beta as well as gamma gene rearrangements. Most CD3+WT31- clones with at least one chromosome rearranged to C gamma 1 exhibited high non-major histocompatibility complex (MHC)-restricted cytotoxic activity, while most of those with two C gamma 2 rearrangements, and therefore expressing a non-disulphide-linked gamma/delta heterodimer, had low activity. Preincubation of effector cells with anti-CD3 strongly inhibited the cytotoxicity of CD3+WT31- clones while that of CD3+WT31+ clones was enhanced. This implicates the CD3-gamma/delta complex in target cell recognition by cytotoxic gamma/delta-bearing T-cell clones. The results show that there is heterogeneity between donors in the relative proportions of CD4-8- -derived clones expressing alpha/beta heterodimers and the different forms of the gamma/delta heterodimer.

  15. 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.

  16. The mineralocorticoid receptor: insights into its molecular and (patho)physiological biology

    PubMed Central

    Viengchareun, Say; Le Menuet, Damien; Martinerie, Laetitia; Munier, Mathilde; Pascual-Le Tallec, Laurent; Lombès, Marc

    2007-01-01

    The last decade has witnessed tremendous progress in the understanding of the mineralocorticoid receptor (MR), its molecular mechanism of action, and its implications for physiology and pathophysiology. After the initial cloning of MR, and identification of its gene structure and promoters, it now appears as a major actor in protein-protein interaction networks. The role of transcriptional coregulators and the determinants of mineralocorticoid selectivity have been elucidated. Targeted oncogenesis and transgenic mouse models have identified unexpected sites of MR expression and novel roles for MR in non-epithelial tissues. These experimental approaches have contributed to the generation of new cell lines for the characterization of aldosterone signaling pathways, and have also facilitated a better understanding of MR physiology in the heart, vasculature, brain and adipose tissues. This review describes the structure, molecular mechanism of action and transcriptional regulation mediated by MR, emphasizing the most recent developments at the cellular and molecular level. Finally, through insights obtained from mouse models and human disease, its role in physiology and pathophysiology will be reviewed. Future investigations of MR biology should lead to new therapeutic strategies, modulating cell-specific actions in the management of cardiovascular disease, neuroprotection, mineralocorticoid resistance, and metabolic disorders. PMID:18174920

  17. cDNA cloning of a serotonin 5-HT1C receptor by electrophysiological assays of mRNA-injected Xenopus oocytes.

    PubMed Central

    Lübbert, H; Hoffman, B J; Snutch, T P; van Dyke, T; Levine, A J; Hartig, P R; Lester, H A; Davidson, N

    1987-01-01

    We describe a strategy for the cloning of neurotransmitter-receptor and ion-channel cDNAs that is based on electrophysiological assays of mRNA-injected Xenopus oocytes. This procedure circumvents the purification of these membrane proteins, which is hindered by their low abundance and their hydrophobic nature. It involves methods for RNA fractionation by high-resolution gel electrophoresis, directional cDNA cloning in a single-stranded vector, and screening of the cDNA library by voltage-clamp measurements of currents induced by serotonin in mRNA-injected oocytes. The applicability of our approach is demonstrated by the isolation of a serotonin receptor cDNA clone from a mouse choroid plexus papilloma. The clone was identified by hybrid-depletion and hybrid-selection procedures. The receptor expressed in oocytes injected with hybrid-selected RNA is fully functional, indicating that it is composed of a single subunit encoded by a 5-kilobase RNA. The pharmacology of the hybrid-selected receptor confirms that we have successfully cloned a serotonin 5-HT1C receptor cDNA. Images PMID:3473504

  18. Molecular Insights into the Transmembrane Domain of the Thyrotropin Receptor

    PubMed Central

    Chantreau, Vanessa; Taddese, Bruck; Munier, Mathilde; Gourdin, Louis; Henrion, Daniel; Rodien, Patrice; Chabbert, Marie

    2015-01-01

    The thyrotropin receptor (TSHR) is a G protein-coupled receptor (GPCR) that is member of the leucine-rich repeat subfamily (LGR). In the absence of crystal structure, the success of rational design of ligands targeting the receptor internal cavity depends on the quality of the TSHR models built. In this subfamily, transmembrane helices (TM) 2 and 5 are characterized by the absence of proline compared to most receptors, raising the question of the structural conformation of these helices. To gain insight into the structural properties of these helices, we carried out bioinformatics and experimental studies. Evolutionary analysis of the LGR family revealed a deletion in TM5 but provided no information on TM2. Wild type residues at positions 2.58, 2.59 or 2.60 in TM2 and/or at position 5.50 in TM5 were substituted to proline. Depending on the position of the proline substitution, different effects were observed on membrane expression, glycosylation, constitutive cAMP activity and responses to thyrotropin. Only proline substitution at position 2.59 maintained complex glycosylation and high membrane expression, supporting occurrence of a bulged TM2. The TSHR transmembrane domain was modeled by homology with the orexin 2 receptor, using a protocol that forced the deletion of one residue in the TM5 bulge of the template. The stability of the model was assessed by molecular dynamics simulations. TM5 straightened during the equilibration phase and was stable for the remainder of the simulations. Our data support a structural model of the TSHR transmembrane domain with a bulged TM2 and a straight TM5 that is specific of glycoprotein hormone receptors. PMID:26545118

  19. From molecular phylogeny towards differentiating pharmacology for NMDA receptor subtypes.

    PubMed

    Platt, Randall J; Curtice, Kigen J; Twede, Vernon D; Watkins, Maren; Gruszczyński, Paweł; Bulaj, Grzegorz; Horvath, Martin P; Olivera, Baldomero M

    2014-04-01

    In order to decode the roles that N-methyl-D-aspartate (NMDA) receptors play in excitatory neurotransmission, synaptic plasticity, and neuropathologies, there is need for ligands that differ in their subtype selectivity. The conantokin family of Conus peptides is the only group of peptidic natural products known to target NMDA receptors. Using a search that was guided by phylogeny, we identified new conantokins from the marine snail Conus bocki that complement the current repertoire of NMDA receptor pharmacology. Channel currents measured in Xenopus oocytes demonstrate conantokins conBk-A, conBk-B, and conBk-C have highest potencies for NR2D containing receptors, in contrast to previously characterized conantokins that preferentially block NR2B containing NMDA receptors. Conantokins are rich in γ-carboxyglutamate, typically 17-34 residues, and adopt helical structure in a calcium-dependent manner. As judged by CD spectroscopy, conBk-C adopts significant helical structure in a calcium ion-dependent manner, while calcium, on its own, appears insufficient to stabilize helical conformations of conBk-A or conBk-B. Molecular dynamics simulations help explain the differences in calcium-stabilized structures. Two-dimensional NMR spectroscopy shows that the 9-residue conBk-B is relatively unstructured but forms a helix in the presence of TFE and calcium ions that is similar to other conantokin structures. These newly discovered conantokins hold promise that further exploration of small peptidic antagonists will lead to a set of pharmacological tools that can be used to characterize the role of NMDA receptors in nervous system function and disease.

  20. Molecular Insights into the Transmembrane Domain of the Thyrotropin Receptor.

    PubMed

    Chantreau, Vanessa; Taddese, Bruck; Munier, Mathilde; Gourdin, Louis; Henrion, Daniel; Rodien, Patrice; Chabbert, Marie

    2015-01-01

    The thyrotropin receptor (TSHR) is a G protein-coupled receptor (GPCR) that is member of the leucine-rich repeat subfamily (LGR). In the absence of crystal structure, the success of rational design of ligands targeting the receptor internal cavity depends on the quality of the TSHR models built. In this subfamily, transmembrane helices (TM) 2 and 5 are characterized by the absence of proline compared to most receptors, raising the question of the structural conformation of these helices. To gain insight into the structural properties of these helices, we carried out bioinformatics and experimental studies. Evolutionary analysis of the LGR family revealed a deletion in TM5 but provided no information on TM2. Wild type residues at positions 2.58, 2.59 or 2.60 in TM2 and/or at position 5.50 in TM5 were substituted to proline. Depending on the position of the proline substitution, different effects were observed on membrane expression, glycosylation, constitutive cAMP activity and responses to thyrotropin. Only proline substitution at position 2.59 maintained complex glycosylation and high membrane expression, supporting occurrence of a bulged TM2. The TSHR transmembrane domain was modeled by homology with the orexin 2 receptor, using a protocol that forced the deletion of one residue in the TM5 bulge of the template. The stability of the model was assessed by molecular dynamics simulations. TM5 straightened during the equilibration phase and was stable for the remainder of the simulations. Our data support a structural model of the TSHR transmembrane domain with a bulged TM2 and a straight TM5 that is specific of glycoprotein hormone receptors. PMID:26545118

  1. Modification of Glutamate Receptor Channels: Molecular Mechanisms and Functional Consequences

    NASA Astrophysics Data System (ADS)

    Hatt, Hanns

    Of the many possible mechanisms for modulating the efficiency of ion channels, the phosphorylation of receptor channel proteins may be the primary one. Changes in the set of molecular subunits of which the channels are composed are also important, especially for long-term regulation. In the central nervous system synaptic plasticity may be altered by modulating the ligand-activated neuronal ion channels involved in synaptic transmission; among them are channels gated directly by glutamate, the regulation of which we are only beginning to understand. This paper focuses on modulation of these channels [α-amino-3-hydroxy-5-methyl-4-isoxazoleprionic acid (AMPA), kainate, and N-methyl-d-aspartate (NMDA) types] by phosphorylation and changes in subunit composition. AMPA- and kainate-activated receptors are modulated by adenosine 3, 5-monophosphate (cAMP) dependent protein kinase A (PKA) coupled via D1 dopamine receptors. An increase in the intracellular concentration of cAMP and protein kinase A potentiates kainate-activated currents in α-motoneurons of the spinal cord by increasing the affinity of the ligand (glutamate) for the phosphorylated receptor protein (GluR6 and 7). The rapid desensitization of AMPA-evoked currents normally observed in horizontal cells of the retina is completely blocked by increasing the intracellular concentration of cAMP. The effects of changes in subunit composition were examined in rat hippocampal neurons. The subunit composition of the NMDA receptor determines the kinetic properties of synaptic currents and can be regulated by the type of innervating neuron. Similar changes also occur during development. An important determinant here is the activity of the system. Dynamic regulation of excitatory receptors by both mechanisms may well be associated with some forms of learning and memory in the mammalian brain.

  2. 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

  3. Molecular Vibration-Activity Relationship in the Agonism of Adenosine Receptors

    PubMed Central

    Chee, Hyun Keun

    2013-01-01

    The molecular vibration-activity relationship in the receptor-ligand interaction of adenosine receptors was investigated by structure similarity, molecular vibration, and hierarchical clustering in a dataset of 46 ligands of adenosine receptors. The resulting dendrogram was compared with those of another kind of fingerprint or descriptor. The dendrogram result produced by corralled intensity of molecular vibrational frequency outperformed four other analyses in the current study of adenosine receptor agonism and antagonism. The tree that was produced by clustering analysis of molecular vibration patterns showed its potential for the functional classification of adenosine receptor ligands. PMID:24465242

  4. Molecular Characterization of Vitellogenin and Vitellogenin Receptor of Bemisia tabaci.

    PubMed

    Upadhyay, Santosh Kumar; Singh, Harpal; Dixit, Sameer; Mendu, Venugopal; Verma, Praveen C

    2016-01-01

    Vitellogenin (Vg) plays vital role in oocytes and embryo development in insects. Vg is synthesized in the fat body, moves through haemolymph and accumulates in oocytes. Vitellogenin receptors (VgR) present on the surface of oocytes, are responsible for Vg transportation from haemolymph to oocytes. Here, we cloned and characterized these genes from Bemisia tabaci Asia1 (BtA1) species. The cloned BtA1Vg and BtA1VgR genes consisted of 6,330 and 5,430 bp long open reading frames, which encoded 2,109 and 1,809 amino acid (AA) residues long protein. The BtA1Vg protein comprised LPD_N, DUF1943 and VWFD domains, typical R/KXXR/K, DGXR and GL/ICG motifs, and polyserine tracts. BtA1VgR protein contained 12 LDLa, 10 LDLb and 7 EGF domains, and a trans-membrane and cytoplasmic region at C-terminus. Phylogenetic analyses indicated evolutionary association of BtA1Vg and BtA1VgR with the homologous proteins from various insect species. Silencing of BtA1VgR by siRNA did not affect the transcript level of BtA1Vg. However, BtA1Vg protein accumulation in oocytes was directly influenced with the expression level of BtA1VgR. Further, BtA1VgR silencing caused significant mortality and reduced fecundity in adult whiteflies. The results established the role of BtA1VgR in transportation of BtA1Vg in oocytes. Further, these proteins are essential for fecundity, and therefore these can be potential RNAi targets for insect control in crop plants. PMID:27159161

  5. Molecular Characterization of Vitellogenin and Vitellogenin Receptor of Bemisia tabaci

    PubMed Central

    Upadhyay, Santosh Kumar; Singh, Harpal; Dixit, Sameer; Mendu, Venugopal; Verma, Praveen C.

    2016-01-01

    Vitellogenin (Vg) plays vital role in oocytes and embryo development in insects. Vg is synthesized in the fat body, moves through haemolymph and accumulates in oocytes. Vitellogenin receptors (VgR) present on the surface of oocytes, are responsible for Vg transportation from haemolymph to oocytes. Here, we cloned and characterized these genes from Bemisia tabaci Asia1 (BtA1) species. The cloned BtA1Vg and BtA1VgR genes consisted of 6,330 and 5,430 bp long open reading frames, which encoded 2,109 and 1,809 amino acid (AA) residues long protein. The BtA1Vg protein comprised LPD_N, DUF1943 and VWFD domains, typical R/KXXR/K, DGXR and GL/ICG motifs, and polyserine tracts. BtA1VgR protein contained 12 LDLa, 10 LDLb and 7 EGF domains, and a trans-membrane and cytoplasmic region at C-terminus. Phylogenetic analyses indicated evolutionary association of BtA1Vg and BtA1VgR with the homologous proteins from various insect species. Silencing of BtA1VgR by siRNA did not affect the transcript level of BtA1Vg. However, BtA1Vg protein accumulation in oocytes was directly influenced with the expression level of BtA1VgR. Further, BtA1VgR silencing caused significant mortality and reduced fecundity in adult whiteflies. The results established the role of BtA1VgR in transportation of BtA1Vg in oocytes. Further, these proteins are essential for fecundity, and therefore these can be potential RNAi targets for insect control in crop plants. PMID:27159161

  6. Cloning Expeditions: Risky but Rewarding

    PubMed Central

    2013-01-01

    In the 1980s, a good part of my laboratory was using the then-new recombinant DNA techniques to clone and characterize many important cell surface membrane proteins: GLUT1 (the red cell glucose transporter) and then GLUT2 and GLUT4, the red cell anion exchange protein (Band 3), asialoglycoprotein receptor subunits, sucrase-isomaltase, the erythropoietin receptor, and two of the subunits of the transforming growth factor β (TGF-β) receptor. These cloned genes opened many new fields of basic research, including membrane insertion and trafficking of transmembrane proteins, signal transduction by many members of the cytokine and TGF-β families of receptors, and the cellular physiology of glucose and anion transport. They also led to many insights into the molecular biology of several cancers, hematopoietic disorders, and diabetes. This work was done by an exceptional group of postdocs and students who took exceptionally large risks in developing and using novel cloning technologies. Unsurprisingly, all have gone on to become leaders in the fields of molecular cell biology and molecular medicine. PMID:24061478

  7. Cloning expeditions: risky but rewarding.

    PubMed

    Lodish, Harvey

    2013-12-01

    In the 1980s, a good part of my laboratory was using the then-new recombinant DNA techniques to clone and characterize many important cell surface membrane proteins: GLUT1 (the red cell glucose transporter) and then GLUT2 and GLUT4, the red cell anion exchange protein (Band 3), asialoglycoprotein receptor subunits, sucrase-isomaltase, the erythropoietin receptor, and two of the subunits of the transforming growth factor β (TGF-β) receptor. These cloned genes opened many new fields of basic research, including membrane insertion and trafficking of transmembrane proteins, signal transduction by many members of the cytokine and TGF-β families of receptors, and the cellular physiology of glucose and anion transport. They also led to many insights into the molecular biology of several cancers, hematopoietic disorders, and diabetes. This work was done by an exceptional group of postdocs and students who took exceptionally large risks in developing and using novel cloning technologies. Unsurprisingly, all have gone on to become leaders in the fields of molecular cell biology and molecular medicine.

  8. Molecular evolution of multiple forms of kisspeptins and GPR54 receptors in vertebrates.

    PubMed

    Lee, Yeo Reum; Tsunekawa, Kenta; Moon, Mi Jin; Um, Haet Nim; Hwang, Jong-Ik; Osugi, Tomohiro; Otaki, Naohito; Sunakawa, Yuya; Kim, Kyungjin; Vaudry, Hubert; Kwon, Hyuk Bang; Seong, Jae Young; Tsutsui, Kazuyoshi

    2009-06-01

    Kisspeptin and its receptor GPR54 play important roles in mammalian reproduction and cancer metastasis. Because the KiSS and GPR54 genes have been identified in a limited number of vertebrate species, mainly in mammals, the evolutionary history of these genes is poorly understood. In the present study, we have cloned multiple forms of kisspeptin and GPR54 cDNAs from a variety of vertebrate species. We found that fish have two forms of kisspeptin genes, KiSS-1 and KiSS-2, whereas Xenopus possesses three forms of kisspeptin genes, KiSS-1a, KiSS-1b, and KiSS-2. The nonmammalian KiSS-1 gene was found to be the ortholog of the mammalian KiSS-1 gene, whereas the KiSS-2 gene is a novel form, encoding a C-terminally amidated dodecapeptide in the Xenopus brain. This study is the first to identify a mature form of KiSS-2 product in the brain of any vertebrate. Likewise, fish possess two receptors, GPR54-1 and GPR54-2, whereas Xenopus carry three receptors, GPR54-1a, GPR54-1b, and GPR54-2. Sequence identity and genome synteny analyses indicate that Xenopus GPR54-1a is a human GPR54 ortholog, whereas Xenopus GPR54-1b is a fish GPR54-1 ortholog. Both kisspeptins and GPR54s were abundantly expressed in the Xenopus brain, notably in the hypothalamus, suggesting that these ligand-receptor pairs have neuroendocrine and neuromodulatory roles. Synthetic KiSS-1 and KiSS-2 peptides activated GPR54s expressed in CV-1 cells with different potencies, indicating differential ligand selectivity. These data shed new light on the molecular evolution of the kisspeptin-GPR54 system in vertebrates. PMID:19164475

  9. Molecular Recognition of Natural Products by Resorc[4]arene Receptors.

    PubMed

    D'Acquarica, Ilaria; Ghirga, Francesca; Quaglio, Deborah; Cerreto, Antonella; Ingallina, Cinzia; Tafi, Andrea; Botta, Bruno

    2016-01-01

    This review is aimed at providing an overview of the up-to-now published literature on resorc[4]arene macrocycles exploited as artificial receptors for the molecular recognition of some classes of natural products. A concise illustration of the main synthetic strategies developed to afford the resorc[4]arene scaffold is followed by a report on the principles of the gas-phase investigation of recognition phenomena by mass spectrometry (MS). Emphasis is placed on gas-phase studies of diastereoisomeric complexes generated inside a Fourier transform-ion cyclotron resonance (FT-ICR) mass spectrometer by resorc[4]arene receptors towards a series of natural products, namely amino acids, amphetamine, ethanolamine neurotransmitters, dipeptides, vinca alkaloids and nucleosides. The literature outcomes discussed here, taken largely from our own revisited work, have been completed by references to other studies, in order to draw a broader picture of this rapidly evolving field of research. PMID:26654589

  10. Molecular cloning and analysis of cDNA encoding a plant tryptophan decarboxylase: comparison with animal dopa decarboxylases.

    PubMed Central

    De Luca, V; Marineau, C; Brisson, N

    1989-01-01

    The sequence of a cDNA clone that includes the complete coding region of tryptophan decarboxylase (EC 4.1.1.28, formerly EC 4.1.1.27) from periwinkle (Catharanthus roseus) is reported. The cDNA clone (1747 base pairs) was isolated by antibody screening of a cDNA expression library produced from poly(A)+ RNA found in developing seedlings of C. roseus. The clone hybridized to a 1.8-kilobase mRNA from developing seedlings and from young leaves of mature plants. The identity of the clone was confirmed when extracts of transformed Escherichia coli expressed a protein containing tryptophan decarboxylase enzyme activity. The tryptophan decarboxylase cDNA clone encodes a protein of 500 amino acids with a calculated molecular mass of 56,142 Da. The amino acid sequence shows a high degree of similarity with the aromatic L-amino acid decarboxylase (dopa decarboxylase) and the alpha-methyldopa-hypersensitive protein of Drosophila melanogaster. The tryptophan decarboxylase sequence also showed significant similarity to feline glutamate decarboxylase and mouse ornithine decarboxylase, suggesting a possible evolutionary link between these amino acid decarboxylases. Images PMID:2704736

  11. 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.

  12. Molecular Cloning, Expression Analysis, and Functional Characterization of the H(+)-Pyrophosphatase from Jatropha curcas.

    PubMed

    Yang, Yumei; Luo, Zhu; Zhang, Mengru; Liu, Chang; Gong, Ming; Zou, Zhurong

    2016-04-01

    H(+)-pyrophosphatase (H(+)-PPase) is a primary pyrophosphate (PPi)-energized proton pump to generate electrochemical H(+) gradient for ATP production and substance translocations across membranes. It plays an important role in stress adaptation that was intensively substantiated by numerous transgenic plants overexpressing H(+)-PPases yet devoid of any correlated studies pointing to the elite energy plant, Jatropha curcas. Herein, we cloned the full length of J. curcas H(+)-PPase (JcVP1) complementary DNA (cDNA) by reverse transcription PCR, based on the assembled sequence of its ESTs highly matched to Hevea brasiliensis H(+)-PPase. This gene encodes a polypeptide of 765 amino acids that was predicted as a K(+)-dependent H(+)-PPase evolutionarily closest to those of other Euphorbiaceae plants. Many cis-regulatory elements relevant to environmental stresses, molecular signals, or tissue-specificity were identified by promoter prediction within the 1.5-kb region upstream of JcVP1 coding sequence. Meanwhile, the responses of JcVP1 expression to several common abiotic stresses (salt, drought, heat, cold) were characterized with a considerable accordance with the inherent stress tolerance of J. curcas. Moreover, we found that the heterologous expression of JcVP1 could significantly improve the salt tolerance in both recombinant Escherichia coli and Saccharomyces cerevisiae, and this effect could be further fortified in yeast by N-terminal addition of a vacuole-targeting signal peptide from the H(+)-PPase of Trypanosoma cruzi. PMID:26643082

  13. 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. PMID:27012894

  14. Unveiling aminopeptidase P from Streptomyces lavendulae: molecular cloning, expression and biochemical characterization.

    PubMed

    Nandan, Arya S; Nampoothiri, Kesavan Madhavan

    2014-02-01

    Presence of proline residues in the second position of the N-terminus in peptides restricts the usage of many aminopeptidases; however, aminopeptidase P (APP) is capable of removing this blockage. Based on the N-terminal amino acid sequences of APP from Streptomyces lavendulae, app gene was cloned in pET28a(+) and over expressed as a His-tagged protein with a molecular weight of ≈60 kDa in Escherichia coli BL21 (DE3). Nucleotide sequencing revealed a 1467 bp open reading frame encoding 488 amino acids (NCBI Accession No: GenBank: KC292272.1). The substrate specificity of the recombinant APP was analyzed by the hydrolysis of the Xaa-Pro bond in Gly-Pro dipeptide and bradykinin. K(m) and V(max) of the enzyme were found to be 0.4697 mmol l⁻¹ and 0.6396 μmol min⁻¹, respectively. APP activity was enhanced in the presence of metal ions such as Co²⁺, Mn²⁺, Mg²⁺ and Cu²⁺ ions and was inhibited by 1,10-phenanthroline, EDTA, PMSF and DTT. The atomic absorption studies revealed the presence of Mn²⁺ in the protein as a co-factor. This substrate specific metalloenzyme was found to be a tetramer and optimally active at pH 8 and 37 °C.

  15. 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.

  16. Molecular cloning and characterization of a chlorophyll degradation regulatory gene (ZjSGR) from Zoysia japonica.

    PubMed

    Teng, K; Chang, Z H; Xiao, G Z; Guo, W E; Xu, L X; Chao, Y H; Han, L B

    2016-01-01

    The stay-green gene (SGR) is a key regulatory factor for chlorophyll degradation and senescence. However, to date, little is known about SGR in Zoysia japonica. In this study, ZjSGR was cloned, using rapid amplification of cDNA ends-polymerase chain reaction (PCR). The target sequence is 831 bp in length, corresponding to 276 amino acids. Protein BLAST results showed that ZjSGR belongs to the stay-green superfamily. A phylogenetic analysis implied that ZjSGR is most closely related to ZmSGR1. The subcellular localization of ZjSGR was investigated, using an Agrobacterium-mediated transient expression assay in Nicotiana benthamiana. Our results demonstrated that ZjSGR protein is localized in the chloroplasts. Quantitative real time PCR was carried out to investigate the expression characteristics of ZjSGR. The expression level of ZjSGR was found to be highest in leaves, and could be strongly induced by natural senescence, darkness, abscisic acid (ABA), and methyl jasmonate treatment. Moreover, an in vivo function analysis indicated that transient overexpression of ZjSGR could accelerate chlorophyll degradation, up-regulate the expression of SAG113, and activate ABA biosynthesis. Taken together, these results provide evidence that ZjSGR could play an important regulatory role in leaf chlorophyll degradation and senescence in plants at the molecular level. PMID:27173268

  17. Molecular cloning and characterization of a tropinone reductase from Dendrobium nobile Lindl.

    PubMed

    Chen, Wei; Cheng, Xiaofei; Zhou, Zhenhua; Liu, Junjun; Wang, Huizhong

    2013-02-01

    A cDNA sequence that encodes a peptide with similarity to known tropinone reductases (TR) was cloned from Dendrobium nobile Lindl. The full coding region of the gene (DnTR1) is 804 bp in length which encodes a putative peptide consisting of 268 amino acids. Phylogenetic analysis showed that DnTR1 was a novel member of the TR family and evolutionarily distant from those well-characterized subgroups of TRs, suggesting that DnTR1 may have distinct characteristics. Structural modeling found that DnTR1 had a similar electrostatic environment at the inner molecular surface of the substrate binding pocket with TRI encoded by Datura stramonium (DsTRI). Catalytic activity assay with recombinant protein demonstrated that DnTR1 was able to reduce tropinone, 3-quinuclidinone hydrochloride, and 4-methylcyclohexanone using NADPH as coenzyme. Gene expression profiling by qRT-PCR revealed that the DnTR1 transcript was expressed in all three vegetative organs (leaves, stems and roots) of D. nobile with the highest expression level in roots. The expression of DnTR1 mRNA was enhanced 9.5 times (P < 0.01) by treatment of methyl jasmonate at 24 h, but not affected by salicylic acid and sodium nitroprusside treatments, indicating that DnTR1 regulation may be involved in a jasmonate-dependent pathway. PMID:23104472

  18. Simple and versatile molecular method of copy-number measurement using cloned competitors.

    PubMed

    Kim, Hyun-Kyoung; Hwang, Hai-Li; Park, Seong-Yeol; Lee, Kwang Man; Park, Won Cheol; Kim, Han-Seong; Um, Tae-Hyun; Hong, Young Jun; Lee, Jin Kyung; Joo, Sun-Young; Seoh, Ju-Young; Song, Yeong-Wook; Kim, Soo-Youl; Kim, Yong-Nyun; Hong, Kyeong-Man

    2013-01-01

    Variations and alterations of copy numbers (CNVs and CNAs) carry disease susceptibility and drug responsiveness implications. Although there are many molecular methods to measure copy numbers, sensitivity, reproducibility, cost, and time issues remain. In the present study, we were able to solve those problems utilizing our modified real competitive PCR method with cloned competitors (mrcPCR). First, the mrcPCR for ERBB2 copy number was established, and the results were comparable to current standard methods but with a shorter assay time and a lower cost. Second, the mrcPCR assays for 24 drug-target genes were established, and the results in a panel of NCI-60 cells were comparable to those from real-time PCR and microarray. Third, the mrcPCR results for FCGR3A and the FCGR3B CNVs were comparable to those by the paralog ratio test (PRT), but without PRT's limitations. These results suggest that mrcPCR is comparable to the currently available standard or the most sensitive methods. In addition, mrcPCR would be invaluable for measurement of CNVs in genes with variants of similar structures, because combination of the other methods is not necessary, along with its other advantages such as short assay time, small sample amount requirement, and applicability to all sequences and genes.

  19. Molecular cloning and daily variations of the Period gene in a reef fish Siganus guttatus.

    PubMed

    Park, Ji-Gweon; Park, Yong-Ju; Sugama, Nozomi; Kim, Se-Jae; Takemura, Akihiro

    2007-04-01

    As the first step in understanding the molecular oscillation of the circa rhythms in the golden rabbitfish Siganus guttatus--a reef fish with a definite lunar-related rhythmicity--we cloned and sequenced a Period gene (rfPer). The rfPer gene contained an open reading frame that encodes a protein consisting of 1,452 amino acids; this protein is highly homologous to PER proteins of vertebrates including zebrafish. Phylogenetic analyses indicated that the rfPER protein is related to the zebrafish PER1 and PER4. The expression of rfPer mRNA in the whole brain, retina, and liver under light/dark (LD) conditions increased at 06:00 h and decreased at 18:00 h, suggesting that its robust circadian rhythm occurs in neural and peripheral tissues. When daily variation in the expression in rfPer mRNA in the whole brain and cultured pineal gland were examined under LD conditions, similar expression patterns of the gene were observed with an increase around dawn. Under constant light condition, the increased expression of rfPer mRNA in the whole brain disappeared around dawn. The present results demonstrate that rfPer is related to zPer4 and possibly zPer1. The present study is the first report on the Period gene from a marine fish.

  20. Molecular cloning, overexpression and characterization of a novel feruloyl esterase from a soil metagenomic library.

    PubMed

    Sang, Shu Li; Li, Gang; Hu, Xiao Peng; Liu, Yu Huan

    2011-01-01

    The gene estF27, encoding a protein with feruloyl esterase activity, was cloned through functional screening from a soil metagenomic library and expressed in Escherichiacoli BL21 (DE3) with high solubility. Sequence analysis showed that estF27 encoded a protein of 291 amino acids with a predicted molecular mass of 31.16 kDa. According to the substrate specificity, EstF27 was classified as a type A feruloyl esterase. EstF27 displayed optimal activity at 40°C and pH 6.8. This enzyme was stable in a broad pH range of 5.0-10.0 over 24 h, and retained more than 50% of its activity after 96 or 120 h incubation in the presence of 3 M KCl or 5 M NaCl. The enzyme activity was slightly enhanced by the addition of Mg(2+) and Fe(3+) at a low concentration, and completely inhibited by Cu(2+). In the enzymatic hydrolysis of destarched wheat bran, EstF27 could release ferulic acid from it in the presence of xylanase from Thermomyces lanuginosus. Given its alkalitolerance, halotolerance and highly soluble expression, EstF27 is a promising candidate for industrial applications.

  1. Molecular cloning and characterization of a new and highly thermostable esterase from Geobacillus sp. JM6.

    PubMed

    Zhu, Yanbing; Zheng, Wenguang; Ni, Hui; Liu, Han; Xiao, Anfeng; Cai, Huinong

    2015-10-01

    A new lipolytic enzyme gene was cloned from a thermophile Geobacillus sp. JM6. The gene contained 750 bp and encoded a 249-amino acid protein. The recombinant enzyme was expressed and purified from Escherichia coli BL21 (DE3) with a molecular mass of 33.6 kDa. Enzyme assays using p-nitrophenyl esters with different acyl chain lengths as the substrates confirmed its esterase activity, yielding the highest activity with p-nitrophenyl butyrate. When p-nitrophenyl butyrate was used as a substrate, the optimum reaction temperature and pH for the enzyme were 60 °C and pH 7.5, respectively. Geobacillus sp. JM6 esterase showed excellent thermostability with 68% residual activity after incubation at 100 °C for 18 h. A theoretical structural model of strain JM6 esterase was developed with a monoacylglycerol lipase from Bacillus sp. H-257 as a template. The predicted core structure exhibits an α/β hydrolase fold, and a putative catalytic triad (Ser97, Asp196, and His226) was identified. Inhibition assays with PMSF indicated that serine residue is involved in the catalytic activity of strain JM6 esterase. The recombinant esterase showed a relatively good tolerance to the detected detergents and denaturants, such as SDS, Chaps, Tween 20, Tween 80, Triton X-100, sodium deoxycholate, urea, and guanidine hydrochloride.

  2. Molecular cloning and purification of an endochitinase from Serratia marcescens (Nima).

    PubMed

    Ruiz-Sanchez, Alejandro; Cruz-Camarillo, Ramon; Salcedo-Hernandez, Ruben; Ibarra, Jorge E; Barboza-Corona, Jose Eleazar

    2005-10-01

    An endochitinase gene from the Serratia marcescens Nima strain (chiA Nima) was cloned, sequenced, and expressed in Escherichia coli DH5alphaF', and the recombinant protein (ChiA Nima) was purified by hydrophobic interaction chromatography. chiA Nima contains an open reading frame (ORF) that encodes an endochitinase with a deduced molecular weight and an isoelectric point of 61 kDa and 6.84, respectively. A sequence at the 5'-end was identified as a signal peptide, recognized by Gram-negative bacteria transport mechanism. Comparison of ChiA Nima with other chitinases revealed a modular structure formed by the catalytic domain and a putative chitin-binding domain. The purified chitinase was able to hydrolyze both trimeric and tetrameric fluorogenic substrates, but not a chitobiose analog substrate. ChiA Nima showed high enzymatic activity within a broad pH range (pH 4.0-10.0), with a peak activity at pH 5.5. The optimal temperature for enzymatic activity was detected at 55 degrees C.

  3. Simple and Versatile Molecular Method of Copy-Number Measurement Using Cloned Competitors

    PubMed Central

    Kim, Hyun-Kyoung; Hwang, Hai-Li; Park, Seong-Yeol; Lee, Kwang Man; Park, Won Cheol; Kim, Han-Seong; Um, Tae-Hyun; Hong, Young Jun; Lee, Jin Kyung; Joo, Sun-Young; Seoh, Ju-Young; Song, Yeong-Wook; Kim, Soo-Youl; Kim, Yong-Nyun; Hong, Kyeong-Man

    2013-01-01

    Variations and alterations of copy numbers (CNVs and CNAs) carry disease susceptibility and drug responsiveness implications. Although there are many molecular methods to measure copy numbers, sensitivity, reproducibility, cost, and time issues remain. In the present study, we were able to solve those problems utilizing our modified real competitive PCR method with cloned competitors (mrcPCR). First, the mrcPCR for ERBB2 copy number was established, and the results were comparable to current standard methods but with a shorter assay time and a lower cost. Second, the mrcPCR assays for 24 drug-target genes were established, and the results in a panel of NCI-60 cells were comparable to those from real-time PCR and microarray. Third, the mrcPCR results for FCGR3A and the FCGR3B CNVs were comparable to those by the paralog ratio test (PRT), but without PRT's limitations. These results suggest that mrcPCR is comparable to the currently available standard or the most sensitive methods. In addition, mrcPCR would be invaluable for measurement of CNVs in genes with variants of similar structures, because combination of the other methods is not necessary, along with its other advantages such as short assay time, small sample amount requirement, and applicability to all sequences and genes. PMID:23936009

  4. Novel glucose dehydrogenase from Mucor prainii: Purification, characterization, molecular cloning and gene expression in Aspergillus sojae.

    PubMed

    Satake, Ryoko; Ichiyanagi, Atsushi; Ichikawa, Keiichi; Hirokawa, Kozo; Araki, Yasuko; Yoshimura, Taro; Gomi, Keiko

    2015-11-01

    Glucose dehydrogenase (GDH) is of interest for its potential applications in the field of glucose sensors. To improve the performance of glucose sensors, GDH is required to have strict substrate specificity. A novel flavin adenine dinucleotide (FAD)-dependent GDH was isolated from Mucor prainii NISL0103 and its enzymatic properties were characterized. This FAD-dependent GDH (MpGDH) exhibited high specificity toward glucose. High specificity for glucose was also observed even in the presence of saccharides such as maltose, galactose and xylose. The molecular masses of the glycoforms of GDH ranged from 90 to 130 kDa. After deglycosylation, a single 80 kDa band was observed. The gene encoding MpGDH was cloned and expressed in Aspergillus sojae. The apparent kcat and Km values of recombinant enzyme for glucose were found to be 749.7 s(-1) and 28.3 mM, respectively. The results indicated that the characteristics of MpGDH were suitable for assaying blood glucose levels.

  5. Molecular cloning and characterization of a galectin-1 homolog in orange-spotted grouper, Epinephelus coioides.

    PubMed

    Chen, Xiuli; Wei, Jingguang; Xu, Meng; Yang, Min; Li, Pingfei; Wei, Shina; Huang, Youhua; Qin, Qiwei

    2016-07-01

    As a member of animal lectin family, galectin has the functions of pathogen recognition, anti-bacteria and anti-virus. In the present study, a galectin-1 homolog (EcGel-1) from grouper (Epinephelus coioides) was cloned and its possible role in fish immunity was analyzed. The full length cDNA of EcGel-1 is 504 bp, including a 408 bp open reading frame (ORF) which encodes 135 amino acids with a molecular mass of 15.19 kDa. Quantitative real-time PCR analysis indicated that EcGel-1 was constitutively expressed in all analyzed tissues of healthy grouper. The expression of EcGel-1 in the spleen of grouper was differentially up-regulated challenged with Singapore grouper iridovirus (SGIV), poly (I:C), and LPS. EcGel-1 was abundantly distributed in the cytoplasm in GS cells. Recombinant EcGel-1(rEcGel-1) protein can make chicken erythrocyte aggregation, and combine with gram negative bacteria and gram positive bacteria in the presence of 2-Mercaptoethanol (β-ME). Taken together, the results showed that EcGel-1 may be an important molecule involved in pathogen recognition and pathogen elimination in the innate immunity of grouper.

  6. Sea bass ghrelin: molecular cloning and mRNA quantification during fasting and refeeding.

    PubMed

    Terova, Genciana; Rimoldi, Simona; Bernardini, Giovanni; Gornati, Rosalba; Saroglia, Marco

    2008-01-15

    Ghrelin is a novel appetite-inducing peptide hormone secreted by the stomach. The purpose of this study was first to identify the cDNA encoding sequence for ghrelin in sea bass (Dicentrarchus labrax). Using molecular cloning techniques we sequenced the cDNA corresponding to sea bass ghrelin mRNA. A total of 798 bases including a 5'-untranslated region (89 bp), an open reading frame (ORF) (324 bp), and a 3'-untranslated region (385 bp) were detected. Nucleotide sequence (ORF) encoded a 108 amino acid prepropeptide that demonstrated complete conservation of the N-terminal "biological active core" (GSSF) of the predicted mature ghrelin peptide. We also analyzed fasting-induced changes in the expression of ghrelin mRNA, using a one-tube two-temperature real-time RT-PCR with which the gene expression can be absolutely quantified using the standard curve method. Our results revealed that ghrelin was highly expressed in the stomach with much lower levels of expression in the proximal intestine and brain. Levels of ghrelin mRNA in the stomach were upregulated under conditions of negative energy balance, such as starvation, and downregulated during positive energy balance, such as refeeding. These findings offer new information about the sea bass ghrelin gene and support a role of this orexigenic hormone in the regulation of food intake in sea bass.

  7. Molecular cloning, genomic organization, and expression of a testicular isoform of hormone-sensitive lipase

    SciTech Connect

    Holst, L.S.; Laurell, H.; Holm, C.

    1996-08-01

    By catalyzing the rate-limiting step in adipose tissue lipolysis, hormone-sensitive lipase (HSL) is an important regulator of energy homeostasis. The role and importance of HSL in tissues other than adipose are poorly understood. We report here the cloning and expression of a testicular isoform, designated HSL{sub tes}. Due to an addition of amino acids at the NH{sub 2}-termini, rat and human HSL{sub tes} consist of 1068 and 1076 amino acids, respectively, compared to the 768 and 775 amino acids, respectively, of the adipocyte isoform (HSL{sub adi}). A novel exon of 1.2 kb, encoding the human testis-specific amino acids, was isolated and mapped to the HSL gene, 16 kb upstream of the exons encoding HSL{sub adi}. The transcribed mRNA of 3.9 kb was specifically expressed in testis. No significant similarity with other known proteins was found for the testis-specific sequence. The amino acid composition differs from the HSL{sub adi} sequence, with a notable hydrophilic character and a high content of prolines and glutamines. COS cells, transfected by the 3.9-kb human testis cDNA, expressed a protein of the expected molecular mass (M{sub r} {approximately}120,000) that exhibited catalytic activity similar to that of HSL{sub adi}. Immunocytochemistry localized HSL to elongating spermatids and spermatozoa; HSL was not detected in interstitial cells. 34 refs., 5 figs.

  8. 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. PMID:25641188

  9. Molecular cloning and characterization of a Bombyx mori gene encoding the transcription factor Atonal.

    PubMed

    Hu, Ping; Feng, Fan; Xia, Hengchuan; Chen, Liang; Yao, Qin; Chen, Keping

    2014-01-01

    The atonal genes are an evolutionarily conserved group of genes encoding regulatory basic helix-loop-helix (bHLH) transcription factors. These transcription factors have a critical antioncogenic function in the retina, and are necessary for cell fate determination through the regulation of the cell signal pathway. In this study, the atonal gene was cloned from Bombyx mori, and the transcription factor was named BmAtonal. Sequence analysis showed that the BmAtonal protein shares extensive homology with other invertebrate Atonal proteins with the bHLH motif. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analyses revealed that BmAtonal was expressed in all developmental stages of B. mori and various larval tissues. The BmAtonal protein was expressed in Escherichia coli, and polyclonal antibodies were raised against the purified protein. By immunofluorescence, the BmAtonal protein was localized to both the nucleus and cytoplasm of BmN cells. After knocking out nuclear localization signals (NLS), the BmAtonal protein was only detected in the cytoplasm. In addition, using the B. mori nuclear polyhedrosis virus (BmNPV) baculovirus expression system, the recombinant BmAtonal protein was successfully expressed in the B. mori cell line BmN. This work lays the foundation for exploring the biological functions of the BmAtonal protein, such as identifying its potential binding partners and understanding the molecular control of the formation of sensory organs. PMID:24873037

  10. Molecular cloning and characterization of a soluble inorganic pyrophosphatase in potato.

    PubMed Central

    du Jardin, P; Rojas-Beltran, J; Gebhardt, C; Brasseur, R

    1995-01-01

    A cDNA clone encoding a soluble inorganic pyrophosphatase (EC 3.6.1.1) of potato (Solanum tuberosum L.) was isolated by screening a developing tuber library with a heterologous probe. The central domain of the encoded polypeptide is nearly identical at the sequence level with its Arabidopsis homolog (J.J. Kieber and E.R. Signer [1991] Plant Mol Biol 16: 345-348). Computer-assisted analysis of the potato, Arabidopsis, and Escherichia coli soluble pyrophosphatases indicated a remarkably conserved organization of the hydrophobic protein domains. The enzymatic function of the potato protein could be deduced from the presence of amino acid residues highly conserved in soluble pyrophosphatases and was confirmed by its capacity to complement a thermosensitive pyrophosphatase mutation in E. coli. The potato polypeptide was purified from complemented bacterial cells and its pyrophosphatase activity was shown to be strictly dependent on Mg2+ and strongly inhibited by Ca2+. The subcellular location of the potato pyrophosphatase is unknown. Structure analysis of the N-terminal protein domain failed to recognize typical transit peptides and the calculated molecular mass of the polypeptide (24 kD) is significantly inferior to the values reported for the plastidic (alkaline) or mitochondrial pyrophosphatases in plants (28-42 kD). Two unlinked loci could be mapped by restriction fragment length polymorphism analysis in the potato genome using the full-length cDNA as probe. PMID:8552717

  11. Molecular Cloning, Expression Analysis, and Functional Characterization of the H(+)-Pyrophosphatase from Jatropha curcas.

    PubMed

    Yang, Yumei; Luo, Zhu; Zhang, Mengru; Liu, Chang; Gong, Ming; Zou, Zhurong

    2016-04-01

    H(+)-pyrophosphatase (H(+)-PPase) is a primary pyrophosphate (PPi)-energized proton pump to generate electrochemical H(+) gradient for ATP production and substance translocations across membranes. It plays an important role in stress adaptation that was intensively substantiated by numerous transgenic plants overexpressing H(+)-PPases yet devoid of any correlated studies pointing to the elite energy plant, Jatropha curcas. Herein, we cloned the full length of J. curcas H(+)-PPase (JcVP1) complementary DNA (cDNA) by reverse transcription PCR, based on the assembled sequence of its ESTs highly matched to Hevea brasiliensis H(+)-PPase. This gene encodes a polypeptide of 765 amino acids that was predicted as a K(+)-dependent H(+)-PPase evolutionarily closest to those of other Euphorbiaceae plants. Many cis-regulatory elements relevant to environmental stresses, molecular signals, or tissue-specificity were identified by promoter prediction within the 1.5-kb region upstream of JcVP1 coding sequence. Meanwhile, the responses of JcVP1 expression to several common abiotic stresses (salt, drought, heat, cold) were characterized with a considerable accordance with the inherent stress tolerance of J. curcas. Moreover, we found that the heterologous expression of JcVP1 could significantly improve the salt tolerance in both recombinant Escherichia coli and Saccharomyces cerevisiae, and this effect could be further fortified in yeast by N-terminal addition of a vacuole-targeting signal peptide from the H(+)-PPase of Trypanosoma cruzi.

  12. Molecular cloning and characterization of amylase from soil metagenomic library derived from Northwestern Himalayas.

    PubMed

    Sharma, Sarika; Khan, Farrah Gul; Qazi, Ghulam Nabi

    2010-05-01

    The increasing demand for novel biocatalysts stimulates exploration of resources from soil. Metagenomics, a culture independent approach, represent a sheer unlimited resource for discovery of novel biocatalysts from uncultured microorganisms. In this study, a soil-derived metagenomic library containing 90,700 recombinants was constructed and screened for lipase, cellulase, protease and amylase activity. A gene (pAMY) of 909 bp encoding for amylase was found after the screening of 35,000 Escherichia coli clones. Amino acid sequence comparison and phylogenetic analysis indicated that pAMY was closely related to uncultured bacteria. The molecular mass of pAMY was estimated about 38 kDa by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Amylase activity was determined using soluble starch, amylose, glycogen and maltose as substrates. The maximal activity (2.46 U/mg) was observed at 40 degrees C under nearly neutral pH conditions with amylose; whereas it retains 90% of its activity at low temperature with all the substrates used in this study. The ability of pAMY to work at low temperature is unique for amylases reported so far from microbes of cultured and uncultured division.

  13. kappa-Opioid receptor in humans: cDNA and genomic cloning, chromosomal assignment, functional expression, pharmacology, and expression pattern in the central nervous system.

    PubMed Central

    Simonin, F; Gavériaux-Ruff, C; Befort, K; Matthes, H; Lannes, B; Micheletti, G; Mattéi, M G; Charron, G; Bloch, B; Kieffer, B

    1995-01-01

    Using the mouse delta-opioid receptor cDNA as a probe, we have isolated genomic clones encoding the human mu- and kappa-opioid receptor genes. Their organization appears similar to that of the human delta receptor gene, with exon-intron boundaries located after putative transmembrane domains 1 and 4. The kappa gene was mapped at position q11-12 in human chromosome 8. A full-length cDNA encoding the human kappa-opioid receptor has been isolated. The cloned receptor expressed in COS cells presents a typical kappa 1 pharmacological profile and is negatively coupled to adenylate cyclase. The expression of kappa-opioid receptor mRNA in human brain, as estimated by reverse transcription-polymerase chain reaction, is consistent with the involvement of kappa-opioid receptors in pain perception, neuroendocrine physiology, affective behavior, and cognition. In situ hybridization studies performed on human fetal spinal cord demonstrate the presence of the transcript specifically in lamina II of the dorsal horn. Some divergences in structural, pharmacological, and anatomical properties are noted between the cloned human and rodent receptors. Images Fig. 3 Fig. 4 PMID:7624359

  14. Molecular cloning and expression analysis of cytochrome c oxidase subunit II from Sitophilus zeamais.

    PubMed

    Hou, Chang-Liang; Wang, Jing-Bo; Wu, Hua; Liu, Jia-Yu; Ma, Zhi-Qing; Feng, Jun-Tao; Zhang, Xing

    2016-09-30

    Cytochrome c oxidase subunit II (COX II) containing a dual core CuA active site is one of the core subunits of mitochondrial Cytochrome c oxidase (Cco), which plays a significant role in the physiological process. In this report, the full-length cDNA of COXII gene was cloned from Sitophilus zeamais, which had an open reading frame (ORF) of 684 bp encoding 227 amino acids residues. The predicted COXII protein had a molecular mass of 26.2 kDa with pI value of 6.37. multiple sequence alignment and phylogenetic analysis indicated that Sitophilus zeamais COXII had high sequence identity with the COXII of other insect species. The gene was subcloned into the expression vector pET-32a, and induced by isopropyl β-d-thiogalactopyranoside (IPTG) in E. coli Transetta (DE3) expression system. Finally the recombinant COXII with 6-His tag was purified using affinity chromatography with Ni(2+)-NTA agarose. Western Blotting (WB) showed the recombinant protein was about 44 kD, and the concentration of fusion protein was 50 μg/mL. UV-spectrophotometer and infrared spectrometer analysis showed that recombinant COXII could catalyze the oxidation of substrate Cytochrome C (Cyt c), and influenced by allyl isothiocyanate (AITC). By using molecular docking method, It was found that a sulfur atom of AITC structure could form a length of 2.9 Å hydrogen bond with Leu-31. These results suggested that tag-free COXII was functional and one of the action sites of AITC, which will be helpful to carry out a point mutation in binding sites for the future research. PMID:27614312

  15. Molecular cloning, purification, and serological characterization of MPT63, a novel antigen secreted by Mycobacterium tuberculosis.

    PubMed Central

    Manca, C; Lyashchenko, K; Wiker, H G; Usai, D; Colangeli, R; Gennaro, M L

    1997-01-01

    Proteins that are actively secreted by Mycobacterium tuberculosis generate immune responses in the infected host. This has prompted the characterization of protein components of mycobacterial culture filtrates to develop subunit vaccines and immunodiagnostic reagents. Fractionation of filtrates of M. tuberculosis cultures has yielded an abundant protein called MPT63, which has an apparent molecular mass of 18 kDa. We report the molecular cloning and nucleotide sequence of the mpt63 gene, purification of recombinant MPT63 antigen from Escherichia coli cells, and serological characterization of MPT63. Nucleotide sequence analysis of mpt63 identified an open reading frame encoding a protein of 159 amino acids (aa) consisting of a 29-aa secretion signal peptide and a 130-aa mature MPT63 protein. Recombinant MPT63 protein, purified from E. coli cells, and native MPT63, purified from M. tuberculosis culture filtrates, were indistinguishable in serological assays. Thus, the recombinant protein constitutes a valuable reagent for immunological studies. MPT63 evoked humoral immune responses in guinea pigs infected with virulent M. tuberculosis by the aerosol route. The mpt63 gene is found only in species of the M. tuberculosis complex, as shown by DNA hybridization experiments. Moreover, polyclonal antibody against MPT63 does not cross-react with proteins of a common environmental mycobacterial species, Mycobacterium avium. The absence of cross-reactive epitopes makes MPT63 an attractive candidate as an M. tuberculosis complex-specific diagnostic reagent. In particular, evaluation of MPT63 as an M. tuberculosis complex-specific reagent for diagnostic skin testing is under way. PMID:8975887

  16. Human phenol sulfotransferase STP2 gene: Molecular cloning, structural characterization, and chromosomal localization

    SciTech Connect

    Her, C.; Raftogianis, R.; Weinshilboum, R.M.

    1996-05-01

    Sulfonation is an important pathway in the biotransformation of many drugs, xenobiotics, neurotransmitters, and steroid hormones. The thermostable (TS) form of phenol sulfotransferase (PST) preferentially catalyzes the sulfonation of {open_quotes}simple{close_quotes} planar phenols, and levels of activity of TS PST in human tissues are controlled by inheritance. Two different human liver TS PST cDNAs have been cloned that encode proteins with amino acid sequences that are 96% identical. We have determined the structure and chromosomal localization of the gene for one of these two cDNAs, STP2, as a step toward understanding molecular genetic mechanisms involved in the regulation of this enzyme activity in humans. STP2 spans approximately 5.1 kb and contains nine exons that range in length from 74 to 347 bp. The locations of most STP2 exon-intron splice junctions are identical to those of a gene for the thermolabile form of PST in humans, STM; a rat PST gene; a human estrogen ST (EST) gene, STE; and a guinea pig EST gene. The two initial STP2 exons, IA and IB, were identified by performing 5{prime}-rapid amplification of cDNA ends with human liver cDNA as template. Exons IA and IB are noncoding and represent two different human liver TS PST cDNA 5{prime}untranslated region sequences. The two apparent 5{prime}-ons IA and IB, contain no canonical TATA boxes, but do contain CCAAT elements. STP2 was localized to human chromosome 16 by performing the PCR with DNA from NIGMS human/rodent somatic cell hybrids as template. Structural characterization of STP2 will make it possible to begin to study molecular genetic mechanisms involved in the regulation of TS PST activity in human tissues. 63 refs., 7 figs., 1 tab.

  17. Molecular Cloning and Expression of Pro J 1: A New Allergen of Prosopis Juliflora Pollen.

    PubMed

    Dousti, Fatemeh; Assarehzadegan, Mohammad-Ali; Morakabati, Payam; Khosravi, Gholam Reza; Akbari, Bahareh

    2016-04-01

    Pollen from mesquite (Prosopis juliflora) is one of the important causes of immediate hypersensitivity reactions in the arid and semi-arid regions of the world. The aim of present study is to produce and purify the recombinant form of allergenic Ole e 1-like protein from the pollen of this allergenic tree. Immunological and cross-inhibition assays were performed for the evaluation of IgE-binding capacity of purified recombinant protein. For molecular cloning, the coding sequence of the mesquite Ole e 1-like protein was inserted into pTZ57R/T vector and expressed in Escherichia coli using the vector pET-21b(+). After purification of the recombinant protein, its immunoreactivity was analysed by in vitro assays using sera from twenty one patients with an allergy to mesquite pollen. The purified recombinant allergen was a member of Ole e 1-like protein family and consisted of 150 amino acid residues, with a predicted molecular mass of 16.5 kDa and a calculated isoelectric point (pI) of 4.75. Twelve patients (57.14%) had significant specific IgE levels for this recombinant allergen. Immunodetection and inhibition assays indicated that the purified recombinant allergen might be the same as that in the crude extract. Herein, we introduce an important new allergen from P. juliflora pollen (Pro j 1), which is a member of the Ole e 1-like protein family and exhibits significant identity and similarity to other allergenic members of this family.

  18. Molecular cloning, characterization, and expression of Cuc m 2, a major allergen in Cucumis melo

    PubMed Central

    Sankian, Mojtaba; Mahmoudi, Mahmoud; Varasteh, Abdol-Reza

    2013-01-01

    Background: Several studies reported the clinical features of IgE-mediated hypersensitivity after ingestion of melon. Melon allergy is a common IgE-mediated fruit allergy in Iran. This prompted us to investigate immunochemical and molecular properties of the major allergen in melon fruit, to compare the IgE-binding capacity of the natural protein with the recombinant allergen, and to determine cross-reactivity of the major allergen with closely-related allergens from other plants displaying clinical cross-reactivity with melon. Methods: Identification and molecular characterization of the major melon allergen were performed using IgE immunoblotting, allergen-specific ELISA, affinity-based purifications, cross-inhibition assays, cloning, and expression of the allergen in Escherichia coli. Results: Melon profilin was identified and isolated as a major IgE-binding component and designated as Cuc m 2. Sequencing corresponding cDNA revealed an open reading frame of 363 bp coding for 131 amino acid residues and two fragments of 171 bp and 383 bps for the 5’and 3’ UTRs, respectively. Significant cross-reactivity was found between melon profilin and Cynodon dactylon, tomato, peach, and grape profilins in cross-inhibition assays. Although the highest degree of amino acid identity was revealed with watermelon profilin, there was no significant cross-reactivity between melon and watermelon profilins. Conclusion: Melon profilin is the major IgE-binding component in melon extract, and the recombinant and natural forms exhibited similar IgE-binding capacities. A part of the fruit-fruit and pollen-fruit cross-reactions could be explained by the presence of this conserved protein; however, sequence homology provides insufficient information to predict IgE cross-reactivity of profilins. PMID:26989709

  19. Cloning the promoter for transforming growth factor-beta type III receptor. Basal and conditional expression in fetal rat osteoblasts

    NASA Technical Reports Server (NTRS)

    Ji, C.; Chen, Y.; McCarthy, T. L.; Centrella, M.

    1999-01-01

    Transforming growth factor-beta binds to three high affinity cell surface molecules that directly or indirectly regulate its biological effects. The type III receptor (TRIII) is a proteoglycan that lacks significant intracellular signaling or enzymatic motifs but may facilitate transforming growth factor-beta binding to other receptors, stabilize multimeric receptor complexes, or segregate growth factor from activating receptors. Because various agents or events that regulate osteoblast function rapidly modulate TRIII expression, we cloned the 5' region of the rat TRIII gene to assess possible control elements. DNA fragments from this region directed high reporter gene expression in osteoblasts. Sequencing showed no consensus TATA or CCAAT boxes, whereas several nuclear factors binding sequences within the 3' region of the promoter co-mapped with multiple transcription initiation sites, DNase I footprints, gel mobility shift analysis, or loss of activity by deletion or mutation. An upstream enhancer was evident 5' proximal to nucleotide -979, and a silencer region occurred between nucleotides -2014 and -2194. Glucocorticoid sensitivity mapped between nucleotides -687 and -253, whereas bone morphogenetic protein 2 sensitivity co-mapped within the silencer region. Thus, the TRIII promoter contains cooperative basal elements and dispersed growth factor- and hormone-sensitive regulatory regions that can control TRIII expression by osteoblasts.

  20. Cloning of two putative ecdysteroid receptor isoforms from Tenebrio molitor and their developmental expression in the epidermis during metamorphosis.

    PubMed

    Mouillet, J F; Delbecque, J P; Quennedey, B; Delachambre, J

    1997-09-15

    Using the Drosophila EcR-B1 cDNA as a probe, we have cloned the putative ecdysteroid receptor from the mealworm Tenebrio molitor. We have isolated two cDNAs with different 5' termini that contain a complete open reading frame. These two cDNAs encode two proteins with distinct N-terminal regions corresponding to two isoforms. The coleopteran receptor is obviously related to the ecdysteroid receptor of other insects, but shares only 89% and 61% amino acid identities with the DNA-binding and ligand-binding domains of the Drosophila receptor, respectively. Its expression pattern has been examined in the epidermis during the last larval instar and pupal stage of T. molitor, in correlation with the hemolymph ecdysteroid titer. Hybridizations revealed two transcripts of 7 kb and 6.5 kb detected in most stages during metamorphosis and corresponding to the A and B1 isoforms. These two mRNAs are highly evident just before the rise of each ecdysteroid peak both in prepupae and in pupae. They show almost the same expression pattern in epidermis except for the second part of the pupal stage, during which only the A isoform is detected.

  1. Molecular and biochemical analysis of symbiotic plant receptor kinase complexes

    SciTech Connect

    Cook, Douglas R; Riely, Brendan K

    2010-09-01

    DE-FG02-01ER15200 was a 36-month project, initiated on Sept 1, 2005 and extended with a one-year no cost extension to August 31, 2009. During the project period we published seven manuscripts (2 in review). Including the prior project period (2002-2005) we published 12 manuscripts in journals that include Science, PNAS, The Plant Cell, Plant Journal, Plant Physiology, and MPMI. The primary focus of this work was to further elucidate the function of the Nod factor signaling pathway that is involved in initiation of the legume-rhizobium symbiosis and in particular to explore the relationship between receptor kinase-like proteins and downstream effectors of symbiotic development. During the project period we have map-base cloned two additional players in symbiotic development, including an ERF transcription factor and an ethylene pathway gene (EIN2) that negatively regulates symbiotic signaling; we have also further characterized the subcellular distribution and function of a nuclear-localized symbiosis-specific ion channel, DMI1. The major outcome of the work has been the development of systems for exploring and validating protein-protein interactions that connect symbiotic receptor-like proteins to downstream responses. In this regard, we have developed both homologous (i.e., in planta) and heterologous (i.e., in yeast) systems to test protein interactions. Using yeast 2-hybrid screens we isolated the only known interactor of the nuclear-localized calcium-responsive kinase DMI3. We have also used yeast 2-hybrid methodology to identify interactions between symbiotic signaling proteins and certain RopGTPase/RopGEF proteins that regulate root hair polar growth. More important to the long-term goals of our work, we have established a TAP tagging system that identifies in planta interactions based on co-immuno precipitation and mass spectrometry. The validity of this approach has been shown using known interactors that either co-iummnoprecipate (i.e., remorin) or co

  2. Molecular characterization of prostaglandin F receptor (FP) and E receptor subtype 1 (EP₁) in zebrafish.

    PubMed

    Kwok, Amy H Y; Wang, Yajun; Leung, Frederick C

    2012-09-01

    Prostaglandins E (PGE) and F (PGF) mediate diverse physiological functions via their cell surface receptors - prostaglandin E receptor (EP) subtypes 1, 2, 3 and 4 (EP(1); EP(2); EP(3); EP(4)) and F receptor (FP). In teleost fishes, PGE was implicated in gill epithelium ion transport, while both PGE and PGF were involved in oocyte maturation, follicular rupture and coordination of reproductive behaviors. However, little is known about the mechanisms behind their actions. In present study, we first identified the full-length ORF cDNA clones of three zebrafish prostaglandin E receptor subtype 1 (zEP(1)) isoforms - zEP(1a), zEP(1b) and zEP(1c) - and FP (zFP) from adult ovary. RT-PCR showed that zEP(1a), zEP(1b) and zFP are widely expressed in adult tissues, while zEP(1c) mRNA expression is mainly confined in brain and kidney. Using a pGL3-NFAT-RE luciferase reporter system, both zEP(1a) and zEP(1b) expressed in DF-1 cells were shown to be activated by PGE(2) potently while zEP(1c) and zFP were activated by PGF(2a) effectively, suggesting that the four receptors are functionally coupled to intracellular Ca(2+)-signaling pathway. Furthermore, EP1a and EP1b, but not EP1c were suggested to couple to cAMP-PKA signaling pathway using a pGL3-CRE luciferase reporter assay. Although zEP(1c) might originate as a paralog to zEP(1a) and zEP(1b), its functional coupling to PGF(2α) instead of PGE(2) suggested that zEP(1) isoforms might have sub-functionalized in their ligand binding and G protein coupling specificity, in addition to differential tissue distribution. Characterization of these receptors undoubtedly furthered our understanding on the diverse yet highly target-specific responses of prostaglandins in teleosts. PMID:22617193

  3. Cloning of a buffalo (Bubalus bubalis) prostaglandin F(2alpha) receptor: changes in its expression and concentration in the buffalo cow corpus luteum.

    PubMed

    Verma-Kumar, Shalu; Srinivas, S V; Muraly, P; Yadav, Vijay K; Medhamurthy, R

    2004-06-01

    Acting primarily through its specific G protein-coupled receptor termed FPr, prostaglandin (PG) F(2alpha) induces regression of the corpus luteum (CL) at the end of a non-fertile oestrous cycle. This study was aimed at cloning a full-length cDNA for FPr and determining its expression and protein concentrations during different stages of CL development in the water buffalo. Serum progesterone and StAR expression were determined to establish temporal relationships between indices of steroidogenesis and changes in FPr expression at different stages of CL development. In contrast to the dairy cow, the stage IV CL (day 20 of the oestrous cycle) did not appear to be functionally regressed in the buffalo. Molecular cloning of a cDNA encoding the buffalo FPr yielded a full length 2193 bp FPr cDNA containing a single open reading frame encoding a 362 amino acid protein with seven putative membrane-spanning domains. The deduced buffalo FPr amino acid sequence possesses a high degree of identity with the other mammalian homologues. Steady state concentration of buffalo FPr transcript increased (P > 0.05) from stage I to stage II/III, and declined at 18 h post PGF(2alpha) injection. The FPr concentration expressed as fmol/microg of plasma membrane protein showed an increase (P > 0.05) from stage I (1.98 +/- 0.10), through stage II/III (2.42 +/- 0.48) to stage IV (2.77 +/- 0.18). High affinity FPr was observed in stage I (K(d) 4.86 nmol) and stage II/III (K(d) 6.28 nmol) while low affinity FPr (K(d) 19.44 nmol) was observed in stage IV. In conclusion, we have cloned a full length FPr cDNA from buffalo cow CL and observed that FPr mRNA expression, receptor number and affinity did not vary significantly (P > 0.05) within the luteal phase of the oestrous cycle.

  4. Rat pristanoyl-CoA oxidase. cDNA cloning and recognition of its C-terminal (SQL) by the peroxisomal-targeting signal 1 receptor.

    PubMed

    Vanhooren, J C; Fransen, M; de Béthune, B; Baumgart, E; Baes, M; Torrekens, S; Van Leuven, F; Mannaerts, G P; Van Veldhoven, P P

    1996-07-15

    The composite pristanoyl-CoA oxidase cDNA sequence, derived from two overlapping clones from a rat liver cDNA library and a 5'-RACE (rapid amplification of cDNA ends) PCR fragment, consisted of 2600 bases and contained an open reading frame of 2100 bases, encoding a protein of 700 amino acids with a calculated molecular mass of 78445 Da. This value is somewhat larger than the reported molecular mass of 70 kDa as determined earlier by SDS-gel electrophoresis. The amino acid identity with rat palmitoyl-CoA oxidase was rather low (28%) and barely higher than that with the yeast acyl-CoA oxidases (20%), suggesting that the palmitoyl-CoA oxidase/pristanoyl-CoA oxidase duplication occurred early in evolution. The carboxy-terminal tripeptide of pristanoyl-CoA oxidase was SQL. In vitro studies with the bacterially expressed human peroxisomal-targeting signal-1 import receptor indicated that SQL functions as a peroxisome-targeting signal. Northern analysis of tissues from control and clofibrate treated rats demonstrated that the pristanoyl-CoA oxidase gene is transcribed in liver and extrahepatic tissues and that transcription is not enhanced by treatment of rats with peroxisome proliferators. No mRNA could be detected by northern analysis of human tissues, suggesting that the human pristanoyl-CoA oxidase gene, if present, is only poorly or not transcribed.

  5. Generation of a molecular clone of an attenuated lentivirus, a first step in understanding cytopathogenicity and virulence.

    PubMed

    Blatti-Cardinaux, Laure; Pisoni, Giuliano; Stoffel, Michael H; Zanoni, Reto; Zahno, Marie-Luise; Bertoni, Giuseppe

    2016-01-01

    Small ruminant lentiviruses infect goats and sheep, inducing clinical disease in a minority of infected animals. Following an eradication campaign, clinical cases may disappear in a population. The complete elimination of these lentiviruses is however difficult to achieve and the spreading of less virulent strains often parallels the elimination of their virulent counterparts. Here, we characterized three such strains isolated from a flock in the post-eradication phase. We completely sequenced their genomes, showing that one of the isolates was most probably the product of a recombination event between the other two viruses. By comparing the sequences of these isolates with those of virulent strains, we found evidence that particular LTR mutations may explain their attenuated phenotype. Finally, we constructed an infectious molecular clone representative of these viruses, analyzing its replication characteristics in different target cells. This clone will permit us to explore the molecular correlates of cytopathogenicity and virulence.

  6. The high-affinity interleukin 8 receptor gene (IL8RA) maps to the 2q33-q36 region of the human genome: Cloning of a pseudogene (IL8RBP) for the low-affinity receptor

    SciTech Connect

    Mollereau, C. Laboratoire de Pharmacologie et Toxicologie Fondamentale du CNRS, Toulouse ); Muscatelli, F.; Mattei, M.G. ); Vassart, G. Universite libre de Bruxelles ); Parmentier, M. )

    1993-04-01

    The selective amplification by polymerase chain reaction (PCR) of gene fragments corresponding to new G-protein-coupled receptors resulted in the cloning of 18 orphan members of this gene family. Of these, three human clones amplified from genomic DNA (HGMP03, HGMP04, and HGMP05) were shown to be structurally related. Genomic clones corresponding to HGMP03 and HGMP05 were isolated and their putative coding region sequenced. Following the characterization of two interleukin 8 (IL-8) receptors, HGMP03 appeared to encode the high-affinity IL-8 receptor, whereas the partial clone HGMP04 encodes the low-affinity IL-8 receptor. Comparison with the cDNA sequence suggests that the high-affinity receptor gene is split by an intron in the 5[prime] untranslated region. The high-affinity receptor gene was mapped by in situ hybridization to the 2q33-q36 region of the human genome. The HGMP05 locus turned out to be a pseudogene for the low-affinity IL-8 receptor (87% identity), with multiple frameshifts and point mutations introducing stop codons. Southern blotting on genomic DNA did not allow the further detection of related loci in the human genome. 12 refs., 2 figs.

  7. Molecular cloning of covalently closed circular DNA of bovine leukemia virus.

    PubMed Central

    Kashmiri, S V; Mehdi, R; Ferrer, J F

    1984-01-01

    The two species of covalently closed circular DNA molecules of bovine leukemia virus were cloned in the lambda phage vector lambda gtWES X lambda B. Of the nine independent recombinant lambda-bovine leukemia virus clones that were analyzed, three were derived from the small and six were derived from the large circular molecules carrying, respectively, one and two copies of the long terminal repeat sequences. Comprehensive restriction endonuclease mapping of the unintegrated bovine leukemia virus and the cloned DNA molecules showed that eight of the nine clones carried viral information without any detectable deletions or insertions of more than ca. 50 base pairs. One of the nine clones, which carries a retroviral insert with one copy of the long terminal repeat, had a deletion of ca. 150 base pairs. Images PMID:6319758

  8. The Molecular Mechanism of P2Y1 Receptor Activation

    PubMed Central

    Chan, H. C. Stephen; Vogel, Horst; Filipek, Slawomir

    2016-01-01

    Human purinergic G protein-coupled receptor P2Y1 (P2Y1R) is activated by adenosine 5’-diphosphate (ADP) to induce platelet activation and thereby serves as an important antithrombotic drug target. Crystal structures of P2Y1R revealed that one ligand (MRS2500) binds to the extracellular vestibule of this GPCR, whereas another (BPTU) occupies the surface between transmembrane (TM) helices TM2 and TM3. We introduced a total of 20 µs all-atom long-timescale molecular dynamic (MD) simulations to inquire why two molecules in completely different locations both serve as antagonists while ADP activates the receptor. Our results indicate that BPTU acts as an antagonist by stabilizing extracellular helix bundles leading to an increase of the lipid order, whereas MRS2500 blocks signaling by occupying the ligand binding site. Both antagonists stabilize an ionic lock within the receptor. However, binding of ADP breaks this ionic lock, forming a continuous water channel that leads to P2Y1R activation. PMID:27460867

  9. Production of interferon-gamma and tumour necrosis factor-alpha by human T-cell clones expressing different forms of the gamma delta receptor.

    PubMed

    Christmas, S E; Meager, A

    1990-12-01

    Panels of human T-cell clones bearing the gamma delta T-cell receptor (TcR) were obtained from peripheral blood and decidual tissue and maintained in the presence of interleukin-2 (IL-2). TcR V gamma and V delta gene expression was determined in 40 TcR delta 1+ clones using the gamma delta T-cell subset markers Ti gamma A and delta TCS1, in conjunction with Southern blot analysis using TcR J gamma and J delta probes. gamma delta T-cell clones, together with control alpha beta T-cell clones derived from the same lymphocyte populations, were stimulated with phytohaemagglutinin (PHA) and their ability to produce interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha) tested using specific ELISA. Many clones representative of the major peripheral V gamma 9/V delta 2J1 subset produced high amounts of both cytokines and mean levels were not significantly different from those produced by alpha beta T-cell clones. Panels of clones expressing V gamma 9 and V delta 2J1 produced significantly higher levels of TNF-alpha than clones not expressing V delta 2J1 and those expressing V delta 1J1. There was no relationship between levels of IFN-gamma and TNF-alpha produced by individual gamma delta T-cell clones and also no relationship between their non-major histocompatibility complex (MHC)-restricted cytotoxic activity and levels of either cytokine. There was a significant tendency for gamma delta T-cell clones to produce more TNF-alpha than IFN-gamma in comparison to alpha beta T-cell clones. The significance of these findings is discussed in the light of the reported differences in distribution in vivo of V delta 1J1+ and V delta 2J1+ cells.

  10. Molecular details of the activation of the μ opioid receptor.

    PubMed

    Shim, Jihyun; Coop, Andrew; MacKerell, Alexander D

    2013-07-01

    Molecular details of μ opioid receptor activations were obtained using molecular dynamics simulations of the receptor in the presence of three agonists, three antagonists, and a partial agonist and on the constitutively active T279K mutant. Agonists have a higher probability of direct interactions of their basic nitrogen (N) with Asp147 as compared with antagonists, indicating that direct ligand-Asp147 interactions modulate activation. Medium-size substituents on the basic N of antagonists lead to steric interactions that perturb N-Asp147 interactions, while additional favorable interactions occur with larger basic N substituents, such as in N-phenethylnormorphine, restoring N-Asp147 interactions, leading to agonism. With the orvinols, the increased size of the C19 substituent in buprenorphine over diprenorphine leads to increased interactions with residues adjacent to Asp147, partially overcoming the presence of the cyclopropyl N substituent, such that buprenorphine is a partial agonist. Results also indicate different conformational properties of the intracellular regions of the transmembrane helices in agonists versus antagonists. PMID:23758404

  11. Identification and cloning of molecular markers for UV-B tolerant gene in wild sugarcane (Saccharum spontaneum L.).

    PubMed

    Li, Yuan; He, Yongmei; Zu, Yanqun; Zhan, Fangdong

    2011-11-01

    Previously we have selected wild sugarcane (Saccharum spontaneum L.) sterile lines that are tolerant or susceptible to UV-B radiation based on response index (RI) in a field screening test. The RI was established according to plant height, tiller number, leaf index, total biomass and brix under enhanced ultraviolet-B (UV-B, 280-310 nm) radiation. In this experiment, molecular markers linked to the UV-B tolerant and susceptible genes were identified and cloned. RAPD (Randomly amplified polymorphic DNAs) assay using 100 arbitrary primers followed by clustering analysis separated the tolerant and susceptible lines into two groups at the genetic distance of 0.380. The UV-B tolerant and susceptible gene pools were constructed and compared using the Bulked Segregate Analysis (BSA) approach. Of the 100 arbitrary RAPD primers, primer OPR16 produced polymorphic DNA banding patterns from both gene pools. The OPR16-1200 bp DNA fragment was only amplified from the tolerant lines and the OPR16-800 bp from the susceptible ones. These two PCR fragments were cloned onto T-vector. DNA sequence alignment analysis determined that 42% homology existed between the reverse and forward sequences of the OPR16-1200 bp clone, and 36% homology between the forward sequences of the OPR16-800 bp and OPR16-1200 bp clones. The two DNA clones were determined to be linked to the UV-B tolerant and susceptible genes, and they can be used to develop molecular markers for the associated traits.

  12. Uncovering Molecular Bases Underlying Bone Morphogenetic Protein Receptor Inhibitor Selectivity

    PubMed Central

    Alsamarah, Abdelaziz; LaCuran, Alecander E.; Oelschlaeger, Peter; Hao, Jijun; Luo, Yun

    2015-01-01

    Abnormal alteration of bone morphogenetic protein (BMP) signaling is implicated in many types of diseases including cancer and heterotopic ossifications. Hence, small molecules targeting BMP type I receptors (BMPRI) to interrupt BMP signaling are believed to be an effective approach to treat these diseases. However, lack of understanding of the molecular determinants responsible for the binding selectivity of current BMP inhibitors has been a big hindrance to the development of BMP inhibitors for clinical use. To address this issue, we carried out in silico experiments to test whether computational methods can reproduce and explain the high selectivity of a small molecule BMP inhibitor DMH1 on BMPRI kinase ALK2 vs. the closely related TGF-β type I receptor kinase ALK5 and vascular endothelial growth factor receptor type 2 (VEGFR2) tyrosine kinase. We found that, while the rigid docking method used here gave nearly identical binding affinity scores among the three kinases; free energy perturbation coupled with Hamiltonian replica-exchange molecular dynamics (FEP/H-REMD) simulations reproduced the absolute binding free energies in excellent agreement with experimental data. Furthermore, the binding poses identified by FEP/H-REMD led to a quantitative analysis of physical/chemical determinants governing DMH1 selectivity. The current work illustrates that small changes in the binding site residue type (e.g. pre-hinge region in ALK2 vs. ALK5) or side chain orientation (e.g. Tyr219 in caALK2 vs. wtALK2), as well as a subtle structural modification on the ligand (e.g. DMH1 vs. LDN193189) will cause distinct binding profiles and selectivity among BMP inhibitors. Therefore, the current computational approach represents a new way of investigating BMP inhibitors. Our results provide critical information for designing exclusively selective BMP inhibitors for the development of effective pharmacotherapy for diseases caused by aberrant BMP signaling. PMID:26133550

  13. Uncovering Molecular Bases Underlying Bone Morphogenetic Protein Receptor Inhibitor Selectivity.

    PubMed

    Alsamarah, Abdelaziz; LaCuran, Alecander E; Oelschlaeger, Peter; Hao, Jijun; Luo, Yun

    2015-01-01

    Abnormal alteration of bone morphogenetic protein (BMP) signaling is implicated in many types of diseases including cancer and heterotopic ossifications. Hence, small molecules targeting BMP type I receptors (BMPRI) to interrupt BMP signaling are believed to be an effective approach to treat these diseases. However, lack of understanding of the molecular determinants responsible for the binding selectivity of current BMP inhibitors has been a big hindrance to the development of BMP inhibitors for clinical use. To address this issue, we carried out in silico experiments to test whether computational methods can reproduce and explain the high selectivity of a small molecule BMP inhibitor DMH1 on BMPRI kinase ALK2 vs. the closely related TGF-β type I receptor kinase ALK5 and vascular endothelial growth factor receptor type 2 (VEGFR2) tyrosine kinase. We found that, while the rigid docking method used here gave nearly identical binding affinity scores among the three kinases; free energy perturbation coupled with Hamiltonian replica-exchange molecular dynamics (FEP/H-REMD) simulations reproduced the absolute binding free energies in excellent agreement with experimental data. Furthermore, the binding poses identified by FEP/H-REMD led to a quantitative analysis of physical/chemical determinants governing DMH1 selectivity. The current work illustrates that small changes in the binding site residue type (e.g. pre-hinge region in ALK2 vs. ALK5) or side chain orientation (e.g. Tyr219 in caALK2 vs. wtALK2), as well as a subtle structural modification on the ligand (e.g. DMH1 vs. LDN193189) will cause distinct binding profiles and selectivity among BMP inhibitors. Therefore, the current computational approach represents a new way of investigating BMP inhibitors. Our results provide critical information for designing exclusively selective BMP inhibitors for the development of effective pharmacotherapy for diseases caused by aberrant BMP signaling. PMID:26133550

  14. Vitamin D receptor alleles: Cloning and characterization of the VDR gene and RT-PCR of VDR cDNA

    SciTech Connect

    Javed, A.A.; Huang, Y.; Bombard, A.T.

    1994-09-01

    Vitamin D{sub 3} receptors (VDR) function as regulators through the action of the ligand 1{alpha}, 25-dihydroxy vitamin D{sub 3}. The receptor specifically finds its ligand and exerts it effect on the regulation of the expression of target genes. It has been shown that mutations in the VDR gene affect the function of the receptors and cause a corresponding disorder state. Recently, it has been reported that common allelic variations found normally in the Caucasian (Australian) population pose varying degrees of risk for osteoporosis. We present here the cloning of the VDR gene and RT-PCR of VDR cDNA. Studies are in progress to establish allele frequency in the Black, Hispanic and Caucasian populations to systematically study the influence of allele types and to develop a risk profile for osteoporosis. The present method for detection of various alleles is based on RFLP analysis. We are developing PCR-based methods for the rapid detection and typing of alleles.

  15. Identification, molecular cloning and functional characterization of a novel NADH kinase from Arabidopsis thaliana (thale cress)

    PubMed Central

    2004-01-01

    NADH kinase (NADHK; ATP:NADH 2′-phosphotransferase; EC 2.7.1.86), an enzyme that preferentially utilizes NADH as the diphosphonicotinamide nucleotide donor, has been identified for the first time in plants. Low activity (0.4 nmol of NADPH produced/min per mg of protein) was observed in clarified protein extracts from Arabidopsis thaliana (thale cress) cell suspension cultures. However, unlike an NADHK from yeast (Saccharomyces cerevisiae) (POS5), the enzyme from Arabidopsis did not associate with the mitochondria. NADHK was cloned (gi:30699338) from Arabidopsis and studied as a recombinant protein following affinity purification from Escherichia coli. The enzyme had a pH optimum for activity of 7.9 and a subunit molecular mass of 35 kDa. Analytical gel filtration demonstrated that the recombinant enzyme exists as a dimer. Hyperbolic saturation kinetics were observed for the binding of NADH, ATP, free Mg2+ and NAD+, with respective Km values of 0.042, 0.062, 1.16, and 2.39 mM. While NADHK could phosphorylate NADH or NAD+, the specificity constant (Vmax/Km) for NADH was 100-fold greater than for NAD+. The enzyme could utilize UTP, GTP and CTP as alternative nucleotides, although ATP was the preferred substrate. PPi or poly-Pi could not substitute as phospho donors. PPi acted as a mixed inhibitor with respect to both NADH and ATP. NADHK was inactivated by thiol-modifying reagents, with inactivation being decreased in the presence of NADH or ATP, but not NAD+. This study suggests that, in Arabidopsis, NADP+/NADPH biosynthetic capacity could, under some circumstances, become uncoupled from the redox status of the diphosphonicotinamide nucleotide pool. PMID:15347288

  16. Molecular cloning, expression and phylogenetic analyses of parvalbumin in tilapia, Oreochromis mossambicus.

    PubMed

    Lee, Shyh-Jye; Ju, Chi-Ching; Chu, Shian-Ling; Chien, Ming-Shan; Chan, Tun-Hao; Liao, Wen-Liang

    2007-01-01

    The gene expression of parvalbumin (Pvalb), a high-affinity calcium-binding protein and the major fish allergen, was significantly increased in the tilapia fry treated with methyltestosterone (MT) as examined using a subtractive hybridization assay. Using the real-time quantitative PCR, we further confirmed the increased Pvalb expression in the MT-treated tilapia fry. The 568 base pairs (bp) tilapia Pvalb (tPvalb) cDNA clone was fully sequenced and found to contain a coding region of 330 bp, which encodes a 108 amino acids protein with a molecular weight of 11,370.5 and an calculated isoelectric point of 4.56. The predicted secondary structure of tPvalb is comprised of seven alpha helices. It contains two characteristic EF-hand calcium-binding motifs, one PKC and five casein kinase II consensus phosphorylation sites. The tPvalb is highly homologous to the selected fish Pvalbs at a similarity ranging from 53% to 80%. The phylogenetic tree analysis showed that the tPvalb is closest to the Scomber japonicus Pvalb. The tPvalb was found to express in the heart, muscle, gill, kidney, brain and ovary of adult fish by RT-PCR analysis. In situ hybridization also revealed that the tPvalb was highly expressed in the hypothalamus and sarcoplasmic reticulum. A tPvalb glutathione S-transferase (GST) fusion protein was generated and digested by thrombin to remove the GST moiety. Further Western analysis showed that the tPvalb protein was cross-reacted to an anti-rat Pvalb antibody. Those results suggest that Pvalb is evolutionally conserved in tilapia. PMID:17094115

  17. Molecular cloning and expression analysis of the sucrose transporter gene family from Theobroma cacao L.

    PubMed

    Li, Fupeng; Wu, Baoduo; Qin, Xiaowei; Yan, Lin; Hao, Chaoyun; Tan, Lehe; Lai, Jianxiong

    2014-08-10

    In this study, we performed cloning and expression analysis of six putative sucrose transporter genes, designated TcSUT1, TcSUT2, TcSUT3, TcSUT4, TcSUT5 and TcSUT6, from the cacao genotype 'TAS-R8'. The combination of cDNA and genomic DNA sequences revealed that the cacao SUT genes contained exon numbers ranging from 1 to 14. The average molecular mass of all six deduced proteins was approximately 56 kDa (range 52 to 66 kDa). All six proteins were predicted to exhibit typical features of sucrose transporters with 12 trans-membrane spanning domains. Phylogenetic analysis revealed that TcSUT2 and TcSUT4 belonged to Group 2 SUT and Group 4 SUT, respectively, and the other TcSUT proteins were belonging to Group 1 SUT. Real-time PCR was conducted to investigate the expression pattern of each member of the SUT family in cacao. Our experiment showed that TcSUT1 was expressed dominantly in pods and that, TcSUT3 and TcSUT4 were highly expressed in both pods and in bark with phloem. Within pods, TcSUT1 and TcSUT4 were expressed more in the seed coat and seed from the pod enlargement stage to the ripening stage. TcSUT5 expression sharply increased to its highest expression level in the seed coat during the ripening stage. Expression pattern analysis indicated that TcSUT genes may be associated with photoassimilate transport into developing seeds and may, therefore, have an impact on seed production.

  18. Molecular cloning and characterization of crustin from mud crab Scylla paramamosain.

    PubMed

    Imjongjirak, Chanprapa; Amparyup, Piti; Tassanakajon, Anchalee; Sittipraneed, Siriporn

    2009-05-01

    Antimicrobial peptides (AMPs) are important components of the host innate immune response against microbial invasion. In the present study, we report the identification and characterization of a crustin (CrusSp) from the hemocyte of mud crab, Scylla paramamosain using an expressed sequence tag (EST) and rapid amplification cDNA end (RACE) approaches. Analysis of the nucleotide sequence revealed seven different variances of the CrusSp cDNA in mud crab. The open reading frame encodes a protein of 111 amino acids with 21 residues signal sequence. The predicted molecular mass of the mature protein (90 amino acids) is 10.27 kDa with an estimated pI of 8.54. Analysis of the protein domain features indicated typical conserved cysteine residues containing a single whey acidic protein (WAP) domain at the C-terminus. A neighbour-joining tree showed that S. paramamosain crustin is closely related to other crustin homologues, and displays the highest similarity to crustin antimicrobial peptide in shore crab Carcinus maenas. Four exons and three introns were identified within the 999 bp genomic DNA sequence of CrusSp. Tissue distribution analysis showed that CrusSp was highly expressed in hemocytes, gills, intestines and muscle but it was not expressed in hepatopancreas and eyestalks. To gain insight into the in vitro antimicrobial activities of CrusSp, the mature peptide coding region was cloned into E. coli for heterologous expression. The recombinant CrusSp could inhibit the growth of gram-positive bacteria but had no inhibition activity against gram-negative bacteria. These results indicated the involvement of CrusSp in the innate immunity of S. paramamosain. PMID:18425600

  19. Molecular cloning of rhamnose-binding lectin gene and its promoter region from snakehead Channa argus.

    PubMed

    Jia, W Z; Shang, N; Guo, Q L

    2010-09-01

    Lectins are sugar-binding proteins that mediate pathogen recognition and cell-cell interactions. A rhamnose-binding lectin (RBL) gene and its promoter region have been cloned and characterized from snakehead Channa argus. From the transcription initiation site, snakehead rhamnose-binding lectin (SHL) gene extends 2,382 bp to the end of the 3' untranslated region (UTR), and contains nine exons and eight introns. The open reading frame (ORF) of the SHL transcript has 675 bp which encodes 224 amino acids. The molecular structure of SHL is composed of two tandem repeat carbohydrate recognition domains (CRD) with 35% internal identity. Analysis of the gene organization of SHL indicates that the ancestral gene of RBL may diverge and evolve by exon shuffling and gene duplication, producing new forms to play their own roles in various organisms. The characteristics of SHL gene 5' flanking region are the presence of consensus nuclear factor of interleukin 6 (NF-IL6) and IFN-gamma activation (GAS) sites. The results provide indirect evidence that up-regulation of SHL expression may be induced in response to inflammatory stimuli, such as lipopolysaccharide (LPS), interleukin 6 (IL-6), and interferon gamma (IFN-gamma). The transcript of SHL mRNA was expressed in the head kidney, posterior kidney, spleen, liver, intestine, heart, muscle, and ovary. No tissue-specific expressive pattern is different from reported STLs, WCLs, and PFLs, suggesting that different types of RBLs exist in species-specific fish that have evolved and adapted to their surroundings.

  20. Molecular cloning of a novel multidomain Kunitz-type proteinase inhibitor from the hookworm Ancylostoma caninum.

    PubMed

    Hawdon, John M; Datu, Bennett; Crowell, Melissa

    2003-04-01

    Degenerate oligonucleotide primers derived from conserved serine protease inhibitors were used to amplify a 90-base pair (bp) amplicon from an Ancylostoma caninum adult-stage complementary deoxyribonucleic acid (cDNA) library by polymerase chain reaction (PCR). The amplicon was labeled and used as a probe to screen the library, and a 2,300-bp cDNA clone was identified. The 5' end of the molecule was obtained from adult cDNA by 5'-RACE. The complete sequence named A. caninum Kunitz-type protease inhibitor (Ac-kpi-1) was 2,371 bp and encoded a 759-amino acid open reading frame. The deduced amino acid sequence had a calculated molecular weight of 84,886 Da and contained an amino terminal signal peptide, suggesting that the protein is secreted. Analysis of the predicted protein sequence indicates 12 highly conserved Kunitz-type serine protease inhibitor domains connected by short, conserved spacers. On the basis of sequence analysis, the first 11 domains are predicted to be active serine protease inhibitors based on the P1 amino acid. Domains 5-8 have identical amino acid sequences, and the remaining domains are 38-88% identical. Domain 12 lacks several of the conserved cysteine residues and has an atypical amino acid in the P1 position, suggesting that it is nonfunctional. Reverse transcriptase-PCR indicated that the Ac-kpi-1 messenger ribonucleic acid is present in egg, L1, L3, and adult stages but is most abundant in the adult stage. Ac-KPI-1 is most similar in domain architecture to several extracellular matrix proteins involved in cellular remodeling during insect development. In addition, there are 44 nematode proteins containing one or more Kunitz domains in GenBank, including several with multiple domains.

  1. Molecular cloning, characterization and expression profiles of thioredoxin 1 and thioredoxin 2 genes in Mytilus galloprovincialis

    NASA Astrophysics Data System (ADS)

    Wang, Qing; Ning, Xuanxuan; Pei, Dong; Zhao, Jianmin; You, Liping; Wang, Chunyan; Wu, Huifeng

    2013-05-01

    Thioredoxin (Trx) proteins are involved in many biological processes especially the regulation of cellular redox homeostasis. In this study, two Trx cDNAs were cloned from the mussel Mytilus galloprovincialis using rapid amplifi cation of cDNA ends-polymerase chain reaction (RACE-PCR). The two cDNAs were named MgTrx1 and MgTrx2, respectively. The open reading frames of MgTrx1 and MgTrx2 were 318 and 507 base pairs (bp) and they encoded proteins of 105 and 168 amino acids with estimated molecular masses of 11.45 and 18.93 kDa, respectively. Sequence analysis revealed that both proteins possessed the conserved active site dithiol motif Cys-Gly-Pro-Cys. In addition, MgTrx2 also possessed a putative mitochondrial targeting signal suggesting that it is located in the mitochondria. Quantitative real-time polymerase chain reaction (qPCR) revealed that both MgTrx1 and MgTrx2 were constitutively expressed in all tissues examined. The MgTrx1 transcript was most abundant in hemocytes and gills, whereas the MgTrx2 transcript was most abundant in gonad, hepatopancreas, gill and hemocytes. Following Vibrio anguillarum challenge, the expression of MgTrx1 was up-regulated and reached its peak, at a value 10-fold the initial value, at 24 h. Subsequently, expression returned back to the original level. In contrast, the expression level of MgTrx2 was down-regulated following bacterial stimulation, with one fi fth of the control level evident at 12 h post challenge. These results suggest that MgTrx1 and MgTrx2 may play important roles in the response of M. galloprovincialis to bacterial challenge.

  2. Molecular cloning and identification of the laspartomycin biosynthetic gene cluster from Streptomyces viridochromogenes

    PubMed Central

    Wang, Yang; Chen, Ying; Shen, Qirong; Yin, Xihou

    2011-01-01

    The biosynthetic gene cluster for laspartomycins, a family of 11 amino acid peptide antibiotics, has been cloned and sequenced from Streptomyces viridochromogenes ATCC 29814. Annotation of a segment of 88912 bp of S. viridochromogenes genomic sequence revealed the putative las cluster and its flanking regions which harbor 43 open reading frames. The lpm cluster, which spans approximately 60 kb, consists of 21 open reading frames. Those include four NRPS genes (lpmA/orf18, lpmB/orf25, lpmC/orf26 and lpmD/orf27), four genes (orfs 21, 22, 24 and 29) involved in the lipid tail biosynthesis and attachment, four regulatory genes (orfs 13, 19, 32 and 33) and three putative exporters or self-resistance genes (orfs 14, 20 and 30). In addition, the gene involved in the biosynthesis of the nonproteinogenic amino acid Pip was also identified in the lpm cluster while the genes necessary for the biosynthesis of the rare residue diaminopropionic acid (Dap) were found to reside elsewhere on the chromosome. Interestingly, the dabA, dabB and dabC genes predicted to code for the biosynthesis of the unusual amino acid diaminobutyric acid (Dab) are organized into the lpm cluster even though the Dab residue was not found in the laspartomycins. Disruption of the NRPS lpmC gene completely abolished laspartomycin production in the corresponding mutant strain. These findings will allow molecular engineering and combinatorial biosynthesis approaches to expand the structural diversity of the amphomycin-group peptide antibiotics including the laspartomycins and friulimicins. PMID:21640802

  3. CLONING, EXPRESSION AND CHARACTERIZATION OF THE ANDROGEN RECEPTOR AND ISOLATION OF ESTROGEN RECEPTOR ALPHA FROM THE FATHEAD MINNOW (PIMEPHALES PROMELAS)

    EPA Science Inventory

    In vitro screening assays designed to identify hormone mimics or antagonists, including those recommended for use in the EPA's Tier 1 screening battery, typically use mammalian estrogen (ER) and androgen receptors (AR) such as rat or human. Although we know that the amino acid s...

  4. Molecular characterization, expression profile, and polymorphism of goose dopamine D1 receptor gene.

    PubMed

    Wang, Cui; Liu, Yi; Wang, Huiying; Wu, Huali; Gong, Shaoming; He, Daqian

    2014-05-01

    Dopamine D1 receptor (DRD1) is one of the dopamine receptors with seven transmembrane domains that are coupled to the G protein. In the present study, we cloned the full coding region of DRD1 gene by the reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends from the goose hypothalamus tissues. Results showed that the goose DRD1 cDNA (GenBank: KF156790) contained a 1,356 bp open reading frame encoding a protein 452 amino acid with a molecular weight of 50.52 kDa and a isoelectric point of 6.96. Bioinformatics analysis indicated that the deduced amino acid sequence was 71-98% identical to the DRD1 protein of other species, contained seven transmembrane domains and four N-glycosylation sites. A phylogenetic tree analysis revealed that the deduced goose DRD1 protein had a close genetic relationship and evolutional distance with that of duck, chicken, and zebra finch. The semi-quantitative RT-PCR analysis displayed goose DRD1 gene was widely expressed in all detected tissues, including heart, lung, liver, spleen, kidney, breast muscle, duodenum, sebum, pituitary, hypothalamus, ovary and oviduct. Eighteen single nucleotide polymorphisms were indentified in 3,169 bp length of this gene. For G90A mutation, the genotyping analysis of PCR-TspRI-RFLP showed the allele G was in dominance in all detected goose breeds, and the allele frequencies of this polymorphism were significantly different between Chinese goose breeds and foreign breeds (P<0.01). These findings will help us understand the functions of the DRD1 gene and the molecular breeding in geese.

  5. Comparative Transcriptome Analysis of Latex Reveals Molecular Mechanisms Underlying Increased Rubber Yield in Hevea brasiliensis Self-Rooting Juvenile Clones

    PubMed Central

    Li, Hui-Liang; Guo, Dong; Zhu, Jia-Hong; Wang, Ying; Chen, Xiong-Ting; Peng, Shi-Qing

    2016-01-01

    Rubber tree (Hevea brasiliensis) self-rooting juvenile clones (JCs) are promising planting materials for rubber production. In a comparative trial between self-rooting JCs and donor clones (DCs), self-rooting JCs exhibited better performance in rubber yield. To study the molecular mechanism associated with higher rubber yield in self-rooting JCs, we sequenced and comparatively analyzed the latex of rubber tree self-rooting JCs and DCs at the transcriptome level. Total raw reads of 34,632,012 and 35,913,020 bp were obtained from the library of self-rooting JCs and DCs, respectively, by using Illumina HiSeq 2000 sequencing technology. De novo assemblies yielded 54689 unigenes from the library of self-rooting JCs and DCs. Among 54689 genes, 1716 genes were identified as differentially expressed between self-rooting JCs and DCs via comparative transcript profiling. Functional analysis showed that the genes related to the mass of categories were differentially enriched between the two clones. Several genes involved in carbohydrate metabolism, hormone metabolism and reactive oxygen species scavenging were up-regulated in self-rooting JCs, suggesting that the self-rooting JCs provide sufficient molecular basis for the increased rubber yielding, especially in the aspects of improved latex metabolisms and latex flow. Some genes encoding epigenetic modification enzymes were also differentially expressed between self-rooting JCs and DCs. Epigenetic modifications may lead to gene differential expression between self-rooting JCs and DCs. These data will provide new cues to understand the molecular mechanism underlying the improved rubber yield of H. brasiliensis self-rooting clones. PMID:27555864

  6. Comparative Transcriptome Analysis of Latex Reveals Molecular Mechanisms Underlying Increased Rubber Yield in Hevea brasiliensis Self-Rooting Juvenile Clones.

    PubMed

    Li, Hui-Liang; Guo, Dong; Zhu, Jia-Hong; Wang, Ying; Chen, Xiong-Ting; Peng, Shi-Qing

    2016-01-01

    Rubber tree (Hevea brasiliensis) self-rooting juvenile clones (JCs) are promising planting materials for rubber production. In a comparative trial between self-rooting JCs and donor clones (DCs), self-rooting JCs exhibited better performance in rubber yield. To study the molecular mechanism associated with higher rubber yield in self-rooting JCs, we sequenced and comparatively analyzed the latex of rubber tree self-rooting JCs and DCs at the transcriptome level. Total raw reads of 34,632,012 and 35,913,020 bp were obtained from the library of self-rooting JCs and DCs, respectively, by using Illumina HiSeq 2000 sequencing technology. De novo assemblies yielded 54689 unigenes from the library of self-rooting JCs and DCs. Among 54689 genes, 1716 genes were identified as differentially expressed between self-rooting JCs and DCs via comparative transcript profiling. Functional analysis showed that the genes related to the mass of categories were differentially enriched between the two clones. Several genes involved in carbohydrate metabolism, hormone metabolism and reactive oxygen species scavenging were up-regulated in self-rooting JCs, suggesting that the self-rooting JCs provide sufficient molecular basis for the increased rubber yielding, especially in the aspects of improved latex metabolisms and latex flow. Some genes encoding epigenetic modification enzymes were also differentially expressed between self-rooting JCs and DCs. Epigenetic modifications may lead to gene differential expression between self-rooting JCs and DCs. These data will provide new cues to understand the molecular mechanism underlying the improved rubber yield of H. brasiliensis self-rooting clones. PMID:27555864

  7. Comparative Transcriptome Analysis of Latex Reveals Molecular Mechanisms Underlying Increased Rubber Yield in Hevea brasiliensis Self-Rooting Juvenile Clones.

    PubMed

    Li, Hui-Liang; Guo, Dong; Zhu, Jia-Hong; Wang, Ying; Chen, Xiong-Ting; Peng, Shi-Qing

    2016-01-01

    Rubber tree (Hevea brasiliensis) self-rooting juvenile clones (JCs) are promising planting materials for rubber production. In a comparative trial between self-rooting JCs and donor clones (DCs), self-rooting JCs exhibited better performance in rubber yield. To study the molecular mechanism associated with higher rubber yield in self-rooting JCs, we sequenced and comparatively analyzed the latex of rubber tree self-rooting JCs and DCs at the transcriptome level. Total raw reads of 34,632,012 and 35,913,020 bp were obtained from the library of self-rooting JCs and DCs, respectively, by using Illumina HiSeq 2000 sequencing technology. De novo assemblies yielded 54689 unigenes from the library of self-rooting JCs and DCs. Among 54689 genes, 1716 genes were identified as differentially expressed between self-rooting JCs and DCs via comparative transcript profiling. Functional analysis showed that the genes related to the mass of categories were differentially enriched between the two clones. Several genes involved in carbohydrate metabolism, hormone metabolism and reactive oxygen species scavenging were up-regulated in self-rooting JCs, suggesting that the self-rooting JCs provide sufficient molecular basis for the increased rubber yielding, especially in the aspects of improved latex metabolisms and latex flow. Some genes encoding epigenetic modification enzymes were also differentially expressed between self-rooting JCs and DCs. Epigenetic modifications may lead to gene differential expression between self-rooting JCs and DCs. These data will provide new cues to understand the molecular mechanism underlying the improved rubber yield of H. brasiliensis self-rooting clones.

  8. Molecular cloning and characterization of two thermostable carboxyl esterases from Geobacillus stearothermophilus.

    PubMed

    Ewis, Hosam E; Abdelal, Ahmed T; Lu, Chung-Dar

    2004-03-31

    Screening of the genomic libraries of Geobacillus stearothermophilus ATCC12980 and ATCC7954 for esterase/lipase activity led to the isolation of two positive clones. The results of subclonings and sequence analyses identified two genes, est30 and est55, encoding two different carboxylesterases, and genetic rearrangement in the est55 locus was revealed from genomic comparison. The est30 gene encodes a polypeptide of 248 amino acids with a calculated molecular mass of 28338 Da, and the est55 gene encodes a polypeptide of 499 amino acids with a calculated molecular mass of 54867 Da. Both enzymes were purified to near homogeneity from recombinant strains of Escherichia coli. The results of enzyme characterization showed that while both enzymes possess optimal activities with short chain acyl derivatives, Est55 has a broader pH tolerance (pH 8-9) and optimal temperature range (30-60 degrees C) than Est30. The activation energy of Est55 (35.7 kJ/mol) was found to be significantly lower than that of Est30 (101.9 kJ/mol). Both enzymes were stable at 60 degrees C for more than 2 h; at 70 degrees C, the half-life for thermal inactivation was 40 and 180 min for Est55 and Est30, respectively. With p-nitrophenyl caproate as the substrate and assayed at 60 degrees C, Est55 had K(m) and k(cat) values of 0.5 microM and 39758 s(-1) while Est30 exhibited values of 2.16 microM and 38 s(-1). Inhibition studies indicated that both Est30 and Est55 were strongly inhibited by phenylmethanesulfonyl fluoride, p-hydroxymercuribenzoate, and tosyl-l-phenylalanine, consistent with the proposed presence of Ser-His-Glu catalytic triad of the alpha/beta hydrolase family. The enzymatic properties of Est30 and Est55 reported here warrant the potential applications of these enzymes in biotechnological industries. PMID:15033540

  9. Molecular cloning and characterization of a complement-depleting factor from king cobra, Ophiophagus hannah.

    PubMed

    Zeng, Lin; Sun, Qian-Yun; Jin, Yang; Zhang, Yong; Lee, Wen-Hui; Zhang, Yun

    2012-09-01

    Cobra venom factor (CVF) is an anti-complement factor existing in cobra venom. CVF proteins have been purified from the venoms of Naja haje, Naja siamensis, Naja atra, Naja kaouthia, Naja naja, Naja melanoleuca and Austrelaps superbus, but only three full-length cDNA sequences of CVF are available. In the present work, a cobra venom factor termed OVF was purified from the crude venom of Ophiophagus hannah by successive gel filtration, ion-exchange and heparin affinity chromatography steps. The purified OVF was homogenous on the SDS-PAGE gel with an apparent molecular weight of 140 kDa under non-reducing conditions. Under reducing conditions, OVF was divided into three bands with apparent molecular weight of 72 kDa (α chain), 45 kDa (β chain) and 32 kDa (γ chain), respectively. OVF consumed complement components with anti-complement activity of 154 units per mg. By using Reverse transcription-PCR and 5'-RACE assay, the open reading frame of OVF was obtained. MALDI-TOF and protein sequencing assays confirmed the cloned cDNA coding for OVF protein. The cDNA sequence of OVF is conservative when aligned with that of other CVFs. Phylogenetic analysis revealed OVF is closer to CVF from N. kaouthia than to AVF-1 and AVF-2 from A. superbus. Our results demonstrated that OVF has its unique features as following: 1) The N-terminal amino acid sequence of OVF γ chain is different from that of other known CVFs, suggesting that the OVF γ chain might be further processed; 2) Unlike N. kaouthia CVF and A. superbus AVF-1, which have potential N-linked glycosylation sites located in both α and β chain, OVF only has N-linked glycosylation site in its α chain as revealed by Schiff's reagent staining and protein sequence analysis; 3) In addition to the 27 well conserved cysteine residues in all known CVFs, OVF have an additional cysteine residue in its γ chain. Understanding the importance of above mentioned specific characteristics might provide useful information on structure

  10. Molecular cloning and characterization of a complement-depleting factor from king cobra, Ophiophagus hannah.

    PubMed

    Zeng, Lin; Sun, Qian-Yun; Jin, Yang; Zhang, Yong; Lee, Wen-Hui; Zhang, Yun

    2012-09-01

    Cobra venom factor (CVF) is an anti-complement factor existing in cobra venom. CVF proteins have been purified from the venoms of Naja haje, Naja siamensis, Naja atra, Naja kaouthia, Naja naja, Naja melanoleuca and Austrelaps superbus, but only three full-length cDNA sequences of CVF are available. In the present work, a cobra venom factor termed OVF was purified from the crude venom of Ophiophagus hannah by successive gel filtration, ion-exchange and heparin affinity chromatography steps. The purified OVF was homogenous on the SDS-PAGE gel with an apparent molecular weight of 140 kDa under non-reducing conditions. Under reducing conditions, OVF was divided into three bands with apparent molecular weight of 72 kDa (α chain), 45 kDa (β chain) and 32 kDa (γ chain), respectively. OVF consumed complement components with anti-complement activity of 154 units per mg. By using Reverse transcription-PCR and 5'-RACE assay, the open reading frame of OVF was obtained. MALDI-TOF and protein sequencing assays confirmed the cloned cDNA coding for OVF protein. The cDNA sequence of OVF is conservative when aligned with that of other CVFs. Phylogenetic analysis revealed OVF is closer to CVF from N. kaouthia than to AVF-1 and AVF-2 from A. superbus. Our results demonstrated that OVF has its unique features as following: 1) The N-terminal amino acid sequence of OVF γ chain is different from that of other known CVFs, suggesting that the OVF γ chain might be further processed; 2) Unlike N. kaouthia CVF and A. superbus AVF-1, which have potential N-linked glycosylation sites located in both α and β chain, OVF only has N-linked glycosylation site in its α chain as revealed by Schiff's reagent staining and protein sequence analysis; 3) In addition to the 27 well conserved cysteine residues in all known CVFs, OVF have an additional cysteine residue in its γ chain. Understanding the importance of above mentioned specific characteristics might provide useful information on structure

  11. Molecular cloning, expression, and enzymatic analysis of cathepsin X from starfish (Asterina pectinifera).

    PubMed

    Bak, Hye Jin; Kim, Moo-Sang; Kim, Na Young; Go, Hye-Jin; Han, Jin Woo; In Jo, Hyae; Ahn, Sang Jung; Park, Nam Gyu; Chung, Joon Ki; Lee, Hyung Ho

    2013-02-01

    Cathepsin X, also known as cathepsin Z, is referred to as a "lysosomal proteolytic enzyme" and a member of the peptidase C1 family, which is involved in various biological processes such as immune response, cell adhesion, and proliferation. In the present study, the cDNA of starfish (Asterina pectinifera), which is known to cause serious damage to commercial shellfish mariculture, cathepsin X (ApCtX) was isolated through the combination of homology molecular cloning and rapid amplification of cDNA ends (RACE) methods for the application to find a way to reduce/control starfish densities. The full-length of ApCtX gene was determined to consist of the 2,240 bp nucleotide sequence, which encoded for a preproprotein of 296 amino acids with a molecular mass of about 32.7 kDa. The tissue type expression of ApCtX was determined in various tissues of A. pectinifera and was shown most abundantly in the liver. The cDNA encoding pro-mature enzyme of ApCtX was expressed in Escherichia coli BL21 (DE3) using the pGEX-4T-1 expression vector. Its activity was quantified by cleaving the synthetic peptide Z-Phe-Arg-AMC. The optimal pH for the protease activity was 6.5. The enzymatic activity of proApCtX was reduced by antipain, NEM, EDTA, EGTA, and 1,10-phenanthroline, and the proApCtX enzyme was significantly inhibited by CuSO₄, HgCl₂, CoCl₂, and SDS whereas Triton X-100 and Brij 35 might have potentially acted as an activator. Here, we demonstrated for the first time that the structural features and enzymatic characteristics of Echinoderms cathepsin X are similar to those of the other mammalian and piscine cathepsin X except its pH optimum, and the results of tissue-specific expression might explain their importance in food digestion by hepatic cecain starfish.

  12. Cloning and initial characterization of nuclear and membrane progesterone receptors in the Fathead Minnow, Pimephales promelas

    EPA Science Inventory

    Both native progestagens and synthetic progestins have important effects on reproduction that are mediated through progesterone receptors (PRs). They regulate gamete maturation and can serve as precursors for other steroid hormones in vertebrates and act as reproductive pheromone...

  13. Molecular cloning and characterization of the DNA of a new human papillomavirus (HPV 30) from a laryngeal carcinoma.

    PubMed

    Kahn, T; Schwarz, E; zur Hausen, H

    1986-01-15

    DNA from a human laryngeal carcinoma was molecularly cloned in Lambda L47. The gene library was screened for human papillomavirus (HPV)-related sequences by hybridization analysis with 32P-labelled HPV 16 DNA at conditions of low stringency (Tm -40 degrees C). One of the clones (4-5) with an insert of 7.8 kb showed cross-hybridization with most of the known HPV types (Tm -40 degrees C), and with several of them even under more stringent conditions (Tm -30 degrees C). No signal was detected under high-stringency conditions (Tm -20 degrees C). The co-linear alignment of clone 4-5 with HPV 16 DNA could be demonstrated by hybridization experiments and also by partial DNA sequence analysis. We conclude that clone 4-5 represents a new HPV type tentatively designated HPV 30. HPV 30 DNA was also detected in 2 genital lesions but not in 41 laryngeal carcinomas analyzed so far. Its presence in other tumor DNA is now under investigation. PMID:3000955

  14. Isolation, molecular cloning and expression of cellobiohydrolase B (CbhB) from Aspergillus niger in Escherichia coli

    NASA Astrophysics Data System (ADS)

    Woon, J. S. K.; Murad, A. M. A.; Abu Bakar, F. D.

    2015-09-01

    A cellobiohydrolase B (CbhB) from Aspergillus niger ATCC 10574 was cloned and expressed in E. coli. CbhB has an open reading frame of 1611 bp encoding a putative polypeptide of 536 amino acids. Analysis of the encoded polypeptide predicted a molecular mass of 56.2 kDa, a cellulose binding module (CBM) and a catalytic module. In order to obtain the mRNA of cbhB, total RNA was extracted from A. niger cells induced by 1% Avicel. First strand cDNA was synthesized from total RNA via reverse transcription. The full length cDNA of cbhB was amplified by PCR and cloned into the cloning vector, pGEM-T Easy. A comparison between genomic DNA and cDNA sequences of cbhB revealed that the gene is intronless. Upon the removal of the signal peptide, the cDNA of cbhB was cloned into the expression vector pET-32b. However, the recombinant CbhB was expressed in Escherichia coli Origami DE3 as an insoluble protein. A homology model of CbhB predicted the presence of nine disulfide bonds in the protein structure which may have contributed to the improper folding of the protein and thus, resulting in inclusion bodies in E. coli.

  15. Isolation, molecular cloning and expression of cellobiohydrolase B (CbhB) from Aspergillus niger in Escherichia coli

    SciTech Connect

    Woon, J. S. K. Murad, A. M. A. Abu Bakar, F. D.

    2015-09-25

    A cellobiohydrolase B (CbhB) from Aspergillus niger ATCC 10574 was cloned and expressed in E. coli. CbhB has an open reading frame of 1611 bp encoding a putative polypeptide of 536 amino acids. Analysis of the encoded polypeptide predicted a molecular mass of 56.2 kDa, a cellulose binding module (CBM) and a catalytic module. In order to obtain the mRNA of cbhB, total RNA was extracted from A. niger cells induced by 1% Avicel. First strand cDNA was synthesized from total RNA via reverse transcription. The full length cDNA of cbhB was amplified by PCR and cloned into the cloning vector, pGEM-T Easy. A comparison between genomic DNA and cDNA sequences of cbhB revealed that the gene is intronless. Upon the removal of the signal peptide, the cDNA of cbhB was cloned into the expression vector pET-32b. However, the recombinant CbhB was expressed in Escherichia coli Origami DE3 as an insoluble protein. A homology model of CbhB predicted the presence of nine disulfide bonds in the protein structure which may have contributed to the improper folding of the protein and thus, resulting in inclusion bodies in E. coli.

  16. Rapid cloning, expression, and functional characterization of paired αβ and γδ T-cell receptor chains from single-cell analysis

    PubMed Central

    Guo, Xi-zhi J; Dash, Pradyot; Calverley, Matthew; Tomchuck, Suzanne; Dallas, Mari H; Thomas, Paul G

    2016-01-01

    Transgenic expression of antigen-specific T-cell receptor (TCR) genes is a promising approach for immunotherapy against infectious diseases and cancers. A key to the efficient application of this approach is the rapid and specific isolation and cloning of TCRs. Current methods are often labor-intensive, nonspecific, and/or relatively slow. Here, we describe an efficient system for antigen-specific αβTCR cloning and CDR3 substitution. We demonstrate the capability of cloning influenza-specific TCRs within 10 days using single-cell polymerase chain reaction (PCR) and Gibson Assembly techniques. This process can be accelerated to 5 days by generating receptor libraries, requiring only the exchange of the antigen-specific CDR3 region into an existing backbone. We describe the construction of this library for human γδ TCRs and report the cloning and expression of a TRGV9/TRDV2 receptor that is activated by zoledronic acid. The functional activity of these αβ and γδ TCRs can be characterized in a novel reporter cell line (Nur77-GFP Jurkat 76 TCRα–β–) for screening of TCR specificity and avidity. In summary, we provide a rapid method for the cloning, expression, and functional characterization of human and mouse TCRs that can assist in the development of TCR-mediated therapeutics. PMID:26858965

  17. Molecular cloning and functional analysis of SUT-1, a sulfate transporter from human high endothelial venules

    PubMed Central

    Girard, Jean-Philippe; Baekkevold, Espen S.; Feliu, Jacques; Brandtzaeg, Per; Amalric, François

    1999-01-01

    High endothelial venules (HEV) are specialized postcapillary venules found in lymphoid organs and chronically inflamed tissues that support high levels of lymphocyte extravasation from the blood. One of the major characteristics of HEV endothelial cells (HEVEC) is their capacity to incorporate large amounts of sulfate into sialomucin-type counter-receptors for the lymphocyte homing receptor L-selectin. Here, we show that HEVEC express two functional classes of sulfate transporters defined by their differential sensitivity to the anion-exchanger inhibitor 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS), and we report the molecular characterization of a DIDS-resistant sulfate transporter from human HEVEC, designated SUT-1. SUT-1 belongs to the family of Na+-coupled anion transporters and exhibits 40–50% amino acid identity with the rat renal Na+/sulfate cotransporter, NaSi-1, as well as with the human and rat Na+/dicarboxylate cotransporters, NaDC-1/SDCT1 and NaDC-3/SDCT2. Functional expression studies in cRNA-injected Xenopus laevis oocytes showed that SUT-1 mediates high levels of Na+-dependent sulfate transport, which is resistant to DIDS inhibition. The SUT-1 gene mapped to human chromosome 7q33. Northern blotting analysis revealed that SUT-1 exhibits a highly restricted tissue distribution, with abundant expression in placenta. Reverse transcription–PCR analysis indicated that SUT-1 and the diastrophic dysplasia sulfate transporter (DTD), one of the two known human DIDS-sensitive sulfate transporters, are coexpressed in HEVEC. SUT-1 and DTD could correspond, respectively, to the DIDS-resistant and DIDS-sensitive components of sulfate uptake in HEVEC. Together, these results demonstrate that SUT-1 is a distinct human Na+-coupled sulfate transporter, likely to play a major role in sulfate incorporation in HEV. PMID:10535998

  18. Adoptive transfer of chimeric antigen receptor re-directed cytolytic T lymphocyte clones in patients with neuroblastoma.

    PubMed

    Park, Julie R; Digiusto, David L; Slovak, Marilyn; Wright, Christine; Naranjo, Araceli; Wagner, Jamie; Meechoovet, Hunsar B; Bautista, Cherrilyn; Chang, Wen-Chung; Ostberg, Julie R; Jensen, Michael C

    2007-04-01

    Metastatic neuroblastoma is a poor-prognosis malignancy arising during childhood that overexpresses the L1-cell adhesion molecule (CD171). We have previously described a tumor L1-cell adhesion molecule-specific, single chain antibody-derived, chimeric antigen receptor designated CE7R for re-directing the antigen-specific effector functioning of cytolytic T lymphocytes. Here, we report on the feasibility of isolating, and the safety of infusing, autologous CD8(+) cytolytic T lymphocyte clones co-expressing CE7R and the selection-suicide expression enzyme HyTK in children with recurrent/refractory neuroblastoma. The cytolytic T lymphocyte products were derived from peripheral blood mononuclear cells that were subjected to polyclonal activation, plasmid vector electrotransfer, limiting dilution hygromycin selection, and expansion to numbers sufficient for adoptive transfer. In total, 12 infusions (nine at 10(8) cells/m(2), three at 10(9) cells/m(2)) were administered to six patients. No overt toxicities to tissues known to express L1-cell adhesion molecule (e.g., central nervous system, adrenal medulla, and sympathetic ganglia) were observed. The persistence of cytolytic T lymphocyte clones in the circulation, measured by vector-specific quantitative polymerase chain reaction, was short (1-7 days) in patients with bulky disease, but significantly longer (42 days) in a patient with a limited disease burden. This first-in-humans pilot study sets the stage for clinical trials employing adoptive transfer in the context of minimal residual disease.

  19. Molecular cloning of the breakpoints of a complex Philadelphia chromosome translocation: identification of a repeated region on chromosome 17.

    PubMed Central

    McKeithan, T W; Warshawsky, L; Espinosa, R; LeBeau, M M

    1992-01-01

    Complex translocations in chronic myelogenous leukemia involve various chromosomes, in addition to chromosomes 9 and 22, in a nonrandom fashion. We have analyzed the DNA from leukemia cells characterized by a complex translocation, t(9;22;10;17)(q34;q11;p13;q21), by using the techniques of Southern blot hybridization, in situ hybridization, and molecular cloning; one of the breakpoints is at 17q21, a band that is frequently involved in complex 9;22 translocations. All of the breakpoint junctions and the corresponding normal sequences from the four involved chromosomes have been molecularly cloned. Restriction mapping is consistent with a simple concerted exchange of chromosomal material among the four chromosomes, except that additional changes appeared to have occurred within the chromosome 17 sequences. The cloned sequences on chromosome 17 at band q21 were found to be repeated in normal cells. By fluorescence in situ hybridization, a strong signal is seen at 17q21, but a weaker signal is also present at 17q23. By comparison with other primate species, an inversion in chromosome 17 during evolution appears to be responsible for the splitting of the cluster of repeat units in normal human cells. Images PMID:1594595

  20. Development of RAPD-SCAR markers for different Ganoderma species authentication by improved RAPD amplification and molecular cloning.

    PubMed

    Fu, J J; Mei, Z Q; Tania, M; Yang, L Q; Cheng, J L; Khan, M A

    2015-05-25

    The sequence-characterized amplified region (SCAR) is a valuable molecular technique for the genetic identification of any species. This method is mainly derived from the molecular cloning of the amplified DNA fragments achieved from the random amplified polymorphic DNA (RAPD). In this study, we collected DNA from 10 species of Ganoderma mushroom and amplified the DNA using an improved RAPD technique. The amplified fragments were then cloned into a T-vector, and positive clones were screened, indentified, and sequenced for the development of SCAR markers. After designing PCR primers and optimizing PCR conditions, 4 SCAR markers, named LZ1-4, LZ2-2, LZ8-2, and LZ9-15, were developed, which were specific to Ganoderma gibbosum (LZ1-4 and LZ8-2), Ganoderma sinense (LZ2-2 and LZ8-2), Ganoderma tropicum (LZ8-2), and Ganoderma lucidum HG (LZ9-15). These 4 novel SCAR markers were deposited into GenBank with the accession Nos. KM391935, KM391936, KM391937, and KM391938, respectively. Thus, in this study we developed specific SCAR markers for the identification and authentication of different Ganoderma species.

  1. Molecular cloning and functional expression of the rfaE gene required for lipopolysaccharide biosynthesis in Salmonella typhimurium.

    PubMed

    Jin, U H; Chung, T W; Lee, Y C; Ha, S D; Kim, C H

    2001-10-01

    The rfaE (WaaE) gene of Salmonella typhimurium is known to be located at 76min on the genetic map outside of the rfa gene cluster encoding core oligosaccharide biosynthesis of lipopolysaccharide(LPS). The rfaE mutant synthesizes heptose-deficient LPS; its LPS consists of only lipid A and 3-deoxy-D-manno-octulosonic acid (KDO), and the rfaE gene is believed to be involved in the formation of ADP-L-glycero-D-manno-heptose. Mutants, which make incomplete LPS, are known as rough mutants. Salmonella typhimurium deep-rough mutants affected in the heptose region of the inner core often show reduced growth rate, sensitivity to high temperature and hypersensitivity to hydrophobic antibiotics. We have cloned the rfaE gene of S. typhimurium. The chromosomal region carrying this gene was isolated by screening a genomic library of S. typhimurium using the complementation of S. typhimurium rfaE mutant. The 2.6-Kb insert in the plasmid pHEPs appears to carry a functional rfaE gene. SL1102 (rfaE543) makes heptose-deficient LPS and has a deep rough phenotype, but pHEPs complement the rfaE543 mutation to give the smooth phenotype. The sensitivity of SL1102 to bacteriophages (P22.c2, Felix-O, Br60) which use LPS as their receptor for adsorption is changed to that of wild-type strain. The permeability barrier of SL1102 to hydrophobic antibiotics (novobiocin) is restored to that of wild-type. LPS produced by SL1102 (rfaE543) carrying pHEPs makes LPS indistinguishable from that of smooth strains. The rfaE gene encoded a polypeptide of 477 amino acid residues highly homologous to the S. enterica rfaE protein (98% identity), E. coli (93% identity), Yersenia pestis (85% identity), Haemophilus influenzae (70% identity) and Helicobacter pyroli (41% identity) with a molecular weight 53 kDa. PMID:12441667

  2. Molecular cloning, characterization, and expression analysis of fatty acid translocase (FAT/CD36) in the pigeon (Columba livia domestica).

    PubMed

    Xie, P; Zhang, A T; Wang, C; Azzam, M M M; Zou, X T

    2012-07-01

    Fatty acid translocase (FAT/CD36) is a transmembrane glycoprotein that plays an important role in transporting long-chain fatty acids. In the current study, a full-length cDNA of FAT/CD36 was first cloned from the intestine of White King pigeon by rapid amplification of cDNA ends (RACE) method. The full-length cDNA of pigeon FAT/CD36 was 2,282 bp, including a 5'-untranslated region of 224 bp, a 3'-untranslated region of 642 bp, and an open reading frame of 1,416 bp encoding a protein of 471 amino acids with the predicted molecular weight of 52.7 kDa. Sequence comparison indicated that FAT/CD36 of pigeon had high identity with other avian FAT/CD36. Using quantitative real-time PCR, expression of FAT/CD36 was the greatest in the duodenum at 28 d posthatch, and in the jejunum, the expression of FAT/CD36 at 14 d posthatch was greater than at 8 d but the same as 28 d posthatch. However, in the ileum, expression of FAT/CD36 peaked at embryonic d 15 and 8 d posthatch. The effects of long-chain fatty acids on pigeon FAT/CD36 and peroxisome proliferator activated receptor γ (PPARγ) mRNA expression were also investigated in vitro. It showed that a low concentration (5 μM) of oleic acid, palmitic acid, and linoleic acid can significantly increase FAT/CD36 and PPARγ mRNA level in pigeon jejunum. However, for linolenic acid or arachidonic acid, the induction of both gene expressions needed a higher concentration (50 μM or 250 μM). Two hundred and 50 μM palmitic acid was shown to suppress FAT/CD36 gene expression. The results suggest that FAT/CD36 may be a representative of intestine development in pigeon, and it could be regulated by long-chain fatty acids via PPARγ pathway.

  3. Molecular cloning, expression and characterization of albolamin: a type P-IIa snake venom metalloproteinase from green pit viper (Cryptelytrops albolabris).

    PubMed

    Jangprasert, Panchalee; Rojnuckarin, Ponlapat

    2014-03-01

    Snake venom metalloproteinases (SVMPs) can damage vessel wall, degrade clotting factors, inhibit integrins and block platelet functions. Studying them not only gives us deeper insights in pathogenesis of snakebites, but also potentially yields novel therapeutic agents. Here, we discovered a clone of an RGD-containing SVMP from the green pit viper (Cryptelytrops albolabris) venom gland cDNA library. Sequence analysis revealed that it belonged to the P-IIa subclass of SVMP comprising signal peptide, prodomain, metalloproteinase and disintegrin. Compared with other P-II SVMPs, it contained 2 additional conserved cysteines that were predicted to prevent the release of disintegrin from the metalloproteinase domain in the mature protein. The N-terminal histidine-tagged construct of metalloproteinase and disintegrin domains of albolamin was inserted into the pPICZαA vector and expressed in Pichia pastoris. The recombinant protein molecular weight was approximately 35 kDa on Western blot probed with anti-polyhistidine antibody. The recombinant albolamin could digest human type IV collagen starting within 15 min after incubation. In addition, it dose-dependently inhibited collagen-induced platelet aggregation with the IC50 of 1.8 μM. However, there was no effect on ADP-induced platelet aggregation. Therefore, the inhibition mechanism is probably through blocking collagen receptor(s). Albolamin activities probably contributed to pathology of green pit viper bites. Its disintegrin domain deserves further studies for the potential to be a useful agent affecting platelet functions. PMID:24380672

  4. Melatonin receptors in a pleuronectiform species, Solea senegalensis: Cloning, tissue expression, day-night and seasonal variations.

    PubMed

    Confente, Francesca; Rendón, María Carmen; Besseau, Laurence; Falcón, Jack; Muñoz-Cueto, José A

    2010-06-01

    Melatonin receptors are expressed in neural and peripheral tissues and mediate melatonin actions on the synchronization of circadian and circannual rhythms. In this study we have cloned three melatonin receptor subtypes (MT1, MT2 and Mel1c) in the Senegalese sole and analyzed their central and peripheral tissue distribution. The full-length MT1 (1452 nt), MT2 (1728 nt) and Mel1c (1980 nt) cDNAs encode different proteins of 345, 373, 355 amino acids, respectively. They were mainly expressed in retina, brain and pituitary, but MT1 was also expressed in gill, liver, intestine, kidney, spleen, heart and skin. At peripheral level, MT2 expression was only evident in gill, kidney and skin whereas Mel1c expression was restricted to the muscle and skin. This pattern of expression was not markedly different between sexes or among the times of day analyzed. The real-time quantitative PCR analyses showed that MT1 displayed higher expression at night than during the day in the retina and optic tectum. Seasonal MT1 expression was characterized by higher mRNA levels in spring and autumn equinoxes for the retina, and in winter and summer solstices for the optic tectum. An almost similar expression profile was found for MT2, but differences were less conspicuous. No day-night differences in MT1 and MT2 expression were observed in the pituitary but a seasonal variation was detected, being mRNA levels higher in summer for both receptors. Mel1c expression did not exhibit significant day-night variation in retina and optic tectum but showed seasonal variations, with higher transcript levels in summer (optic tectum) and autumn (retina). Our results suggest that day-night and seasonal variations in melatonin receptor expression could also be mediating circadian and circannual rhythms in sole.

  5. Expression cloning and chromosomal mapping of the leukocyte activation antigen CD97, a new seven-span transmembrane molecule of the secretin receptor superfamily with an unusual extracellular domain

    SciTech Connect

    Hamann, J. |; Hamann, D.; Lier, R.A.W.

    1995-08-15

    CD97 is a monomeric glycoprotein of 75 to 85 kDa that is induced rapidly on the surface of most leukocytes upon activation. We herein report the isolation of a cDNA encoding human CD97 by expression cloning in COS cells. The 3-kb cDNA clone encodes a mature polypeptide chain of 722 amino acids with a predicted molecular mass of 79 kDa. Within the C-terminal part of the protein, a region with seven hydrophobic segments was identified, suggesting that CD97 is a seven-span transmembrane molecule. Sequence comparison indicates that CD97 is the first leukocyte Ag in a recently described superfamily that includes the receptors for secretin, calcitonin, and other mammalian and insect peptide hormones. Different from these receptors, CD97 has an extended extracellular region of 433 amino acids that possesses three N-terminal epidermal growth factor-like domains, two of them with a calcium-binding site, and single Arg-Gly-Asp (RGD) motif. The existence of structural elements characteristic for extracellular matrix proteins in a seven-span transmembrane molecule makes CD97 a receptor potentially involved in both adhesion and signaling processes early after leukocyte activation. The gene encoding CD97 is localized on chromosome 19 (19p13.12-13.2).

  6. Directed Molecular Evolution of an Engineered Gammaretroviral Envelope Protein with Dual Receptor Use Shows Stable Maintenance of Both Receptor Specificities

    PubMed Central

    Friis, Kristina Pagh; Iturrioz, Xavier; Thomsen, Jonas; Alvear-Perez, Rodrigo; Bahrami, Shervin; Llorens-Cortes, Catherine

    2015-01-01

    ABSTRACT We have previously reported the construction of a murine leukemia virus-based replication-competent gammaretrovirus (SL3-AP) capable of utilizing the human G protein-coupled receptor APJ (hAPJ) as its entry receptor and its natural receptor, the murine Xpr1 receptor, with equal affinities. The apelin receptor has previously been shown to function as a coreceptor for HIV-1, and thus, adaptation of the viral vector to this receptor is of significant interest. Here, we report the molecular evolution of the SL3-AP envelope protein when the virus is cultured in cells harboring either the Xpr1 or the hAPJ receptor. Interestingly, the dual receptor affinity is maintained even after 10 passages in these cells. At the same time, the chimeric viral envelope protein evolves in a distinct pattern in the apelin cassette when passaged on D17 cells expressing hAPJ in three separate molecular evolution studies. This pattern reflects selection for reduced ligand-receptor interaction and is compatible with a model in which SL3-AP has evolved not to activate hAPJ receptor internalization. IMPORTANCE Few successful examples of engineered retargeting of a retroviral vector exist. The engineered SL3-AP envelope is capable of utilizing either the murine Xpr1 or the human APJ receptor for entry. In addition, SL3-AP is the first example of an engineered retrovirus retaining its dual tropism after several rounds of passaging on cells expressing only one of its receptors. We demonstrate that the virus evolves toward reduced ligand-receptor affinity, which sheds new light on virus adaptation. We provide indirect evidence that such reduced affinity leads to reduced receptor internalization and propose a novel model in which too rapid receptor internalization may decrease virus entry. PMID:26608314

  7. Molecular analysis of the bovine anaphylatoxin C5a receptor

    PubMed Central

    Nemali, Sailasree; Siemsen, Daniel W.; Nelson, Laura K.; Bunger, Peggy L.; Faulkner, Craig L.; Rainard, Pascal; Gauss, Katherine A.; Jutila, Mark A.; Quinn, Mark T.

    2008-01-01

    Recruitment of phagocytes to inflammatory sites involves the coordinated action of several chemoattractants, including the anaphylatoxin C5a. While the C5a receptor (C5aR) has been well characterized in humans and rodents, little is known about the bovine C5aR. Here, we report cloning of bovine C5R1, the gene encoding bovine C5aR. We also analyzed genomic sequence upstream of the C5R1 translation start site. Although the bovine C5aR amino acid sequence was well conserved among species, significant differences in conserved features were found, including major differences in the N terminus, intracellular loop 3, and transmembrane domain VII. Analysis of C5aR expression by flow cytometry and confocal microscopy demonstrated high levels of C5aR on all bovine neutrophils and a subset of bovine monocytes. C5aR was not expressed on resting or activated bovine lymphocytes, although C5aR message was present in these cells. C5aR was also expressed on a small subset of bovine mammary epithelial cells. Pharmacological analysis of bovine C5aR-mediated responses showed that bovine C5a and C5adesArg both induced dose-dependent calcium fluxes and chemotaxis in bovine neutrophils, with similar efficacy for both agonists. Treatment of bovine neutrophils with C5a or C5adesArg resulted in homologous desensitization of bovine C5aR and cross-desensitization to interleukin 8 (IL-8) and platelet-activating factor (PAF); whereas, treatment with IL-8 or PAF did not cross-desensitize the cells to C5a or C5adesArg. Overall, these studies provide important information regarding distinct structural and functional features that may contribute to the unique pharmacological properties of bovine C5aR. PMID:18480166

  8. Molecular cloning and characterization of a C-type lectin in yellow catfish Tachysurus fulvidraco.

    PubMed

    Ke, F; Zhang, H B; Wang, Y; Hou, L F; Dong, H J; Wang, Z F; Pan, G W; Cao, X Y

    2016-09-01

    This study represents the first report of a C-type lectin (ctl) in yellow catfish Tachysurus fulvidraco. The complete sequence of ctl complementary (c)DNA consisted of 685 nucleotides. The open reading frame potentially encoded a protein of 177 amino acids with a calculated molecular mass of c.y 20.204 kDa. The deduced amino-acid sequence contained a signal peptide and a single carbohydrate recognition domain with four cysteine residues and GlnProAsp (QPD) and TrpAsnAsp (WND) motifs. Ctl showed the highest identity (56.0%) to the predicted lactose binding lectin from channel catfish Ictalurus punctatus. Quantitative real-time (qrt)-PCR analysis showed that ctl messenger (m)RNA was constitutively expressed in all examined tissues in normal fish, with high expression in trunk kidney and head kidney, which was increased following Aeromonas hydrophila challenge in a duration-dependent manner. Purified recombinant Ctl (rCtl) from Escherichia coli BL21 was able to bind and agglutinate Gram-positive and Gram-negative bacteria in a calcium-dependent manner. These results suggested that Ctl might be a C-type lectin of T. fulvidraco involved in innate immune responses as receptors (PRR). PMID:27418461

  9. Molecular and functional characterization of the first tick CAP2b (periviscerokinin) receptor from Rhipicephalus (Boophilus) microplus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The cDNA of the receptor for CAP2b/periviscerokinin (PVK) neuropeptides, designated Rhimi-CAP2b-R, was cloned from synganglia of tick Rhipicephalus (Boophilus) microplus. This receptor is the ortholog of the insect CAP2b/PVK receptor, as concluded from analyses of the predicted protein sequence, ph...

  10. Retinoic acid receptors: from molecular mechanisms to cancer therapy.

    PubMed

    di Masi, Alessandra; Leboffe, Loris; De Marinis, Elisabetta; Pagano, Francesca; Cicconi, Laura; Rochette-Egly, Cécile; Lo-Coco, Francesco; Ascenzi, Paolo; Nervi, Clara

    2015-02-01

    Retinoic acid (RA), the major bioactive metabolite of retinol or vitamin A, induces a spectrum of pleiotropic effects in cell growth and differentiation that are relevant for embryonic development and adult physiology. The RA activity is mediated primarily by members of the retinoic acid receptor (RAR) subfamily, namely RARα, RARβ and RARγ, which belong to the nuclear receptor (NR) superfamily of transcription factors. RARs form heterodimers with members of the retinoid X receptor (RXR) subfamily and act as ligand-regulated transcription factors through binding specific RA response elements (RAREs) located in target genes promoters. RARs also have non-genomic effects and activate kinase signaling pathways, which fine-tune the transcription of the RA target genes. The disruption of RA signaling pathways is thought to underlie the etiology of a number of hematological and non-hematological malignancies, including leukemias, skin cancer, head/neck cancer, lung cancer, breast cancer, ovarian cancer, prostate cancer, renal cell carcinoma, pancreatic cancer, liver cancer, glioblastoma and neuroblastoma. Of note, RA and its derivatives (retinoids) are employed as potential chemotherapeutic or chemopreventive agents because of their differentiation, anti-proliferative, pro-apoptotic, and anti-oxidant effects. In humans, retinoids reverse premalignant epithelial lesions, induce the differentiation of myeloid normal and leukemic cells, and prevent lung, liver, and breast cancer. Here, we provide an overview of the biochemical and molecular mechanisms that regulate the RA and retinoid signaling pathways. Moreover, mechanisms through which deregulation of RA signaling pathways ultimately impact on cancer are examined. Finally, the therapeutic effects of retinoids are reported. PMID:25543955

  11. Molecular cloning and characterization of multiple isoforms of the snowdrop (Galanthus nivalis L.) lectin.

    PubMed

    Van Damme, E J; De Clercq, N; Claessens, F; Hemschoote, K; Peeters, B; Peumans, W J

    1991-12-01

    Screening of a copy-DNA (cDNA) library constructed from RNA isolated from young developing ovaries of snowdrop (Galanthus nivalis) resulted in the isolation of five lectin clones which clearly differed from each other with regard to their nucleotide sequence and deduced amino-acid sequence. Sequence comparison between the coding regions of different lectin cDNAs revealed the highest homology between lectin clones LECGNA 3 and LECGNA 5, showing 96.4% and 93.6% similarity at the nucleotide level and at the deduced amino-acid level, respectively, whereas lectin clones LECGNA 1 and LECGNA 3 showed the lowest homology of 81.6% and 68.6% for the nucleotide sequence and the amino-acid sequence, respectively. Only very few lectin cDNA clones containing a polyadenylated tail could be isolated. Moreover all these cDNA clones were derived from isolectin 3 and showed some variability within the length of the 3' untranslated region. The major transcription initiation site was located 30 bases upstream from the AUG codon as could be deduced from primer-extension analysis. Taking into account the small 5' untranslated region of the lectin clones, the size of the lectin mRNA, which is approx. 780 nucleotides as determined by Northern blot analysis, is in good agreement with the length of the cDNA clones isolated. Besides the ovary tissue, both the leaf and the flower tissue were also shown to express the lectin mRNA in a flowering snowdrop plant.

  12. Molecular mechanisms of gonadotropin-releasing hormone receptor gene regulation.

    PubMed

    Norwitz, E R; Jeong, K H; Chin, W W

    1999-01-01

    GnRH plays a critical role in regulating mammalian reproductive development and function. At the level of the anterior pituitary, GnRH binds to the GnRH receptor (GnRHR) on the cell surface of pituitary gonadotropes. Here, it activates intracellular signal transduction pathways to effect both the synthesis and intermittent release of the gonadotropins LH and FSH. These hormones then enter the systemic circulation to regulate gonadal function, including steroid hormone synthesis and gametogenesis. The response of pituitary gonadotropes to GnRH correlates directly with the concentration of GnRHR on the cell surface, which is mediated, at least in part, at the level of gene expression. A number of endocrine, paracrine, and autocrine factors are known to regulate GnRHR gene expression. This article reviews in detail the role of the GnRHR in the hypothalamic-pituitary-gonadal axis and the factors mediating expression of this gene. A better understanding of the molecular mechanisms that regulate transcription of the GnRHR gene will further our knowledge about the role of this receptor in mammalian reproductive physiology in health and disease.

  13. Molecular Insights for Optimizing T Cell Receptor Specificity Against Cancer

    PubMed Central

    Hebeisen, Michael; Oberle, Susanne G.; Presotto, Danilo; Speiser, Daniel E.; Zehn, Dietmar; Rufer, Nathalie

    2013-01-01

    Cytotoxic CD8 T cells mediate immunity to pathogens and they are able to eliminate malignant cells. Immunity to viruses and bacteria primarily involves CD8 T cells bearing high affinity T cell receptors (TCRs), which are specific to pathogen-derived (non-self) antigens. Given the thorough elimination of high affinity self/tumor-antigen reactive T cells by central and peripheral tolerance mechanisms, anti-cancer immunity mostly depends on TCRs with intermediate-to-low affinity for self-antigens. Because of this, a promising novel therapeutic approach to increase the efficacy of tumor-reactive T cells is to engineer their TCRs, with the aim to enhance their binding kinetics to pMHC complexes, or to directly manipulate the TCR-signaling cascades. Such manipulations require a detailed knowledge on how pMHC-TCR and co-receptors binding kinetics impact the T cell response. In this review, we present the current knowledge in this field. We discuss future challenges in identifying and targeting the molecular mechanisms to enhance the function of natural or TCR-affinity optimized T cells, and we provide perspectives for the development of protective anti-tumor T cell responses. PMID:23801991

  14. Molecular evolution of the mammalian alpha 2B adrenergic receptor.

    PubMed

    Madsen, Ole; Willemsen, Diederik; Ursing, Björn M; Arnason, Ulfur; de Jong, Wilfried W

    2002-12-01

    The alpha 2B adrenergic receptor (A2AB) is a heptahelical G protein-coupled receptor for catecholamines. We compared the almost complete coding region (about 1,175 bp) of the A2AB gene from 48 mammalian species, including eight newly determined sequences, representing all the 18 eutherian and two marsupial orders. Comparison of the encoded proteins reveals that residues thought to be involved in agonist binding are highly conserved, as are the regions playing a role in G protein-coupling. The three extracellular loops are generally more variable than the transmembrane domains and two of the intracellular loops, indicating a lower functional constraint. However, the greatest variation is observed in the very long, third intracellular loop, where only a few residues and a polyglutamyl tract are preserved. Although this polyglutamyl domain displays a great variation in length, its presence in all described A2ABs confirms its proposed role in agonist-dependent phosphorylation of the third intracellular loop. Phylogenetic analyses of the A2AB data set, including Bayesian methods, recognized the superordinal clades Afrotheria, Laurasiatheria, and Euarchontoglires, in agreement with recent molecular evidence, albeit with lower support. Within Afrotheria, A2AB strongly supports the paenungulate clade and the association of the continental African otter shrew with Malagasy tenrecs. Among Laurasiatheria, A2AB confirms the nesting of whales within the artiodactyls, as a sister group to hippopotamus. Within the Euarchontoglires, there is constant support for rodent monophyly. PMID:12446807

  15. Molecular cloning and analysis of Ancylostoma ceylanicum glutamate-cysteine ligase.

    PubMed

    Wiśniewski, Marcin; Lapiński, Maciej; Zdziarska, Anna; Długosz, Ewa; Bąska, Piotr

    2014-08-01

    Glutamate-cysteine ligase (GCL) is a heterodimer enzyme composed of a catalytic subunit (GCLC) and a modifier subunit (GCLM). This enzyme catalyses the synthesis of γ-glutamylcysteine, a precursor of glutathione. cDNAs of the putative glutamate-cysteine ligase catalytic (Ace-GCLC) and modifier subunits (Ace-GCLM) of Ancylostoma ceylanicum were cloned using the RACE-PCR amplification method. The Ace-gclc and Ace-gclm cDNAs encode proteins with 655 and 254 amino acids and calculated molecular masses of 74.76 and 28.51kDa, respectively. The Ace-GCLC amino acid sequence shares about 70% identity and 80% sequence similarity with orthologs in Loa loa, Onchocerca volvulus, Brugia malayi, and Ascaris suum, whereas the Ace-GCLM amino acid sequence has only about 30% sequence identity and 50% similarity to homologous proteins in those species. Real-time PCR analysis of mRNA expression in L3, serum stimulated L3 and adult stages of A. ceylanicum showed the highest level of Ace-GCLC and Ace-GCLM expression occurred in adult worms. No differences were detected among adult hookworms harvested 21 and 35dpi indicating expression of Ace-gclc and Ace-gclm in adult worms is constant during the course of infection. Positive interaction between two subunits of glutamate-cysteine ligase was detected using the yeast two-hybrid system, and by specific enzymatic reaction. Ace-GCL is an intracellular enzyme and is not exposed to the host immune system. Thus, as expected, we did not detect IgG antibodies against Ace-GCLC or Ace-GCLM on days 21, 60 and 120 of A. ceylanicum infection in hamsters. Furthermore, vaccination with one or both antigens did not reduce worm burdens, and resulted in no improvement of clinical parameters (hematocrit and hemoglobin) of infected hamsters. Therefore, due to the significant role of the enzyme in parasite metabolism, our analyses raises hope for the development of a successful new drug against ancylostomiasis based on the specific GCL inhibitor. PMID

  16. SLC26 anion exchangers of guinea pig pancreatic duct: molecular cloning and functional characterization

    PubMed Central

    Stewart, Andrew K.; Shmukler, Boris E.; Vandorpe, David H.; Reimold, Fabian; Heneghan, John F.; Nakakuki, M.; Akhavein, Arash; Ko, Shigeru; Ishiguro, Hiroshi

    2011-01-01

    The secretin-stimulated human pancreatic duct secretes HCO3−-rich fluid essential for normal digestion. Optimal stimulation of pancreatic HCO3− secretion likely requires coupled activities of the cystic fibrosis transmembrane regulator (CFTR) anion channel and apical SLC26 Cl−/HCO3− exchangers. However, whereas stimulated human and guinea pig pancreatic ducts secrete ∼140 mM HCO3− or more, mouse and rat ducts secrete ∼40–70 mM HCO3−. Moreover, the axial distribution and physiological roles of SLC26 anion exchangers in pancreatic duct secretory processes remain controversial and may vary among mammalian species. Thus the property of high HCO3− secretion shared by human and guinea pig pancreatic ducts prompted us to clone from guinea pig pancreatic duct cDNAs encoding Slc26a3, Slc26a6, and Slc26a11 polypeptides. We then functionally characterized these anion transporters in Xenopus oocytes and human embryonic kidney (HEK) 293 cells. In Xenopus oocytes, gpSlc26a3 mediated only Cl−/Cl− exchange and electroneutral Cl−/HCO3− exchange. gpSlc26a6 in Xenopus oocytes mediated Cl−/Cl− exchange and bidirectional exchange of Cl− for oxalate and sulfate, but Cl−/HCO3− exchange was detected only in HEK 293 cells. gpSlc26a11 in Xenopus oocytes exhibited pH-dependent Cl−, oxalate, and sulfate transport but no detectable Cl−/HCO3− exchange. The three gpSlc26 anion transporters exhibited distinct pharmacological profiles of 36Cl− influx, including partial sensitivity to CFTR inhibitors Inh-172 and GlyH101, but only Slc26a11 was inhibited by PPQ-102. This first molecular and functional assessment of recombinant SLC26 anion transporters from guinea pig pancreatic duct enhances our understanding of pancreatic HCO3− secretion in species that share a high HCO3− secretory output. PMID:21593449

  17. Molecular cloning and characterization of cathepsin L from freshwater mussel, Cristaria plicata.

    PubMed

    Hu, Xiaojuan; Hu, Xiangping; Hu, Baoqing; Wen, Chungen; Xie, Yanhai; Wu, Dan; Tao, Zhiying; Li, Aihua; Gao, Qian

    2014-10-01

    Cathepsin L is one of the crucial enzyme superfamilies and involved in the immune responses. The Cathepsin L cDNA and genome of Cristaria plicata(CpCL) was cloned from the hemocytes using degenerate primers by the rapid amplification of cDNA ends (RACE) PCR. The genomic DNA was 9353 bp long and had a total of six introns and seven exons. The full-length cDNA of CpCL was 1144 bp, the cDNA contained a 5' untranslated region (UTR) of 34 nucleotides, the 3' UTR of 108 bp with a canonical polyadenylation signal sequence AATAAA and a polyA tail, and an open reading frame (ORF) of 1002 bp, encoding 333 amino acid residues with 37.65 kDa predicted molecular weight. The theoretical isoelectric point was 8.61. The prepro-cathepsin L was consisted of a typical signal peptide (Met1-Gly20), a pro-region peptide (Leu21-Glu116) and a mature peptide (Tyr117-Val333). Many members of the papain family possessed of a proline residue at position 2 in the mature enzymem, this was also observed in CpCL. The preproprotein included an oxyanion hole (Gln 135), the active center formed by Cys141, His280 and Asn 300, the potential N-glycosylation site (Asn38, Asn 113 and Asn 272) and the conserved GCXGG motifs, which was characteristic of cathepsin, the conserved ERWNIN and GNFD motifs, which were characteristic for cathepsin L. Homology analysis revealed that the CpCL shared 49-87% identity to other known cathepsin L sequences. The phylogenetic tree showed that the CpCL clustered with the invertebrate cathepsin L cysteine proteases, and was closely related to the cathepsin L of Hyriopsis cumingii. The expression of CpCL mRNA was detected in hepatopancreas, hemocytes, mantle, gills and adductor muscle, and the higher expression level was in hepatopancreas. After A. hydrophila stimulation, the expression of the CpCL mRNA was up-regulated in hemocytes and hepatopancreas, and the expression level was significantly lower in gill than one after PBS challenge group. PMID:25038281

  18. Purification, characterization and molecular cloning of human hepatic lysosomal acid lipase.

    PubMed

    Ameis, D; Merkel, M; Eckerskorn, C; Greten, H

    1994-02-01

    Lysosomal acid lipase (LAL) is a hydrolase essential for the intracellular degradation of cholesteryl esters and triacylglycerols. This report describes a multi-step procedure for the purification of LAL from human liver. After solubilization with non-ionic detergent, acid hydrolase activity was purified 17000-fold to apparent homogeneity by sequential chromatography on Concanavalin A Sepharose, carboxymethyl-cellulose, phenyl Superose, Mono S cation exchange and Superose 12 gel-filtration columns. This procedure yielded two silver-staining protein bands of 56 kDa and 41 kDa on SDS/PAGE. Size-exclusion chromatography of the 41-kDa protein indicated that the enzyme was catalytically competent as a monomer of approximately 38 kDa. When assayed in the presence of cholesteryl oleate or trioleoylglycerol, purified acid lipase had Vmax values of 4390 nmol fatty acid.min-1.mg protein and 4756 nmol fatty acid.min-1.mg protein-1, and apparent Km values of 0.142 mM and 0.138 mM, respectively. The purified enzyme was most active at low pH (4.5-5.0) and required non-ionic detergent and ethylene glycol for optimal stability. Incubation of the 41-kDa acid lipase with endoglucosaminidase H reduced the molecular mass by 4-6 kDa, demonstrating Asn-linked glycosylation with high-mannose oligosaccharides. Deglycosylation did not affect enzymic activity, indicating that carbohydrates are not required for LAL activity. Based on partial peptide sequence, an oligonucleotide was synthesized and utilized to isolate LAL cDNA clones from a human liver cDNA library. A full-length LAL cDNA contained 2626 nucleotides and coded for a predicted protein of 372 amino acids, preceded by a 27 residue hydrophobic signal peptide. Hepatic LAL differed from fibroblast acid lipase at the N-terminus and revealed extensive similarities with human gastric lipase and rat lingual lipase, confirming a gene family of acid lipases. Northern hybridization using the complete LAL cDNA as a radiolabeled probe

  19. Molecular cloning and characterization of potato spindle tuber viroid cDNA sequences

    PubMed Central

    Owens, Robert A.; Cress, Dean E.

    1980-01-01

    Double-stranded cDNA has been synthesized from a polyadenylylated potato spindle tuber viroid (PSTV) template and inserted in the Pst I endonuclease site of plasmid pBR322 by using the oligo(dC)·oligo(dG)-tailing procedure. Tetracycline-resistant ampicillin-sensitive transformants contained sequences complementary to PSTV [32P]cDNA, and one recombinant clone (pDC-29) contains a 460-base-pair insert. This cloned double-stranded PSTV cDNA contains the cleavage sites for six restriction endonucleases predicted by the published primary sequence of PSTV as well as one additional site each for Ava I, Hae III, Hpa II, and Sma I. The additional Ava I, Hpa II, and Sma I sites are explained by the presence of a second C-C-C-G-G-G sequence in the cloned double-stranded cDNA. The largest fragment released by Hae III digestion contains approximately 360 base pairs. These results suggest that we have cloned almost the entire sequence of PSTV, but the sequence cloned differs slightly from that published. Hybridization probes derived from pDC-29 insert have allowed detection and preliminary characterization of RNA molecules having the same size as PSTV but the opposite polarity. This RNA is present during PSTV replication in infected tomato cells. Images PMID:16592877

  20. Cloning and characterization of new orphan nuclear receptors and their developmental profiles during Tenebrio metamorphosis.

    PubMed

    Mouillet, J F; Bousquet, F; Sedano, N; Alabouvette, J; Nicolaï, M; Zelus, D; Laudet, V; Delachambre, J

    1999-11-01

    Five PCR fragments corresponding to a part of the DNA-binding domain of different hormone nuclear receptors were isolated from Tenebrio molitor mRNAs. The sequence identity of three of them with known Drosophila nuclear receptors strongly suggests that they are the Tenebrio orthologs of seven-up, DHR3 and beta-FTZ-F1, and thus named Tmsvp, TmHR3 and TmFTZ-F1. The full-length sequences of the other two were established. TmHR78 is either a new receptor of the DHR78 family or the same gene which has evolved rapidly, particularly in the E domain. TmGRF belongs to the GCNF1 family and its in vitro translated product binds to the extended half site TCAAGGTCA with high affinity. The periods of expression of the corresponding transcripts in epidermal cells during Tenebrio metamorphosis were analyzed as a function of 20-hydroxyecdysone titers measured in the hemolymph of the animals taken for RNA extraction. Comparison of the expression profiles of these nuclear receptors with those observed during Drosophila metamorphosis revealed similar temporal correlations as a function of ecdysteroid variations, which further supported the sequence identity data for TmSVP, TmHR3, TmFTZ-F1 and TmHR78.

  1. Molecular cloning of L-JAK, a Janus family protein-tyrosine kinase expressed in natural killer cells and activated leukocytes.

    PubMed Central

    Kawamura, M; McVicar, D W; Johnston, J A; Blake, T B; Chen, Y Q; Lal, B K; Lloyd, A R; Kelvin, D J; Staples, J E; Ortaldo, J R

    1994-01-01

    Protein-tyrosine kinases (PTKs) are critical enzymes for receptor-mediated signaling in lymphocytes. Because natural killer (NK) cells are large granular lymphocytes with specialized effector function, we set out to identify PTKs preferentially expressed in these cells. One such PTK was identified and molecularly cloned. The predicted amino acid sequence shows that this kinase lacks SH2 or SH3 domains typical of src family kinases but has tandem nonidentical catalytic domains, indicating that it is a member of the Janus family of PTKs. Immunoprecipitation using antiserum generated against a peptide corresponding to the deduced amino acid sequence of this gene revealed a kinase with a molecular weight of approximately 125,000. The pattern of expression of this kinase contrasted sharply with that of other Janus kinases, which are ubiquitously expressed. The kinase described in the present study was found to be more limited in its expression; expression was found in NK cells and an NK-like cell line but not in resting T cells or in other tissues. In contrast, stimulated and transformed T cells expressed the gene, suggesting a role in lymphoid activation. Because of its homology and tissue expression, we have tentatively termed this PTK gene L-JAK for leukocyte Janus kinase. Images PMID:8022790

  2. Cloning and expression analysis of nonspecific cytotoxic cell receptor 1 (Ls-NCCRP1) from red snapper (Lutjanus sanguineus).

    PubMed

    Cai, Jia; Wei, Shina; Wang, Bei; Huang, Yucong; Tang, Jufen; Lu, Yishan; Wu, Zaohe; Jian, Jichang

    2013-09-01

    It is well known that nonspecific cytotoxic cells (NCCs) are kinds of natural killer cell mediated innate immune responses in teleosts. The nonspecific cytotoxic cell receptor protein 1 (NCCRP-1) is an important cell surface protein on NCC, which serves crucial functions in target cell recognition and cytotoxicity activation. In the present study, a nonspecific cytotoxic cell receptor protein NCCRP-1 (Ls-NCCRP1) was cloned from red snapper, Lutjanus sanguineus. The Ls-NCCRP1 cDNA is composed of 986bp with a 43bp of 5'-UTR, 702bp open reading frame (ORF) and 241bp 3'-UTR, encoding a polypeptide of 233 amino acids (GenBank accession no: ADK32635). Phylogenetic analysis revealed that Ls-NCCRP1 showed highest similarity to sea bream NCCRP-1. Quantitative real-time PCR (qRT-PCR) analysis showed that Ls-NCCRP1 had relatively high expression level in the head kidney, spleen and liver. After Vibrio alginolyticus infection, transcripts of Ls-NCCRP1 increased and reached its peak at 4h p.i. These results indicated that Ls-NCCRP1 may play an important role in innate immune response to bacteria.

  3. All five cloned human somatostatin receptors (hSSTR1-5) are functionally coupled to adenylyl cyclase.

    PubMed

    Patel, Y C; Greenwood, M T; Warszynska, A; Panetta, R; Srikant, C B

    1994-01-28

    Recent reports have suggested that only some of the cloned somatostatin receptors (SSTRs) are coupled to adenylyl cyclase. These studies have used both stable and transiently transfected cells or cells lacking appropriate Gi alpha and are controversial. To investigate SSTR signalling mechanisms, we have established stably transfected CHO-K1 cells expressing human genes for SSTR1-5. The effect of 0.1-100 nM SST-14 and SST-28 on forskolin (1 microM) stimulated cAMP accumulation was determined and compared to their receptor binding affinities. The 5 expressed hSSTRs bound SST-14 and SST-28 with high affinity (IC50 1.1-2.1 nM for SST-14; IC50 0.25-5.4 nM for SST-28). hSSTR1-4 bound SST-14 > SST-28 whereas hSSTR5 bound SST-28 > SST-14. Radioligand binding to hSSTR1-5 was significantly inhibited by GTP, GTP gamma S and pertussis toxin. Both SST-14 and SST-28 inhibited forskolin-induced cAMP stimulation with ED50 values which paralleled their binding affinities for the individual hSSTR subtypes. These results demonstrate that all 5 human SSTRs are functionally coupled to inhibition of adenylyl cyclase in CHO-K1 cells via pertussis toxin sensitive G proteins.

  4. Leptin receptor gene in the European sea bass (Dicentrarchus labrax): Cloning, phylogeny, tissue distribution and neuroanatomical organization.

    PubMed

    Escobar, Sebastián; Rocha, Ana; Felip, Alicia; Carrillo, Manuel; Zanuy, Silvia; Kah, Olivier; Servili, Arianna

    2016-04-01

    In this study, we report the cloning of three transcripts for leptin receptor in the European sea bass, a marine teleost of economic interest. The two shortest variants, generated by different splice sites, encode all functional extracellular and intracellular domains but missed the transmembrane domain. The resulting proteins are therefore potential soluble binding proteins for leptin. The longest transcript (3605bp), termed sblepr, includes all the essential domains for binding and transduction of the signal. Thus, it is proposed as the ortholog for the human LEPR gene, the main responsible for leptin signaling. Phylogenetic analysis shows the sblepr clustered within the teleost leptin receptor group in 100% of the bootstrap replicates. The neuroanatomical localization of sblepr expressing cells has been assessed by in situ hybridization in brains of sea bass of both sexes during their first sexual maturation. At histological level, the distribution pattern of sblepr expressing cells in the brain shows no clear differences regarding sex or reproductive season. Transcripts of the sblepr have a widespread distribution throughout the forebrain and midbrain until the caudal portion of the hypothalamus. A high hybridization signal is detected in the telencephalon, preoptic area, medial basal and caudal hypothalamus and in the pituitary gland. In a more caudal region, sblepr expressing cells are identified in the longitudinal torus. The expression pattern observed for sblepr suggests that in sea bass, leptin is very likely to be involved in the control of food intake, energy reserves and reproduction. PMID:26979276

  5. Construction and characterization of a human T-cell lymphotropic virus type 3 infectious molecular clone.

    PubMed

    Chevalier, Sébastien Alain; Ko, Nga Ling; Calattini, Sara; Mallet, Adeline; Prévost, Marie-Christine; Kehn, Kylene; Brady, John N; Kashanchi, Fatah; Gessain, Antoine; Mahieux, Renaud

    2008-07-01

    We and others have uncovered the existence of human T-cell lymphotropic virus type 3 (HTLV-3). We have now generated an HTLV-3 proviral clone. We established that gag, env, pol, pro, and tax/rex as well as minus-strand mRNAs are present in cells transfected with the HTLV-3 clone. HTLV-3 p24(gag) protein is detected in the cell culture supernatant. Transfection of 293T-long terminal repeat (LTR)-green fluorescent protein (GFP) cells with the HTLV-3 clone promotes formation of syncytia, a hallmark of Env expression, together with the appearance of fluorescent cells, demonstrating that Tax is expressed. Viral particles are visible by electron microscopy. These particles are infectious, as demonstrated by infection experiments with purified virions.

  6. Molecular cloning and characterization of the human WISP-2/CCN5 gene promoter reveal its upregulation by oestrogens.

    PubMed

    Fritah, Asmaà; Redeuilh, Gérard; Sabbah, Michèle

    2006-12-01

    Wnt-1-induced signalling pathway protein-2 (WISP-2)/connective tissue growth factor/cysteine-rich 61/nephroblastoma overexpressed (CCN)5 is a member of the CCN family of growth factors and was identified as an oestrogen- inducible gene in the MCF-7 cell line. However, the role of WISP-2/CCN5 in breast carcinogenesis remains unclear. In this study, we examined the mechanism by which oestrogens regulate the expression of human (h) Wnt-1 induced signalling pathway protein (WISP-2)/CCN5. Real-time RT-PCR showed that hWISP-2/CCN5 mRNA transcripts level is upregulated by oestrogens in the oestrogen receptor-positive human breast cancer cell lines MCF-7, T47D and ZR-75.1. Cloning of a 1.9 kb fragment of the hWISP-2/CCN5 5'-flanking sequence and subsequent analysis of potential transcription factor-binding sites identified a functional oestrogen response element site located between - 581 and - 569 upstream from the oestrogen-induced transcription start site. Transient transfections of MCF-7 cells with the cloned fragment showed that oestradiol caused an increase in reporter gene activity, which was inhibited by anti-oestrogens ICI 182 780 and 4-hydroxytamoxifen. Chromatin immunoprecipitation analysis revealed an oestradiol-dependent recruitment of the oestrogen receptor alpha to the oestrogen- responsive region of the hWISP-2/CCN5 gene promoter. We also showed that endogenous CREB-binding protein (CBP) and p21(WAF1/CIP1) are recruited to the chromosomal hWISP-2/CCN5 promoter in MCF-7 cells in an oestrogen-dependent manner, suggesting that CBP and p21(WAF1/CIP1) participate in the oestrogen receptor alpha-mediated transcriptional control of the hWISP-2/CCN5 gene.

  7. Molecular cloning and expression analysis of a gene for sucrose transporter from pear (Pyrus bretschneideri Rehd.) fruit.

    PubMed

    Zhang, Huping; Zhang, Shujun; Qin, Gaihua; Wang, Lifen; Wu, Tao; Qi, Kaijie; Zhang, Shaoling

    2013-12-01

    Here we report the cloning of a sucrose transporter cDNA from pear (Pyrus bretschneideri Rehd. cv 'Yali') fruit and an analysis of the expression of the gene. A cDNA clone, designated PbSUT1 was identified as a sucrose transporter cDNA from its sequence homology at the amino acid level to sucrose transporters that have been cloned from other higher plant species. PbSUT1 potentially encoded a protein of 499 amino acid residues with a predicted molecular mass of 53.4 kDa and an isoelectric point (pI) of 9.21. Phylogenetic analysis revealed that the PbSUT1 belonged to type III SUTs and was more closely related to the MdSUT1 from apple fruit. Some major facilitator superfamily (MFS)-specific sequence motifs were found in the predicted PbSUT1 peptides, and an MFS_1 domain was located at the amino acid positions of 29-447 of the sequence. A study of gene expression along fruit development showed that PbSUT1 transcripts are present at all stages but significantly increase before fruit enlargement and during the ripening process with increasing sucrose levels. In contrast, the expression levels don't change much during the period of rapid fruit growth. This work shows that sucrose transporter may play a role in the accumulation of sugars during maturation and in maintaining the internal cellular distribution.

  8. Molecular Cloning, Expression and Genome Organization of Channel Catfish (Ictalurus punctatus) Matrix Metalloproteinase-9

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In the course of studying pathogenesis of enteric septicemia of catfish, we noted that channel catfish matrix metalloproteinase-9 (MMP-9) gene was up-regulated after Edwardsiella ictaluri infection. In this study, we cloned, sequenced using the RACE (rapid amplification of cDNA ends) method and cha...

  9. Molecular Cloning and Sequencing of Hemoglobin-Beta Gene of Channel Catfish, Ictalurus Punctatus Rafinesque

    Technology Transfer Automated Retrieval System (TEKTRAN)

    : Hemoglobin-y gene of channel catfish , lctalurus punctatus, was cloned and sequenced . Total RNA from head kidneys was isolated, reverse transcribed and amplified . The sequence of the channel catfish hemoglobin-y gene consists of 600 nucleotides . Analysis of the nucleotide sequence reveals one o...

  10. Molecular cloning of Reteplase and its expression in E. coli using tac promoter

    PubMed Central

    Aghaabdollahian, Safieh; Rabbani, Mohammad; Ghaedi, Kamran; Sadeghi, Hamid Mir Mohammad

    2014-01-01

    Background and Aims: This study aimed to clone and express the reteplase cDNA, a thrombolytic agent used for the treatment of acute myocardial infarction and stroke, in E. coli, utilizing tac promoter for its expression. Materials and Methods: Reteplase cDNA was amplified by polymerase chain reaction (PCR) with designed primers. The product was then cloned into pTZ57R plasmid. The cloned cDNA was digested out and ligated into pGEX-5x-1 expression vector. The presence of the insert was confirmed by restriction digestion. By using 0.2, 0.5 and 1 mM isopropyl beta-D thiogalactopyranoside (IPTG), expression of reteplase was induced in E. coli TOP10 cells and analyzed by SDS-PAGE. Results: Electrophoresis of PCR product and also double digested recombinant pTZ57R plasmid, also, pGEX-5x-1 vector, showed a 1068bp band of reteplase. SDS-PAGE analysis showed a 60 KDa band of protein product induced with different concentrations of IPTG. Conclusion: In the present study, reteplase cDNA was successfully cloned and expressed using tac promoter. This vector will be used for the optimization of the expression of reteplase in E. coli. PMID:25298959

  11. Molecular Cloning and Characterisation of Heparanase mRNA in Porcine Placenta Throughout Gestation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The placenta contains a complex extracellular matrix composed of several glycosaminoglycans including heparan sulfate (HS). Heparanase (HPSE) is an endoglycosidase that specifically degrades HS. The objective of this study was to clone cDNA encoding porcine HPSE and characterize the expression lev...

  12. Molecular evidence for the existence of an aryl hydrocarbon receptor pathway in scallops Chlamys farreri.

    PubMed

    Cai, Yuefeng; Pan, Luqing; Miao, Jingjing

    2016-01-01

    The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that controls the expression of a diverse set of genes. In this study we cloned full-length cDNAs encoding an AhR homologue (designated CfAhR, Accession number: FJ588640) from scallop Chlamys farreri. The CfAhR sequence was constituted by an open reading frame (ORF) of 2466bp encoding 821 amino acids. The predicted molecular weight was 93.0kDa. The CfAhR showed a high conservation of the residues and domains essential to the function of AhR, including basic helix-loop-helix (bHLH) and Per-ARNT-Sim (PAS) domains. Phylogenetic analysis demonstrated that it was clustered within the invertebrate AhR branch. CfAhR expression was detected in gill, digestive gland, ovary, spermary, mantle and adductor, and the highest transcription level was observed in gill. Recombinant plasmid CfAhR-pET32a (designated rCfAhR) was successfully expressed in Escherichia coli BL21. To investigate the molecular detoxification mechanism of benzo(a)pyrene (BaP) detoxification-related genes (AhR; aryl hydrocarbon receptor nuclear translocator, ARNT; heat shock protein 90, HSP90; cytochrome P450 1A1, CYP1A1; glutathione S-transferase pi, GST-pi and P glycoprotein, Pgp) in C. farreri gill, real-time quantitative PCR analysis revealed that the mRNA expression level of CfAhR, xenobiotic-metabolizing enzymes and efflux transporters was induced by BaP and was sensitive to BaP exposure time and concentration, suggesting that BaP influenced the expression of a putative AhR/ARNT signaling pathway in scallops. Our results support the possibility that CfAhR genes are early molecular indicators of BaP through a putative CYP signaling pathway in marine bivalves.

  13. Molecular Recognition of Paired Receptors in the Immune System

    PubMed Central

    Kuroki, Kimiko; Furukawa, Atsushi; Maenaka, Katsumi

    2012-01-01

    Cell surface receptors are responsible for regulating cellular function on the front line, the cell membrane. Interestingly, accumulating evidence clearly reveals that the members of cell surface receptor families have very similar extracellular ligand-binding regions but opposite signaling systems, either inhibitory or stimulatory. These receptors are designated as paired receptors. Paired receptors often recognize not only physiological ligands but also non-self ligands, such as viral and bacterial products, to fight infections. In this review, we introduce several representative examples of paired receptors, focusing on two major structural superfamilies, the immunoglobulin-like and the C-type lectin-like receptors, and explain how these receptors distinguish self and non-self ligands to maintain homeostasis in the immune system. We further discuss the evolutionary aspects of these receptors as well as the potential drug targets for regulating diseases. PMID:23293633

  14. Subtype selectivity of peptide analogs for all five cloned human somatostatin receptors (hsstr 1-5).

    PubMed

    Patel, Y C; Srikant, C B

    1994-12-01

    Recent reports (Raynor et al) have claimed the identification of potent somatostatin (SST) agonists exhibiting binding affinities of 1-2 pM and up to 30,000 fold binding selectivity for several of the 5 cloned sstr subtypes. These conclusions, however, are based on binding comparisons of sstr subtypes from different species expressed in different cell lines and studied with different radioligands. To eliminate the effect of species and/or methodological variations, we have investigated agonist selectivity of 32 synthetic SST analogs for all 5 hsstrs stably expressed in CHO-K1 cells under identical binding conditions. We show that hsstr2, 3, 5 react potently with hexapeptide as well as cyclic and linear octapeptide analogs and belong to a similar sstr subclass. hsstr1 and 4 react poorly with these analogs and belong to a separate subclass. The present generation of SST analogs exhibit a modest-50 fold increase in binding potency compared to SST-14 for 2 subtypes (hsstr2, 3), and relative selectivity for only 1 subtype (hsstr2) which is at best only 35 fold. The potency and degree of selectivity of these analogs is several orders of magnitude less than that reported earlier and suggests the need for caution in using these compounds as putative superagonists or subtype selective compounds for any of the individual sstrs.

  15. Estrogen Receptor-β Expression in Invasive Breast Cancer in Relation to Molecular Phenotype: Results from the Nurses’ Health Study

    PubMed Central

    Marotti, Jonathan; Collins, Laura C.; Hu, Rong; Tamimi, Rulla M.

    2011-01-01

    The expression of estrogen receptor-alpha (ER-α) and related genes has emerged as one of the major determinants of molecular classification of invasive breast cancers. However, patterns of expression of a second estrogen receptor, estrogen receptor-beta (ER-β), have not been previously evaluated in a large population-based study. Therefore, we examined ER-β expression in a large population of women with breast cancer to assess its relationship to molecular categories of invasive breast cancer. We constructed tissue microarrays from paraffin blocks of 3,093 breast cancers that developed in women enrolled in the Nurses' Health Study. Tissue microarray sections were immunostained for ER-α, progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), cytokeratin 5/6 and epidermal growth factor receptor (EGFR). Cancers were categorized as luminal A (ER-α+ and/or PR+ and HER2−); luminal B (ER-α+ and/or PR+ and HER2+); HER2 (ER-α− and PR− and HER2+); and basal-like (ER-α−, PR−, HER2− and EGFR or cytokeratin 5/6+). Tissue microarray sections were also immunostained with a monoclonal antibody to ER-β (ER-β1, clone PPG5/1/0, Serotec). The relationship between expression of ER-β to molecular class of invasive breast cancer was analyzed. Overall, 68% of the invasive breast carcinomas were ER-β positive. Expression of ER-β was significantly associated with expression of ER-α (p<0.0001) and PR (p<0.0001), and was inversely related to expression of HER2 (p=0.004), CK5/6 (p=0.02) and EGFR (p=0.006). Among 2,170 invasive cancers with complete immunophenotypic data, 73% were luminal A, 5% luminal B, 6 % HER2 and 11% basal-like. ER-β expression was significantly related to molecular category (p<0.0001) and was more common in luminal A (72% of cases) and B (68% of cases) than in HER2 or basal-like types. However, despite their being defined by the absence of ER-α expression, 55% of HER2-type and 60% of basal-like cancers showed expression of

  16. Molecular cloning, genomic organization, chromosomal mapping and subcellular localization of mouse PAP7: a PBR and PKA-RIalpha associated protein.

    PubMed

    Liu, Jun; Cavalli, Luciane R; Haddad, Bassem R; Papadopoulos, Vassilios

    2003-04-10

    A mouse protein that interacts with the peripheral-type benzodiazepine receptor (PBR) and the cAMP-dependent protein kinase A (PKA) regulatory subunit RIalpha (PKA-RIalpha), named PBR and PKA associated protein 7 (PAP7) was identified and shown to be involved in hormone-induced steroid biosynthesis in testicular Leydig cells. In the present study, mouse PAP7 cDNA was extended by 5'-rapid amplification of cDNA ends; and a 3432 bp sequence, encoding a 525-amino-acid protein with a calculated molecular weight of 60 kDa, was re-assembled. Mouse and human PAP7 share an 85% amino acid identity and contain a conserved acyl-CoA-binding protein/diazepam binding inhibitor (ACBP/DBI) motif. ACBP/DBI has been identified as the endogenous PBR ligand able to stimulate mitochondrial steroid formation in all steroidogenic cells. The full-length mouse PAP7 gene was cloned and assembled by screening a BAC clone, polymerase chain reaction and searching the mouse genome database. The gene is approximately 29 kb in length and includes eight exons and seven introns. Although it is shorter than the human PAP7 gene, all exons are conserved between the mouse and human. The mouse PAP7 gene was mapped to chromosome 1H3-5 by fluorescence in situ hybridization in agreement with in silico search of the mouse genome database that mapped the PAP7 cDNA sequence to the 1H4 area. Immunofluorescence confocal microscopy demonstrated that PAP7 is mainly localized in the trans-Golgi apparatus and mitochondria in mouse tumor Leydig cells, in agreement with its proposed function in targeting the PKA isoenzyme to organelles rich in PBR, i.e. mitochondria, where phosphorylation of specific protein substrates mediates the hormone-induced steroid formation.

  17. Cloning of murine IL-22 receptor alpha 2 and comparison with its human counterpart.

    PubMed

    Weiss, B; Wolk, K; Grünberg, B H; Volk, H-D; Sterry, W; Asadullah, K; Sabat, R

    2004-08-01

    We have identified the mouse and rat homologs of human interleukin-22 receptor alpha 2 (IL-22R alpha 2) and compared the localization, structure, and expression of the encoding murine and human genes. The mouse IL-22R alpha 2-encoding gene is located on chromosome 10A3 between, like in human, the genes for interferon-gamma R1 and IL-20R1. It spans a region of approximately 10 kb therefore being three times shorter than the human gene. Although the overall gene structure in both species is similar, the mouse gene lacks a counterpart to the third coding exon of the human gene known to be alternatively spliced. Like in human, mouse and rat IL-22R alpha 2 exist only as soluble receptors as deduced from the lack of transmembrane and intracellular domains encoding sequences. Quantitative expression analyses showed, analogically to the human system, a limited tissue distribution of mouse IL-22R alpha 2 mRNA. Differential modulation of IL-22R alpha 2 mRNA expression was observed upon systemic inflammation in mice in spleen, thymus, and lymph node.

  18. Cloning, characterization, expression, and feeding response of thyrotropin receptor in largemouth bass (Micropterus salmoides).

    PubMed

    Gao, Y L; Song, W; Jiang, L L; Mao, M X; Wang, C L; Ge, C T; Qian, G Y

    2016-01-01

    Thyrotropin receptor (TSHR) is a G-protein-coupled receptor that regulates the synthesis, storage, and secretion of thyroid hormones in the thyroid tissue. The aims of the present study were to characterize the full-length TSHR cDNA in largemouth bass (Micropterus salmoides), and to determine the TSHR gene transcription levels in different tissues. In addition, the response of TSHR transcription levels to daily feeding in thyroid tissue was investigated. The results showed that the full-length cDNA sequence was 2743 bp with an open reading frame of 2340 bp encoding a 779-amino acid peptide. BLAST analysis indicated that the amino acid sequence displayed 58.4-90.2% identity and 5.6-125.8 divergence, compared with other known fish species. The most abundant TSHR transcription levels were found in the spleen, head kidney, and kidney. Feeding did not affect the transcription level of TSHR in thyroid tissue over the course of the day. Thus, the current study suggests that there was no relationship between daily nutritional status and TSHR transcription level in the thyroid tissue of largemouth bass. The spleen, head kidney, and kidney exhibited the most abundant TSHR transcription levels.

  19. Cloning, characterization, expression, and feeding response of thyrotropin receptor in largemouth bass (Micropterus salmoides).

    PubMed

    Gao, Y L; Song, W; Jiang, L L; Mao, M X; Wang, C L; Ge, C T; Qian, G Y

    2016-01-01

    Thyrotropin receptor (TSHR) is a G-protein-coupled receptor that regulates the synthesis, storage, and secretion of thyroid hormones in the thyroid tissue. The aims of the present study were to characterize the full-length TSHR cDNA in largemouth bass (Micropterus salmoides), and to determine the TSHR gene transcription levels in different tissues. In addition, the response of TSHR transcription levels to daily feeding in thyroid tissue was investigated. The results showed that the full-length cDNA sequence was 2743 bp with an open reading frame of 2340 bp encoding a 779-amino acid peptide. BLAST analysis indicated that the amino acid sequence displayed 58.4-90.2% identity and 5.6-125.8 divergence, compared with other known fish species. The most abundant TSHR transcription levels were found in the spleen, head kidney, and kidney. Feeding did not affect the transcription level of TSHR in thyroid tissue over the course of the day. Thus, the current study suggests that there was no relationship between daily nutritional status and TSHR transcription level in the thyroid tissue of largemouth bass. The spleen, head kidney, and kidney exhibited the most abundant TSHR transcription levels. PMID:27525899

  20. Cloning, genomic organization, and expression analysis of zebrafish nuclear receptor coactivator, TIF2.

    PubMed

    Tan, Jee-Hian; Quek, Sue-Ing; Chan, Woon-Khiong

    2005-01-01

    Thyroid hormone receptors (TRs) are involved in numerous diverse biological processes such as growth and differentiation, thermogenesis, neurulation, homeostasis, and metamorphosis. In zebrafish, TRbeta1 has been implicated to be involved in the obligatory embryonic-to-larval transitory phase. In order to understand if nuclear receptor coactivators could modulate the transcriptional activities of TRs during this transitory phase, the transcriptionary intermediary factor 2 (TIF2), a member of the p160 coactivator, was isolated from zebrafish. The zebrafish tif2 cDNA encodes a polypeptide of 1,505 amino acids. The tif2 gene is made up of 23 exons with the AUG and stop codon located in Exon IV and XXIII, respectively. The overall genomic organization of human and zebrafish tif2 genes are very similar. Four tif2 isoforms were identified by RT-PCR. The N-terminus mRNA variants are generated as a result of multiple initiation start sites located upstream of the noncoding Exon I and Exon II. The C-terminus isoforms, E20a and E20b, resulted from the alternative splicing of Exon XX. Although E20a and E20b isoforms were ubiquitously expressed, they were very highly expressed in reproductive tissues. The availability of TIF2 cDNA will allow the analysis of its functional roles in mediating the actions of TRs in various aspects of zebrafish developmental biology.

  1. Molecular pharmacology of the mineralocorticoid receptor: prospects for novel therapeutics.

    PubMed

    Kolkhof, Peter; Borden, Steffen A

    2012-03-24

    The blockade of mineralocorticoid receptors (MR) has been shown to be an invaluable therapy in heart failure and hypertension. To date, only two steroidal antimineralocorticoids, spironolactone (and its active metabolite canrenone) and eplerenone, have been approved, whereas novel non-steroidal compounds are in preclinical and early development. The careful investigation of the efficacy and tolerance of spironolactone in essential hypertension initially supported the idea that a more selective second generation of MR antagonists is desired for chronic treatment of cardiovascular diseases. More than 40 years went by between the approval of the first MR antagonist spironolactone and the market introduction of its sole successor, eplerenone. The molecular pharmacology of MR antagonists may be addressed at different levels. Available preclinical and clinical data of the two approved steroidal antimineralocorticoids allow a good comparison of potency and selectivity of MR antagonists and their pharmacokinetic properties. The search for novel generations of MR antagonists with the ultimate goal of a more tissue selective mode of action may require novel compounds that are differentiated with respect to the binding mode to the MR. Other factors that may contribute to tissue selectivity as e.g. the physicochemical properties of a drug and how they influence the resulting pharmacology in the context of tissue selective co-factor expression are even less well understood. In the following we will review these aspects and demonstrate that the molecular pharmacology of current MR antagonists is on the one hand far from well understood and, on the other hand, still offers room for improvements. PMID:21771637

  2. Molecular cloning of Bacillus sphaericus penicillin V amidase gene and its expression in Escherichia coli and Bacillus subtilis.

    PubMed Central

    Olsson, A; Hagström, T; Nilsson, B; Uhlén, M; Gatenbeck, S

    1985-01-01

    The Bacillus sphaericus gene coding for penicillin V amidase, which catalyzes the hydrolysis of penicillin V to yield 6-aminopenicillanic acid and phenoxyacetic acid, has been isolated by molecular cloning in Escherichia coli. The gene is contained within a 2.2-kilobase HindIII-PstI fragment and is expressed when transferred into E. coli and Bacillus subtilis. The expression in B. subtilis carrying the recombinant plasmid is approximately two times higher than in the original B. sphaericus strain. A comparison of the purified enzyme from B. sphaericus and the expressed gene product in E. coli minicells suggests that the native enzyme consists of four identical subunits, each with a molecular weight of 35,000. Images PMID:3923928

  3. Molecular cloning and characterization of the light-harvesting chlorophyll a/b gene from the pigeon pea (Cajanus cajan).

    PubMed

    Qiao, Guang; Wen, Xiao-Peng; Zhang, Ting

    2015-12-01

    Light-harvesting chlorophyll a/b-binding proteins (LHCB) have been implicated in the stress response. In this study, a gene encoding LHCB in the pigeon pea was cloned and characterized. Based on the sequence of a previously obtained 327 bp Est, a full-length 793 bp cDNA was cloned using the rapid amplification of cDNA ends (RACE) method. It was designated CcLHCB1 and encoded a 262 amino acid protein. The calculated molecular weight of the CcLHCB1 protein was 27.89 kDa, and the theoretical isoelectric point was 5.29. Homology search and sequence multi-alignment demonstrated that the CcLHCB1 protein sequence shared a high identity with LHCB from other plants. Bioinformatics analysis revealed that CcLHCB1 was a hydrophobic protein with three transmembrane domains. By fluorescent quantitative real-time polymerase chain reaction (PCR), CcLHCB1 mRNA transcripts were detectable in different tissues (leaf, stem, and root), with the highest level found in the leaf. The expression of CcLHCB1 mRNA in the leaves was up-regulated by drought stimulation and AM inoculation. Our results provide the basis for a better understanding of the molecular organization of LCHB and might be useful for understanding the interaction between plants and microbes in the future. PMID:26329890

  4. Molecular cloning, sequence analysis and phylogeny of first caudata g-type lysozyme in axolotl (Ambystoma mexicanum).

    PubMed

    Yu, Haining; Gao, Jiuxiang; Lu, Yiling; Guang, Huijuan; Cai, Shasha; Zhang, Songyan; Wang, Yipeng

    2013-11-01

    Lysozymes are key proteins that play important roles in innate immune defense in many animal phyla by breaking down the bacterial cell-walls. In this study, we report the molecular cloning, sequence analysis and phylogeny of the first caudate amphibian g-lysozyme: a full-length spleen cDNA library from axolotl (Ambystoma mexicanum). A goose-type (g-lysozyme) EST was identified and the full-length cDNA was obtained using RACE-PCR. The axolotl g-lysozyme sequence represents an open reading frame for a putative signal peptide and the mature protein composed of 184 amino acids. The calculated molecular mass and the theoretical isoelectric point (pl) of this mature protein are 21523.0 Da and 4.37, respectively. Expression of g-lysozyme mRNA is predominantly found in skin, with lower levels in spleen, liver, muscle, and lung. Phylogenetic analysis revealed that caudate amphibian g-lysozyme had distinct evolution pattern for being juxtaposed with not only anura amphibian, but also with the fish, bird and mammal. Although the first complete cDNA sequence for caudate amphibian g-lysozyme is reported in the present study, clones encoding axolotl's other functional immune molecules in the full-length cDNA library will have to be further sequenced to gain insight into the fundamental aspects of antibacterial mechanisms in caudate.

  5. Human uroporphyrinogen III synthase: Molecular cloning, nucleotide sequence, and expression of a full-length cDNA

    SciTech Connect

    Tsai, Shihfeng; Bishop, D.F.; Desnick, R.J. )

    1988-10-01

    Uroporphyrinogen III synthase, the fourth enzyme in the heme biosynthetic pathway, is responsible for conversion of the linear tetrapyrrole, hydroxymethylbilane, to the cyclic tetrapyrrole, uroporphyrinogen III. The deficient activity of URO-synthase is the enzymatic defect in the autosomal recessive disorder congenital erythropoietic porphyria. To facilitate the isolation of a full-length cDNA for human URO-synthase, the human erythrocyte enzyme was purified to homogeneity and 81 nonoverlapping amino acids were determined by microsequencing the N terminus and four tryptic peptides. Two synthetic oligonucleotide mixtures were used to screen 1.2 {times} 10{sup 6} recombinants from a human adult liver cDNA library. Eight clones were positive with both oligonucleotide mixtures. Of these, dideoxy sequencing of the 1.3 kilobase insert from clone pUROS-2 revealed 5' and 3' untranslated sequences of 196 and 284 base pairs, respectively, and an open reading frame of 798 base pairs encoding a protein of 265 amino acids with a predicted molecular mass of 28,607 Da. The isolation and expression of this full-length cDNA for human URO-synthase should facilitate studies of the structure, organization, and chromosomal localization of this heme biosynthetic gene as well as the characterization of the molecular lesions causing congenital erythropoietic porphyria.

  6. Molecular cloning, sequence analysis and phylogeny of first caudata g-type lysozyme in axolotl (Ambystoma mexicanum).

    PubMed

    Yu, Haining; Gao, Jiuxiang; Lu, Yiling; Guang, Huijuan; Cai, Shasha; Zhang, Songyan; Wang, Yipeng

    2013-11-01

    Lysozymes are key proteins that play important roles in innate immune defense in many animal phyla by breaking down the bacterial cell-walls. In this study, we report the molecular cloning, sequence analysis and phylogeny of the first caudate amphibian g-lysozyme: a full-length spleen cDNA library from axolotl (Ambystoma mexicanum). A goose-type (g-lysozyme) EST was identified and the full-length cDNA was obtained using RACE-PCR. The axolotl g-lysozyme sequence represents an open reading frame for a putative signal peptide and the mature protein composed of 184 amino acids. The calculated molecular mass and the theoretical isoelectric point (pl) of this mature protein are 21523.0 Da and 4.37, respectively. Expression of g-lysozyme mRNA is predominantly found in skin, with lower levels in spleen, liver, muscle, and lung. Phylogenetic analysis revealed that caudate amphibian g-lysozyme had distinct evolution pattern for being juxtaposed with not only anura amphibian, but also with the fish, bird and mammal. Although the first complete cDNA sequence for caudate amphibian g-lysozyme is reported in the present study, clones encoding axolotl's other functional immune molecules in the full-length cDNA library will have to be further sequenced to gain insight into the fundamental aspects of antibacterial mechanisms in caudate. PMID:24199859

  7. Molecular cloning and characterization of the light-harvesting chlorophyll a/b gene from the pigeon pea (Cajanus cajan).

    PubMed

    Qiao, Guang; Wen, Xiao-Peng; Zhang, Ting

    2015-12-01

    Light-harvesting chlorophyll a/b-binding proteins (LHCB) have been implicated in the stress response. In this study, a gene encoding LHCB in the pigeon pea was cloned and characterized. Based on the sequence of a previously obtained 327 bp Est, a full-length 793 bp cDNA was cloned using the rapid amplification of cDNA ends (RACE) method. It was designated CcLHCB1 and encoded a 262 amino acid protein. The calculated molecular weight of the CcLHCB1 protein was 27.89 kDa, and the theoretical isoelectric point was 5.29. Homology search and sequence multi-alignment demonstrated that the CcLHCB1 protein sequence shared a high identity with LHCB from other plants. Bioinformatics analysis revealed that CcLHCB1 was a hydrophobic protein with three transmembrane domains. By fluorescent quantitative real-time polymerase chain reaction (PCR), CcLHCB1 mRNA transcripts were detectable in different tissues (leaf, stem, and root), with the highest level found in the leaf. The expression of CcLHCB1 mRNA in the leaves was up-regulated by drought stimulation and AM inoculation. Our results provide the basis for a better understanding of the molecular organization of LCHB and might be useful for understanding the interaction between plants and microbes in the future.

  8. Molecular cloning and expression analysis of GhLOF genes in upland cotton (Gossypium hirsutum L.).

    PubMed

    Dai, T C; Wang, Z M

    2015-05-04

    Shoot branching, i.e., the timing and position of shoot growth, determines to a large extend the pattern of plant architecture, and is the result of the integration of a plant's genetic background and environmental cues. Many genes that are involved in the formation and outgrowth of axillary buds have been cloned, but the exact mechanism is still unclear. Branching pattern is an important agronomic trait in many crops, including cotton. In the present study, we cloned four genes from cotton, and designated them as GhLOF1/2/3/4. Sequence analysis revealed that all four genes shared conserved protein domains with LATERAL ORGAN FUSION (LOF) from Arabidopsis and TRIFOLIATE (Tf) from tomato. Phylogenetic analysis revealed that GhLOF3 and GhLOF4 were close to Tf because of their similar expression patterns, whereas GhLOF1 and GhLOF2 were differentially expressed.

  9. Molecular Cloning and Heterologous Expression of the Dehydrophos Biosynthetic Gene Cluster

    PubMed Central

    Circello, Benjamin T.; Eliot, Andrew C.; Lee, Jin-Hee; van der Donk, Wilfred A.; Metcalf, William W.

    2010-01-01

    Summary Dehydrophos is a vinyl phosphonate tripeptide produced by Streptomyces luridus with demonstrated broad spectrum antibiotic activity. To identify genes necessary for biosynthesis of this unusual compound we screened a fosmid library of S. luridus for the presence of the phosphoenolpyruvate mutase gene, which is required for biosynthesis of most phosphonates. Integration of one such fosmid clone into the chromosome of Streptomyces lividans led to heterologous production of dehydrophos. Deletion analysis of this clone allowed identification of the minimal contiguous dehydrophos cluster, which contained 17 open reading frames (ORFs). Bioinformatic analyses of these ORFs are consistent with a proposed biosynthetic pathway that generates dehydrophos from phosphoenolpyruvate. The early steps of this pathway are supported by analysis of intermediates accumulated by blocked mutants and in vitro biochemical experiments. PMID:20416511

  10. Molecular cloning and characterization of two novel cellulase genes from the mollusc Ampullaria crossean.

    PubMed

    Guo, Rui; Ding, Ming; Zhang, Si-Liang; Xu, Gen-Jun; Zhao, Fu-Kun

    2008-02-01

    Cellulase genes have been reported not only from fungi, bacteria and plant, but also from some invertebrate animals. Here, two cellulase (endo-beta-1,4-glucanase, EC 3.2.1.4) genes, eg27I and eg27II, were cloned from the freshwater snail Ampullaria crossean cDNA using degenerate primers. The nucleotide sequences of the two genes shared 94.5% identity. The open reading frames of both genes consisted of 588 bp, encoding 195 amino acids. Both EG27I and EG27II belong to the glycoside hydrolase family 45, and each lacks a carbohydrate-binding module. The presence of introns demonstrated a eukaryotic origin of the EG27 gene, and, in addition, successful cloning of EG27 cDNA supported endogenous production of EG27 cellulase by Ampullaria crossean. Investigation of the EG27 cDNA from A. crossean will provide further information on GHF45 cellulases.

  11. Molecular cloning, DNA structure and expression of the Escherichia coli D-xylose isomerase.

    PubMed Central

    Briggs, K A; Lancashire, W E; Hartley, B S

    1984-01-01

    The D-xylose isomerase (EC 5.3.1.5) gene from Escherichia coli was cloned and isolated by complementation of an isomerase-deficient E. coli strain. The insert containing the gene was restriction mapped and further subcloning located the gene in a 1.6-kb Bg/II fragment. This fragment was sequenced by the chain termination method, and showed the gene to be 1002 bp in size. The Bg/II fragment was cloned into a yeast expression vector utilising the CYCl yeast promoter. This construct allowed expression in E. coli grown on xylose but not glucose suggesting that the yeast promoter is responding to the E. coli catabolite repression system. No expression was detected in yeast from this construct and this is discussed in terms of the upstream region in the E. coli insert with suggestions of how improved constructs may permit achievement of the goal of a xylose-fermenting yeast. PMID:6325179

  12. Molecular cloning and expression of a human heat shock factor, HSF1

    SciTech Connect

    Rabindran, S.K.; Giorgi, G.; Clos, J.; Wu, C. )

    1991-08-15

    Human cells respond to heat stress by inducing the binding of a preexisting transcriptional activator (heat shock factor, HSF) to DNA. The authors isolated recombinant DNA clones for a human cDNA fragment. The human HSF1 probe was produced by the PCR with primers deduced from conserved amino acids in the Drosophila and yeast HSF sequences. The human HSF1 mRNA is constitutively expressed in HeLa cells under nonshock conditions and encodes a protein with four conserved leucine zipper motifs. Like its counterpart in Drosophila, human HSF1 produced in Escherichia coli in the absence of heat shock is active as a DNA binding transcription factor, suggesting that the intrinsic activity of HSF is under negative control in human cells. Surprisingly, an independently isolated human HSF clone, HSF2, is related to but significantly different from HSF.

  13. Molecular cloning and in vitro expression of a silent phenoxazinone synthase gene from Streptomyces lividans.

    PubMed

    Madu, A C; Jones, G H

    1989-12-14

    Phenoxazinone synthase (PHS) catalyzes a step in actinomycin D biosynthesis in Streptomyces antibioticus. Two sequences from Streptomyces lividans that hybridize to the phs gene of S. antibioticus have been cloned in Escherichia coli K-12 using the plasmid pBR322. Although there was some similarity in the restriction maps of the two cloned fragments, neither insert appeared to be a direct subset of the other nor of the S. antibioticus phs gene. In vitro expression studies, in a streptomycete coupled transcription-translation system, showed that a 3.98-kb SphI fragment encoded a PHS-related protein. These observations provide additional support for the existence of silent genes for antibiotic production in streptomycetes.

  14. Molecular cloning of Phaseolus vulgaris cDNA encoding proliferating cell nuclear antigen.

    PubMed

    Strzalka, Wojciech; Ziemienowicz, Alicja

    2007-02-01

    A cDNA fragment encoding a common bean (Phaseolus vulgaris) proliferating cell nuclear antigen (PCNA) was isolated using rapid amplification of cDNA 3' end (3' RACE) method, cloned and sequenced. The nucleotide sequence of this clone contains an open reading frame of 798 nucleotides encoding a protein of 265 amino acids. Alignment of the common bean PCNA predicted sequence shows its high degree of identity with PCNA from other plant species. Analysis of PCNA content in the germinating embryos of common bean showed a decrease in the protein level after 60h of germination. Moreover, PCNA was not detected in the tested plant organs (root, stem, leaf and flower). The presence of PCNA in the germinating seeds and its absence from mature plants suggests that this protein plays a crucial role during early stages of plant development.

  15. Molecular cloning and characterization of human pyruvate dehydrogenase. beta. subunit gene

    SciTech Connect

    Koike, Kichiko; Urata, Yoshishige; Koike, Masahiko )

    1990-08-01

    A genomic clone encompassing the entire gene for the human pyruvate dehydrogenase {beta} subunit (PDH{beta}) has been isolated by screening a leukocyte genomic library with a nick-translated human foreskin fibroblast PDH{beta} cDNA probe. The 18-kilobase clone was characterized by restriction enzyme analysis, extensive DNA sequencing, and primer-extension analysis. The PDH{beta} structural gene is composed of 10 exons and 9 introns. All intron-exon splice junctions follow the GT/AG rule. The Alu family was found in introns 2 and 8. The 5{prime} flanking region of the PDH{beta} gene contains a CAAT consensus promoter sequence but no TATA sequence. Primer-extension analysis indicated the PDH{beta} gene transcription start site is an adenine residue located 132 bases upstream from the initiation codon in exon 1.

  16. Molecular cloning of chitinase 33 (chit33) gene from Trichoderma atroviride

    PubMed Central

    Matroudi, S.; Zamani, M.R.; Motallebi, M.

    2008-01-01

    In this study Trichoderma atroviride was selected as over producer of chitinase enzyme among 30 different isolates of Trichoderma sp. on the basis of chitinase specific activity. From this isolate the genomic and cDNA clones encoding chit33 have been isolated and sequenced. Comparison of genomic and cDNA sequences for defining gene structure indicates that this gene contains three short introns and also an open reading frame coding for a protein of 321 amino acids. The deduced amino acid sequence includes a 19 aa putative signal peptide. Homology between this sequence and other reported Trichoderma Chit33 proteins are discussed. The coding sequence of chit33 gene was cloned in pEt26b(+) expression vector and expressed in E. coli. PMID:24031242

  17. Nuclear estrogen receptor molecular heterogeneity in the mouse uterus.

    PubMed Central

    Golding, T S; Korach, K S

    1988-01-01

    Holomeric estrogen receptor (ER) prepared from ovariectomized mouse uteri displays heterogeneous electrophoretic mobility when analyzed by NaDodSO4/PAGE. ER derived from nuclei (ERn) appears as a closely spaced doublet having apparent molecular masses of 66.4 and 65 kDa, while ER from the cytosolic compartment (ERc) has a single band of 65 kDa. Both partially purified ERc and the 8S form of unactivated ERc show only the 65-kDa band. The appearance of the ERn doublet is hormonally inducible, and the relative proportions of the two doublet bands are influenced by the type of hormone treatment, with weakly estrogenic compounds yielding the lower band as predominant while potent estrogens increase the proportion of the upper band. Steroid binding of the ERn doublet was determined by [3H]tamoxifen aziridine affinity labeling of both the 66.4- and the 65-kDa peptides; binding to the 65-kDa peptide was predominant. The ERn doublet displays a time dependency after estrogen administration with maximal amounts occurring in a bimodal fashion at 1 and 8 hr. Images PMID:3422428

  18. Molecular Cooperativity Governs Diverse and Monoallelic Olfactory Receptor Expression

    NASA Astrophysics Data System (ADS)

    Xing, Jianhua; Tian, Xiaojun; Zhang, Hang; Sannerud, Jens

    Multiple-objective optimization is common in biological systems. In the mammalian olfactory system, each sensory neuron stochastically expresses only one out of up to thousands of olfactory receptor (OR) gene alleles; at organism level the types of expressed ORs need to be maximized. The molecular mechanism of this Nobel-Prize winning puzzle remains unresolved after decades of extensive studies. Existing models focus only on monoallele activation, and cannot explain recent observations in mutants, especially the reduced global diversity of expressed ORs in G9a/GLP knockouts. In this work we integrated existing information on OR expression, and proposed an evolutionarily optimized three-layer regulation mechanism, which includes zonal segregation, epigenetic and enhancer competition coupled to a negative feedback loop. This model not only recapitulates monoallelic OR expression, but also elucidates how the olfactory system maximizes and maintains the diversity of OR expression. The model is validated by several experimental results, and particularly underscores cooperativity and synergy as a general design principle of multi-objective optimization in biology. The work is supported by the NIGMS/DMS Mathematical Biology program.

  19. Nuclear estrogen receptor molecular heterogeneity in the mouse uterus

    SciTech Connect

    Golding, T.S.; Korach, K.S.

    1988-01-01

    Holomeric estrogen receptor (ER) prepared from ovariectomized mouse uteri displays heterogeneous electrophoretic mobility when analyzed by NaDodSO/sub 4//PAGE. ER derived from nuclei (ER/sub n/) appears as a closely spaced doublet having apparent molecular masses of 66.4 and 65 kDa, while ER from the cytosolic compartment (ER/sub c/) has a single band of 65 kDa. Both partially purified ER/sub c/ and the 8S form of unactivated ER/sub c/ show only the 65-kDa band. The appearance of the ER/sub n/ doublet is hormonally inducible, and the relative proportions of the two doublet bands are influenced by the type of hormone treatment, with weakly estrogenic compounds yielding the lower band as predominant while potent estrogens increase the proportion of the upper band. Steroid binding of the ER/sub n/ doublet was determined by (/sup 3/H)tamoxifen aziridine affinity labeling of both the 66.4- and the 65-kDa peptides; binding to the 65-kDa peptide was predominant. The ER/sub n/ doublet displays a time dependency after estrogen administration with maximal amounts occurring in a bimodal fashion at 1 and 8 hr.

  20. Molecular cloning and synthesis of biologically active human tissue inhibitor of metalloproteinases in yeast

    SciTech Connect

    Kaczorek, M.; Honore, N.; Ribes, V.; Dehoux, P.; Cornet, P.; Cartwright, T.; Streeck, R.E.

    1987-06-01

    Tissue inhibitor of metalloproteinases (TIMP) is a widely distributed glycoprotein that stochiometrically inactivates metalloproteinases involved in connective tissue catabolism. Here they report the cDNA cloning of TIMP from human fibroblastic MRC5 cells using a single 42-base oligonucleotide probe. Expression in S. cerevisiae of complete TIMP cDNA yielded insoluble protein aggregates. Biologically active TIMP was reconstituted from the yeast product by a denaturation/renaturation procedure.

  1. Molecular cloning, characterization, and overexpression of ERG7, the Saccharomyces cerevisiae gene encoding lanosterol synthase.

    PubMed Central

    Corey, E J; Matsuda, S P; Bartel, B

    1994-01-01

    We report the cloning, characterization, and overexpression of Saccharomyces cerevisiae ERG7, which encodes lanosterol synthase [(S)-2,3-epoxysqualene mutase (cyclizing, lanosterol forming), EC 5.4.99.7], the enzyme responsible for the complex cyclization/rearrangement step in sterol biosynthesis. Oligonucleotide primers were designed corresponding to protein sequences conserved between Candida albicans ERG7 and the related Arabidopsis thaliana cycloartenol synthase [(S)-2,3-epoxysqualene mutase (cyclizing, cycloartenol forming), EC 5.4.99.8]. A PCR product was amplified from yeast genomic DNA using these primers and was used to probe yeast libraries by hybridization. Partial-length clones homologous to the two known epoxysqualene mutases were isolated, but a full-length sequence was found neither in cDNA nor genomic libraries, whether in phage or plasmids. Two overlapping clones were assembled to make a functional reconstruction of the gene, which contains a 2196-bp open reading frame capable of encoding an 83-kDa protein. The reconstruction complemented the erg7 mutation when driven from either its native promoter or the strong ADH1 promoter. Images PMID:8134375

  2. Molecular cloning and characterization of an N-acetylglucosamine-6-O-sulfotransferase.

    PubMed

    Uchimura, K; Muramatsu, H; Kadomatsu, K; Fan, Q W; Kurosawa, N; Mitsuoka, C; Kannagi, R; Habuchi, O; Muramatsu, T

    1998-08-28

    We isolated a cDNA clone encoding mouse N-acetylglucosamine-6-O-sulfotransferase based on sequence homology to the previously cloned mouse chondroitin 6-sulfotransferase. The cDNA clone contained an open reading frame that predicts a type II transmembrane protein composed of 483 amino acid residues. The expressed enzyme transferred sulfate to the 6 position of nonreducing GlcNAc in GlcNAcbeta1-3Galbeta1-4GlcNAc. Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAc and various glycosaminoglycans did not serve as acceptors. Expression of the cDNA in COS-7 cells resulted in production of a cell-surface antigen, the epitope of which was NeuAcalpha2-3Galbeta1-4(SO4-6)GlcNAc; double transfection with fucosyltransferase IV yielded Galbeta1-4(Fucalpha1-3)(SO4-6)GlcNAc antigen. The sulfotransferase mRNA was strongly expressed in the cerebrum, cerebellum, eye, pancreas, and lung of adult mice. In situ hybridization revealed that the mRNA was localized in high endothelial venules of mesenteric lymph nodes. The sulfotransferase was concluded to be involved in biosynthesis of glycoconjugates bearing the 6-sulfo N-acetyllactosamine structure such as 6-sulfo sialyl Lewis X. The products of the sulfotransferase probably include glycoconjugates with intercellular recognition signals; one candidate of such a glycoconjugate is an L-selectin ligand.

  3. Nitrocellulose immunoblotting for identification and molecular gene cloning of Eimeria maxima antigens that stimulate lymphocyte proliferation.

    PubMed Central

    Bumstead, J M; Dunn, P P; Tomley, F M

    1995-01-01

    An immunoblotting technique was used to identify lymphostimulatory antigens within sized polypeptide fractions of Eimeria maxima sporozoites. Six fractions contained polypeptides that specifically stimulated the proliferation of immune lymphocytes in an in vitro assay, and polyclonal antisera were made in rabbits against these fractions. cDNA clones, isolated with antisera against a lymphostimulatory fraction of around 70 kDa, were found to encode four different antigens including a classical hsp70, a molecule homologous to an endoplasmic reticulum chaperonin (BiP/GRP), and a calcium-dependent serine/threonine protein kinase that appears homologous to a recently described molecule from Plasmodium falciparum. The protein kinase cDNA clone was overexpressed in Escherichia coli, and the recombinant antigen was found to induce both antibody and lymphoproliferative responses in chickens when administered subcutaneously. Thus, immunoblotting, in combination with in vitro lymphoproliferation assays, can be used as an initial screen for the identification of lymphostimulatory antigens from a complex pool of polypeptides, and a combination of cDNA cloning, expression, and immunization allows assessment of the lymphostimulatory activity of individual polypeptides. These studies should facilitate further evaluation of antigens that are potential candidates for inclusion in a recombinant vaccine against poultry coccidiosis. PMID:8548529

  4. Cloning, sequence analysis and expression profiles of Toll-like receptor 7 from Chinese giant salamander Andrias davidianus.

    PubMed

    Huang, Lili; Fan, Yuding; Zhou, Yong; Jiang, Nan; Liu, Wenzhi; Meng, Yan; Zeng, Lingbing

    2015-06-01

    The Chinese giant salamander, Andrias davidianus, is the largest extant amphibian species in the world, which is of significance due to its specific position in the evolutionary history of vertebrates. Currently, limited information about the innate immune system of this animal is known. In this study, the toll-like receptor 7 (TLR7), designated CgsTLR7, was cloned from Chinese giant salamander, A. davidianus. The full-length cDNA of CgsTLR7 is 3747 bp, with an open reading frame of 3150 bp, encoding 1049 amino acids. The TLR family motifs, including the leucine-rich repeat (LRR) and Toll/interleukin (IL)-1 receptor (TIR) domain are conserved in CgsTLR7, which includes 19 LRRs and a TIR domain. The predicted amino acid sequence of CgsTLR7 has 71%, 65%, 63% and 55% identity with turtle, chicken, human and fugu TLR7 homologues, respectively. Phylogenetic analysis showed that CgsTLR7 is closest to that of frog TLR7 among the examined species. Quantitative real-time PCR analysis revealed broad expression of CgsTLR7 in tissues from apparently healthy Chinese giant salamanders with the highest expression in the liver and the lowest expression in the intestine. The mRNA expression was up-regulated and reached a peak level in the kidney, liver and spleen at 12 h, 24 h and 48 h after infecting the animals with the giant salamander iridovirus (GSIV), respectively. These results suggest that CgsTLR7 has a conserved gene structure and might play an important role in immune regulation against viral infections in the Chinese giant salamander.

  5. Cloning and expression analysis of a Toll-like receptor 22 (tlr22) gene from turbot, Scophthalmus maximus.

    PubMed

    Hu, Guo-Bin; Zhang, Shou-Feng; Yang, Xi; Liu, Da-Hai; Liu, Qiu-Ming; Zhang, Shi-Cui

    2015-06-01

    Toll-like receptor 22 (TLR22) exists exclusively in aquatic animals and recognizes double stranded RNA (dsRNA). In the present study, a tlr22 gene and its 5'-flanking sequence were cloned from turbot, Scophthalmus maximus, its immune responsive expression was subsequently studied in vivo. The turbot (sm)tlr22 gene spans over 5.6 kb with a structure of 4 exon-3 intron and encodes 962 amino acids. The deduced protein shows the highest sequence identity (76.7%) to Japanese flounder Tlr22 and possesses a signal peptide sequence, a leucine-rich repeat (LRR) domain composed of 27 LRR motifs, a transmembrane region and a Toll/interleukin-1 receptor (TIR) domain. Phylogenetic analysis grouped it with other teleost Tlr22s. The interferon-stimulated response element (ISRE) and signal transducer and activator of transcription (STAT) binding site important for the basal transcriptional activity of TLR3 were predicted in the 5'-flanking sequence of smtlr22 gene. Quantitative real-time PCR (qPCR) analysis demonstrated the constitutive expression of smtlr22 mRNA in all examined tissues with higher levels in the head kidney, kidney and spleen. Further, smtlr22 expression was significantly up-regulated following challenge with polyinosinic: polycytidylic acid (poly I:C), lipopolysaccharide (LPS) or turbot reddish body iridovirus (TRBIV) in the gills, head kidney, spleen and muscle, with maximum increases ranging from 2.56 to 6.24 fold upon different immunostimulants and organs. These findings suggest a possible role of Smtlr22 in the immune responses to the infections of a broad range of pathogens that include DNA and RNA viruses and Gram-negative bacteria. PMID:25770871

  6. Molecular cloning of bovine lymphocyte activation gene-3 and its expression characteristics in bovine leukemia virus-infected cattle.

    PubMed

    Shirai, Tatsuya; Konnai, Satoru; Ikebuchi, Ryoyo; Okagawa, Tomohiro; Suzuki, Saori; Sunden, Yuji; Onuma, Misao; Murata, Shiro; Ohashi, Kazuhiko

    2011-12-15

    Lymphocyte activation gene-3 (LAG-3), a major histocompatibility complex (MHC) class II binding CD4 homologue has recently been shown as one of the mechanisms for down-regulating immune responses during chronic disease progression. For the first time, we cloned LAG-3 from two breeds of cattle (Holstein and Japanese Black), and analyzed its expression levels in cattle infected with bovine leukemia virus (BLV), a chronic viral infection that leads to immuno-suppression. The cloned cDNA of bovine LAG-3 have an open reading frame of 1551 nucleotides, encoding a polypeptide of 515 amino acids in length. Similar to the swine LAG-3, the bovine LAG-3 protein sequence consisted of four extracellular domains, a transmembrane domain and an inhibitory motif, KTGELE. We found that the bovine LAG-3 mRNA transcripts were expressed predominantly on T-cells such as CD4(+) and CD8(+) cells, among peripheral blood mononuclear cells. In subsequent expression analysis, LAG-3 mRNA expression on CD4(+) T-cells from BLV-infected cattle was upregulated compared to that in normal cattle. Comparable results were obtained with CD8(+) T-cells from cattle infected with BLV. We further observed strong upregualtion of MHC class II molecule, the ligand for LAG-3 in BLV-infected cattle. These findings indicate an important role for inhibitory receptor molecules such as LAG-3 in chronic bovine infections and future studies will elucidate the specific role of LAG-3 in bovine diseases.

  7. Molecular characterizat