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Sample records for encoding laminin-binding protein

  1. Purification, crystallization and preliminary crystallographic analysis of Streptococcus pyogenes laminin-binding protein Lbp

    SciTech Connect

    Linke, Christian; Caradoc-Davies, Tom T.; Proft, Thomas; Baker, Edward N.

    2008-02-01

    The S. pyogenes laminin-binding protein Lbp, which is essential for adhesion to human laminin, has been expressed, purified and crystallized. The laminin-binding protein Lbp (Spy2007) from Streptococcus pyogenes (a group A streptococcus) mediates adhesion to the human basal lamina glycoprotein laminin. Accordingly, Lbp is essential in in vitro models of cell adhesion and invasion. However, the molecular and structural basis of laminin binding by bacteria remains unknown. Therefore, the lbp gene has been cloned for recombinant expression in Escherichia coli. Lbp has been purified and crystallized from 30%(w/v) PEG 1500 by the sitting-drop vapour-diffusion method. The crystals belonged to the monoclinic space group P2{sub 1}, with unit-cell parameters a = 42.62, b = 92.16, c = 70.61 Å, β = 106.27°, and diffracted to 2.5 Å resolution.

  2. Identification of the N-acetylneuraminyllactose-specific laminin-binding protein of Helicobacter pylori.

    PubMed Central

    Valkonen, K H; Wadström, T; Moran, A P

    1997-01-01

    The interaction of the gastroduodenal pathogen Helicobacter pylori with the glycoprotein laminin was investigated. Binding of 125I-radiolabelled laminin in a liquid-phase assay by both hemagglutinating and poorly hemagglutinating strains was rapid, saturable, specific, partially reversible, of high affinity, and insensitive to pH. Inhibition of laminin binding by fetuin, but not asialofetuin, and reduced bacterial binding to periodate- or sialidase-treated laminin indicated that glycosylation, particularly sialylation, was important for laminin binding by H. pylori. Inhibition experiments with monosaccharides, disaccharides, and trisaccharides showed that the strains bound to a region spanning a trisaccharide. In particular, inhibition and displacement studies showed that binding to the trisaccharide N-acetylneuraminyl-alpha(2-3)-lactose [NeuAc(2-3)Lac] was preferential to that to the NeuAc(2-6)Lac isomer. Complete inhibition of laminin binding by both hemagglutinating and poorly hemagglutinating strains was achieved only when isolated lipopolysaccharide (LPS) was used as an inhibitor in combination with heat or protease treatment of H. pylori cells, thereby confirming the involvement of both LPS and a protein adhesin in laminin binding. Further inhibition experiments indicated that the protein receptor, rather than LPS, on H. pylori bound NeuAc(2-3)Lac. By using a Western blotting procedure, a 25-kDa outer membrane protein was identified as mediating laminin binding by both hemagglutinating and poorly hemagglutinating H. pylori strains. The specificity of binding was confirmed by complete inhibition of laminin binding by the 25-kDa protein with NeuAc(2-3)Lac. The data collectively suggest that a 25-kDa outer membrane protein acts in a lectin-like manner with LPS to mediate attachment of H. pylori to laminin. PMID:9038297

  3. Expression and identification of a laminin-binding protein in Aspergillus fumigatus conidia.

    PubMed Central

    Tronchin, G; Esnault, K; Renier, G; Filmon, R; Chabasse, D; Bouchara, J P

    1997-01-01

    Adhesion of Aspergillus fumigatus, the causative agent of human aspergillosis, to the extracellular matrix protein laminin has been previously demonstrated. This study investigated the expression of laminin receptors during swelling of conidia, a step leading to germination and subsequent colonization of tissues. Scanning electron microscopy showed that the laminin binding sites were distributed over the external rodlet layer of resting conidia. During swelling, the characteristic rodlet layer progressively disintegrated and conidia surrounded by a smooth cell wall layer appeared. Flow cytometry using fluorescein isothiocyanate-conjugated laminin demonstrated that expression of laminin receptors at the surface of conidia was swelling dependent. Resting conidia expressed high levels of laminin receptors on their surface. A gradual decrease of laminin binding was then observed as swelling occurred, reaching a minimum for 4-h-swollen conidia. This correlated with a loss of adherence of swollen conidia to laminin immobilized on microtiter plates. Trypsin pretreatment of conidia reduced laminin binding. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and ligand blotting with laminin identified in a cell wall extract a major 72-kDa cell wall glycoprotein which binds laminin. Thus, one of the initial events in the host colonization may be the recognition of basement membrane laminin by this 72-kDa cell wall surface component. PMID:8975886

  4. Induction of a putative laminin-binding protein of Streptococcus gordonii in human infective endocarditis.

    PubMed Central

    Sommer, P; Gleyzal, C; Guerret, S; Etienne, J; Grimaud, J A

    1992-01-01

    There is evidence to suggest that the virulence of Streptococcus strains in infective endocarditis might be due to the expression of binding sites for the extracellular matrix proteins of damaged valves. In this communication, we draw attention to one laminin-binding protein from a strain of Streptococcus gordonii isolated from a patient with human endocarditis. This 145-kDa protein was found on the cell wall of the bacterium. The level of expression of this binding protein might be regulated by the presence of extracellular matrix proteins: the protein was lacking after in vitro selection of laminin, collagen I, and fibronectin nonbinding variants, and it was recovered after growth of the variants when laminin or collagen I was added to the growth medium. It was also missing after 10 subcultures in minimal medium, indicating some positive control. Furthermore, the 145-kDa protein was recognized as a major antigen by sera from patients treated for streptococcal infective endocarditis, while sera from patients with valvulopathies gave only slight recognition, suggesting an increase of the expression of this protein during infective endocarditis. It was also shown that the 145-kDa protein carried a collagen I-like determinant detected with anti-human collagen I antibodies. Images PMID:1530927

  5. Scaffold-forming and Adhesive Contributions of Synthetic Laminin-binding Proteins to Basement Membrane Assembly.

    PubMed

    McKee, Karen K; Capizzi, Stephanie; Yurchenco, Peter D

    2009-03-27

    Laminins that possess three short arms contribute to basement membrane assembly by anchoring to cell surfaces, polymerizing, and binding to nidogen and collagen IV. Although laminins containing the alpha4 and alpha5 subunits are expressed in alpha2-deficient congenital muscular dystrophy, they may be ineffective substitutes because they bind weakly to cell surfaces and/or because they lack the third arm needed for polymerization. We asked whether linker proteins engineered to bind to deficient laminins that provide such missing activities would promote basement membrane assembly in a Schwann cell model. A chimeric fusion protein (alphaLNNd) that adds a short arm terminus to laminin through the nidogen binding locus was generated and compared with the dystrophy-ameliorating protein miniagrin (mAgrin) that binds to the laminin coiled-coil dystroglycan and sulfatides. alphaLNNd was found to mediate laminin binding to collagen IV, to bind to galactosyl sulfatide, and to selectively convert alpha-short arm deletion-mutant laminins LmDeltaalphaLN and LmDeltaalphaLN-L4b into polymerizing laminins. This protein enabled polymerization-deficient laminin but not an adhesion-deficient laminin lacking LG domains (LmDeltaLG) to assemble an extracellular matrix on Schwann cell surfaces. mAgrin, on the other hand, enabled LmDeltaLG to form an extracellular matrix on cell surfaces without increasing accumulation of non-polymerizing laminins. These gain-of-function studies reveal distinct polymerization and anchorage contributions to basement membrane assembly in which the three different LN domains mediate the former, and the LG domains provide primary anchorage with secondary contributions from the alphaLN domain. These findings may be relevant for an understanding of the pathogenesis and treatment of laminin deficiency states. PMID:19189961

  6. Inverse expression of two laminin binding proteins, 67LR and galectin-3, correlates with the invasive phenotype of trophoblastic tissue.

    PubMed

    van den Brûle, F A; Price, J; Sobel, M E; Lambotte, R; Castronovo, V

    1994-05-30

    Tumor invasion of host tissues and trophoblastic penetration of the endometrium share common biological features. Both processes involve the invasion of basement membranes, an event that is initiated by adhesion of cancer or trophoblast cells to basement membrane components and particularly to laminin. Adhesion to this latter glycoprotein is mediated through a variety of cell surface receptors. We have previously shown that the 67 kD Laminin Receptor (67LR) and a 31 kD Human Laminin Binding Protein, recently renamed galectin-3, are inversely modulated as the invasive phenotype of cancer cells progresses, with up regulation of the former, and down regulation of the latter, respectively. In this study, we examined the expression of these two proteins in 27 human trophoblastic specimens at different gestational ages using Northern and Western blot techniques. Expression of the 67LR increased from 7 weeks to a maximum at 12 weeks, when invasion is maximal, and then decreased. Expression of galectin-3 was inversely modulated by the gestational age, with a minimum expression at 12 weeks. Our data demonstrate that invasive trophoblast displays the same pattern of laminin binding proteins expression than invasive cancer cells, and further demonstrates that invasion of the extracellular matrix by trophoblast and cancer cells share common molecular mechanisms. PMID:8198600

  7. Characterization of the Mycobacterium avium subsp. paratuberculosis laminin-binding/histone-like protein (Lbp/Hlp) which reacts with sera from patients with Crohn's disease.

    PubMed

    Lefrançois, Louise H; Pujol, Céline; Bodier, Christelle C; Teixeira-Gomez, Ana Paula; Drobecq, Hervé; Rosso, Marie-Laure; Raze, Dominique; Dias, André Alves; Hugot, Jean-Pierre; Chacon, Ofelia; Barletta, Raul G; Locht, Camille; Vidal Pessolani, Maria Cristina; Biet, Franck

    2011-06-01

    Mycobacterium avium subsp. paratuberculosis (Map) causes a chronic enteric disease in ruminants, called paratuberculosis or Johne's disease. The current model proposes that after ingestion by the host, Map crosses the intestinal barrier via internalization by the M cells. Experimental observations suggest, however, that Map may also transcytose the intestinal wall via the enterocytes, but the mechanisms involved in this process remain poorly understood. Cytoadherence assays performed on epithelial cells with Map revealed that the addition of laminin to the cell culture increases adhesion. A Map protein was isolated by heparin-Sepharose chromatography and identified as a laminin-binding protein like. The gene encoding this protein named Lbp/Hlp was identified in the Map genome sequence at locus MAP3024 (annotated Hup B). The deduced Map Lbp/Hlp amino acid sequence reveals 80% identity with that reported for other mycobacteria. The C-terminal domain involved in adhesion is mainly composed of arginine and lysine residues modified by methylation. In vitro tests demonstrated that recombinant Lbp/Hlp binds laminin, heparin, collagen and epithelial cells. Interestingly, we found that this adhesin corresponds to the antigen described as the target of pANCA and serum antibodies of patients with Crohn's disease. PMID:21334452

  8. Force-induced globule-coil transition in laminin binding protein and its role for viral-cell membrane fusion.

    PubMed

    Zaitsev, Boris N; Benedetti, Fabrizio; Mikhaylov, Andrey G; Korneev, Denis V; Sekatskii, Sergey K; Karakouz, Tanya; Belavin, Pavel A; Netesova, Nina A; Protopopova, Elena V; Konovalova, Svetlana N; Dietler, Giovanni; Loktev, Valery B

    2014-12-01

    The specific interactions of the pairs laminin binding protein (LBP)-purified tick-borne encephalitis viral surface protein E and certain recombinant fragments of this protein, as well as West Nile viral surface protein E and certain recombinant fragments of that protein, are studied by combined methods of single-molecule dynamic force spectroscopy (SMDFS), enzyme immunoassay and optical surface waves-based biosensor measurements. The experiments were performed at neutral pH (7.4) and acid pH (5.3) conditions. The data obtained confirm the role of LBP as a cell receptor for two typical viral species of the Flavivirus genus. A comparison of these data with similar data obtained for another cell receptor of this family, namely human αVβ3 integrin, reveals that both these receptors are very important. Studying the specific interaction between the cell receptors in question and specially prepared monoclonal antibodies against them, we could show that both interaction sites involved in the process of virus-cell interaction remain intact at pH 5.3. At the same time, for these acid conditions characteristic for an endosome during flavivirus-cell membrane fusion, SMDFS data reveal the existence of a force-induced (effective already for forces as small as 30-70 pN) sharp globule-coil transition for LBP and LBP-fragments of protein E complexes. We argue that this conformational transformation, being an analog of abrupt first-order phase transition and having similarity with the famous Rayleigh hydrodynamic instability, might be indispensable for the flavivirus-cell membrane fusion process. PMID:25319621

  9. High Throughput Screening for Compounds That Alter Muscle Cell Glycosylation Identifies New Role for N-Glycans in Regulating Sarcolemmal Protein Abundance and Laminin Binding*

    PubMed Central

    Cabrera, Paula V.; Pang, Mabel; Marshall, Jamie L.; Kung, Raymond; Nelson, Stanley F.; Stalnaker, Stephanie H.; Wells, Lance; Crosbie-Watson, Rachelle H.; Baum, Linda G.

    2012-01-01

    Duchenne muscular dystrophy is an X-linked disorder characterized by loss of dystrophin, a cytoskeletal protein that connects the actin cytoskeleton in skeletal muscle cells to extracellular matrix. Dystrophin binds to the cytoplasmic domain of the transmembrane glycoprotein β-dystroglycan (β-DG), which associates with cell surface α-dystroglycan (α-DG) that binds laminin in the extracellular matrix. β-DG can also associate with utrophin, and this differential association correlates with specific glycosylation changes on α-DG. Genetic modification of α-DG glycosylation can promote utrophin binding and rescue dystrophic phenotypes in mouse dystrophy models. We used high throughput screening with the plant lectin Wisteria floribunda agglutinin (WFA) to identify compounds that altered muscle cell surface glycosylation, with the goal of finding compounds that increase abundance of α-DG and associated sarcolemmal glycoproteins, increase utrophin usage, and increase laminin binding. We identified one compound, lobeline, from the Prestwick library of Food and Drug Administration-approved compounds that fulfilled these criteria, increasing WFA binding to C2C12 cells and to primary muscle cells from wild type and mdx mice. WFA binding and enhancement by lobeline required complex N-glycans but not O-mannose glycans that bind laminin. However, inhibiting complex N-glycan processing reduced laminin binding to muscle cell glycoproteins, although O-mannosylation was intact. Glycan analysis demonstrated a general increase in N-glycans on lobeline-treated cells rather than specific alterations in cell surface glycosylation, consistent with increased abundance of multiple sarcolemmal glycoproteins. This demonstrates the feasibility of high throughput screening with plant lectins to identify compounds that alter muscle cell glycosylation and identifies a novel role for N-glycans in regulating muscle cell function. PMID:22570487

  10. Mapping the laminin-binding and adhesive domain of the cell surface-associated Hlp/LBP protein from Mycobacterium leprae.

    PubMed

    Soares de Lima, Cristiana; Zulianello, Laurence; Marques, Maria Angela de Melo; Kim, Heejin; Portugal, Michelle Iespa; Antunes, Sérgio Luiz; Menozzi, Franco Dante; Ottenhoff, Tom Henricus Maria; Brennan, Patrick Joseph; Pessolani, Maria Cristina Vidal

    2005-07-01

    Binding of Mycobacterium leprae to and invasion of Schwann cells (SC) represent a crucial step that initiates nerve damage in leprosy. We and others have described that M. leprae colonization of the peripheral nerve system may be mediated in part by a surface-exposed histone-like protein (Hlp), characterized as a laminin-binding protein (LBP). Hlp/LBP has also been shown to play a role in the binding of mycobacteria to alveolar epithelial cells and macrophages. In the present study we report that M. leprae expresses Hlp/LBP protein during the course of human infection. Additionally, we analyzed the interaction of Hlp/LBP with the extracellular matrix and host cell surface. We show that Hlp/LBP, besides laminin, also binds heparin and heparan sulfate. Testing truncated recombinant Hlp molecules corresponding to the N-terminal (rHlp-N) and the C-terminal (rHlp-C) domains of the protein, we established that interaction of Hlp/LBP with laminin-2 and heparin is mainly mediated by the C-terminal domain of the protein. Moreover, the same domain was found to be involved in Hlp/LBP-mediating bacterial binding to human SC. Finally, evidence is shown suggesting that M. leprae produces a post-translationally modified Hlp/LBP containing methyllysine residues. Methylation of the lysine residues, however, seems not to affect the adhesive properties of Hlp/LBP. Taken together, our observations reinforce the involvement of Hlp/LBP as an adhesin in mycobacterial infections and define its highly positive C-terminal region as the major adhesive domain of this protein. PMID:15919224

  11. Borrelia burgdorferi BmpA Is a Laminin-Binding Protein▿

    PubMed Central

    Verma, Ashutosh; Brissette, Catherine A.; Bowman, Amy; Stevenson, Brian

    2009-01-01

    The Borrelia burgdorferi BmpA outer surface protein plays a significant role in mammalian infection by the Lyme disease spirochete and is an important antigen for the serodiagnosis of human infection. B. burgdorferi adheres to host extracellular matrix components, including laminin. The results of our studies indicate that BmpA and its three paralogous proteins, BmpB, BmpC, and BmpD, all bind to mammalian laminin. BmpA did not bind mammalian type I or type IV collagens or fibronectin. BmpA-directed antibodies significantly inhibited the adherence of live B. burgdorferi to laminin. The laminin-binding domain of BmpA was mapped to the carboxy-terminal 80 amino acids. Solubilized collagen inhibited BmpA-laminin binding, suggesting interactions through the collagen-binding domains of laminin. These results, together with previous data, indicate that BmpA and its paralogs are targets for the development of preventative and curative therapies for Lyme disease. PMID:19703983

  12. Sarcospan integration into laminin-binding adhesion complexes that ameliorate muscular dystrophy requires utrophin and α7 integrin

    PubMed Central

    Marshall, Jamie L.; Oh, Jennifer; Chou, Eric; Lee, Joy A.; Holmberg, Johan; Burkin, Dean J.; Crosbie-Watson, Rachelle H.

    2015-01-01

    Duchenne muscular dystrophy (DMD) is caused by mutations in the dystrophin gene that result in loss of the dystrophin–glycoprotein complex, a laminin receptor that connects the myofiber to its surrounding extracellular matrix. Utrophin, a dystrophin ortholog that is normally localized to the neuromuscular junction, is naturally upregulated in DMD muscle, which partially compensates for the loss of dystrophin. Transgenic overexpression of utrophin causes broad sarcolemma localization of utrophin, restoration of laminin binding and amelioration of disease in the mdx mouse model of DMD. We previously demonstrated that overexpression of sarcospan, a dystrophin- and utrophin-binding protein, ameliorates mdx muscular dystrophy. Sarcospan boosts levels of utrophin to therapeutic levels at the sarcolemma, where attachment to laminin is restored. However, understanding the compensatory mechanism is complicated by concomitant upregulation of α7β1 integrin, which also binds laminin. Similar to the effects of utrophin, transgenic overexpression of α7 integrin prevents DMD disease in mice and is accompanied by increased abundance of utrophin around the extra-synaptic sarcolemma. In order to investigate the mechanisms underlying sarcospan ‘rescue’ of muscular dystrophy, we created double-knockout mice to test the contributions of utrophin or α7 integrin. We show that sarcospan-mediated amelioration of muscular dystrophy in DMD mice is dependent on the presence of both utrophin and α7β1 integrin, even when they are individually expressed at therapeutic levels. Furthermore, we found that association of sarcospan into laminin-binding complexes is dependent on utrophin and α7β1 integrin. PMID:25504048

  13. Laminin-binding integrin gene copy number alterations in distinct epithelial-type cancers

    PubMed Central

    Harryman, William L; Pond, Erika; Singh, Parminder; Little, Andrew S; Eschbacher, Jennifer M; Nagle, Raymond B; Cress, Anne E

    2016-01-01

    Background: The laminin-binding integrin (LBI) family are cell adhesion molecules that are essential for invasion and metastasis of human epithelial cancers and cell adhesion mediated drug resistance. We investigated whether copy number alteration (CNA) or mutations of a five-gene signature (ITGB4, ITGA3, LAMB3, PLEC, and SYNE3), representing essential genes for LBI adhesion, would correlate with patient outcomes within human epithelial-type tumor data sets currently available in an open access format. Methods: We investigated the relative alteration frequency of an LBI signature panel (integrin β4 (ITGB4), integrin α3 (ITGA3), laminin β3 chain (LAMB3), plectin (PLEC), and nesprin 3 (SYNE3)), independent of the epithelial cancer type, within publically available and published data using cBioPortal and Oncomine software. We rank ordered the results using a 20% alteration frequency cut-off and limited the analysis to studies containing at least 100 samples. Kaplan-Meier survival curves were analyzed to determine if alterations in the LBI signature correlated with patient survival. The Oncomine data mining tool was used to compare the heat map expression of the LBI signature without SYNE3 (as this was not included in the Oncomine database) to drug resistance patterns. Results: Twelve different cancer types, representing 5,647 samples, contained at least a 20% alteration frequency of the five-gene LBI signature. The frequency of alteration ranged from 38.3% to 19.8%. Within the LBI signature, PLEC was the most commonly altered followed by LAMB3, ITGB4, ITGA3, and SYNE3 across all twelve cancer types. Within cancer types, there was little overlap of the individual amplified genes from each sample, suggesting different specific amplicons may alter the LBI adhesion structures. Of the twelve cancer types, overall survival was altered by CNA presence in bladder urothelial carcinoma (p=0.0143*) and cervical squamous cell carcinoma and endocervical adenocarcinoma (p=0

  14. The SpeB virulence factor of Streptococcus pyogenes, a multifunctional secreted and cell surface molecule with strepadhesin, laminin-binding and cysteine protease activity.

    PubMed

    Hytönen, J; Haataja, S; Gerlach, D; Podbielski, A; Finne, J

    2001-01-01

    The interactions between pathogenic bacteria and the host need to be resolved at the molecular level in order to develop novel vaccines and drugs. We have previously identified strepadhesin, a novel glycoprotein-binding activity in Streptococcus pyogenes, which is regulated by Mga, a regulator of streptococcal virulence factors. We have now identified the protein responsible for the strepadhesin activity and find that (i) strepadhesin activity is carried by SpeB, streptococcal pyrogenic exotoxin with cysteine protease activity; (ii) SpeB carries laminin-binding activity of the bacteria; and (iii) SpeB is not only a secreted molecule but also occurs unexpectedly tightly bound to the bacterial cell surface. Thus, in contrast to the previous view of SpeB as mainly an extracellular protease, it is also present as a streptococcal surface molecule with binding activity to laminin and other glycoproteins. PMID:11136470

  15. Residual laminin-binding activity and enhanced dystroglycan glycosylation by LARGE in novel model mice to dystroglycanopathy

    PubMed Central

    Kanagawa, Motoi; Nishimoto, Akemi; Chiyonobu, Tomohiro; Takeda, Satoshi; Miyagoe-Suzuki, Yuko; Wang, Fan; Fujikake, Nobuhiro; Taniguchi, Mariko; Lu, Zhongpeng; Tachikawa, Masaji; Nagai, Yoshitaka; Tashiro, Fumi; Miyazaki, Jun-Ichi; Tajima, Youichi; Takeda, Shin'ichi; Endo, Tamao; Kobayashi, Kazuhiro; Campbell, Kevin P.; Toda, Tatsushi

    2009-01-01

    Hypoglycosylation and reduced laminin-binding activity of α-dystroglycan are common characteristics of dystroglycanopathy, which is a group of congenital and limb-girdle muscular dystrophies. Fukuyama-type congenital muscular dystrophy (FCMD), caused by a mutation in the fukutin gene, is a severe form of dystroglycanopathy. A retrotransposal insertion in fukutin is seen in almost all cases of FCMD. To better understand the molecular pathogenesis of dystroglycanopathies and to explore therapeutic strategies, we generated knock-in mice carrying the retrotransposal insertion in the mouse fukutin ortholog. Knock-in mice exhibited hypoglycosylated α-dystroglycan; however, no signs of muscular dystrophy were observed. More sensitive methods detected minor levels of intact α-dystroglycan, and solid-phase assays determined laminin binding levels to be ∼50% of normal. In contrast, intact α-dystroglycan is undetectable in the dystrophic Largemyd mouse, and laminin-binding activity is markedly reduced. These data indicate that a small amount of intact α-dystroglycan is sufficient to maintain muscle cell integrity in knock-in mice, suggesting that the treatment of dystroglycanopathies might not require the full recovery of glycosylation. To examine whether glycosylation defects can be restored in vivo, we performed mouse gene transfer experiments. Transfer of fukutin into knock-in mice restored glycosylation of α-dystroglycan. In addition, transfer of LARGE produced laminin-binding forms of α-dystroglycan in both knock-in mice and the POMGnT1 mutant mouse, which is another model of dystroglycanopathy. Overall, these data suggest that even partial restoration of α-dystroglycan glycosylation and laminin-binding activity by replacing or augmenting glycosylation-related genes might effectively deter dystroglycanopathy progression and thus provide therapeutic benefits. PMID:19017726

  16. Molecular mechanisms for protein-encoded inheritance

    SciTech Connect

    Wiltzius, Jed J.W.; Landau, Meytal; Nelson, Rebecca; Sawaya, Michael R.; Apostol, Marcin I.; Goldschmidt, Lukasz; Soriaga, Angela B.; Cascio, Duilio; Rajashankar, Kanagalaghatta; Eisenberg, David

    2009-12-01

    In prion inheritance and transmission, strains are phenotypic variants encoded by protein 'conformations'. However, it is unclear how a protein conformation can be stable enough to endure transmission between cells or organisms. Here we describe new polymorphic crystal structures of segments of prion and other amyloid proteins, which offer two structural mechanisms for the encoding of prion strains. In packing polymorphism, prion strains are encoded by alternative packing arrangements (polymorphs) of {beta}-sheets formed by the same segment of a protein; in segmental polymorphism, prion strains are encoded by distinct {beta}-sheets built from different segments of a protein. Both forms of polymorphism can produce enduring conformations capable of encoding strains. These molecular mechanisms for transfer of protein-encoded information into prion strains share features with the familiar mechanism for transfer of nucleic acid-encoded information into microbial strains, including sequence specificity and recognition by noncovalent bonds.

  17. Functional regeneration of the transected recurrent laryngeal nerve using a collagen scaffold loaded with laminin and laminin-binding BDNF and GDNF.

    PubMed

    Wang, Baoxin; Yuan, Junjie; Chen, Xinwei; Xu, Jiafeng; Li, Yu; Dong, Pin

    2016-01-01

    Recurrent laryngeal nerve (RLN) injury remains a challenge due to the lack of effective treatments. In this study, we established a new drug delivery system consisting of a tube of Heal-All Oral Cavity Repair Membrane loaded with laminin and neurotrophic factors and tested its ability to promote functional recovery following RLN injury. We created recombinant fusion proteins consisting of brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) fused to laminin-binding domains (LBDs) in order to prevent neurotrophin diffusion. LBD-BDNF, LBD-GDNF, and laminin were injected into a collagen tube that was fitted to the ends of the transected RLN in rats. Functional recovery was assessed 4, 8, and 12 weeks after injury. Although vocal fold movement was not restored until 12 weeks after injury, animals treated with the collagen tube loaded with laminin, LBD-BDNF and LBD-GDNF showed improved recovery in vocalisation, arytenoid cartilage angles, compound muscle action potentials and regenerated fibre area compared to animals treated by autologous nerve grafting (p < 0.05). These results demonstrate the drug delivery system induced nerve regeneration following RLN transection that was superior to that induced by autologus nerve grafting. It may have potential applications in nerve regeneration of RLN transection injury. PMID:27558932

  18. Functional regeneration of the transected recurrent laryngeal nerve using a collagen scaffold loaded with laminin and laminin-binding BDNF and GDNF

    PubMed Central

    Wang, Baoxin; Yuan, Junjie; Chen, Xinwei; Xu, Jiafeng; Li, Yu; Dong, Pin

    2016-01-01

    Recurrent laryngeal nerve (RLN) injury remains a challenge due to the lack of effective treatments. In this study, we established a new drug delivery system consisting of a tube of Heal-All Oral Cavity Repair Membrane loaded with laminin and neurotrophic factors and tested its ability to promote functional recovery following RLN injury. We created recombinant fusion proteins consisting of brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) fused to laminin-binding domains (LBDs) in order to prevent neurotrophin diffusion. LBD-BDNF, LBD-GDNF, and laminin were injected into a collagen tube that was fitted to the ends of the transected RLN in rats. Functional recovery was assessed 4, 8, and 12 weeks after injury. Although vocal fold movement was not restored until 12 weeks after injury, animals treated with the collagen tube loaded with laminin, LBD-BDNF and LBD-GDNF showed improved recovery in vocalisation, arytenoid cartilage angles, compound muscle action potentials and regenerated fibre area compared to animals treated by autologous nerve grafting (p < 0.05). These results demonstrate the drug delivery system induced nerve regeneration following RLN transection that was superior to that induced by autologus nerve grafting. It may have potential applications in nerve regeneration of RLN transection injury. PMID:27558932

  19. DNA encoding a DNA repair protein

    DOEpatents

    Petrini, John H.; Morgan, William Francis; Maser, Richard Scott; Carney, James Patrick

    2006-08-15

    An isolated and purified DNA molecule encoding a DNA repair protein, p95, is provided, as is isolated and purified p95. Also provided are methods of detecting p95 and DNA encoding p95. The invention further provides p95 knock-out mice.

  20. Gene encoding herbicide safener binding protein

    SciTech Connect

    Walton, J.D.; Scott-Craig, J.S.

    1999-10-26

    The cDNA encoding safener binding protein (SafBP), also referred to as SBP1, is presented. The deduced amino acid sequence is provided. Methods of making and using SBP1 and SafBP to alter a plant's sensitivity to certain herbicides or a plant's responsiveness to certain safeners are also provided, as well as expression vectors, transgenic plants or other organisms transfected with vectors and seeds from the plants.

  1. Nucleic acids encoding human trithorax protein

    DOEpatents

    Evans, Glen A.; Djabali, Malek; Selleri, Licia; Parry, Pauline

    2001-01-01

    In accordance with the present invention, there is provided an isolated peptide having the characteristics of human trithorax protein (as well as DNA encoding same, antisense DNA derived therefrom and antagonists therefor). The invention peptide is characterized by having a DNA binding domain comprising multiple zinc fingers and at least 40% amino acid identity with respect to the DNA binding domain of Drosophila trithorax protein and at least 70% conserved sequence with respect to the DNA binding domain of Drosophila trithorax protein, and wherein said peptide is encoded by a gene located at chromosome 11 of the human genome at q23. Also provided are methods for the treatment of subject(s) suffering from immunodeficiency, developmental abnormality, inherited disease, or cancer by administering to said subject a therapeutically effective amount of one of the above-described agents (i.e., peptide, antagonist therefor, DNA encoding said peptide or antisense DNA derived therefrom). Also provided is a method for the diagnosis, in a subject, of immunodeficiency, developmental abnormality, inherited disease, or cancer associated with disruption of chromosome 11 at q23.

  2. Polynucleotides encoding TRF1 binding proteins

    DOEpatents

    Campisi, Judith; Kim, Sahn-Ho

    2002-01-01

    The present invention provides a novel telomere associated protein (Trf1-interacting nuclear protein 2 "Tin2") that hinders the binding of Trf1 to its specific telomere repeat sequence and mediates the formation of a Tin2-Trf1-telomeric DNA complex that limits telomerase access to the telomere. Also included are the corresponding nucleic acids that encode the Tin2 of the present invention, as well as mutants of Tin2. Methods of making, purifying and using Tin2 of the present invention are described. In addition, drug screening assays to identify drugs that mimic and/or complement the effect of Tin2 are presented.

  3. Gene encoding herbicide safener binding protein

    SciTech Connect

    Walton, Jonathan D.; Scott-Craig, John S.

    1999-01-01

    The cDNA encoding safener binding protein (SafBP), also referred to as SBP1, is set forth in FIG. 5 and SEQ ID No. 1. The deduced amino acid sequence is provided in FIG. 5 and SEQ ID No. 2. Methods of making and using SBP1 and SafBP to alter a plant's sensitivity to certain herbicides or a plant's responsiveness to certain safeners are also provided, as well as expression vectors, transgenic plants or other organisms transfected with said vectors and seeds from said plants.

  4. Schwann Cells Increase Prostate and Pancreatic Tumor Cell Invasion Using Laminin Binding A6 Integrin

    PubMed Central

    Sroka, Isis C.; Chopra, Harsharon; Das, Lipsa; Gard, Jaime M.C.; Nagle, Raymond B.; Cress, Anne E.

    2016-01-01

    Human pancreatic and prostate cancers metastasize along nerve axons during perineural invasion. The extracellular matrix laminin class of proteins is an abundant component of both myelinated and non-myelinated nerves. Analysis of human pancreatic and prostate tissue revealed both perineural and endoneural invasion with Schwann cells surrounded or disrupted by tumor, respectively. Tumor and nerve cell co-culture conditions were used to determine if myelinating or non-myelinating Schwann cell (S16 and S16Y, respectively) phenotype was equally likely to promote integrin-dependent cancer cell invasion and migration on laminin. Conditioned medium from S16 cells increased tumor cell (DU145, PC3, and CFPAC1) invasion into laminin approximately 1.3–2.0 fold compared to fetal bovine serum (FBS) treated cells. Integrin function (e.g., ITGA6p formation) increased up to 1.5 fold in prostate (DU145, PC3, RWPE-1) and pancreatic (CFPAC1) cells, and invasion was dependent on ITGA6p formation and ITGB1 as determined by function-blocking antibodies. In contrast, conditioned medium isolated from S16Y cells (non-myelinating phenotype) decreased constitutive levels of ITGA6p in the tumor cells by 50% compared to untreated cells and decreased ITGA6p formation 3.0 fold compared to S16 treated cells. Flow cytometry and western blot analysis revealed loss of ITGA6p formation as reversible and independent of overall loss of ITGA6 expression. These results suggest that the myelinating phenotype of Schwann cells within the tumor microenvironment increased integrin-dependent tumor invasion on laminin. PMID:26239765

  5. The use of laminin modified linear ordered collagen scaffolds loaded with laminin-binding ciliary neurotrophic factor for sciatic nerve regeneration in rats.

    PubMed

    Cao, Jiani; Sun, Changkai; Zhao, Hui; Xiao, Zhifeng; Chen, Bing; Gao, Jian; Zheng, Tiezheng; Wu, Wei; Wu, Shuang; Wang, Jingyu; Dai, Jianwu

    2011-06-01

    Nerve conduit provides a promising strategy for nerve injury repair in the peripheral nervous system (PNS). However, simply bridging the transected nerve with an empty conduit is hard to satisfy functional recovery. The regenerated axons may disperse during regeneration in the empty lumen, limiting the functional recovery. Our previous work had reported that linear ordered collagen scaffold (LOCS) could be used as a nerve guidance material. Here we cross-linked LOCS fibers with laminin which was a major component of the extracellular matrix in nervous system. Ciliary neurotrophic factor (CNTF) plays a critical role in peripheral nerve regeneration. But the lack of efficient CNTF delivery approach limits its clinical applications. To retain CNTF on the scaffold, a laminin binding domain (LBD) was fused to the N-terminal of CNTF. Compared with NAT-CNTF, LBD-CNTF exhibited specific laminin-binding ability and comparable neurotrophic bioactivity. We combined LBD-CNTF with the laminin modified LOCS fibers to construct a double-functional bio-scaffold. The functional scaffold was filled in silicon conduit and tested in the rat sciatic nerve transection model. Results showed that this functional biomaterial could guide the axon growth, retain more CNTF on the scaffolds and enhance the nerve regeneration as well as functional recovery. PMID:21397941

  6. Selection for genes encoding secreted proteins and receptors.

    PubMed Central

    Klein, R D; Gu, Q; Goddard, A; Rosenthal, A

    1996-01-01

    Extracellular proteins play an essential role in the formation, differentiation, and maintenance of multicellular organisms. Despite that, the systematic identification of genes encoding these proteins has not been possible. We describe here a highly efficient method to isolate genes encoding secreted and membrane-bound proteins by using a single-step selection in yeast. Application of this method, termed signal peptide selection, to various tissues yielded 559 clones that appear to encode known or novel extracellular proteins. These include members of the transforming growth factor and epidermal growth factor protein families, endocrine hormones, tyrosine kinase receptors, serine/threonine kinase receptors, seven transmembrane receptors, cell adhesion molecules, extracellular matrix proteins, plasma proteins, and ion channels. The eventual identification of most, or all, extracellular signaling molecules will advance our understanding of fundamental biological processes and our ability to intervene in disease states. Images Fig. 1 PMID:8692953

  7. Nucleic acid compositions and the encoding proteins

    DOEpatents

    Preston, III, James F.; Chow, Virginia; Nong, Guang; Rice, John D.; St. John, Franz J.

    2014-09-02

    The subject invention provides at least one nucleic acid sequence encoding an aldouronate-utilization regulon isolated from Paenibacillus sp. strain JDR-2, a bacterium which efficiently utilizes xylan and metabolizes aldouronates (methylglucuronoxylosaccharides). The subject invention also provides a means for providing a coordinately regulated process in which xylan depolymerization and product assimilation are coupled in Paenibacillus sp. strain JDR-2 to provide a favorable system for the conversion of lignocellulosic biomass to biobased products. Additionally, the nucleic acid sequences encoding the aldouronate-utilization regulon can be used to transform other bacteria to form organisms capable of producing a desired product (e.g., ethanol, 1-butanol, acetoin, 2,3-butanediol, 1,3-propanediol, succinate, lactate, acetate, malate or alanine) from lignocellulosic biomass.

  8. Proteins encoded near the adenovirus late messenger RNA leader segments

    SciTech Connect

    Lewis, J.B.; Anderson, C.W.

    1983-01-01

    Small fragments of adenovirus 2 DNA cloned into the single-strand phage M13 were used to select adenoviral messenger RNAs transcribed from the R-strand between map positions 16 and 30. Cell-free translation of these mRNAs produced proteins of 13.5K, 13.6K, and 11.5K, respectively encoded between the first and second segments of the tripartite major late leader, within the ''i''-leader segment, and immediately preceding the third leader segment. Partial sequence analysis of the 13.6K protein is consistent with the hypothesis that it is encoded within the i-leader segment.

  9. Evolutionary relationship of nuclear genes encoding mitochondrial proteins across grasses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Comparative genome studies were done across taxa to provide a basic understanding of genome evolution regarding nuclear genes encoding for mitochondrial proteins and their conservation in grass species. Two different mitochondria-related gene sets, one from rice and another from Arabidopsis, were us...

  10. On the Encoding of Proteins for Disordered Regions Prediction

    PubMed Central

    Becker, Julien; Maes, Francis; Wehenkel, Louis

    2013-01-01

    Disordered regions, i.e., regions of proteins that do not adopt a stable three-dimensional structure, have been shown to play various and critical roles in many biological processes. Predicting and understanding their formation is therefore a key sub-problem of protein structure and function inference. A wide range of machine learning approaches have been developed to automatically predict disordered regions of proteins. One key factor of the success of these methods is the way in which protein information is encoded into features. Recently, we have proposed a systematic methodology to study the relevance of various feature encodings in the context of disulfide connectivity pattern prediction. In the present paper, we adapt this methodology to the problem of predicting disordered regions and assess it on proteins from the 10th CASP competition, as well as on a very large subset of proteins extracted from PDB. Our results, obtained with ensembles of extremely randomized trees, highlight a novel feature function encoding the proximity of residues according to their accessibility to the solvent, which is playing the second most important role in the prediction of disordered regions, just after evolutionary information. Furthermore, even though our approach treats each residue independently, our results are very competitive in terms of accuracy with respect to the state-of-the-art. A web-application is available at http://m24.giga.ulg.ac.be:81/x3Disorder. PMID:24358161

  11. On the encoding of proteins for disordered regions prediction.

    PubMed

    Becker, Julien; Maes, Francis; Wehenkel, Louis

    2013-01-01

    Disordered regions, i.e., regions of proteins that do not adopt a stable three-dimensional structure, have been shown to play various and critical roles in many biological processes. Predicting and understanding their formation is therefore a key sub-problem of protein structure and function inference. A wide range of machine learning approaches have been developed to automatically predict disordered regions of proteins. One key factor of the success of these methods is the way in which protein information is encoded into features. Recently, we have proposed a systematic methodology to study the relevance of various feature encodings in the context of disulfide connectivity pattern prediction. In the present paper, we adapt this methodology to the problem of predicting disordered regions and assess it on proteins from the 10th CASP competition, as well as on a very large subset of proteins extracted from PDB. Our results, obtained with ensembles of extremely randomized trees, highlight a novel feature function encoding the proximity of residues according to their accessibility to the solvent, which is playing the second most important role in the prediction of disordered regions, just after evolutionary information. Furthermore, even though our approach treats each residue independently, our results are very competitive in terms of accuracy with respect to the state-of-the-art. A web-application is available at http://m24.giga.ulg.ac.be:81/x3Disorder. PMID:24358161

  12. Identification of Sequences Encoding Symbiodinium minutum Mitochondrial Proteins

    PubMed Central

    Butterfield, Erin R.; Howe, Christopher J.; Nisbet, R. Ellen R.

    2016-01-01

    The dinoflagellates are an extremely diverse group of algae closely related to the Apicomplexa and the ciliates. Much work has previously been undertaken to determine the presence of various biochemical pathways within dinoflagellate mitochondria. However, these studies were unable to identify several key transcripts including those encoding proteins involved in the pyruvate dehydrogenase complex, iron–sulfur cluster biosynthesis, and protein import. Here, we analyze the draft nuclear genome of the dinoflagellate Symbiodinium minutum, as well as RNAseq data to identify nuclear genes encoding mitochondrial proteins. The results confirm the presence of a complete tricarboxylic acid cycle in the dinoflagellates. Results also demonstrate the difficulties in using the genome sequence for the identification of genes due to the large number of introns, but show that it is highly useful for the determination of gene duplication events. PMID:26798115

  13. Identification of Sequences Encoding Symbiodinium minutum Mitochondrial Proteins.

    PubMed

    Butterfield, Erin R; Howe, Christopher J; Nisbet, R Ellen R

    2016-02-01

    The dinoflagellates are an extremely diverse group of algae closely related to the Apicomplexa and the ciliates. Much work has previously been undertaken to determine the presence of various biochemical pathways within dinoflagellate mitochondria. However, these studies were unable to identify several key transcripts including those encoding proteins involved in the pyruvate dehydrogenase complex, iron-sulfur cluster biosynthesis, and protein import. Here, we analyze the draft nuclear genome of the dinoflagellate Symbiodinium minutum, as well as RNAseq data to identify nuclear genes encoding mitochondrial proteins. The results confirm the presence of a complete tricarboxylic acid cycle in the dinoflagellates. Results also demonstrate the difficulties in using the genome sequence for the identification of genes due to the large number of introns, but show that it is highly useful for the determination of gene duplication events. PMID:26798115

  14. Biosensor studies of collagen and laminin binding with immobilized Escherichia coli O157:H7 and inhibition with naturally occurring food additives

    NASA Astrophysics Data System (ADS)

    Medina, Marjorie B.

    1999-01-01

    Escherichia coli O157:H7 outbreaks were mostly due to consumption of undercooked contaminated beef which resulted in severe illness and several fatalities. Recalls of contaminated meat are costly for the meat industry. Our research attempts to understand the mechanisms of bacterial adhesion on animal carcass in order to eliminate or reduce pathogens in foods. We have reported the interactions of immobilized E. coli O157:H7 cells with extracellular matrix (ECM) components using a surface plasmon resonance biosensor (BIAcore). These studies showed that immobilized bacterial cells allowed the study of real-time binding interactions of bacterial surface with the ECM compounds, collagen I, laminin and fibronectin. Collagen I and laminin bound to the E. coli sensor surface with dissociation and association rates ranging from 106 to 109. Binding of collagen I and laminin mixture resulted in synergistic binding signals. An inhibition model was derived using collagen-laminin as the ligand which binds with E. coli sensor. A select group of naturally occurring food additives was evaluated by determining their effectivity in inhibiting the collagen-laminin binding to the bacterial sensor. Bound collagen-laminin was detached from the E. coli sensor surface with the aid of an organic acid. The biosensor results were verified with cell aggregation assays which were observed with optical and electron microscopes. These biosensor studies provided understanding of bacterial adhesion to connective tissue macromolecules. It also provided a model system for the rapid assessment of potential inhibitors that can be used in carcass treatment to inhibit or reduce bacterial contamination.

  15. Neutron-Encoded Protein Quantification by Peptide Carbamylation

    NASA Astrophysics Data System (ADS)

    Ulbrich, Arne; Merrill, Anna E.; Hebert, Alexander S.; Westphall, Michael S.; Keller, Mark P.; Attie, Alan D.; Coon, Joshua J.

    2014-01-01

    We describe a chemical tag for duplex proteome quantification using neutron encoding (NeuCode). The method utilizes the straightforward, efficient, and inexpensive carbamylation reaction. We demonstrate the utility of NeuCode carbamylation by accurately measuring quantitative ratios from tagged yeast lysates mixed in known ratios and by applying this method to quantify differential protein expression in mice fed a either control or high-fat diet.

  16. Neutron-encoded protein quantification by peptide carbamylation.

    PubMed

    Ulbrich, Arne; Merrill, Anna E; Hebert, Alexander S; Westphall, Michael S; Keller, Mark P; Attie, Alan D; Coon, Joshua J

    2014-01-01

    We describe a chemical tag for duplex proteome quantification using neutron encoding (NeuCode). The method utilizes the straightforward, efficient, and inexpensive carbamylation reaction. We demonstrate the utility of NeuCode carbamylation by accurately measuring quantitative ratios from tagged yeast lysates mixed in known ratios and by applying this method to quantify differential protein expression in mice fed a either control or high-fat diet. PMID:24178922

  17. Neutron-encoded protein quantification by peptide carbamylation

    PubMed Central

    Ulbrich, Arne; Merrill, Anna E.; Hebert, Alexander S.; Westphall, Michael S.; Keller, Mark P.; Attie, Alan D.; Coon, Joshua J.

    2013-01-01

    We describe a chemical tag for duplex proteome quantification using neutron encoding (NeuCode). The method utilizes the straightforward, efficient, and inexpensive carbamylation reaction. We demonstrate the utility of NeuCode carbamylation by accurately measuring quantitative ratios from tagged yeast lysates mixed in known ratios and by applying this method to quantify differential protein expression in mice fed a either control or high-fat diet. PMID:24178922

  18. Transport of nuclear-encoded proteins into secondarily evolved plastids.

    PubMed

    Hempel, Franziska; Bozarth, Andrew; Sommer, Maik S; Zauner, Stefan; Przyborski, Jude M; Maier, Uwe-G

    2007-09-01

    Many algal groups evolved by engulfment and intracellular reduction of a eukaryotic phototroph within a heterotrophic cell. Via this process, so-called secondary plastids evolved, surrounded by three or four membranes. In these organisms most of the genetic material encoding plastid functions is localized in the cell nucleus, with the result that many proteins have to pass three, four, or even five membranes to reach their final destination within the plastid. In this article, we review recent models and findings that help to explain important cellular mechanisms involved in the complex process of protein transport into secondary plastids. PMID:17696773

  19. Expression of genes encoding extracellular matrix proteins: A macroarray study

    PubMed Central

    FUTYMA, KONRAD; MIOTŁA, PAWEŁ; RÓŻYŃSKA, KRYSTYNA; ZDUNEK, MAŁGORZATA; SEMCZUK, ANDRZEJ; RECHBERGER, TOMASZ; WOJCIEROWSKI, JACEK

    2014-01-01

    Endometrial cancer (EC) is one of the most common gynecological malignancies in Poland, with well-established risk factors. Genetic instability and molecular alterations responsible for endometrial carcinogenesis have been systematically investigated. The aim of the present study was to investigate, by means of cDNA macroarrays, the expression profiles of genes encoding extracellular matrix (ECM) proteins in ECs. Tissue specimens were collected during surgical procedures from 40 patients with EC, and control tissue was collected from 9 patients with uterine leiomyomas. RNA was isolated and RT-PCR with radioisotope-labeled cDNA was performed. The levels of ECM protein gene expression in normal endometrial tissues were compared to the expression of these genes in EC specimens. Statistically significant differences in gene expression, stratified by clinical stage of the ECs, were detected for aggrecan, vitronectin, tenascin R, nidogen and two collagen proteins: type VIII chain α1 and type XI chain α2. All of these proteins were overexpressed in stage III endometrial carcinomas compared to levels in stage I and II uterine neoplasms. In conclusion, increased expression of genes encoding ECM proteins may play an important role in facilitating accelerated disease progression of human ECs. PMID:25231141

  20. Genetically encoded sensors of protein hydrodynamics and molecular proximity

    PubMed Central

    Hoepker, Alexander C.; Wang, Ariel; Le Marois, Alix; Suhling, Klaus; Yan, Yuling; Marriott, Gerard

    2015-01-01

    The specialized light organ of the ponyfish supports the growth of the bioluminescent symbiont Photobacterium leiognathi. The bioluminescence of P. leiognathi is generated within a heteromeric protein complex composed of the bacterial luciferase and a 20-kDa lumazine binding protein (LUMP), which serves as a Förster resonance energy transfer (FRET) acceptor protein, emitting a cyan-colored fluorescence with an unusually long excited state lifetime of 13.6 ns. The long fluorescence lifetime and small mass of LUMP are exploited for the design of highly optimized encoded sensors for quantitative fluorescence anisotropy (FA) measurements of protein hydrodynamics. In particular, large differences in the FA values of the free and target-bound states of LUMP fusions appended with capture sequences of up to 20 kDa are used in quantitative FA imaging and analysis of target proteins. For example, a fusion protein composed of LUMP and a 5-kDa G protein binding domain is used as an FA sensor to quantify the binding of the GTP-bound cell division control protein 42 homolog (Cdc42) (21 kDa) in solution and within Escherichia coli. Additionally, the long fluorescence lifetime and the surface-bound fluorescent cofactor 6,7-dimethyl-8- (1′-dimethyl-ribityl) lumazine in LUMP are utilized in the design of highly optimized FRET probes that use Venus as an acceptor probe. The efficiency of FRET in a zero-length LUMP-Venus fusion is 62% compared to ∼31% in a related CFP-Venus fusion. The improved FRET efficiency obtained by using LUMP as a donor probe is used in the design of a FRET-optimized genetically encoded LUMP-Venus substrate for thrombin. PMID:25931526

  1. Adenovirus type 2 encoded early 11 kDa protein

    SciTech Connect

    Murthy, S.V.K.N.; Kapoor, Q.S.

    1986-05-01

    Several adenovirus type 2 (Ad2) encoded early proteins have been identified in viral infected human KB cells. These proteins are of great interest as they play key roles in cell transformation, viral DNA synthesis and gene expression. They have partially purified an AD2 encoded early polypeptide of an apparent molecular weight of 11 kilodaltons from the nuclei of viral infected cells labelled with /sup 35/S-methionine. After DNA removal from the nuclear extracts, the polypeptide was isolated using DEAE-Sephacel anion exchange and Biogel P-10 gel filtration columns. This simple two step procedure yielded several fold purification of the polypeptide. Antisera raised in mice against an Ad2 transformed rat cell line 8617 was found to immunoprecipitate the 11 kDa polypeptide from the nuclear extract of Ad2 infected KB cells. After relating this protein to an open reading frame of an Ad2 early gene block by matching the amino acid sequences to the nucleotide sequences of early genes, they plan to functionally characterize this protein by using monoclonal antibodies in in vivo and in vitro experiments.

  2. Kinetoplast DNA-encoded ribosomal protein S12

    PubMed Central

    Aphasizheva, Inna; Maslov, Dmitri A; Aphasizhev, Ruslan

    2013-01-01

    Mitochondrial ribosomes of Trypanosoma brucei are composed of 9S and 12S rRNAs, which are encoded by the kinetoplast genome, and more than 150 proteins encoded in the nucleus and imported from the cytoplasm. However, a single ribosomal protein RPS12 is encoded by the kinetoplast DNA (kDNA) in all trypanosomatid species examined. As typical for these organisms, the gene itself is cryptic and its transcript undergoes an extensive U-insertion/deletion editing. An evolutionary trend to reduce or eliminate RNA editing could be traced with other cryptogenes, but the invariably pan-edited RPS12 cryptogene is apparently spared. Here we inquired whether editing of RPS12 mRNA is essential for mitochondrial translation. By RNAi-mediated knockdowns of RNA editing complexes and inducible knock-in of a key editing enzyme in procyclic parasites, we could reversibly downregulate production of edited RPS12 mRNA and, by inference, synthesis of this protein. While inhibition of editing decreased edited mRNA levels, the translation of edited (Cyb) and unedited (COI) mRNAs was blocked. Furthermore, the population of SSU-related 45S complexes declined upon inactivation of editing and so did the amount of mRNA-bound ribosomes. In bloodstream parasites, which lack active electron transport chain but still require translation of ATP synthase subunit 6 mRNA (A6), both edited RPS12 and A6 mRNAs were detected in translation complexes. Collectively, our results indicate that a single ribosomal protein gene retained by the kinetoplast mitochondrion serves as a possible functional link between editing and translation processes and provide the rationale for the evolutionary conservation of RPS12 pan-editing. PMID:24270388

  3. (Genetic engineering with a gene encoding a soybean storage protein)

    SciTech Connect

    Beachy, R.N.

    1985-12-18

    We have isolated and characterized a gene which encodes the alpha prime subunit of beta conglycinin. This gene was fully sequenced by DNA sequence analysis and a report of that work was prepared and submitted for publication in early November 1985. This represented the culmination of several years of research effort by several scientists. A preprint of that work is attached to this report and has been offered by Dr. J.J. Doyle, Dr. Mary A. Schuler and Dr. Jerry Slighton, as well as myself. This paper is a comparison of the alpha prime subunit gene with a similar gene from phaseolus vulgaris, the common garden bean. In this paper we compare the sequences that are 5' of the gene, and which would represent the transcriptional promoter, as well as the sequences within the structural region of the gene. The sequence paper also compares the amino acid sequence of these two genes with that of other genes from Phaseolus, peas and from soybeans. On the basis of this comparison, we predict evolutionary trends within the multigene families which encode these proteins in the various plants, as well as to look at the protein itself to try to predict regions of the protein that might have functional significance. All of this work was done on a prior DOE-BER grant and has simply been reported here for the first time.

  4. Mitochondrial heat shock protein 70, a molecular chaperone for proteins encoded by mitochondrial DNA

    PubMed Central

    1994-01-01

    Mitochondrial heat shock protein 70 (mt-Hsp70) has been shown to play an important role in facilitating import into, as well as folding and assembly of nuclear-encoded proteins in the mitochondrial matrix. Here, we describe a role for mt-Hsp70 in chaperoning proteins encoded by mitochondrial DNA and synthesized within mitochondria. The availability of mt-Hsp70 function influences the pattern of proteins synthesized in mitochondria of yeast both in vivo and in vitro. In particular, we show that mt-Hsp70 acts in maintaining the var1 protein, the only mitochondrially encoded subunit of mitochondrial ribosomes, in an assembly competent state, especially under heat stress conditions. Furthermore, mt-Hsp70 helps to facilitate assembly of mitochondrially encoded subunits of the ATP synthase complex. By interacting with the ATP-ase 9 oligomer, mt-Hsp70 promotes assembly of ATP-ase 6, and thereby protects the latter protein from proteolytic degradation. Thus mt-Hsp70 by acting as a chaperone for proteins encoded by the mitochondrial DNA, has a critical role in the assembly of supra- molecular complexes. PMID:7962074

  5. Bioinformatics Annotation of Human Y Chromosome-Encoded Protein Pathways and Interactions.

    PubMed

    Rengaraj, Deivendran; Kwon, Woo-Sung; Pang, Myung-Geol

    2015-09-01

    We performed a comprehensive analysis of human Y chromosome-encoded proteins, their pathways, and their interactions using bioinformatics tools. From the NCBI annotation release 107 of human genome, we retrieved a total of 66 proteins encoded on Y chromosome. Most of the retrieved proteins were also matched with the proteins listed in the core databases of the Human Proteome Project including neXtProt, PeptideAtlas, and the Human Protein Atlas. When we examined the pathways of human Y-encoded proteins through KEGG database and Pathway Studio software, many of proteins fall into the categories related to cell signaling pathways. Using the STRING program, we found a total of 49 human Y-encoded proteins showing strong/medium interaction with each other. While using the Pathway studio software, we found that a total of 16 proteins interact with other chromosome-encoded proteins. In particular, the SRY protein interacted with 17 proteins encoded on other chromosomes. Additionally, we aligned the sequences of human Y-encoded proteins with the sequences of chimpanzee and mouse Y-encoded proteins using the NCBI BLAST program. This analysis resulted in a significant number of orthologous proteins between human, chimpanzee, and mouse. Collectively, our findings provide the scientific community with additional information on the human Y chromosome-encoded proteins. PMID:26279084

  6. A new heterogeneous family of telomerically encoded Cryptosporidium proteins

    PubMed Central

    Bouzid, Maha; Hunter, Paul R; McDonald, Vincent; Elwin, Kristin; Chalmers, Rachel M; Tyler, Kevin M

    2013-01-01

    Cryptosporidiosis is predominantly caused by two closely related species of protozoan parasites the zoonotic Cryptosporidium parvum and anthroponotic Cryptosporidium hominis which diverge phenotypically in respect to host range and virulence. Using comparative genomics we identified two genes displaying overt heterogeneity between species. Although initial work suggested both were species specific, Cops-1 for C. parvum and Chos-1 for C. hominis, subsequent study identified an abridged ortholog of Cops-1 in C. hominis. Cops-1 and Chos-1 showed limited, but significant, similarity to each other and share common features: (i) telomeric location: Cops-1 is the last gene on chromosome 2, whilst Chos-1 is the first gene on chromosome 5, (ii) encode circa 50-kDa secreted proteins with isoelectric points above 10, (iii) are serine rich, and (iv) contain internal nucleotide repeats. Importantly, Cops-1 sequence contains specific SNPs with good discriminatory power useful epidemiologically. C. parvum-infected patient sera recognized a 50-kDa protein in antigen preparations of C. parvum but not C. hominis, consistent with Cops-1 being antigenic for patients. Interestingly, anti-Cops-1 monoclonal antibody (9E1) stained oocyst content and sporozoite surface of C. parvum only. This study provides a new example of protozoan telomeres as rapidly evolving contingency loci encoding putative virulence factors. PMID:23467513

  7. Expression cloning of genes encoding human peroxisomal proteins

    SciTech Connect

    Spathaky, J.M.; Tate, A.W.; Cox, T.M.

    1994-09-01

    Numerous metabolic disorders associated with diverse peroxisomal defects have been identified but their molecular characterization has been hampered by difficulties associated with the purification of proteins from this fragile organelle. We have utilized antibodies directed against the C-terminal tripeptide peroxisomal targeting signal to detect hitherto unknown peroxisomal proteins in tissue fractions and to isolate genes encoding peroxisonal proteins from human expression libraries. We immunized rabbits with a peptide conjugate encompassing the C-terminal nine amino acids of rat peroxisomal acyl CoA oxidase. Immunoprecipitation assays using radio-labelled peptide showed that the antibody specifically recognizes the terminal SKL motif as well as C-terminal SHL and SRL but not SHL at an internal position. Affinity-purified antibody was used to probe Western blots of crude and peroxisome-enriched monkey liver preparations and detected 8-10 proteins specifically in the peroxisome fractions. 100 positive clones were identified on screening a human liver cDNA expression library in {lambda}-gt11. Sequence analysis has confirmed the identity of cDNA clones for human acyl CoA oxidase and epoxide hydrolase. Four clones show no sequence identity and their putative role in the human peroxisome is being explored.

  8. Identification of three transforming proteins encoded by cottontail rabbit papillomavirus.

    PubMed Central

    Meyers, C; Harry, J; Lin, Y L; Wettstein, F O

    1992-01-01

    Cottontail rabbit papillomavirus (CRPV) provides an animal model for human papillomaviruses associated with a high risk of cancer development. So far, nothing is known about the transforming functions of CRPV genes because of the lack of an assay system. We have recently developed two systems to assay for CRPV transforming functions. One is based on the finding that transformation of NIH 3T3 cells by CRPV is considerably increased by deleting sequences in open reading frame L2. The second one is based on the use of a cottontail rabbit skin epithelial cell line, sf1Ep (C. Meyers and F. O. Wettstein, Virology 181:637-646, 1991). Mutations were introduced which abolished expression of the full-length E6 protein (LE6), the short E6 protein (SE6) initiated at the second ATG of E6, the E7 protein, or the E5 protein. Mutations affecting LE6 or E7, but not SE6, reduced transformation of NIH 3T3 and sf1Ep cells. Transformed NIH 3T3 cell lines with mutations in LE6 and E7 did not grow in soft agar, while those with mutations in SE6 and E5 grew with a reduced efficiency. The cell lines with mutations in LE6, SE6, or E7 still did induce tumors in nude mice. These mutations, however, abolished the ability to induce papillomas in rabbits. When expressed individually with a retroviral vector, LE6, SE6, or E7, but not E5, conferred anchorage-independent growth. The level of viral protein expression in these cell lines was generally low, and a comparison of the abundance of virus-specific mRNA showed that cell lines contained 20 to 50 times less mRNA than a cottontail rabbit papilloma. These data demonstrate that CRPV encodes at least three transforming proteins. Images PMID:1310771

  9. Human cytoplasmic actin proteins are encoded by a multigene family

    SciTech Connect

    Engel, J.; Gunning, P.; Kedes, L.

    1982-06-01

    The authors characterized nine human actin genes that they isolated from a library of cloned human DNA. Measurements of the thermal stability of hybrids formed between each cloned actin gene and ..cap alpha..-, ..beta..-, and ..gamma..-actin mRNA demonstrated that only one of the clones is most homologous to sarcomeric actin mRNA, whereas the remaining eight clones are most homologous to cytoplasmic actin mRNA. By the following criteria they show that these nine clones represent nine different actin gene loci rather than different alleles or different parts of a single gene: (i) the restriction enzyme maps of the coding regions are dissimilar; (ii) each clone contains sufficient coding region to encode all or most of an entire actin gene; and (iii) each clone contains sequences homologous to both the 5' and 3' ends of the coding region of a cloned chicken ..beta..-actin cDNA. They conclude, therefore, that the human cytoplasmic actin proteins are encoded by a multigene family.

  10. Genetically encoded force sensors for measuring mechanical forces in proteins

    PubMed Central

    Wang, Yuexiu; Sachs, Frederick

    2011-01-01

    There are three sources of free energy for cells: chemical potential, electrical potential and mechanical potential. There is little known about the last one since there have not been simple ways to measure stress in proteins in cells. we have now developed genetically encoded force sensors to assess the stress in fibrous proteins in living cells. These FReT based fluorescence sensors can be read out at video rates and provide real time maps of the stress distribution in cells, tissues and animals. The sensors can be inserted into specific proteins and in general do not disturb the normal function or anatomy. The original sensors used mutant GFPs linked by elastic linkers. These sensors provide a linear output with applied stress but the response is linear in strain. To improve contrast and dynamic range we have now developed a new class of sensors that are smaller making them less invasive, and have much higher intrinsic sensitivity since force modulates the angle between the donor and acceptor much more than the distance between them. Known as cpstFRET, the probe shows improved biocompatibility, wider dynamic range and higher sensitivity. PMID:21966553

  11. Hepatitis E virus ORF1 encoded non structural protein-host protein interaction network.

    PubMed

    Ojha, Nishant Kumar; Lole, Kavita S

    2016-02-01

    Hepatitis E virus ORF1 encoded non-structural polyprotein (nsP) consist of multiple domains, namely: Methyltransferase, Y-domain, Protease, X-domain, Helicase and RNA dependent RNA polymerase. We have attempted to identify human liver cell proteins that are interacting with HEV ORF1 encoded functional domains by using Y2H screening. A total of 155 protein-protein interactions between HEV-ORF1 and human proteins were identified. Comparative analysis of the HEV-ORF1-Human interaction network with reconstructed human interactome showed that the cellular proteins interacting with HEV-ORF1 are central and interconnected. Enrichment analysis of Gene Ontology and cellular pathways showed that the viral proteins preferentially interacted with the proteins of metabolism and energy generation along with host immune response and ubiquitin proteasomal pathways. The mTOR and focal adhesion pathways were also targeted by the virus. These interactions suggest that HEV probably utilizes important proteins in carbohydrate metabolism, energy generation and iron homoeostasis in the host cells during its establishment. PMID:26689634

  12. Fluorescent Proteins as Genetically Encoded FRET Biosensors in Life Sciences

    PubMed Central

    Hochreiter, Bernhard; Pardo Garcia, Alan; Schmid, Johannes A.

    2015-01-01

    Fluorescence- or Förster resonance energy transfer (FRET) is a measurable physical energy transfer phenomenon between appropriate chromophores, when they are in sufficient proximity, usually within 10 nm. This feature has made them incredibly useful tools for many biomedical studies on molecular interactions. Furthermore, this principle is increasingly exploited for the design of biosensors, where two chromophores are linked with a sensory domain controlling their distance and thus the degree of FRET. The versatility of these FRET-biosensors made it possible to assess a vast amount of biological variables in a fast and standardized manner, allowing not only high-throughput studies but also sub-cellular measurements of biological processes. In this review, we aim at giving an overview over the recent advances in genetically encoded, fluorescent-protein based FRET-biosensors, as these represent the largest and most vividly growing group of FRET-based sensors. For easy understanding, we are grouping them into four categories, depending on their molecular mechanism. These are based on: (a) cleavage; (b) conformational-change; (c) mechanical force and (d) changes in the micro-environment. We also address the many issues and considerations that come with the development of FRET-based biosensors, as well as the possibilities that are available to measure them. PMID:26501285

  13. Sequence heuristics to encode phase behaviour in intrinsically disordered protein polymers.

    PubMed

    Quiroz, Felipe García; Chilkoti, Ashutosh

    2015-11-01

    Proteins and synthetic polymers that undergo aqueous phase transitions mediate self-assembly in nature and in man-made material systems. Yet little is known about how the phase behaviour of a protein is encoded in its amino acid sequence. Here, by synthesizing intrinsically disordered, repeat proteins to test motifs that we hypothesized would encode phase behaviour, we show that the proteins can be designed to exhibit tunable lower or upper critical solution temperature (LCST and UCST, respectively) transitions in physiological solutions. We also show that mutation of key residues at the repeat level abolishes phase behaviour or encodes an orthogonal transition. Furthermore, we provide heuristics to identify, at the proteome level, proteins that might exhibit phase behaviour and to design novel protein polymers consisting of biologically active peptide repeats that exhibit LCST or UCST transitions. These findings set the foundation for the prediction and encoding of phase behaviour at the sequence level. PMID:26390327

  14. Sequence heuristics to encode phase behaviour in intrinsically disordered protein polymers

    NASA Astrophysics Data System (ADS)

    Quiroz, Felipe García; Chilkoti, Ashutosh

    2015-11-01

    Proteins and synthetic polymers that undergo aqueous phase transitions mediate self-assembly in nature and in man-made material systems. Yet little is known about how the phase behaviour of a protein is encoded in its amino acid sequence. Here, by synthesizing intrinsically disordered, repeat proteins to test motifs that we hypothesized would encode phase behaviour, we show that the proteins can be designed to exhibit tunable lower or upper critical solution temperature (LCST and UCST, respectively) transitions in physiological solutions. We also show that mutation of key residues at the repeat level abolishes phase behaviour or encodes an orthogonal transition. Furthermore, we provide heuristics to identify, at the proteome level, proteins that might exhibit phase behaviour and to design novel protein polymers consisting of biologically active peptide repeats that exhibit LCST or UCST transitions. These findings set the foundation for the prediction and encoding of phase behaviour at the sequence level.

  15. Sequence heuristics to encode phase behaviour in intrinsically disordered protein polymers

    PubMed Central

    Quiroz, Felipe García; Chilkoti, Ashutosh

    2015-01-01

    Proteins and synthetic polymers that undergo aqueous phase transitions mediate self-assembly in nature and in man-made material systems. Yet little is known about how the phase behaviour of a protein is encoded in its amino acid sequence. Here, by synthesizing intrinsically disordered, repeat proteins to test motifs that we hypothesized would encode phase behaviour, we show that the proteins can be designed to exhibit tunable lower or upper critical solution temperature (LCST and UCST, respectively) transitions in physiological solutions. We also show that mutation of key residues at the repeat level abolishes phase behaviour or encodes an orthogonal transition. Furthermore, we provide heuristics to identify, at the proteome level, proteins that might exhibit phase behaviour and to design novel protein polymers consisting of biologically active peptide repeats that exhibit LCST or UCST transitions. These findings set the foundation for the prediction and encoding of phase behaviour at the sequence level. PMID:26390327

  16. Comparative differential gene expression analysis of nucleus-encoded proteins for Rafflesia cantleyi against Arabidopsis thaliana

    NASA Astrophysics Data System (ADS)

    Ng, Siuk-Mun; Lee, Xin-Wei; Wan, Kiew-Lian; Firdaus-Raih, Mohd

    2015-09-01

    Regulation of functional nucleus-encoded proteins targeting the plastidial functions was comparatively studied for a plant parasite, Rafflesia cantleyi versus a photosynthetic plant, Arabidopsis thaliana. This study involved two species of different feeding modes and different developmental stages. A total of 30 nucleus-encoded proteins were found to be differentially-regulated during two stages in the parasite; whereas 17 nucleus-encoded proteins were differentially-expressed during two developmental stages in Arabidopsis thaliana. One notable finding observed for the two plants was the identification of genes involved in the regulation of photosynthesis-related processes where these processes, as expected, seem to be present only in the autotroph.

  17. Cloning and sequencing of a cDNA encoding a taste-modifying protein, miraculin.

    PubMed

    Masuda, Y; Nirasawa, S; Nakaya, K; Kurihara, Y

    1995-08-19

    A cDNA clone encoding a taste-modifying protein, miraculin (MIR), was isolated and sequenced. The encoded precursor to MIR was composed of 220 amino acid (aa) residues, including a possible signal sequence of 29 aa. Northern blot analysis showed that the mRNA encoding MIR was already expressed in fruits of Richadella dulcifica at 3 weeks after pollination and was present specifically in the pulp. PMID:7665074

  18. Experimental strategies for cloning or identifying genes encoding DNA-binding proteins.

    PubMed

    Carey, Michael F; Peterson, Craig L; Smale, Stephen T

    2012-02-01

    This article describes experimental strategies for cloning or identifying genes encoding DNA-binding proteins. DNA-binding proteins are most commonly identified by electrophoretic mobility-shift assay (EMSA) or DNase I footprinting. To identify the gene encoding a protein detected by EMSA or DNase footprinting, the protein often needs to be purified and its sequence analyzed, as described here. Other methods are also available which do not resort to protein purification, including the one-hybrid screen, in vitro expression library screen, and mammalian expression cloning. These methods are outlined, and their advantages and disadvantages are discussed. PMID:22301659

  19. (Genetic engineering with a gene encoding a soybean storage protein). Progress report

    SciTech Connect

    Beachy, R.N.

    1985-01-01

    Progress is reported on research directed toward introducing a gene (Gmg 17.1) encoding the ..cap alpha..'-subunit of ..beta..-conglycinin, a soybean seed protein, into petunia plants using gene transfer mechanisms. (ACR)

  20. A genetically encoded aldehyde for rapid protein labelling.

    PubMed

    Tuley, Alfred; Lee, Yan-Jiun; Wu, Bo; Wang, Zhiyong U; Liu, Wenshe R

    2014-07-18

    Using a mutant pyrrolysyl-tRNA synthetase-tRNA(Pyl)(CUA) pair, 3-formyl-phenylalanine is genetically incorporated into proteins at amber mutation sites in Escherichia coli. This non-canonical amino acid readily reacts with hydroxylamine dyes, leading to rapid and site-selective protein labelling. PMID:24756176

  1. Isotope-Encoded Carboxyl Group Footprinting for Mass Spectrometry-Based Protein Conformational Studies

    NASA Astrophysics Data System (ADS)

    Zhang, Hao; Liu, Haijun; Blankenship, Robert E.; Gross, Michael L.

    2016-01-01

    We report an isotope-encoding method coupled with carboxyl-group footprinting to monitor protein conformational changes. The carboxyl groups of aspartic/glutamic acids and of the C-terminus of proteins can serve as reporters for protein conformational changes when labeled with glycine ethyl ester (GEE) mediated by carbodiimide. In the new development, isotope-encoded "heavy" and "light" GEE are used to label separately the two states of the orange carotenoid protein (OCP) from cyanobacteria. Two samples are mixed (1:1 ratio) and analyzed by a single LC-MS/MS experiment. The differences in labeling extent between the two states are represented by the ratio of the "heavy" and "light" peptides, providing information about protein conformational changes. Combining isotope-encoded MS quantitative analysis and carboxyl-group footprinting reduces the time of MS analysis and improves the sensitivity of GEE and other footprinting.

  2. SurfaceomeDB: a cancer-orientated database for genes encoding cell surface proteins.

    PubMed

    de Souza, Jorge Estefano Santana; Galante, Pedro Alexandre Favoretto; de Almeida, Renan Valieris Bueno; da Cunha, Julia Pinheiro Chagas; Ohara, Daniel Takatori; Ohno-Machado, Lucila; Old, Lloyd J; de Souza, Sandro José

    2012-01-01

    Cell surface proteins (CSPs) are excellent targets for the development of diagnostic and therapeutic reagents, and it is estimated that 10-20% of all genes in the human genome encode CSPs. In an effort to integrate all data publicly available for genes encoding cell surface proteins, a database (SurfaceomeDB) was developed. SurfaceomeDB is a gene-centered portal containing different types of information, including annotation for gene expression, protein domains, somatic mutations in cancer, and protein-protein interactions for all human genes encoding CSPs. SurfaceomeDB was implemented as an integrative and relational database in a user-friendly web interface, where users can search for gene name, gene annotation, or keywords. There is also a streamlined graphical representation of all data provided and links to the most important data repositories and databases, such as NCBI, UCSC Genome Browser, and EBI. PMID:23390370

  3. Isotope-Encoded Carboxyl Group Footprinting for Mass Spectrometry-Based Protein Conformational Studies.

    PubMed

    Zhang, Hao; Liu, Haijun; Blankenship, Robert E; Gross, Michael L

    2016-01-01

    We report an isotope-encoding method coupled with carboxyl-group footprinting to monitor protein conformational changes. The carboxyl groups of aspartic/glutamic acids and of the C-terminus of proteins can serve as reporters for protein conformational changes when labeled with glycine ethyl ester (GEE) mediated by carbodiimide. In the new development, isotope-encoded "heavy" and "light" GEE are used to label separately the two states of the orange carotenoid protein (OCP) from cyanobacteria. Two samples are mixed (1:1 ratio) and analyzed by a single LC-MS/MS experiment. The differences in labeling extent between the two states are represented by the ratio of the "heavy" and "light" peptides, providing information about protein conformational changes. Combining isotope-encoded MS quantitative analysis and carboxyl-group footprinting reduces the time of MS analysis and improves the sensitivity of GEE and other footprinting. PMID:26384685

  4. Multifractals, encoded walks and the ergodicity of protein sequences.

    PubMed

    Dewey, T G; Strait, B J

    1996-01-01

    A variety of statistical methods have been developed to explore correlations in protein and nucleic acid sequences. Such correlations have important implications for the evolution and stability of these macromolecules. Recently, a number of fractal analyses of sequence data have been developed. These analyses have considerable appeal as they are extremely sensitive to long range correlations and to hierarchical structures. One such analysis decodes sequence information into a random walk and the statistics of the resulting random walk is investigated. Anomalous scaling of such walks has been interpreted as indicative of a fractal structure. Alternatively, a generalized box counting analysis of decoded sequences can be used to establish multifractal properties. In this work, the connection between these two seemingly disparate approaches is established. This connection is exploited to investigate correlations in protein sequences. An ensemble consisting of a comprehensive data set of representative protein sequences is analyzed to establish the ergodicity of protein sequences. The implications of this ergodicity for information theoretical approaches to protein structure prediction is explored. PMID:9390234

  5. Genes encoding calmodulin-binding proteins in the Arabidopsis genome

    NASA Technical Reports Server (NTRS)

    Reddy, Vaka S.; Ali, Gul S.; Reddy, Anireddy S N.

    2002-01-01

    Analysis of the recently completed Arabidopsis genome sequence indicates that approximately 31% of the predicted genes could not be assigned to functional categories, as they do not show any sequence similarity with proteins of known function from other organisms. Calmodulin (CaM), a ubiquitous and multifunctional Ca(2+) sensor, interacts with a wide variety of cellular proteins and modulates their activity/function in regulating diverse cellular processes. However, the primary amino acid sequence of the CaM-binding domain in different CaM-binding proteins (CBPs) is not conserved. One way to identify most of the CBPs in the Arabidopsis genome is by protein-protein interaction-based screening of expression libraries with CaM. Here, using a mixture of radiolabeled CaM isoforms from Arabidopsis, we screened several expression libraries prepared from flower meristem, seedlings, or tissues treated with hormones, an elicitor, or a pathogen. Sequence analysis of 77 positive clones that interact with CaM in a Ca(2+)-dependent manner revealed 20 CBPs, including 14 previously unknown CBPs. In addition, by searching the Arabidopsis genome sequence with the newly identified and known plant or animal CBPs, we identified a total of 27 CBPs. Among these, 16 CBPs are represented by families with 2-20 members in each family. Gene expression analysis revealed that CBPs and CBP paralogs are expressed differentially. Our data suggest that Arabidopsis has a large number of CBPs including several plant-specific ones. Although CaM is highly conserved between plants and animals, only a few CBPs are common to both plants and animals. Analysis of Arabidopsis CBPs revealed the presence of a variety of interesting domains. Our analyses identified several hypothetical proteins in the Arabidopsis genome as CaM targets, suggesting their involvement in Ca(2+)-mediated signaling networks.

  6. Relating sequence encoded information to form and function of intrinsically disordered proteins

    PubMed Central

    Das, Rahul K.; Ruff, Kiersten M.; Pappu, Rohit V.

    2015-01-01

    Intrinsically disordered proteins (IDPs) showcase the importance of conformational plasticity and heterogeneity in protein function. We summarize recent advances that connect information encoded in IDP sequences to their conformational properties and functions. We focus on insights obtained through a combination of atomistic simulations and biophysical measurements that are synthesized into a coherent framework using polymer physics theories. PMID:25863585

  7. The virally encoded killer proteins from Ustilago maydis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Several strains of Ustilago maydis, a causal agent of corn smut disease, exhibit a 'killer' phenotype that is due to persistent infection by double-stranded RNA Totiviruses. These viruses produce potent killer proteins that are secreted by the host. This is a rare example of virus/host symbiosis in ...

  8. Identification of Two Laminin-Binding Fimbriae, the Type 1 Fimbria of Salmonella enterica Serovar Typhimurium and the G Fimbria of Escherichia coli, as Plasminogen Receptors

    PubMed Central

    Kukkonen, Maini; Saarela, Sirkku; Lähteenmäki, Kaarina; Hynönen, Ulla; Westerlund-Wikström, Benita; Rhen, Mikael; Korhonen, Timo K.

    1998-01-01

    Escherichia coli strains carrying recombinant plasmids encoding either the type 1 fimbria of Salmonella enterica serovar Typhimurium or the G fimbria of E. coli exhibited binding of human 125I-Glu-plasminogen and enhanced the tissue-type plasminogen activator-catalyzed conversion of plasminogen to plasmin. Purified type 1 or G fimbriae similarly bound plasminogen and enhanced its activation. The binding of plasminogen did not involve the characteristic carbohydrate-binding property of the fimbriae but was inhibited at low concentrations by the lysine analog ɛ-aminocaproic acid. Because these fimbrial types bind to laminin of basement membranes (M. Kukkonen et al., Mol. Microbiol. 7:229–237, 1993; S. Saarela et al., Infect. Immun. 64:2857–2860, 1996), the results demonstrate a structural unity in the creation and targeting of bacterium-bound proteolytic plasmin activity to basement membranes. PMID:9746604

  9. Chimeric proteins define variable and essential regions of Ha-ras-encoded protein

    SciTech Connect

    Lowe, D.G.; Ricketts, M.; Levinson, A.D.; Goeddel, D.V.

    1988-02-01

    The biological role of amino acid differences between the human 21-kDa Ha-ras protein (p21) and the human 23-kDa R-ras protein (p23) was investigated by engineering mutant Ha-ras p21 molecules containing divergent amino acid sequences from R-ras p23. Variant amino acids from R-ras p23 regions 1-30, 52-57, 67-78, 1-30 and 67-78 together, and 112-124 were substituted for the corresponding Ha-ras p21 amino acid regions 1-4, 26-31, 41-52, 1-4 and 41-52 together, and 86-98, respectively. Rat fibroblasts transfected with genes encoding these position-12 valine-substituted chimeric Ha-ras proteins displayed the same properties of morphological transformation and anchorage-independent growth as Ha-ras T24 oncogene-transformed fibroblasts. However, substitution of variant amino acids from the 80 C-terminal residues (amino acids 138-218) of R-ras p23 for the corresponding p21 amino acids (residues 112-189) inactivated the transforming activity of position-12 valine-substituted p21. The converse substitution of Ha-ras p21 C-terminal residues into R-ras p23 did not result in transformation by position-38 valine-substituted p232. These data are discussed in terms of the structure of ras proteins and the nature of interactions determining the specificity of effector function.

  10. Apolipoprotein A-I mutant proteins having cysteine substitutions and polynucleotides encoding same

    DOEpatents

    Oda, Michael N.; Forte, Trudy M.

    2007-05-29

    Functional Apolipoprotein A-I mutant proteins, having one or more cysteine substitutions and polynucleotides encoding same, can be used to modulate paraoxonase's arylesterase activity. These ApoA-I mutant proteins can be used as therapeutic agents to combat cardiovascular disease, atherosclerosis, acute phase response and other inflammatory related diseases. The invention also includes modifications and optimizations of the ApoA-I nucleotide sequence for purposes of increasing protein expression and optimization.

  11. Synthesis of Non-linear Protein Dimers through a Genetically Encoded Thiol-ene Reaction

    PubMed Central

    Torres-Kolbus, Jessica; Chou, Chungjung; Liu, Jihe; Deiters, Alexander

    2014-01-01

    Site-specific incorporation of bioorthogonal unnatural amino acids into proteins provides a useful tool for the installation of specific functionalities that will allow for the labeling of proteins with virtually any probe. We demonstrate the genetic encoding of a set of alkene lysines using the orthogonal PylRS/PylTCUA pair in Escherichia coli. The installed double bond functionality was then applied in a photoinitiated thiol-ene reaction of the protein with a fluorescent thiol-bearing probe, as well as a cysteine residue of a second protein, showing the applicability of this approach in the formation of heterogeneous non-linear fused proteins. PMID:25181502

  12. Nucleic acid encoding DS-CAM proteins and products related thereto

    SciTech Connect

    Korenberg, Julie R.

    2005-11-01

    In accordance with the present invention, there are provided Down Syndrome-Cell Adhesion Molecule (DS-CAM) proteins. Nucleic acid sequences encoding such proteins and assays employing same are also disclosed. The invention DS-CAM proteins can be employed in a variety of ways, for example, for the production of anti-DS-CAM antibodies thereto, in therapeutic compositions and methods employing such proteins and/or antibodies. DS-CAM proteins are also useful in bioassays to identify agonists and antagonists thereto.

  13. Learning from Bacteriophages - Advantages and Limitations of Phage and Phage-Encoded Protein Applications

    PubMed Central

    Drulis-Kawa, Zuzanna; Majkowska-Skrobek, Grażyna; Maciejewska, Barbara; Delattre, Anne-Sophie; Lavigne, Rob

    2012-01-01

    The emergence of bacteria resistance to most of the currently available antibiotics has become a critical therapeutic problem. The bacteria causing both hospital and community-acquired infections are most often multidrug resistant. In view of the alarming level of antibiotic resistance between bacterial species and difficulties with treatment, alternative or supportive antibacterial cure has to be developed. The presented review focuses on the major characteristics of bacteriophages and phage-encoded proteins affecting their usefulness as antimicrobial agents. We discuss several issues such as mode of action, pharmacodynamics, pharmacokinetics, resistance and manufacturing aspects of bacteriophages and phage-encoded proteins application. PMID:23305359

  14. IQCJ-SCHIP1, a novel fusion transcript encoding a calmodulin-binding IQ motif protein

    SciTech Connect

    Kwasnicka-Crawford, Dorota A. . E-mail: dakc@yorku.ca; Carson, Andrew R.; Scherer, Stephen W.

    2006-12-01

    The existence of transcripts that span two adjacent, independent genes is considered rare in the human genome. This study characterizes a novel human fusion gene named IQCJ-SCHIP1. IQCJ-SCHIP1 is the longest isoform of a complex transcriptional unit that bridges two separate genes that encode distinct proteins, IQCJ, a novel IQ motif containing protein and SCHIP1, a schwannomin interacting protein that has been previously shown to interact with the Neurofibromatosis type 2 (NF2) protein. IQCJ-SCHIP1 is located on the chromosome 3q25 and comprises a 1692-bp transcript encompassing 11 exons spanning 828 kb of the genomic DNA. We show that IQCJ-SCHIP1 mRNA is highly expressed in the brain. Protein encoded by the IQCJ-SCHIP1 gene was localized to cytoplasm and actin-rich regions and in differentiated PC12 cells was also seen in neurite extensions.

  15. Genetically Encoding an Electrophilic Amino Acid for Protein Stapling and Covalent Binding to Native Receptors

    PubMed Central

    2015-01-01

    Covalent bonds can be generated within and between proteins by an unnatural amino acid (Uaa) reacting with a natural residue through proximity-enabled bioreactivity. Until now, Uaas have been developed to react mainly with cysteine in proteins. Here we genetically encoded an electrophilic Uaa capable of reacting with histidine and lysine, thereby expanding the diversity of target proteins and the scope of the proximity-enabled protein cross-linking technology. In addition to efficient cross-linking of proteins inter- and intramolecularly, this Uaa permits direct stapling of a protein α-helix in a recombinant manner and covalent binding of native membrane receptors in live cells. The target diversity, recombinant stapling, and covalent targeting of endogenous proteins enabled by this versatile Uaa should prove valuable in developing novel research tools, biological diagnostics, and therapeutics by exploiting covalent protein linkages for specificity, irreversibility, and stability. PMID:25010185

  16. Orf-I and Orf-II-Encoded Proteins in HTLV-1 Infection and Persistence

    PubMed Central

    Edwards, Dustin; Fenizia, Claudio; Gold, Heather; de Castro-Amarante, Maria Fernanda; Buchmann, Cody; Pise-Masison, Cynthia A.; Franchini, Genoveffa

    2011-01-01

    The 3′ end of the human T-cell leukemia/lymphoma virus type-1 (HTLV-1) genome contains four overlapping open reading frames (ORF) that encode regulatory proteins. Here, we review current knowledge of HTLV-1 orf-I and orf-II protein products. Singly spliced mRNA from orf-I encodes p12, which can be proteolytically cleaved to generate p8, while differential splicing of mRNA from orf-II results in production of p13 and p30. These proteins have been demonstrated to modulate transcription, apoptosis, host cell activation and proliferation, virus infectivity and transmission, and host immune responses. Though these proteins are not essential for virus replication in vitro, p8, p12, p13, and p30 have an important role in the establishment and maintenance of HTLV-1 infection in vivo. PMID:21994758

  17. Genetically encoded protein photocrosslinker with a transferable mass spectrometry-identifiable label

    PubMed Central

    Yang, Yi; Song, Haiping; He, Dan; Zhang, Shuai; Dai, Shizhong; Lin, Shixian; Meng, Rong; Wang, Chu; Chen, Peng R.

    2016-01-01

    Coupling photocrosslinking reagents with mass spectrometry has become a powerful tool for studying protein–protein interactions in living systems, but it still suffers from high rates of false-positive identifications as well as the lack of information on interaction interface due to the challenges in deciphering crosslinking peptides. Here we develop a genetically encoded photo-affinity unnatural amino acid that introduces a mass spectrometry-identifiable label (MS-label) to the captured prey proteins after photocrosslinking and prey–bait separation. This strategy, termed IMAPP (In-situ cleavage and MS-label transfer After Protein Photocrosslinking), enables direct identification of photo-captured substrate peptides that are difficult to uncover by conventional genetically encoded photocrosslinkers. Taking advantage of the MS-label, the IMAPP strategy significantly enhances the confidence for identifying protein–protein interactions and enables simultaneous mapping of the binding interface under living conditions. PMID:27460181

  18. Role of Soybean mosaic virus-encoded proteins in seed and aphid transmission in soybean

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Soybean mosaic virus (SMV) is seed and aphid transmitted and can cause significant reductions in yield and seed quality in soybean, Glycine max. The roles in seed and aphid transmission of selected SMV-encoded proteins were investigated by constructing chimeric recombinants between SMV 413 (efficien...

  19. Storable N-Phenylcarbamate Palladacycles for Rapid Functionalization of An Alkyne-Encoded Protein

    PubMed Central

    Cheng, Gang; Lim, Reyna K. V.; Ramil, Carlo P.

    2014-01-01

    Here we report the synthesis of storable N-phenyl-carbamate palladacycles that showed robust reactivity in the cross-coupling reaction with an alkyne-encoded protein with second-order rate constantapproaching 19 770 ± 930 M−1 s−1. PMID:25140915

  20. Post-anthesis Fertilizer Influences Expression of Genes Encoding Allergenic Proteins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Omega-gliadins comprise between 5-10% of the wheat flour protein and have been implicated in food allergies. In particular, omega-gliadins encoded by the 1B chromosome have been associated with wheat-dependent exercise-induced anaphalaxis (WDEIA) and urticaria. Because the omega-gliadins consist alm...

  1. Isolation and analysis of a cDNA clone encoding an S. guttatum alternataive oxidase protein

    SciTech Connect

    Rhoads, D.M.; McIntosh, L. Michigan State Univ., East Lansing )

    1990-05-01

    Antibodies that recognize the 35, 36, and 37 kilodalton (kDa) alternative oxidase proteins were used to isolate a cDNA proteins were used to isolate a cDNA clone of a nuclearly encoded protein of Sauromatum guttatum. The amino acid sequence deduced from clone pAOSG81 revealed a protein with a predicted molecular mass of 44 kDa, while a 42 kDa protein is immunoprecipitated from in vitro translation products made using S. guttatum poly A+ RNA. The protein contains a 60-65 amino acid transit peptide which is predicted to form amphiphilic helices. We have also identified regions of the mature 42 kDa protein which are likely to be membrane associated. Clone pAOSG81 is being used to screen a genomic library. The genomic clone encoding the 42 kDa protein will be used to investigate the salicylic-acid-controlled transcriptional regulation of the S. guttatum alternative oxidase proteins.

  2. Genetically encoded norbornene directs site-specific cellular protein labelling via a rapid bioorthogonal reaction

    NASA Astrophysics Data System (ADS)

    Lang, Kathrin; Davis, Lloyd; Torres-Kolbus, Jessica; Chou, Chungjung; Deiters, Alexander; Chin, Jason W.

    2012-04-01

    The site-specific incorporation of bioorthogonal groups via genetic code expansion provides a powerful general strategy for site-specifically labelling proteins with any probe. However, the slow reactivity of the bioorthogonal functional groups that can be encoded genetically limits the utility of this strategy. We demonstrate the genetic encoding of a norbornene amino acid using the pyrrolysyl tRNA synthetase/tRNACUA pair in Escherichia coli and mammalian cells. We developed a series of tetrazine-based probes that exhibit ‘turn-on’ fluorescence on their rapid reaction with norbornenes. We demonstrate that the labelling of an encoded norbornene is specific with respect to the entire soluble E. coli proteome and thousands of times faster than established encodable bioorthogonal reactions. We show explicitly the advantages of this approach over state-of-the-art bioorthogonal reactions for protein labelling in vitro and on mammalian cells, and demonstrate the rapid bioorthogonal site-specific labelling of a protein on the mammalian cell surface.

  3. Multiple proteins encoded within the urease gene complex of Proteus mirabilis.

    PubMed

    Walz, S E; Wray, S K; Hull, S I; Hull, R A

    1988-03-01

    Chromosomal DNA fragments from a uropathogenic isolate of Proteus mirabilis were inserted into the cosmid vector pHC79 to construct a genomic library in Escherichia coli HB101. A urease-positive recombinant cosmid, designated pSKW1, was recovered. Sequential recombinant manipulation of pSKW1 yielded a 10.2-kilobase plasmid, designated pSKW4, which encoded three urease isozymes with electrophoretic mobilities identical to those of the donor P. mirabilis strain. Plasmid pSKW4 gene sequences encode seven proteins designated 68K (apparent molecular weight, of 68,000), 28K, 25K, 22.5K, 18.5K, 7.5K, and 5.2K within the limits of the urease gene complex. Insertion mutations in genes encoding the 68K, 28K, 25K, 22.5K, 7.5K, and 5.2K proteins resulted in complete or partial (22.5K) loss of urease activity. There was no reduction in urease activity when the gene encoding the 18.5K protein was inactivated. PMID:2830226

  4. Monoclonal antibodies to individual tyrosine-phosphorylated protein substrates of oncogene-encoded tyrosine kinases

    SciTech Connect

    Kanner, S.B.; Reynolds, A.B.; Vines, R.R.; Parsons, J.T. )

    1990-05-01

    Cellular transformation by oncogenic retroviruses encoding protein tyrosine kinases coincides with the tyrosine-specific phosphorylation of multiple protein substrates. Previous studies have shown that tyrosine phosphorylation of a protein of 120 kDa, p120, correlated with src transformation in chicken embryo fibroblasts. Additionally, the authors previously identified two phosphotyrosine-containing cellular proteins, p130 and p110, that formed stable complexes with activated variants of pp60{sup src}, the src-encoded tyrosine kinase. To study transformation-relevant tyrosine kinase substrates, they have generated monoclonal antibodies to individual tyrosine phosphoproteins, including p130, p120, p110, and five additional phosphoproteins (p210, p125, p118, p85, and p185/p64). These antibodies detected several of the same tyrosine phosphoproteins in chicken embryo fibroblasts transformed by avian retroviruses Y73 and CT10, encoding the yes and crk oncogenes, respectively. Protein substrates in mouse, rat, hamster, and human cells overexpressing activated variants of chicken pp60{sup src} were also detected by several of the monoclonal antibodies.

  5. Bacteriophage-encoded cochaperonins can substitute for Escherichia coli’s essential GroES protein

    PubMed Central

    Keppel, France; Rychner, Monique; Georgopoulos, Costa

    2002-01-01

    The Escherichia coli chaperonin machine is composed of two members, GroEL and GroES. The GroEL chaperonin can bind 10–15% of E. coli’s unfolded proteins in one of its central cavities and help them fold in cooperation with the GroES cochaperonin. Both proteins are absolutely essential for bacterial growth. Several large, lytic bacteriophages, such as T4 and RB49, use the host-encoded GroEL in conjunction with their own bacteriophage-encoded cochaperonin for the correct assembly of their major capsid protein, suggesting a cochaperonin specificity for the in vivo folding of certain substrates. Here, we demonstrate that, when the cochaperonin of either bacteriophage T4 (Gp31) or RB49 (CocO) is expressed in E. coli, the otherwise essential groES gene can be deleted. Thus, it appears that, despite very little sequence identity with groES, the bacteriophage-encoded Gp31 and CocO proteins are capable of replacing GroES in the folding of E. coli’s essential, housekeeping proteins. PMID:12189177

  6. Nucleic acids encoding phloem small RNA-binding proteins and transgenic plants comprising them

    DOEpatents

    Lucas, William J.; Yoo, Byung-Chun; Lough, Tony J.; Varkonyi-Gasic, Erika

    2007-03-13

    The present invention provides a polynucleotide sequence encoding a component of the protein machinery involved in small RNA trafficking, Cucurbita maxima phloem small RNA-binding protein (CmPSRB 1), and the corresponding polypeptide sequence. The invention also provides genetic constructs and transgenic plants comprising the polynucleotide sequence encoding a phloem small RNA-binding protein to alter (e.g., prevent, reduce or elevate) non-cell autonomous signaling events in the plants involving small RNA metabolism. These signaling events are involved in a broad spectrum of plant physiological and biochemical processes, including, for example, systemic resistance to pathogens, responses to environmental stresses, e.g., heat, drought, salinity, and systemic gene silencing (e.g., viral infections).

  7. Reduction of a 4q35-encoded nuclear envelope protein in muscle differentiation

    SciTech Connect

    Ostlund, Cecilia; Guan, Tinglu; Figlewicz, Denise A.; Hays, Arthur P.; Worman, Howard J.; Gerace, Larry; Schirmer, Eric C.

    2009-11-13

    Muscular dystrophy and peripheral neuropathy have been linked to mutations in genes encoding nuclear envelope proteins; however, the molecular mechanisms underlying these disorders remain unresolved. Nuclear envelope protein p19A is a protein of unknown function encoded by a gene at chromosome 4q35. p19A levels are significantly reduced in human muscle as cells differentiate from myoblasts to myotubes; however, its levels are not similarly reduced in all differentiation systems tested. Because 4q35 has been linked to facioscapulohumeral muscular dystrophy (FSHD) and some adjacent genes are reportedly misregulated in the disorder, levels of p19A were analyzed in muscle samples from patients with FSHD. Although p19A was increased in most cases, an absolute correlation was not observed. Nonetheless, p19A downregulation in normal muscle differentiation suggests that in the cases where its gene is inappropriately re-activated it could affect muscle differentiation and contribute to disease pathology.

  8. Nucleotide sequence and characterization of peb4A encoding an antigenic protein in Campylobacter jejuni.

    PubMed

    Burucoa, C; Frémaux, C; Pei, Z; Tummuru, M; Blaser, M J; Cenatiempo, Y; Fauchère, J L

    1995-01-01

    The 29-kDa protein PEB4, a major antigen of Campylobacter jejuni, is present in all C. jejuni strains tested and elicits an antibody response in infected patients. By screening a lambda gt11 library of chromosomal DNA fragments of C. jejuni strain 81-176 in Escherichia coli Y1090 cells with antibody raised against purified PEB4, a recombinant phage with a 2-kb insert expressing an immunoreactive protein of 29 kDa was isolated. DNA sequence analysis revealed that the insert contains two complete open reading frames ORF-A and ORF-B. ORF-A (peb4A) encodes a 273-residue protein with a calculated molecular mass of 30,460 daltons. The deduced amino acid sequence, composition and pl of the recombinant mature protein are similar to those determined for purified PEB4. The first 21 residues resemble a signal peptide. Gene bank searches indicated 33.7% identity with protein export protein PrsA of Bacillus subtilis and 23.8% identity with protease maturation protein precursor PrtM of Lactococcus lactis. PCR experiments indicate that peb4A is highly conserved among C. jejuni strains. ORF-B begins 2 bp after the last codon of peb4A and encodes a putative protein of 353 residues with 63.4% identity with E. coli fructose 1,6-biphosphate aldolase. The sequence arrangement suggests that these two genes form an operon. PMID:8525063

  9. Open reading frame 2 of porcine circovirus type 2 encodes a major capsid protein.

    PubMed

    Nawagitgul, P; Morozov, I; Bolin, S R; Harms, P A; Sorden, S D; Paul, P S

    2000-09-01

    Porcine circovirus 2 (PCV2), a single-stranded DNA virus associated with post-weaning multisystemic wasting syndrome of swine, has two potential open reading frames, ORF1 and ORF2, greater than 600 nucleotides in length. ORF1 is predicted to encode a replication-associated protein (Rep) essential for replication of viral DNA, while ORF2 contains a conserved basic amino acid sequence at the N terminus resembling that of the major structural protein of chicken anaemia virus. Thus far, the structural protein(s) of PCV2 have not been identified. In this study, a viral structural protein of 30 kDa was identified in purified PCV2 particles. ORF2 of PCV2 was cloned into a baculovirus expression vector and the gene product was expressed in insect cells. The expressed ORF2 gene product had a molecular mass of 30 kDa, similar to that detected in purified virus particles. The recombinant ORF2 protein self-assembled to form capsid-like particles when viewed by electron microscopy. Antibodies against the ORF2 protein were detected in samples of sera obtained from pigs as early as 3 weeks after experimental infection with PCV2. These results show that the major structural protein of PCV2 is encoded by ORF2 and has a molecular mass of 30 kDa. PMID:10950986

  10. African swine fever virus encodes a serine protein kinase which is packaged into virions.

    PubMed Central

    Baylis, S A; Banham, A H; Vydelingum, S; Dixon, L K; Smith, G L

    1993-01-01

    Nucleotide sequencing of the SalI j region of the virulent Malawi (LIL20/1) strain of African swine fever virus (ASFV) identified an open reading frame (ORF), designated j9L, with extensive similarity to the family of protein kinases. This ORF encodes a 35.1-kDa protein of 299 amino acids which shares 24.6% amino acid identity with the human pim-1 proto-oncogene and 21.0% identity with the vaccinia virus B1R-encoded protein kinase. The ASFV ORF contains the motifs characteristic of serine-threonine protein kinases, with the exception of the presumed ATP-binding site, which is poorly conserved. The ORF was expressed to high levels in Escherichia coli, and the recombinant enzyme phosphorylated a calf thymus histone protein on serine residues in vitro. An antibody raised to an amino-terminal peptide of the ASFV protein kinase was reactive with the recombinant protein in Western immunoblot analyses and was used to demonstrate the presence of the protein kinase in ASF virions. Images PMID:8331722

  11. Newly identified RNAs of raspberry leaf blotch virus encoding a related group of proteins.

    PubMed

    Lu, Yuwen; McGavin, Wendy; Cock, Peter J A; Schnettler, Esther; Yan, Fei; Chen, Jianping; MacFarlane, Stuart

    2015-11-01

    Members of the genus Emaravirus, including Raspberry leaf blotch virus (RLBV), are enveloped plant viruses with segmented genomes of negative-strand RNA, although the complete genome complement for any of these viruses is not yet clear. Currently, wheat mosaic virus has the largest emaravirus genome comprising eight RNAs. Previously, we identified five genomic RNAs for RLBV; here, we identify a further three RNAs (RNA6-8). RNA6-8 encode proteins that have clear homologies to one another, but not to any other emaravirus proteins. The proteins self-interacted in yeast two-hybrid and bimolecular fluorescence complementation (BiFC) experiments, and the P8 protein interacted with the virus nucleocapsid protein (P3) using BiFC. Expression of two of the proteins (P6 and P7) using potato virus X led to an increase in virus titre and symptom severity, suggesting that these proteins may play a role in RLBV pathogenicity; however, using two different tests, RNA silencing suppression activity was not detected for any of the RLBV proteins encoded by RNA2-8. PMID:26358478

  12. Francisella novicida pathogenicity island encoded proteins were secreted during infection of macrophage-like cells.

    PubMed

    Hare, Rebekah F; Hueffer, Karsten

    2014-01-01

    Intracellular pathogens and other organisms have evolved mechanisms to exploit host cells for their life cycles. Virulence genes of some intracellular bacteria responsible for these mechanisms are located in pathogenicity islands, such as secretion systems that secrete effector proteins. The Francisella pathogenicity island is required for phagosomal escape, intracellular replication, evasion of host immune responses, virulence, and encodes a type 6 secretion system. We hypothesize that some Francisella novicida pathogenicity island proteins are secreted during infection of host cells. To test this hypothesis, expression plasmids for all Francisella novicida FPI-encoded proteins with C-terminal and N-terminal epitope FLAG tags were developed. These plasmids expressed their respective epitope FLAG-tagged proteins at their predicted molecular weights. J774 murine macrophage-like cells were infected with Francisella novicida containing these plasmids. The FPI proteins expressed from these plasmids successfully restored the intramacrophage growth phenotype in mutants of the respective genes that were deficient for intramacrophage growth. Using these expression plasmids, the localization of the Francisella pathogenicity island proteins were examined via immuno-fluorescence microscopy within infected macrophage-like cells. Several Francisella pathogenicity island encoded proteins (IglABCDEFGHIJ, PdpACE, DotU and VgrG) were detected extracellularly and they were co-localized with the bacteria, while PdpBD and Anmk were not detected and thus remained inside bacteria. Proteins that were co-localized with bacteria had different patterns of localization. The localization of IglC was dependent on the type 6 secretion system. This suggests that some Francisella pathogenicity island proteins were secreted while others remain within the bacterium during infection of host cells as structural components of the secretion system and were necessary for secretion. PMID:25158041

  13. Francisella novicida Pathogenicity Island Encoded Proteins Were Secreted during Infection of Macrophage-Like Cells

    PubMed Central

    Hare, Rebekah F.; Hueffer, Karsten

    2014-01-01

    Intracellular pathogens and other organisms have evolved mechanisms to exploit host cells for their life cycles. Virulence genes of some intracellular bacteria responsible for these mechanisms are located in pathogenicity islands, such as secretion systems that secrete effector proteins. The Francisella pathogenicity island is required for phagosomal escape, intracellular replication, evasion of host immune responses, virulence, and encodes a type 6 secretion system. We hypothesize that some Francisella novicida pathogenicity island proteins are secreted during infection of host cells. To test this hypothesis, expression plasmids for all Francisella novicida FPI-encoded proteins with C-terminal and N-terminal epitope FLAG tags were developed. These plasmids expressed their respective epitope FLAG-tagged proteins at their predicted molecular weights. J774 murine macrophage-like cells were infected with Francisella novicida containing these plasmids. The FPI proteins expressed from these plasmids successfully restored the intramacrophage growth phenotype in mutants of the respective genes that were deficient for intramacrophage growth. Using these expression plasmids, the localization of the Francisella pathogenicity island proteins were examined via immuno-fluorescence microscopy within infected macrophage-like cells. Several Francisella pathogenicity island encoded proteins (IglABCDEFGHIJ, PdpACE, DotU and VgrG) were detected extracellularly and they were co-localized with the bacteria, while PdpBD and Anmk were not detected and thus remained inside bacteria. Proteins that were co-localized with bacteria had different patterns of localization. The localization of IglC was dependent on the type 6 secretion system. This suggests that some Francisella pathogenicity island proteins were secreted while others remain within the bacterium during infection of host cells as structural components of the secretion system and were necessary for secretion. PMID:25158041

  14. A Betabaculovirus-Encoded gp64 Homolog Codes for a Functional Envelope Fusion Protein

    PubMed Central

    Ardisson-Araújo, Daniel M. P.; Melo, Fernando L.; Clem, Rollie J.; Wolff, José L. C.

    2015-01-01

    The GP64 envelope fusion protein is a hallmark of group I alphabaculoviruses. However, the Diatraea saccharalis granulovirus genome sequence revealed the first betabaculovirus species harboring a gp64 homolog (disa118). In this work, we have shown that this homolog encodes a functional envelope fusion protein and could enable the infection and fusogenic abilities of a gp64-null prototype baculovirus. Therefore, GP64 may complement or may be in the process of replacing F protein activity in this virus lineage. PMID:26537678

  15. A Betabaculovirus-Encoded gp64 Homolog Codes for a Functional Envelope Fusion Protein.

    PubMed

    Ardisson-Araújo, Daniel M P; Melo, Fernando L; Clem, Rollie J; Wolff, José L C; Ribeiro, Bergmann M

    2016-02-01

    The GP64 envelope fusion protein is a hallmark of group I alphabaculoviruses. However, the Diatraea saccharalis granulovirus genome sequence revealed the first betabaculovirus species harboring a gp64 homolog (disa118). In this work, we have shown that this homolog encodes a functional envelope fusion protein and could enable the infection and fusogenic abilities of a gp64-null prototype baculovirus. Therefore, GP64 may complement or may be in the process of replacing F protein activity in this virus lineage. PMID:26537678

  16. Precursor protein of Alzheimer's disease A4 amyloid is encoded by 16 exons

    SciTech Connect

    Lemaire, H.G.; Kang, J.; Mueller-Hill, B. ); Salbaum, J.M.; Multhaup, G.; Beyreuther, K. ); Bayney, R.M.; Unterbeck, A. )

    1989-01-25

    Alzheimer's disease (AD) is characterized by the cerebral deposition of fibrillar aggregates of the amyloid A4 protein. Complementary DNA's coding for the precursor of the amyloid A4 protein have been described. In order to identify the structure of the precursor gene relevant clones from several human genomic libraries were isolated. Sequence analysis of the various clones revealed 16 exons to encode the 695 residue precursor protein (PreA4{sub 695}) of Alzheimer's disease amyloid A4 protein. The DNA sequence coding for the amyloid A4 protein is interrupted by an intron. This finding supports the idea that amyloid A4 protein arises by incomplete proteolysis of a larger precursor, and not by aberrant splicing.

  17. The Arabidopsis genome encodes structurally and functionally diverse HMGB-type proteins.

    PubMed

    Grasser, Marion; Lentz, Anne; Lichota, Jacek; Merkle, Thomas; Grasser, Klaus D

    2006-05-01

    The high mobility group (HMG) proteins of the HMGB family are chromatin-associated proteins that act as architectural factors in nucleoprotein structures, which regulate DNA-dependent processes including transcription and recombination. In addition to the previously identified HMGB1-HMGB6 proteins, the Arabidopsis genome encodes at least two other candidate family members (encoded by the loci At2g34450 and At5g23405) having the typical overall structure of a central domain displaying sequence similarity to HMG-box DNA binding domains, which is flanked by basic N-terminal and acidic C-terminal regions. Subcellular localisation experiments demonstrate that the At2g34450 protein is a nuclear protein, whereas the At5g23405 protein is found mainly in the cytoplasm. In line with this finding, At5g23405 displays specific interaction with the nuclear export receptor AtXPO1a. According to CD measurements, the HMG-box domains of both proteins have an alpha-helical structure. The HMG-box domain of At2g34450 interacts with linear DNA and binds structure-specifically to DNA minicircles, whereas the HMG-box domain of At5g23405 does not interact with DNA at all. In ligation experiments with short DNA fragments, the At2g34450 HMG-box domain can facilitate the formation of linear oligomers, but it does not promote the formation of DNA minicircles. Therefore, the At2g34450 protein shares several features with HMGB proteins, whereas the At5g23405 protein has different characteristics. Despite the presence of a region with similarity to the nucleosome-binding domain typical of HMGN proteins, At2g34450 does not bind nucleosome particles. In summary, our data demonstrate (i) that plant HMGB-type proteins are functionally variable and (ii) that it is difficult to predict HMG-box function solely based on sequence similarity. PMID:16563436

  18. Cloning and characterization of human liver cDNA encoding a protein S precursor

    SciTech Connect

    Hoskins, J.; Norman, D.K.; Beckmann, R.J.; Long, G.L.

    1987-01-01

    Human liver cDNA encoding a protein S precursor was isolated from two cDNA libraries by two different techniques. Based upon the frequency of positive clones, the abundance of mRNA for protein S is approx. = 0.01%. Blot hybridization of electrophoretically fractionated poly(A)/sup +/ RNA revealed a major mRNA approx. = 4 kilobases long and two minor forms of approx. = 3.1 and approx. = 2.6 kilobases. One of the cDNA clones contains a segment encoding a 676 amino acid protein S precursor, as well as 108 and 1132 nucleotides of 5' and 3' noncoding sequence, respectively, plus a poly(A) region at the 3' end. The cDNAs are adenosine plus thymidine-rich (60%) except for the 5' noncoding region, where 78% of the nucleotides are guanosine or cytosine. The protein precursor consists of a 41 amino acid leader peptide followed by 635 amino acids corresponding to mature protein S. Comparison of the mature protein region with homologous vitamin K-dependent plasma proteins shows that it is composed of the following domains: an amino-terminal ..gamma..-carboxyglutamic acid-rich region of 37 amino acids; a 36 amino acid linker region rich in hydroxy amino acids; four epidermal growth factor-like segments, each approx. = 45 amino acids long; and a 387 amino acid carboxyl-terminal domain of unrecognized structure and unknown function.

  19. Genetically encoded multispectral labeling of proteins with polyfluorophores on a DNA backbone.

    PubMed

    Singh, Vijay; Wang, Shenliang; Kool, Eric T

    2013-04-24

    Genetically encoded methods for protein conjugation are of high importance as biological tools. Here we describe the development of a new class of dyes for genetically encoded tagging that add new capabilities for protein reporting and detection via HaloTag methodology. Oligodeoxyfluorosides (ODFs) are short DNA-like oligomers in which the natural nucleic acid bases are replaced by interacting fluorescent chromophores, yielding a broad range of emission colors using a single excitation wavelength. We describe the development of an alkyl halide dehalogenase-compatible chloroalkane linker phosphoramidite derivative that enables the rapid automated synthesis of many possible dyes for protein conjugation. Experiments to test the enzymatic self-conjugation of nine different DNA-like dyes to proteins with HaloTag domains in vitro were performed, and the data confirmed the rapid and efficient covalent labeling of the proteins. Notably, a number of the ODF dyes were found to increase in brightness or change color upon protein conjugation. Tests in mammalian cellular settings revealed that the dyes are functional in multiple cellular contexts, both on the cell surface and within the cytoplasm, allowing protein localization to be imaged in live cells by epifluorescence and laser confocal microscopy. PMID:23590213

  20. Compensation for differences in gene copy number among yeast ribosomal proteins is encoded within their promoters

    PubMed Central

    Zeevi, Danny; Sharon, Eilon; Lotan-Pompan, Maya; Lubling, Yaniv; Shipony, Zohar; Raveh-Sadka, Tali; Keren, Leeat; Levo, Michal; Weinberger, Adina; Segal, Eran

    2011-01-01

    Coordinate regulation of ribosomal protein (RP) genes is key for controlling cell growth. In yeast, it is unclear how this regulation achieves the required equimolar amounts of the different RP components, given that some RP genes exist in duplicate copies, while others have only one copy. Here, we tested whether the solution to this challenge is partly encoded within the DNA sequence of the RP promoters, by fusing 110 different RP promoters to a fluorescent gene reporter, allowing us to robustly detect differences in their promoter activities that are as small as ∼10%. We found that single-copy RP promoters have significantly higher activities, suggesting that proper RP stoichiometry is indeed partly encoded within the RP promoters. Notably, we also partially uncovered how this regulation is encoded by finding that RP promoters with higher activity have more nucleosome-disfavoring sequences and characteristic spatial organizations of these sequences and of binding sites for key RP regulators. Mutations in these elements result in a significant decrease of RP promoter activity. Thus, our results suggest that intrinsic (DNA-dependent) nucleosome organization may be a key mechanism by which genomes encode biologically meaningful promoter activities. Our approach can readily be applied to uncover how transcriptional programs of other promoters are encoded. PMID:22009988

  1. Virulence Plasmid of Rhodococcus equi Contains Inducible Gene Family Encoding Secreted Proteins

    PubMed Central

    Byrne, Barbara A.; Prescott, John F.; Palmer, Guy H.; Takai, Shinji; Nicholson, Vivian M.; Alperin, Debra C.; Hines, Stephen A.

    2001-01-01

    Rhodococcus equi causes severe pyogranulomatous pneumonia in foals. This facultative intracellular pathogen produces similar lesions in immunocompromised humans, particularly in AIDS patients. Virulent strains of R. equi bear a large plasmid that is required for intracellular survival within macrophages and for virulence in foals and mice. Only two plasmid-encoded proteins have been described previously; a 15- to 17-kDa surface protein designated virulence-associated protein A (VapA) and an antigenically related 20-kDa protein (herein designated VapB). These two proteins are not expressed by the same R. equi isolate. We describe here the substantial similarity between VapA and VapB. Moreover, we identify three additional genes carried on the virulence plasmid, vapC, -D, and -E, that are tandemly arranged downstream of vapA. These new genes are members of a gene family and encode proteins that are approximately 50% homologous to VapA, VapB, and each other. vapC, -D, and -E are found only in R. equi strains that express VapA and are highly conserved in VapA-positive isolates from both horses and humans. VapC, -D, and -E are secreted proteins coordinately regulated by temperature with VapA; the proteins are expressed when R. equi is cultured at 37°C but not at 30°C, a finding that is compatible with a role in virulence. As secreted proteins, VapC, -D, and -E may represent targets for the prevention of rhodococcal pneumonia. An immunologic study using VapA-specific antibodies and recombinant Vap proteins revealed no evidence of cross-reactivity despite extensive sequence similarity over the carboxy terminus of all four proteins. PMID:11159951

  2. The KUP gene, located on human chromosome 14, encodes a protein with two distant zinc fingers.

    PubMed Central

    Chardin, P; Courtois, G; Mattei, M G; Gisselbrecht, S

    1991-01-01

    We have isolated a human cDNA (kup), encoding a new protein with two distantly spaced zinc fingers of the C2H2 type. This gene is highly conserved in mammals and is expressed mainly in hematopoietic cells and testis. Its expression was not higher in the various transformed cells tested than in the normal corresponding tissues. The kup gene is located in region q23-q24 of the long arm of human chromosome 14. The kup protein is 433 a.a. long, has a M.W. close to 50 kD and binds to DNA. Although the structure of the kup protein is unusual, the isolated fingers resemble closely those of the Krüppel family, suggesting that this protein is also a transcription factor. The precise function and DNA motif recognized by the kup protein remain to be determined. Images PMID:2027750

  3. Neurally expressed Drosophila genes encoding homologs of the NSF and SNAP secretory proteins.

    PubMed Central

    Ordway, R W; Pallanck, L; Ganetzky, B

    1994-01-01

    Several lines of investigation have now converged to indicate that the neurotransmitter release apparatus is formed by assembly of cytosolic proteins with proteins of the synaptic vesicle and presynaptic terminal membranes. We are undertaking a genetic approach in Drosophila melanogaster to investigate the functions of two types of cytosolic proteins thought to function in this complex: N-ethylmaleimide-sensitive fusion protein (NSF) and the soluble NSF attachment proteins (SNAPs). We have identified Drosophila homologs of the vertebrate and yeast NSF and SNAP genes. Both Drosophila genes encode polypeptides that closely resemble their vertebrate counterparts and are expressed in the nervous system; neither appears to be in a family of closely related Drosophila genes. These results indicate that the Drosophila NSF and SNAP genes are excellent candidates for mutational analysis of neurotransmitter release. Images PMID:8202553

  4. Cloning and expression analysis of a prion protein encoding gene in guppy ( Poecilia reticulata)

    NASA Astrophysics Data System (ADS)

    Wu, Suihan; Wei, Qiwei; Yang, Guanpin; Wang, Dengqiang; Zou, Guiwei; Chen, Daqing

    2008-11-01

    The full length cDNA of a prion protein (PrP) encoding gene of guppy ( Poecilia reticulata) and the corresponding genomic DNA were cloned. The cDNA was 2245 bp in length and contained an open reading frame (ORF) of 1545 bp encoding a protein of 515 amino acids, which held all typical structural characteristics of the functional PrP. The cloned genomic DNA fragment corresponding to the cDNA was 3720 bp in length, consisting of 2 introns and 2 exons. The 5' untranslated region of cDNA originated from the 2 exons, while the ORF originated from the second exon. Although the gene was transcribed in diverse tissues including brain, eye, liver, intestine, muscle and tail, its transcript was most abundant in the brain. In addition, the transcription of the gene was enhanced by 5 salinity, implying that it was associated with the response of guppy to saline stress.

  5. Characterization of the gene encoding a fibrinogen-related protein expressed in Crassostrea gigas hemocytes.

    PubMed

    Skazina, M A; Gorbushin, A M

    2016-07-01

    Four exons of the CgFrep1 gene (3333 bp long) encode a putative fibrinogen-related protein (324 aa) bearing a single C-terminal FBG domain. Transcripts of the gene obtained from hemocytes of different Pacific oysters show prominent individual variation based on SNP and indels of tandem repeats resulted in polymorphism of N-terminus of the putative CgFrep1 polypeptide. The polypeptide chain bears N-terminal coiled-coil region potentially acting as inter-subunit interface in the protein oligomerization. It is suggested that CgFrep1 gene encodes the oligomeric lectin composed of at least two subunits. PMID:27189918

  6. Cloning and expression of prion protein encoding gene of flounder ( Paralichthys olivaceus)

    NASA Astrophysics Data System (ADS)

    Zhang, Zhiwen; Sun, Xiuqin; Zhang, Jinxing; Zan, Jindong

    2008-02-01

    The prion protein (PrP) encoding gene of flounder ( Paralichthys olivaceus) was cloned. It was not interrupted by an intron. This gene has two promoters in its 5' upstream, indicating that its transcription may be intensive, and should have an important function. It was expressed in all 14 tissues tested, demonstrating that it is a house-keeping gene. Its expression in digestion and reproduction systems implies that the possible prions of fish may transfer horizontally.

  7. Dissecting Protein Function: An Efficient Protocol for Identifying Separation-of-Function Mutations That Encode Structurally Stable Proteins

    PubMed Central

    Lubin, Johnathan W.; Rao, Timsi; Mandell, Edward K.; Wuttke, Deborah S.; Lundblad, Victoria

    2013-01-01

    Mutations that confer the loss of a single biochemical property (separation-of-function mutations) can often uncover a previously unknown role for a protein in a particular biological process. However, most mutations are identified based on loss-of-function phenotypes, which cannot differentiate between separation-of-function alleles vs. mutations that encode unstable/unfolded proteins. An alternative approach is to use overexpression dominant-negative (ODN) phenotypes to identify mutant proteins that disrupt function in an otherwise wild-type strain when overexpressed. This is based on the assumption that such mutant proteins retain an overall structure that is comparable to that of the wild-type protein and are able to compete with the endogenous protein (Herskowitz 1987). To test this, the in vivo phenotypes of mutations in the Est3 telomerase subunit from Saccharomyces cerevisiae were compared with the in vitro secondary structure of these mutant proteins as analyzed by circular-dichroism spectroscopy, which demonstrates that ODN is a more sensitive assessment of protein stability than the commonly used method of monitoring protein levels from extracts. Reverse mutagenesis of EST3, which targeted different categories of amino acids, also showed that mutating highly conserved charged residues to the oppositely charged amino acid had an increased likelihood of generating a severely defective est3− mutation, which nevertheless encoded a structurally stable protein. These results suggest that charge-swap mutagenesis directed at a limited subset of highly conserved charged residues, combined with ODN screening to eliminate partially unfolded proteins, may provide a widely applicable and efficient strategy for generating separation-of-function mutations. PMID:23307900

  8. 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.

  9. Systematic Identification and Characterization of Novel Human Skin-Associated Genes Encoding Membrane and Secreted Proteins

    PubMed Central

    Buhren, Bettina Alexandra; Martinez, Cynthia; Schrumpf, Holger; Gasis, Marcia; Grether-Beck, Susanne; Krutmann, Jean

    2013-01-01

    Through bioinformatics analyses of a human gene expression database representing 105 different tissues and cell types, we identified 687 skin-associated genes that are selectively and highly expressed in human skin. Over 50 of these represent uncharacterized genes not previously associated with skin and include a subset that encode novel secreted and plasma membrane proteins. The high levels of skin-associated expression for eight of these novel therapeutic target genes were confirmed by semi-quantitative real time PCR, western blot and immunohistochemical analyses of normal skin and skin-derived cell lines. Four of these are expressed specifically by epidermal keratinocytes; two that encode G-protein-coupled receptors (GPR87 and GPR115), and two that encode secreted proteins (WFDC5 and SERPINB7). Further analyses using cytokine-activated and terminally differentiated human primary keratinocytes or a panel of common inflammatory, autoimmune or malignant skin diseases revealed distinct patterns of regulation as well as disease associations that point to important roles in cutaneous homeostasis and disease. Some of these novel uncharacterized skin genes may represent potential biomarkers or drug targets for the development of future diagnostics or therapeutics. PMID:23840300

  10. Pheromone binding proteins of Epiphyas postvittana (Lepidoptera: Tortricidae) are encoded at a single locus.

    PubMed

    Newcomb, R D; Sirey, T M; Rassam, M; Greenwood, D R

    2002-11-01

    The light brown apple moth, Epiphyas postvittana (Tortricidae: Lepidoptera) uses a blend of (E)-11-tetradecenyl acetate and (E,E)-9,11-tetradecadienyl acetate as its sex pheromone. Odorant binding proteins, abundant in the antennae of male and female E. postvittana, were separated by native PAGE to reveal four major proteins with distinct mobilities. Microsequencing of their N-terminal residues showed that two were general odorant binding proteins (GOBPs) while two were pheromone binding proteins (PBPs). Full length cDNAs encoding these proteins were amplified using a combination of PCR and RACE-PCR. Sequence of the GOBPs revealed two genes (EposGOBP1, EposGOBP2), similar to orthologues in other species of Lepidoptera. Eleven cDNAs of the PBP gene were amplified, cloned and sequenced revealing two major phylogenetic clusters of PBP sequences differing by six amino acid substitutions. The position of the six amino acid differences on the protein was predicted by mapping onto the three-dimensional structure of PBP of Bombyx mori. All six substitutions were predicted to fall on the outside of the protein away from the inner pheromone binding pocket. One substitution does fall close to the putative dimerisation region of the protein (Ser63Thr). Expression of three of the cDNAs in a baculovirus expression system revealed that one class encodes an electrophoretically slow form (EposPBP1-12) while the other encodes a fast form (EposPBP1-2, EposPBP1-3). A native Western of these expressed proteins compared with antennal protein extracts demonstrated that PBP is also expressed in female antennae and that PBP may be present as a dimer as well as a monomer in E. postvittana. The fast and slow forms of EposPBP1 are allelic. Westerns on single antennal pair protein extracts and allele-specific PCR from genomic DNA both show a segregating pattern of inheritance in laboratory and wild populations. Radio labelled (E)-11-tetradecenyl acetate binds to both fast and slow PBP forms in

  11. Cloning of a complementary DNA encoding an 80 kilodalton nuclear cap binding protein.

    PubMed Central

    Kataoka, N; Ohno, M; Kangawa, K; Tokoro, Y; Shimura, Y

    1994-01-01

    It has been shown that the monomethylated cap structure plays important roles in nuclear events. The cap structure has been implicated in the enhancement of pre-mRNA splicing. More recently, this structure has also been suggested to facilitate RNA transport from the nucleus to the cytoplasm. We have previously identified and purified an 80kD Nuclear Cap Binding Protein (NCBP) from a HeLa cell nuclear extract, which could possibly mediate these nuclear activities. In this report, we describe cloning of complementary DNA (cDNA) encoding NCBP. The partial protein sequences of NCBP were determined, and the full-length cDNA of NCBP was isolated from HeLa cDNA libraries. This cDNA encoded an open reading frame of 790 amino acids with a calculated molecular mass of 91,734 daltons, which contained most of the determined protein sequences. However, the protein sequence had no significant homology to any known proteins. Transfection experiments demonstrated that the epitope-tagged NCBP, transiently expressed in HeLa cells, was localized exclusively in the nucleoplasm. Similar experiments using a truncated NCBP cDNA indicated that this nuclear localization activity is conferred by the N-terminal 70 amino-acid region. Images PMID:7937105

  12. Subcellular localization and immunological detection of proteins encoded by the vir locus of Bordetella pertussis.

    PubMed Central

    Stibitz, S; Yang, M S

    1991-01-01

    The DNA sequence of the central regulatory locus vir of Bordetella pertussis predicts that three gene products, BvgA, BvgB, and BvgC, are encoded. Features of the predicted primary structures of these proteins and their homology to other two-component systems suggest that BvgA is located in the cytoplasm, BvgB is located in the periplasm, and BvgC spans the inner membrane. We have used gene fusions to the phoA and lacZ genes of Escherichia coli to investigate the subcellular localization and membrane topology of these proteins. PhoA fusion proteins were also purified and used to raise antibodies that allowed visualization of the vir-encoded polypeptides by Western immunoblotting. Our results have largely confirmed the predictions of the DNA sequence, with the exception that BvgB and BvgC were found to constitute one larger protein that was homologous to the sensor class of two-component systems. We propose that this protein be named BvgS (for sensor) and that its gene be named bvgS. Images PMID:2066330

  13. The Arabidopsis HUELLENLOS Gene, Which Is Essential for Normal Ovule Development, Encodes a Mitochondrial Ribosomal Protein

    PubMed Central

    Skinner, Debra J.; Baker, Shawn C.; Meister, Robert J.; Broadhvest, Jean; Schneitz, Kay; Gasser, Charles S.

    2001-01-01

    The HUELLENLOS (HLL) gene participates in patterning and growth of the Arabidopsis ovule. We have isolated the HLL gene and shown that it encodes a protein homologous to the L14 proteins of eubacterial ribosomes. The Arabidopsis genome also includes a highly similar gene, HUELLENLOS PARALOG (HLP), and genes for both cytosolic (L23) and chloroplast ribosome L14 proteins. Phylogenetic analysis shows that HLL and HLP differ significantly from these other two classes of such proteins. HLL and HLP fusions to green fluorescent protein were localized to mitochondria. Ectopic expression of HLP complemented the hll mutant, indicating that HLP and HLL share redundant functions. We conclude that HLL and HLP encode L14 subunits of mitochondrial ribosomes. HLL mRNA was at significantly higher levels than HLP mRNA in pistils, with the opposite pattern in leaves. This differential expression can explain the confinement of effects of hll mutations to gynoecia and ovules. Our elucidation of the nature of HLL shows that metabolic defects can have specific effects on developmental patterning. PMID:11752383

  14. A genetically encoded alkyne directs palladium-mediated protein labeling on live mammalian cell surface.

    PubMed

    Li, Nan; Ramil, Carlo P; Lim, Reyna K V; Lin, Qing

    2015-02-20

    The merging of site-specific incorporation of small bioorthogonal functional groups into proteins via amber codon suppression with bioorthogonal chemistry has created exciting opportunities to extend the power of organic reactions to living systems. Here we show that a new alkyne amino acid can be site-selectively incorporated into mammalian proteins via a known orthogonal pyrrolysyl-tRNA synthetase/tRNACUA pair and directs an unprecedented, palladium-mediated cross-coupling reaction-driven protein labeling on live mammalian cell surface. A comparison study with the alkyne-encoded proteins in vitro indicated that this terminal alkyne is better suited for the palladium-mediated cross-coupling reaction than the copper-catalyzed click chemistry. PMID:25347611

  15. Genes encoding FAD-binding proteins in Volvariella volvacea exhibit differential expression in homokaryons and heterokaryons.

    PubMed

    Meng, Li; Yan, Junjie; Xie, Baogui; Li, Yu; Chen, Bingzhi; Liu, Shuyan; Li, Dan; Yang, Zhiyun; Zeng, Xiancheng; Deng, Youjin; Jiang, Yuji

    2013-10-01

    Flavin adenine dinucleotide (FAD)-binding proteins play a vital role in energy transfer and utilization during fungal growth and mycelia aggregation. We sequenced the genome of Volvariella volvacea, an economically important edible fungus, and discovered 41 genes encoding FAD-binding proteins. Gene expression profiles revealed that the expression levels of four distinctly differentially expressed genes in heterokaryotic strain H1521 were higher than in homokaryotic strains PYd15 and PYd21 combined. These observations were validated by quantitative real-time PCR. The results suggest that the differential expression of FAD-binding proteins may be important in revealing the distinction between homokaryons and heterokaryons on the basis of FAD-binding protein functionality. PMID:23570970

  16. Cloning and characterization of the nucleoredoxin gene that encodes a novel nuclear protein related to thioredoxin

    SciTech Connect

    Kurooka, Hisanori; Kato, Keizo; Minoguchi, Shigeru

    1997-02-01

    In a yeast artificial chromosome contig close to the nude locus on mouse chromosome 11, we identified a novel gene, nucleoredoxin, that encodes a protein with similarity to the active site of thioredoxins. Nucleoredoxin is conserved between mammalian species, and two homologous genes were found in Caenorhabditis elegans. The nucleoredoxin transcripts are expressed in all adult tissues examined, but restricted to the nervous system and the limb buds in Day 10.5-11.5 embryos. The nucleoredoxin protein is predominantly localized in the nucleus of cells transfected with the nucleoredoxin expression construct. Since the bacterially expressed protein of nucleoredoxin showed oxidoreductase activity of the insulin disulfide bonds with kinetics similar to that of thioredoxin, it may be a redox regulator of the nuclear proteins, such as transcription factors. 40 refs., 6 figs.

  17. Localization microscopy using noncovalent fluorogen activation by genetically encoded fluorogen activating proteins

    PubMed Central

    Maji, Suvrajit; Huang, Fang; Szent-Gyorgyi, Chris; Lidke, Diane S.; Lidke, Keith A.; Bruchez, Marcel P.

    2014-01-01

    The noncovalent equilibrium activation of a fluorogenic malachite green dye and its cognate fluorogen activating protein has been exploited to produce a sparse labeling distribution of densely tagged genetically encoded proteins, enabling single molecule detection and superresolution imaging in fixed and living cells. These sparse labeling conditions are achieved by control of the dye concentration in the milieu, and do not require any photoswitching or photoactivation. The labeling is achieved using physiological buffers and cellular media, and does not require additives or switching buffer to obtain superresolution images. We evaluate superresolution properties and images obtained from a selected fluorogen activating protein clone fused to actin, and show that the photon counts per object fall between those typically reported for fluorescent proteins and switching dye-pairs, resulting in 10-30 nm localization precision per object. This labeling strategy complements existing approaches, and may simplify multicolor labeling of cellular structures. PMID:24194371

  18. A fully genetically-encoded protein architecture for optical control of peptide ligand concentration

    PubMed Central

    Schmidt, Daniel; Tillberg, Paul W.; Chen, Fei; Boyden, Edward S.

    2014-01-01

    Ion channels are amongst the most important proteins in biology - regulating the activity of excitable cells and changing in diseases. Ideally it would be possible to actuate endogenous ion channels, in a temporally precise and reversible fashion, and without requiring chemical co-factors. Here we present a modular protein architecture for fully genetically encoded, light-modulated control of ligands that modulate ion channels of a targeted cell. Our reagent, which we call a lumitoxin, combines a photoswitch and an ion channel-blocking peptide toxin. Illumination causes the photoswitch to unfold, lowering the toxin’s local concentration near the cell surface, and enabling the ion channel to function. We explore lumitoxin modularity by showing operation with peptide toxins that target different voltage-dependent K+ channels. The lumitoxin architecture may represent a new kind of modular protein engineering strategy for designing light-activated proteins, and thus may enable development of novel tools for modulating cellular physiology. PMID:24407101

  19. A fully genetically encoded protein architecture for optical control of peptide ligand concentration

    NASA Astrophysics Data System (ADS)

    Schmidt, Daniel; Tillberg, Paul W.; Chen, Fei; Boyden, Edward S.

    2014-01-01

    Ion channels are among the most important proteins in biology, regulating the activity of excitable cells and changing in diseases. Ideally it would be possible to actuate endogenous ion channels, in a temporally precise and reversible manner, and without requiring chemical cofactors. Here we present a modular protein architecture for fully genetically encoded, light-modulated control of ligands that modulate ion channels of a targeted cell. Our reagent, which we call a lumitoxin, combines a photoswitch and an ion channel-blocking peptide toxin. Illumination causes the photoswitch to unfold, lowering the toxin's local concentration near the cell surface, and enabling the ion channel to function. We explore lumitoxin modularity by showing operation with peptide toxins that target different voltage-dependent K+ channels. The lumitoxin architecture may represent a new kind of modular protein-engineering strategy for designing light-activated proteins, and thus may enable development of novel tools for modulating cellular physiology.

  20. A fully genetically encoded protein architecture for optical control of peptide ligand concentration.

    PubMed

    Schmidt, Daniel; Tillberg, Paul W; Chen, Fei; Boyden, Edward S

    2014-01-01

    Ion channels are among the most important proteins in biology, regulating the activity of excitable cells and changing in diseases. Ideally it would be possible to actuate endogenous ion channels, in a temporally precise and reversible manner, and without requiring chemical cofactors. Here we present a modular protein architecture for fully genetically encoded, light-modulated control of ligands that modulate ion channels of a targeted cell. Our reagent, which we call a lumitoxin, combines a photoswitch and an ion channel-blocking peptide toxin. Illumination causes the photoswitch to unfold, lowering the toxin's local concentration near the cell surface, and enabling the ion channel to function. We explore lumitoxin modularity by showing operation with peptide toxins that target different voltage-dependent K(+) channels. The lumitoxin architecture may represent a new kind of modular protein-engineering strategy for designing light-activated proteins, and thus may enable development of novel tools for modulating cellular physiology. PMID:24407101

  1. Generation of a panel of antibodies against proteins encoded on human chromosome 21

    PubMed Central

    2010-01-01

    Background Down syndrome (DS) is caused by trisomy of all or part of chromosome 21. To further understanding of DS we are working with a mouse model, the Tc1 mouse, which carries most of human chromosome 21 in addition to the normal mouse chromosome complement. This mouse is a model for human DS and recapitulates many of the features of the human syndrome such as specific heart defects, and cerebellar neuronal loss. The Tc1 mouse is mosaic for the human chromosome such that not all cells in the model carry it. Thus to help our investigations we aimed to develop a method to identify cells that carry human chromosome 21 in the Tc1 mouse. To this end, we have generated a panel of antibodies raised against proteins encoded by genes on human chromosome 21 that are known to be expressed in the adult brain of Tc1 mice Results We attempted to generate human specific antibodies against proteins encoded by human chromosome 21. We selected proteins that are expressed in the adult brain of Tc1 mice and contain regions of moderate/low homology with the mouse ortholog. We produced antibodies to seven human chromosome 21 encoded proteins. Of these, we successfully generated three antibodies that preferentially recognise human compared with mouse SOD1 and RRP1 proteins on western blots. However, these antibodies did not specifically label cells which carry a freely segregating copy of Hsa21 in the brains of our Tc1 mouse model of DS. Conclusions Although we have successfully isolated new antibodies to SOD1 and RRP1 for use on western blots, in our hands these antibodies have not been successfully used for immunohistochemistry studies. These antibodies are freely available to other researchers. Our data high-light the technical difficulty of producing species-specific antibodies for both western blotting and immunohistochemistry. PMID:20727138

  2. The evolution of genes encoding for green fluorescent proteins: insights from cephalochordates (amphioxus)

    NASA Astrophysics Data System (ADS)

    Yue, Jia-Xing; Holland, Nicholas D.; Holland, Linda Z.; Deheyn, Dimitri D.

    2016-06-01

    Green Fluorescent Protein (GFP) was originally found in cnidarians, and later in copepods and cephalochordates (amphioxus) (Branchiostoma spp). Here, we looked for GFP-encoding genes in Asymmetron, an early-diverged cephalochordate lineage, and found two such genes closely related to some of the Branchiostoma GFPs. Dim fluorescence was found throughout the body in adults of Asymmetron lucayanum, and, as in Branchiostoma floridae, was especially intense in the ripe ovaries. Spectra of the fluorescence were similar between Asymmetron and Branchiostoma. Lineage-specific expansion of GFP-encoding genes in the genus Branchiostoma was observed, largely driven by tandem duplications. Despite such expansion, purifying selection has strongly shaped the evolution of GFP-encoding genes in cephalochordates, with apparent relaxation for highly duplicated clades. All cephalochordate GFP-encoding genes are quite different from those of copepods and cnidarians. Thus, the ancestral cephalochordates probably had GFP, but since GFP appears to be lacking in more early-diverged deuterostomes (echinoderms, hemichordates), it is uncertain whether the ancestral cephalochordates (i.e. the common ancestor of Asymmetron and Branchiostoma) acquired GFP by horizontal gene transfer (HGT) from copepods or cnidarians or inherited it from the common ancestor of copepods and deuterostomes, i.e. the ancestral bilaterians.

  3. The evolution of genes encoding for green fluorescent proteins: insights from cephalochordates (amphioxus).

    PubMed

    Yue, Jia-Xing; Holland, Nicholas D; Holland, Linda Z; Deheyn, Dimitri D

    2016-01-01

    Green Fluorescent Protein (GFP) was originally found in cnidarians, and later in copepods and cephalochordates (amphioxus) (Branchiostoma spp). Here, we looked for GFP-encoding genes in Asymmetron, an early-diverged cephalochordate lineage, and found two such genes closely related to some of the Branchiostoma GFPs. Dim fluorescence was found throughout the body in adults of Asymmetron lucayanum, and, as in Branchiostoma floridae, was especially intense in the ripe ovaries. Spectra of the fluorescence were similar between Asymmetron and Branchiostoma. Lineage-specific expansion of GFP-encoding genes in the genus Branchiostoma was observed, largely driven by tandem duplications. Despite such expansion, purifying selection has strongly shaped the evolution of GFP-encoding genes in cephalochordates, with apparent relaxation for highly duplicated clades. All cephalochordate GFP-encoding genes are quite different from those of copepods and cnidarians. Thus, the ancestral cephalochordates probably had GFP, but since GFP appears to be lacking in more early-diverged deuterostomes (echinoderms, hemichordates), it is uncertain whether the ancestral cephalochordates (i.e. the common ancestor of Asymmetron and Branchiostoma) acquired GFP by horizontal gene transfer (HGT) from copepods or cnidarians or inherited it from the common ancestor of copepods and deuterostomes, i.e. the ancestral bilaterians. PMID:27311567

  4. Divergence of genes encoding non-specific lipid transfer proteins in the poaceae family.

    PubMed

    Jang, Cheol Seong; Jung, Jae Hyeong; Yim, Won Cheol; Lee, Byung-Moo; Seo, Yong Weon; Kim, Wook

    2007-10-31

    The genes encoding non-specific lipid transfer proteins (nsLTPs), members of a small multigene family, show a complex pattern of expressional regulation, suggesting that some diversification may have resulted from changes in their expression after duplication. In this study, the evolution of nsLTP genes within the Poaceae family was characterized via a survey of the pseudogenes and unigenes encoding the nsLTP in rice pseudomolecules and the NCBI unigene database. nsLTP-rich regions were detected in the distal portions of rice chromosomes 11 and 12; these may have resulted from the most recent large segmental duplication in the rice genome. Two independent tandem duplications were shown to occur within the nsLTP-rich regions of rice. The genomic distribution of the nsLTP genes in the rice genome differs from that in wheat. This may be attributed to gene migration, chromosomal rearrangement, and/or differential gene loss. The genomic distribution pattern of nsLTP genes in the Poaceae family points to the existence of some differences among cereal nsLTP genes, all of which diverged from an ancient gene. The unigenes encoding nsLTPs in each cereal species are clustered into five groups. The somewhat different distribution of nsLTP-encoding EST clones between the groups across cereal species imply that independent duplication(s) followed by subfunctionalization (and/or neofunctionalization) of the nsLTP gene family in each species occurred during speciation. PMID:17978574

  5. The evolution of genes encoding for green fluorescent proteins: insights from cephalochordates (amphioxus)

    PubMed Central

    Yue, Jia-Xing; Holland, Nicholas D.; Holland, Linda Z.; Deheyn, Dimitri D.

    2016-01-01

    Green Fluorescent Protein (GFP) was originally found in cnidarians, and later in copepods and cephalochordates (amphioxus) (Branchiostoma spp). Here, we looked for GFP-encoding genes in Asymmetron, an early-diverged cephalochordate lineage, and found two such genes closely related to some of the Branchiostoma GFPs. Dim fluorescence was found throughout the body in adults of Asymmetron lucayanum, and, as in Branchiostoma floridae, was especially intense in the ripe ovaries. Spectra of the fluorescence were similar between Asymmetron and Branchiostoma. Lineage-specific expansion of GFP-encoding genes in the genus Branchiostoma was observed, largely driven by tandem duplications. Despite such expansion, purifying selection has strongly shaped the evolution of GFP-encoding genes in cephalochordates, with apparent relaxation for highly duplicated clades. All cephalochordate GFP-encoding genes are quite different from those of copepods and cnidarians. Thus, the ancestral cephalochordates probably had GFP, but since GFP appears to be lacking in more early-diverged deuterostomes (echinoderms, hemichordates), it is uncertain whether the ancestral cephalochordates (i.e. the common ancestor of Asymmetron and Branchiostoma) acquired GFP by horizontal gene transfer (HGT) from copepods or cnidarians or inherited it from the common ancestor of copepods and deuterostomes, i.e. the ancestral bilaterians. PMID:27311567

  6. The herpes simplex virus 1 protein kinase encoded by the US3 gene mediates posttranslational modification of the phosphoprotein encoded by the UL34 gene.

    PubMed Central

    Purves, F C; Spector, D; Roizman, B

    1991-01-01

    Earlier studies have shown that a herpes simplex virus 1 (HSV-1) open reading frame, US3, encodes a novel protein kinase and have characterized the cognate amino acid sequence which is phosphorylated by this enzyme. This report identifies an apparently essential viral phosphoprotein whose posttranslational processing involves the viral protein kinase. Analyses of viral proteins phosphorylated in the course of productive infection revealed a phosphoprotein whose mobility was viral protein kinase and serotype dependent. Thus, the corresponding HSV-1 and HSV-2 phosphoproteins differ in their electrophoretic mobilities, and the phosphoprotein specified by the HSV-1 mutant deleted in US3 (R7041) differs from that of the corresponding HSV-1 and HSV-2 proteins. Analyses of HSV-1 x HSV-2 recombinants mapped the phosphoprotein between 0.42 and 0.47 map units on the prototype HSV-1 DNA map. Within this region, the UL34 open reading frame was predicted to encode a protein of appropriate molecular weight which would also contain the consensus target site for phosphorylation by the viral protein kinase as previously defined with synthetic peptides. Replacement of the native UL34 gene with a UL34 gene tagged with a 17-amino-acid epitope from the alpha 4 protein identified this gene as encoding the phosphoprotein. Finally, mutagenesis of the predicted phosphorylation site on UL34 in the viral genome, and specifically the substitution of threonine or serine with alanine in the product of the UL34 gene, yielded phosphoproteins whose electrophoretic mobilities could not be differentiated from that of the US3- mutant. We conclude that the posttranslational processing of the UL34 gene product to its wild-type phenotype requires the participation of the viral protein kinase. While the viral protein kinase is not essential for viral replication in cells in culture, the UL34 gene product itself may not be dispensable. Images PMID:1656069

  7. Mammalian ets-1 and ets-2 genes encode highly conserved proteins

    SciTech Connect

    Watson, D.K.; McWilliams, M.J.; Lapis, P.; Lautenberger, J.A.; Schweinfest, C.W.; Papas, T.S. )

    1988-11-01

    Cellular ets sequences homologous to v-ets of the avian leukemia virus E26 are highly conserved. In mammals the ets sequences are dispersed on two separate chromosomal loci, called ets-1 and ets-2. To determine the structure of these two genes and identify the open reading frames that code for the putative proteins, the authors have sequenced human ets-1 cDNAs and ets-2 cDNA clones obtained from both human and mouse. The human ETS1 gene is capable of encoding a protein of 441 amino acids. This protein is >95% identical to the chicken c-ets-1 gene product. Thus, the human ETS1 gene is homologous to the chicken c-ets-1 gene, the protooncogene that the E26 virus transduced. Human and mouse ets-2 cDNA clones are closely related and contain open reading frames capable of encoding proteins of 469 and 468 residues, respectively. Direct comparison of these data with previously published finding indicates that ets is a family of genes whose members share distinct domains.

  8. Mammalian ets-1 and ets-2 genes encode highly conserved proteins.

    PubMed Central

    Watson, D K; McWilliams, M J; Lapis, P; Lautenberger, J A; Schweinfest, C W; Papas, T S

    1988-01-01

    Cellular ets sequences homologous to v-ets of the avian leukemia virus E26 are highly conserved. In mammals the ets sequences are dispersed on two separate chromosomal loci, called ets-1 and ets-2. To determine the structure of these two genes and identify the open reading frames that code for the putative proteins, we have sequenced human ets-1 cDNAs and ets-2 cDNA clones obtained from both human and mouse. The human ETS1 gene is capable of encoding a protein of 441 amino acids. This protein is greater than 95% identical to the chicken c-ets-1 gene product. Thus, the human ETS1 gene is homologous to the chicken c-ets-1 gene, the protooncogene that the E26 virus transduced. Human and mouse ets-2 cDNA clones are closely related and contain open reading frames capable of encoding proteins of 469 and 468 residues, respectively. Direct comparison of these data with previously published findings indicates that ets is a family of genes whose members share distinct domains. PMID:2847145

  9. Ectromelia virus encodes an anti-apoptotic protein that regulates cell death.

    PubMed

    Mehta, Ninad; Taylor, John; Quilty, Douglas; Barry, Michele

    2015-01-15

    Apoptosis serves as a powerful defense against damaged or pathogen-infected cells. Since apoptosis is an effective defense against viral infection, many viruses including poxviruses, encode proteins to prevent or delay apoptosis. Here we show that ectromelia virus, the causative agent of mousepox encodes an anti-apoptotic protein EVM025. Here we demonstrate that expression of functional EVM025 is crucial to prevent apoptosis triggered by virus infection and staurosporine. We demonstrate that the expression of EVM025 prevents the conformational activation of the pro-apoptotic proteins Bak and Bax, allowing the maintenance of mitochondrial membrane integrity upon infection with ECTV. Additionally, EVM025 interacted with intracellular Bak. We were able to demonstrate that EVM025 ability to inhibit Bax activation is a function of its ability to inhibit the activity of an upstream BH3 only protein Bim. Collectively, our data indicates that EVM025 inhibits apoptosis by sequestering Bak and inhibiting the activity of Bak and Bax. PMID:25462348

  10. Interaction between the human cytomegalovirus‑encoded UL142 and cellular Snapin proteins.

    PubMed

    Liu, Chang; Qi, Ying; Ma, Yanping; He, Rong; Sun, Zhengrong; Huang, Yujing; Ji, Yaohua; Ruan, Qiang

    2015-02-01

    Human cytomegalovirus (HCMV) infection can cause severe illness in immunocompromised and immunodeficient individuals. As a novel HCMV‑encoded major histocompatibility complex class I‑related molecule, the UL142‑encoded protein (pUL142) is capable of suppressing natural killer (NK) cell recognition in the course of infection. However, no host factors that directly interact with HCMV pUL142 have been reported so far. In order to understand the interactions between HCMV pUL142 and host proteins, the current study used yeast two‑hybrid screening, a GST pull‑down assay and an immunofluorescence assay. A host protein, the SNARE‑associated protein Snapin, was identified to directly interact and colocalize with HCMV pUL142 in transfected human embryonic kidney‑293 cells. Snapin is abundantly expressed in the majority of cells and mediates the release of neurotransmitters through vesicular transport in the nervous system and vesicle fusion in non‑neuronal cells. It is hypothesized that HCMV pUL142 may have an impact on the neurotransmitter release process and viral dissemination via interaction with Snapin. PMID:25369979

  11. RAD6 gene of Saccharomyces cerevisiae encodes a protein containing a tract of 13 consecutive aspartates

    SciTech Connect

    Reynolds, P.; Weber, S.; Prakash, L.

    1985-01-01

    The RAD6 gene of Saccharomyces cerevisiae is required for postreplication repair of UV-damaged DNA, for induced mutagenesis, and for sporulation. The authors have mapped the transcripts and determined the nucleotide sequence of the cloned RAD6 gene. The RAD6 gene encodes two transcripts of 0.98 and 0.86 kilobases which differ only in their 3' termini. The transcribed region contains an open reading frame of 516 nucleotides. The rad6-1 and rad6-3 mutant alleles, which the authors have cloned and sequenced, introduce amber and ochre nonsense mutations, respectively into the open reading frame, proving that it encodes the RAD6 protein. The RAD6 protein predicted by the nucleotide sequence is 172 amino acids long, has a molecular weight of 19,704, and contains 23.3% acidic and 11.6% basic residues. Its most striking feature is the highly acidic carboxyl terminus: 20 of the 23 terminal amino acids are acidic, including 13 consecutive aspartates. RAD6 protein thus resembles high mobility group proteins HMG-1 and HMG-2, which each contain a carboxyl-proximal tract of acidic amino acids. 48 references, 6 figures.

  12. Changes in cellular microRNA expression induced by porcine circovirus type 2-encoded proteins.

    PubMed

    Hong, Jae-Sang; Kim, Nam-Hoon; Choi, Chang-Yong; Lee, Jun-Seong; Na, Dokyun; Chun, Taehoon; Lee, Young Sik

    2015-01-01

    Porcine circovirus type 2 (PCV2) is the primary causative agent of postweaning multisystemic wasting syndrome, which leads to serious economic losses in the pig industry worldwide. While the molecular basis of PCV2 replication and pathogenicity remains elusive, it is increasingly apparent that the microRNA (miRNA) pathway plays a key role in controlling virus-host interactions, in addition to a wide range of cellular processes. Here, we employed Solexa deep sequencing technology to determine which cellular miRNAs were differentially regulated after expression of each of three PCV2-encoded open reading frames (ORFs) in porcine kidney epithelial (PK15) cells. We identified 51 ORF1-regulated miRNAs, 74 ORF2-regulated miRNAs, and 32 ORF3-regulated miRNAs that differed in abundance compared to the control. Gene ontology analysis of the putative targets of these miRNAs identified transcriptional regulation as the most significantly enriched biological process, while KEGG pathway analysis revealed significant enrichment for several pathways including MAPK signaling, which is activated during PCV2 infection. Among the potential target genes of ORF-regulated miRNAs, two genes encoding proteins that are known to interact with PCV2-encoded proteins, zinc finger protein 265 (ZNF265) and regulator of G protein signaling 16 (RGS16), were selected for further analysis. We provide evidence that ZNF265 and RGS16 are direct targets of miR-139-5p and let-7e, respectively, which are both down-regulated by ORF2. Our data will initiate further studies to elucidate the roles of ORF-regulated cellular miRNAs in PCV2-host interactions. PMID:25885539

  13. Sequence and regulation of a gene encoding a human 89-kilodalton heat shock protein

    SciTech Connect

    Hickey, E.; Brandon, S.E.; Weber, L.A.; Lloyd, D.

    1989-06-01

    Vertebrate cells synthesize two forms of the 82- to 90-kilodalton heat shock protein that are encoded by distinct gene families. In HeLa cells, both proteins (hsp89/alpha/ and hspio/beta/) are abundant under normal growth conditions and are synthesized at increased rates in response to heat stress. Only the larger form, hsp89/alpha/, is induced by the adenovirus E1A gene product. The authors have isolated a human hsp89/alpha/ gene that shows complete sequence identity with heat- and E1A-inducible cDNA used as a hybridization probe. The 5'-flanking region contained overlapping and inverted consensus heat shock control elements that can confer heat-inducible expression n a /beta/-globin reporter gene. The gene contained 10 intervening sequences. The first intron was located adjacent to the translation start codon, an arrangement also found in the Drosophila hsp82 gene. The spliced mRNA sequence contained a single open reading frame encoding an 84,564-dalton polypeptide showing high homology with the hsp82 to hsp90 proteins of other organisms. The deduced hsp89/alpha/ protein sequence differed from the human hsp89/beta/ sequence reported elsewhere in at least 99 out of the 732 amino acids. Transcription of the hsp89/alpha/ gene was induced by serum during normal cell growth, but expression did not appear to be restricted to a particular stage of the cell cycles. hsp89/alpha/ mRNA was considerably more stable than the mRNA encoding hsp70, which can account for the higher constitutive rate of hsp89 synthesis in unstressed cells.

  14. sar1, a gene from Schizosaccharomyces pombe encoding a protein that regulates ras1.

    PubMed Central

    Wang, Y; Boguski, M; Riggs, M; Rodgers, L; Wigler, M

    1991-01-01

    Proper ras1 function is required for normal sexual function in the yeast Schizosaccharomyces pombe. We have found a gene in S. pombe, sar1, that encodes a product capable of regulating ras1 function. sar1 is a member of an expanding family of RAS GTPase-activating proteins (GAPs) that includes mammalian GAP, the yeast Saccharomyces cerevisiae IRA proteins, and the product of the human neurofibromatosis locus, NF1 sar1, like these other proteins, can complement the loss of IRA function in S. cerevisiae. Computer analysis shows that the highest degree of sequence conservation is restricted to a very small number of diagnostic residues represented by the motif Phe-Leu-Arg-X-X-X-Pro-Ala-X-X-X-Pro. We find no evidence that sar1 is required for the effector function of ras1. Images PMID:1883874

  15. The yeast SNF3 gene encodes a glucose transporter homologous to the mammalian protein.

    PubMed Central

    Celenza, J L; Marshall-Carlson, L; Carlson, M

    1988-01-01

    The SNF3 gene is required for high-affinity glucose transport in the yeast Saccharomyces cerevisiae and has also been implicated in control of gene expression by glucose repression. We report here the nucleotide sequence of the cloned SNF3 gene. The predicted amino acid sequence shows that SNF3 encodes a 97-kilodalton protein that is homologous to mammalian glucose transporters and has 12 putative membrane-spanning regions. We also show that a functional SNF3-lacZ gene-fusion product cofractionates with membrane proteins and is localized to the cell surface, as judged by indirect immunofluorescence microscopy. Expression of the fusion protein is regulated by glucose repression. Images PMID:3281163

  16. Identification of two proteins encoded by com, a competence control locus of Streptococcus pneumoniae.

    PubMed Central

    Chandler, M S; Morrison, D A

    1988-01-01

    The com locus, which controls competence for genetic transformation in Streptococcus pneumoniae, was analyzed by construction of a series of subclones, insertion mutations, and deletions of the cloned DNA in Escherichia coli. In vitro transcription-translation of these com plasmids revealed two neighboring genes, comA and comB, encoding proteins of 77,000 and 49,000 daltons, respectively. Their map positions and orientations were determined. Insertions in either gene eliminated the corresponding protein and had no effect on the other. In addition, a 15,000-dalton com protein was tentatively identified, although the exact location of this gene remains to be determined. Features of the DNA adjacent to the com locus are also described. Images PMID:3384803

  17. Three abundant germ line-specific transcripts in Volvox carteri encode photosynthetic proteins.

    PubMed

    Choi, G; Przybylska, M; Straus, D

    1996-09-01

    Volvox carteri is a multicellular eukaryotic green alga composed of about 2000 cells of only two differentiated types: somatic and germ line. To understand how embryonic cells are assigned either to somatic or germ line fates, we are investigating the regulation of transcripts that are abundant in only one cell type. Here we report the identity of three transcripts that are coordinately expressed at high levels in germ line cells but not in somatic cells. Surprisingly, all three transcripts encode photosynthetic chloroplast proteins (light-harvesting complex protein, oxygen-evolving enhancer protein 3, and ferredoxin-NADP+ reductase) that are transcribed from nuclear genes. We discuss why these mRNAs might be required at high levels in germ line cells and present a hypothesis, suggested by our results, on the evolution of cell specialization in the Volvocales. PMID:8781179

  18. A FUSCA gene of Arabidopsis encodes a novel protein essential for plant development.

    PubMed Central

    Castle, L A; Meinke, D W

    1994-01-01

    Arabidopsis fusca mutants display striking purple coloration due to anthocyanin accumulation in their cotyledons. We describe six recessive fusca mutants isolated from Agrobacterium-transformed Arabidopsis families. These mutants first become defective during embryogenesis and exhibit limited seedling development. Double mutant constructs revealed that developmental defects were not simply a consequence of anthocyanin accumulation. fusca seedlings showed altered responses to several environmental and endogenous factors. Allelism tests established that three fusca loci are represented by mutants previously described as defective in light-regulated responses. To study the molecular basis of the fusca phenotype, we cloned the FUS6 gene. FUS6 encodes a novel protein that is hydrophilic, alpha-helical, and contains potential protein kinase C phosphorylation sites. The FUSCA proteins appear to act in a network of signal transduction pathways critical for plant development. PMID:8130643

  19. Alternative splicing of the mRNA encoding the human cholesteryl ester transfer protein

    SciTech Connect

    Inazu, Akihiro; Quinet, E.M.; Suke Wang; Brown, M.L.; Stevenson, S.; Barr, M.L.; Moulin, P.; Tall, A.R. )

    1992-03-03

    The plasma cholesteryl ester transfer protein (CETP) is known to facilitate the transfer of lipids between plasma lipoproteins. The human CETP gene is a complex locus encompassing 16 exons. The CETP mRNA is found in liver and small intestine as well as in a variety of peripheral tissues. While the CETP cDNA from human adipose tissue was being cloned, a variant CETP cDNA was discovered which excluded the complete sequence encoded by exon 9, but which was otherwise identical to the full-length CETP cDNA, suggesting modification of the CETP gene transcript by an alternative RNA splicing mechanism. RNase protection analysis of tissue RNA confirmed the presence of exon 9 deleted transcripts and showed that they represented a variable proportion of the total CETP mRNA in various human tissues including adipose tissue (25%), liver (33%), and spleen (46%). Transient expression of the exon 9 deleted cDNA in COS cells or stable expression in CHO cells showed that the protein encoded by the alternatively spliced transcript was inactive in neutral lipid transfer, smaller, and poorly secreted compared to the protein derived from the full-length cDNA. Endo H digestion suggested that the inactive, cell-associated protein was present within the endoplasmic reticulum. The experiments show that the expression of the human CETP gene is modified by alternative splicing of the ninth exon, in a tissue-specific fashion. The function of alternative splicing is unknown but could serve to produce a protein with a function other than plasma neutral lipid transfer, or as an on-off switch to regulate the local concentration of biologically active protein.

  20. Ectromelia virus encodes a family of Ankyrin/F-box proteins that regulate NFκB

    SciTech Connect

    Burles, Kristin Buuren, Nicholas van; Barry, Michele

    2014-11-15

    A notable feature of poxviruses is their ability to inhibit the antiviral response, including the nuclear factor kappa B (NFκB) pathway. NFκB is a transcription factor that is sequestered in the cytoplasm until cell stimulation, and relies on the SCF (Skp1, culllin-1, F-box) ubiquitin ligase to target its inhibitor, IκBα, for degradation. IκBα is recruited to the SCF by the F-box domain-containing protein βTrCP. Here, we show that ectromelia virus, the causative agent of mousepox, encodes four F-box-containing proteins, EVM002, EVM005, EVM154, and EVM165, all of which contain Ankyrin (Ank) domains. The Ank/F-box proteins inhibit NFκB nuclear translocation, and this inhibition is dependent on the F-box domain. We also demonstrate that EVM002, EVM005, EVM154, and EVM165 prevent IκBα degradation, suggesting that they target the SCF. This study identifies a new mechanism by which ectromelia virus inhibits NFκB. - Highlights: • Ectromelia virus encodes four Ank/F-box proteins, EVM002, EVM005, EVM154 and EVM165. • The Ank/F-box proteins inhibit NFκB nuclear translocation, dependent on the F-box. • The Ank/F-box proteins prevent IκBα degradation, suggesting they target the SCF. • Deletion of a single Ank/F-box gene from ECTV does not prevent viral NFκB inhibition. • This study identifies a new mechanism by which ectromelia virus inhibits NFκB.

  1. Genome segment 6 of Antheraea mylitta cypovirus encodes a structural protein with ATPase activity

    SciTech Connect

    Chavali, Venkata R.M.; Madhurantakam, Chaithanya; Ghorai, Suvankar; Roy, Sobhan; Das, Amit K.; Ghosh, Ananta K.

    2008-07-20

    The genome segment 6 (S6) of the 11 double stranded RNA genomes from Antheraea mylitta cypovirus was converted into cDNA, cloned and sequenced. S6 consisted of 1944 nucleotides with an ORF of 607 amino acids and could encode a protein of 68 kDa, termed P68. Motif scan and molecular docking analysis of P68 showed the presence of two cystathionine beta synthase (CBS) domains and ATP binding sites. The ORF of AmCPV S6 was expressed in E. coli as His-tag fusion protein and polyclonal antibody was raised. Immunoblot analysis of virus infected gut cells and purified polyhedra using raised anti-p68 polyclonal antibody showed that S6 encodes a viral structural protein. Fluorescence and ATPase assay of soluble P68 produced in Sf-9 cells via baculovirus expression system showed its ability to bind and cleave ATP. These results suggest that P68 may bind viral RNA through CBS domains and help in replication and transcription through ATP binding and hydrolysis.

  2. Prediction of HIV drug resistance from genotype with encoded three-dimensional protein structure

    PubMed Central

    2014-01-01

    Background Drug resistance has become a severe challenge for treatment of HIV infections. Mutations accumulate in the HIV genome and make certain drugs ineffective. Prediction of resistance from genotype data is a valuable guide in choice of drugs for effective therapy. Results In order to improve the computational prediction of resistance from genotype data we have developed a unified encoding of the protein sequence and three-dimensional protein structure of the drug target for classification and regression analysis. The method was tested on genotype-resistance data for mutants of HIV protease and reverse transcriptase. Our graph based sequence-structure approach gives high accuracy with a new sparse dictionary classification method, as well as support vector machine and artificial neural networks classifiers. Cross-validated regression analysis with the sparse dictionary gave excellent correlation between predicted and observed resistance. Conclusion The approach of encoding the protein structure and sequence as a 210-dimensional vector, based on Delaunay triangulation, has promise as an accurate method for predicting resistance from sequence for drugs inhibiting HIV protease and reverse transcriptase. PMID:25081370

  3. Characterization and Expression of Genes Encoding Three Small Heat Shock Proteins in Sesamia inferens (Lepidoptera: Noctuidae)

    PubMed Central

    Sun, Meng; Lu, Ming-Xing; Tang, Xiao-Tian; Du, Yu-Zhou

    2014-01-01

    The pink stem borer, Sesamia inferens (Walker), is a major pest of rice and is endemic in China and other parts of Asia. Small heat shock proteins (sHSPs) encompass a diverse, widespread class of stress proteins that have not been characterized in S. inferens. In the present study, we isolated and characterized three S. inferens genes that encode members of the α-crystallin/sHSP family, namely, Sihsp21.4, Sihsp20.6, and Sihsp19.6. The three cDNAs encoded proteins of 187, 183 and 174 amino acids with calculated molecular weights of 21.4, 20.6 and 19.6 kDa, respectively. The deduced amino acid sequences of the three genes showed strong similarity to sHSPs identified in other lepidopteran insects. Sihsp21.4 contained an intron, but Sihsp20.6 and Sihsp19.6 lacked introns. Real-time quantitative PCR analyses revealed that Sihsp21.4 was most strongly expressed in S. inferens heads; Whereas expression of Sihsp20.6 and Sihsp19.6 was highest in eggs. The three S. inferens sHSP genes were up-regulated during low temperature stress. In summary, our results show that S. inferens sHSP genes have distinct regulatory roles in the physiology of S. inferens. PMID:25514417

  4. Boymaw, overexpressed in brains with major psychiatric disorders, may encode a small protein to inhibit mitochondrial function and protein translation.

    PubMed

    Ji, Baohu; Kim, Minjung; Higa, Kerin K; Zhou, Xianjin

    2015-06-01

    The t(1,11) chromosome translocation co-segregates with major psychiatric disorders in a large Scottish family. The translocation disrupts the DISC1and Boymaw (DISC1FP1) genes on chromosomes 1 and 11, respectively. After translocation, two fusion genes are generated. Our recent studies found that the DISC1-Boymaw fusion protein is localized in mitochondria and inhibits oxidoreductase activity, rRNA expression, and protein translation. Mice carrying the DISC1-Boymaw fusion genes display intermediate behavioral phenotypes related to major psychiatric disorders. Here, we report that the Boymaw gene may encode a small protein predominantly localized in mitochondria. The Boymaw protein inhibits oxidoreductase activity, rRNA expression, and protein translation in the same way as the DISC1-Boymaw fusion protein. Interestingly, Boymaw expression is up-regulated by different stressors at RNA and/or protein translational levels. In addition, we found that Boymaw RNA expression is significantly increased in the postmortem brains of patients with major psychiatric disorders. Our studies therefore suggest that the Boymaw gene could potentially be a susceptibility gene for major psychiatric disorders in both the Scottish t(1,11) family and the general population of patients. PMID:25943690

  5. An Apicoplast Localized Ubiquitylation System Is Required for the Import of Nuclear-encoded Plastid Proteins

    PubMed Central

    Ponts, Nadia; van Dooren, Giel G.; Prudhomme, Jacques; Brooks, Carrie F.; Rodrigues, Elisadra M.; Tan, John C.; Ferdig, Michael T.; Striepen, Boris; Le Roch, Karine G.

    2013-01-01

    Apicomplexan parasites are responsible for numerous important human diseases including toxoplasmosis, cryptosporidiosis, and most importantly malaria. There is a constant need for new antimalarials, and one of most keenly pursued drug targets is an ancient algal endosymbiont, the apicoplast. The apicoplast is essential for parasite survival, and several aspects of its metabolism and maintenance have been validated as targets of anti-parasitic drug treatment. Most apicoplast proteins are nuclear encoded and have to be imported into the organelle. Recently, a protein translocon typically required for endoplasmic reticulum associated protein degradation (ERAD) has been proposed to act in apicoplast protein import. Here, we show ubiquitylation to be a conserved and essential component of this process. We identify apicoplast localized ubiquitin activating, conjugating and ligating enzymes in Toxoplasma gondii and Plasmodium falciparum and observe biochemical activity by in vitro reconstitution. Using conditional gene ablation and complementation analysis we link this activity to apicoplast protein import and parasite survival. Our studies suggest ubiquitylation to be a mechanistic requirement of apicoplast protein import independent to the proteasomal degradation pathway. PMID:23785288

  6. The A alpha mating locus of Schizophyllum commune encodes two dissimilar multiallelic homeodomain proteins.

    PubMed Central

    Stankis, M M; Specht, C A; Yang, H; Giasson, L; Ullrich, R C; Novotny, C P

    1992-01-01

    The A alpha mating locus is one of four multiallelic loci that govern sexual development in the basidiomycete fungus Schizophyllum commune. We have determined the nucleotide sequence encoding three A alpha mating types, A alpha 1, A alpha 3, and A alpha 4. We have found that the locus for A alpha 3 and A alpha 4 consists of two genes: Y and Z. The locus for A alpha 1 encodes only one gene, Y. The Z polypeptides encoded by different alleles exhibit 42% identity. The Y polypeptides exhibit 49-54% identity. The finding that the deduced Z and Y polypeptides have homeodomain motifs suggests that these polypeptides may be DNA-binding regulatory proteins that control the expression of developmental genes. The deduced Z polypeptide also has acidic regions that might be functionally analogous to the acidic regions in yeast GAL4 and GCN4 that activate transcription. The Y polypeptide has a serine-rich region and a basic region that shows some identity to the lysine-rich region of H1 histones. PMID:1353886

  7. PCF11 encodes a third protein component of yeast cleavage and polyadenylation factor I.

    PubMed Central

    Amrani, N; Minet, M; Wyers, F; Dufour, M E; Aggerbeck, L P; Lacroute, F

    1997-01-01

    Cleavage and polyadenylation factor I (CF I) is one of four factors required in vitro for yeast pre-mRNA 3'-end processing. Two protein components of this factor, encoded by genes RNA14 and RNA15, have already been identified. We describe here another gene, PCF11 (for protein 1 of CF I), that genetically interacts with RNA14 and RNA15 and which presumably codes for a third protein component of CF I. This gene was isolated in a two-hybrid screening designed to identify proteins interacting with Rna14 and Rna15. PCF11 is an essential gene encoding for a protein of 626 amino acids having an apparent molecular mass of 70 kDa. Thermosensitive mutations in PCF11 are synergistically lethal with thermosensitive alleles of RNA14 and RNA15. The Pcf11-2 thermosensitive strain shows a shortening of the poly(A) tails and a strong decrease in the steady-state level of actin transcripts after a shift to the nonpermissive temperature as do the thermosensitive alleles of RNA14 and RNA15. Extracts from the pcf11-1 and pcf11-2 thermosensitive strains and the wild-type strain, when Pcf11 is neutralized by specific antibodies, are deficient in cleavage and polyadenylation. Moreover, fractions obtained by anion-exchange chromatography of extracts from the wild-type strain contain both Pcf11 and Rna15 in the same fractions, as shown by immunoblotting with a Pcf11-specific antibody. PMID:9032237

  8. Expression of Ndufb11 encoding the neuronal protein 15.6 during neurite outgrowth and development.

    PubMed

    Gurok, Ulf; Bork, Kaya; Nuber, Ulrike; Spörle, Ralf; Nöhring, Sabine; Horstkorte, Rüdiger

    2007-01-01

    Neurite outgrowth (e.g. axonal or dendrite outgrowth) of neurons is necessary for the development and functioning of the central nervous system. It is well accepted that the differentiation of neurons and neurite outgrowth involve alterations in gene expression. Furthermore, mitochondria play a role in different aspects of neurite outgrowth. Here we show that the expression of Ndufb11, a gene encoding the mitochondrial protein NP15.6 is decreased in the course of neuronal differentiation. NP15.6 is homologous to the bovine protein ESSS, a component of the mitochondrial complex 1. The homologous human NDUFB11 gene is localized to Xp11.3-Xp11.23, a region associated with neurogenetic disorders. The down-regulation of NP15.6 correlates with neurite outgrowth of PC12 cells induced by nerve growth factor. Furthermore, we analyzed the expression of Ndufb11 in the embryonic and adult mouse. PMID:16962385

  9. Genetic variability in the sable (Martes zibellina L.) with respect to genes encoding blood proteins

    SciTech Connect

    Kashtanov, S.N.; Kazakova, T.I.

    1995-02-01

    Electrophoresis of blood proteins was used to determine, for the first time, the level of genetic variability of certain loci in the sable (Martes zibellina L., Mustelidae). Variation of 23 blood proteins encoded by 25 genes was analyzed. Polymorphism was revealed in six genes. The level of heterozygosity was estimated at 0.069; the proportion of polymorphic loci was 24%. Data on the history of the sable population maintained at the farm, on geographical distribution of natural sable populations, and on the number of animals selected for reproduction in captivity is presented. The great number of animals studies and the extensive range of natural sable populations, on the basis of which the population maintained in captivity was obtained, suggest that the results of this work can be used for estimating the variability of the gene pool of sable as a species. 9 refs., 2 figs., 1 tab.

  10. Homolog detection using global sequence properties suggests an alternate view of structural encoding in protein sequences

    PubMed Central

    Scheraga, Harold A.; Rackovsky, S.

    2014-01-01

    We show that a Fourier-based sequence distance function is able to identify structural homologs of target sequences with high accuracy. It is shown that Fourier distances correlate very strongly with independently determined structural distances between molecules, a property of the method that is not attainable using conventional representations. It is further shown that the ability of the Fourier approach to identify protein folds is statistically far in excess of random expectation. It is then shown that, in actual searches for structural homologs of selected target sequences, the Fourier approach gives excellent results. On the basis of these results, we suggest that the global information detected by the Fourier representation is an essential feature of structure encoding in protein sequences and a key to structural homology detection. PMID:24706836

  11. Modulation of cellular signaling by herpesvirus-encoded G protein-coupled receptors

    PubMed Central

    de Munnik, Sabrina M.; Smit, Martine J.; Leurs, Rob; Vischer, Henry F.

    2015-01-01

    Human herpesviruses (HHVs) are widespread infectious pathogens that have been associated with proliferative and inflammatory diseases. During viral evolution, HHVs have pirated genes encoding viral G protein-coupled receptors (vGPCRs), which are expressed on infected host cells. These vGPCRs show highest homology to human chemokine receptors, which play a key role in the immune system. Importantly, vGPCRs have acquired unique properties such as constitutive activity and the ability to bind a broad range of human chemokines. This allows vGPCRs to hijack human proteins and modulate cellular signaling for the benefit of the virus, ultimately resulting in immune evasion and viral dissemination to establish a widespread and lifelong infection. Knowledge on the mechanisms by which herpesviruses reprogram cellular signaling might provide insight in the contribution of vGPCRs to viral survival and herpesvirus-associated pathologies. PMID:25805993

  12. Localization of a bacterial group II intron-encoded protein in human cells.

    PubMed

    Reinoso-Colacio, Mercedes; García-Rodríguez, Fernando Manuel; García-Cañadas, Marta; Amador-Cubero, Suyapa; García Pérez, José Luis; Toro, Nicolás

    2015-01-01

    Group II introns are mobile retroelements that self-splice from precursor RNAs to form ribonucleoparticles (RNP), which can invade new specific genomic DNA sites. This specificity can be reprogrammed, for insertion into any desired DNA site, making these introns useful tools for bacterial genetic engineering. However, previous studies have suggested that these elements may function inefficiently in eukaryotes. We investigated the subcellular distribution, in cultured human cells, of the protein encoded by the group II intron RmInt1 (IEP) and several mutants. We created fusions with yellow fluorescent protein (YFP) and with a FLAG epitope. We found that the IEP was localized in the nucleus and nucleolus of the cells. Remarkably, it also accumulated at the periphery of the nuclear matrix. We were also able to identify spliced lariat intron RNA, which co-immunoprecipitated with the IEP, suggesting that functional RmInt1 RNPs can be assembled in cultured human cells. PMID:26244523

  13. Early auxin-induced genes encode short-lived nuclear proteins.

    PubMed Central

    Abel, S; Oeller, P W; Theologis, A

    1994-01-01

    The plant growth hormone indoleacetic acid (IAA) transcriptionally activates gene expression in plants. Some of the genes whose expression is induced by IAA encode a family of proteins in pea (PS-IAA4 and PS-IAA6) and Arabidopsis (IAA1 and IAA2) that contain putative nuclear localization signals that direct a beta-glucuronidase reporter protein into the nucleus. Pulse-chase and immunoprecipitation experiments have defined the t1/2 of the PS-IAA4 and PS-IAA6 proteins to be 8 and 6 min, respectively. Their most prominent feature is the presence of a beta alpha alpha motif similar to the beta-sheet DNA-binding domain found in prokaryotic repressors of the Arc family. Based on these data, we suggest that plant tissues express short-lived nuclear proteins as a primary response to IAA. We propose that these proteins act as activators or repressors of genes responsible for mediating the various auxin responses. Images Fig. 2 Fig. 3 Fig. 4 PMID:8278386

  14. Ring Separation Highlights the Protein-Folding Mechanism Used by the Phage EL-Encoded Chaperonin.

    PubMed

    Molugu, Sudheer K; Hildenbrand, Zacariah L; Morgan, David Gene; Sherman, Michael B; He, Lilin; Georgopoulos, Costa; Sernova, Natalia V; Kurochkina, Lidia P; Mesyanzhinov, Vadim V; Miroshnikov, Konstantin A; Bernal, Ricardo A

    2016-04-01

    Chaperonins are ubiquitous, ATP-dependent protein-folding molecular machines that are essential for all forms of life. Bacteriophage φEL encodes its own chaperonin to presumably fold exceedingly large viral proteins via profoundly different nucleotide-binding conformations. Our structural investigations indicate that ATP likely binds to both rings simultaneously and that a misfolded substrate acts as the trigger for ATP hydrolysis. More importantly, the φEL complex dissociates into two single rings resulting from an evolutionarily altered residue in the highly conserved ATP-binding pocket. Conformational changes also more than double the volume of the single-ring internal chamber such that larger viral proteins are accommodated. This is illustrated by the fact that φEL is capable of folding β-galactosidase, a 116-kDa protein. Collectively, the architecture and protein-folding mechanism of the φEL chaperonin are significantly different from those observed in group I and II chaperonins. PMID:26996960

  15. Cloning a cDNA encoding an alternatively spliced protein of BRCA2-associated factor 35.

    PubMed

    Wang, Chiang; McCarty, Ida M; Balazs, Louisa; Li, Yi; Steiner, Mitchell S

    2002-07-01

    Inheritance of mutations in the breast cancer susceptibility gene, BRCA2, predisposes humans to breast and ovarian cancers. Inherited mutations in the BRCA2 gene are also known to cause susceptibility to prostate cancer. BRCA2 protein exists in a large multi-protein complex from which a novel structural DNA binding protein BRCA2-associated factor 35 (BRAF35) has been isolated. We have cloned a novel cDNA encoding an alternatively spliced protein of BRAF35, designated as BRAF25. BRAF25 transcript is present in various human cells. We have precisely mapped the BRAF25 cDNA sequence to the genomic chromosome 19 sequence. Analysis of the predicted sequence of BRAF25 identified a protein of 215 amino acids. BRAF25 contains a truncated high mobility group domain, a kinesin-like coiled-coil domain and multiple Src homology 2 (SH2) motifs. Western blot analysis using antibodies specific for BRAF25 revealed the presence of BRAF25 in human prostate cancer cells. PMID:12083779

  16. RNF38 encodes a nuclear ubiquitin protein ligase that modifies p53

    SciTech Connect

    Sheren, Jamie E.; Kassenbrock, C. Kenneth

    2013-11-01

    Highlights: •RNF38 is shown to be a nuclear protein with a bipartite nuclear localization signal. •RNF38 protein is purified and shown to have ubiquitin protein ligase (E3) activity. •We show that RNF38 binds p53 and can ubiquitinate p53 in vitro. •Overexpression of RNF38 increases p53 ubiquitination in HEK293T cells. •Overexpression of RNF38 in HEK293T cells alters p53 localization. -- Abstract: The RNF38 gene encodes a RING finger protein of unknown function. Here we demonstrate that RNF38 is a functional ubiquitin protein ligase (E3). We show that RNF38 isoform 1 is localized to the nucleus by a bipartite nuclear localization sequence (NLS). We confirm that RNF38 is a binding partner of p53 and demonstrate that RNF38 can ubiquitinate p53 in vitro and in vivo. Finally, we show that overexpression of RNF38 in HEK293T cells results in relocalization of p53 to discrete foci associated with PML nuclear bodies. These results suggest RNF38 is an E3 ubiquitin ligase that may play a role in regulating p53.

  17. Yeast RAD14 and human xeroderma pigmentosum group A DNA-repair genes encode homologous proteins.

    PubMed

    Bankmann, M; Prakash, L; Prakash, S

    1992-02-01

    Xeroderma pigmentosum (XP), a human autosomal recessive disorder, is characterized by extreme sensitivity to sunlight and high incidence of skin cancers. XP cells are defective in the incision step of excision repair of DNA damaged by ultraviolet light. Cell fusion studies have defined seven XP complementation groups, XP-A to XP-G. Similar genetic complexity of excision repair is observed in the yeast Saccharomyces cerevisiae. Mutations in any one of five yeast genes, RAD1, RAD2, RAD3, RAD4, and RAD10, cause a total defect in incision and an extreme sensitivity to ultraviolet light. Here we report the characterization of the yeast RAD14 gene. The available rad14 point mutant is only moderately ultraviolet-sensitive, and it performs a substantial amount of incision of damaged DNA. Our studies with the rad14 deletion (delta) mutation indicate an absolute requirement of RAD14 in incision. RAD14 encodes a highly hydrophilic protein of 247 amino acids containing zinc-finger motifs, and it is similar to the protein encoded by the human XPAC gene that complements XP group A cell lines. PMID:1741034

  18. Cloning, sequencing, and expression of bacteriophage BF23 late genes 24 and 25 encoding tail proteins.

    PubMed Central

    Nakayama, S; Kaneko, T; Ishimaru, H; Moriwaki, H; Mizobuchi, K

    1994-01-01

    Two bacteriophage BF23 late genes, genes 24 and 25, were isolated on a 7.4-kb PstI fragment from the phage DNA, and their nucleotide sequences were determined. Gene 24 encodes a minor tail protein with the expected M(r) of 34,309, and gene 25 located 4 bp upstream of gene 24 encodes a major tail protein with the expected M(r) of 50,329. When total cellular RNA isolated from either phage-infected cells or cells bearing the cloned genes was analyzed by the primer extension method using the primers specific to either gene 25 or gene 24, we identified a possible late gene promoter, designated P25, in the 5'-flanking region of gene 25. This promoter was similar in structure to Escherichia coli promoters for sigma 70. Studies of the translational gene 25- and gene 24-lacZ fusions in the cloned gene system revealed that the promoter P25 was responsible for the expression of both genes 25 and 24 even in the absence of the regulatory genes which were absolutely required for late gene expression in the normal phage-infected cells. These results indicate that the two genes constitute an operon under the control of P25 and that the regulatory gene products of BF23 do not participate directly in specifying the late gene promoter. Images PMID:7961500

  19. Protein Phosphatase 1 β Paralogs Encode the Zebrafish Myosin Phosphatase Catalytic Subunit

    PubMed Central

    Jayashankar, Vaishali; Nguyen, Michael J.; Carr, Brandon W.; Zheng, Dale C.; Rosales, Joseph B.; Rosales, Joshua B.; Weiser, Douglas C.

    2013-01-01

    Background The myosin phosphatase is a highly conserved regulator of actomyosin contractility. Zebrafish has emerged as an ideal model system to study the in vivo role of myosin phosphatase in controlling cell contractility, cell movement and epithelial biology. Most work in zebrafish has focused on the regulatory subunit of the myosin phosphatase called Mypt1. In this work, we examined the critical role of Protein Phosphatase 1, PP1, the catalytic subunit of the myosin phosphatase. Methodology/Principal Findings We observed that in zebrafish two paralogous genes encoding PP1β, called ppp1cba and ppp1cbb, are both broadly expressed during early development. Furthermore, we found that both gene products interact with Mypt1 and assemble an active myosin phosphatase complex. In addition, expression of this complex results in dephosphorylation of the myosin regulatory light chain and large scale rearrangements of the actin cytoskeleton. Morpholino knock-down of ppp1cba and ppp1cbb results in severe defects in morphogenetic cell movements during gastrulation through loss of myosin phosphatase function. Conclusions/Significance Our work demonstrates that zebrafish have two genes encoding PP1β, both of which can interact with Mypt1 and assemble an active myosin phosphatase. In addition, both genes are required for convergence and extension during gastrulation and correct dosage of the protein products is required. PMID:24040418

  20. Transcription of genes encoding iron and heme acquisition proteins of Haemophilus influenzae during acute otitis media.

    PubMed Central

    Whitby, P W; Sim, K E; Morton, D J; Patel, J A; Stull, T L

    1997-01-01

    Unencapsulated Haemophilus influenzae is the second most common etiologic agent of otitis media in children. H. influenzae requires heme for aerobic growth in vitro and is able to utilize hemoglobin and complexes of heme-hemopexin, heme-albumin, and hemoglobin-haptoglobin and ferritransferrin as sources of iron and heme in vitro. Several of the acquisition mechanisms have been characterized and been shown to be heme repressible in vitro. However, little is known about the expression of heme and/or iron acquisition mechanisms during infections in the middle ear. This study was performed to determine if the genes encoding heme and iron acquisition proteins are transcribed during in vivo growth and to compare these findings with those for samples grown in vitro. Reverse transcriptase PCR (RT-PCR) was used to analyze total RNA fractions derived from in vitro- and in vivo-grown H. influenzae. Genes encoding the transferrin-binding proteins TbpA and TbpB, the 100-kDa hemopexin-binding protein HxuA, and the hemoglobin-binding protein HgpA were transcribed during otitis media. Twelve middle ear fluid samples were analyzed by blind RT-PCR to determine the transcriptional status of these genes in H. influenzae during otitis media. Five isolates had transcripts corresponding to tbpA, tbpB, and hxuA. The presence of hgpA transcripts was variable, depending on the presence of hgpA in the genome of the H. influenzae isolate. Samples without H. influenzae gene transcripts contained other etiologic agents commonly causing otitis media. These data demonstrate that H. influenzae iron and/or heme acquisition genes are transcribed during otitis media and suggest that the microenvironment during acute otitis media starves H. influenzae of heme. PMID:9353052

  1. Genetically Encoded Molecular Tension Probe for Tracing Protein-Protein Interactions in Mammalian Cells.

    PubMed

    Kim, Sung Bae; Nishihara, Ryo; Citterio, Daniel; Suzuki, Koji

    2016-02-17

    Optical imaging of protein-protein interactions (PPIs) facilitates comprehensive elucidation of intracellular molecular events. We demonstrate an optical measure for visualizing molecular tension triggered by any PPI in mammalian cells. Twenty-three kinds of candidate designs were fabricated, in which a full-length artificial luciferase (ALuc) was sandwiched between two model proteins of interest, e.g., FKBP and FRB. One of the designs greatly enhanced the bioluminescence in response to varying concentrations of rapamycin. It is confirmed with negative controls that the elevated bioluminescence is solely motivated from the molecular tension. The probe design was further modified toward eliminating the C-terminal end of ALuc and was found to improve signal-to-background ratios, named "a combinational probe". The utilities were elucidated with detailed substrate selectivity, bioluminescence imaging of live cells, and different PPI models. This study expands capabilities of luciferases as a tool for analyses of molecular dynamics and cell signaling in living subjects. PMID:26322739

  2. Lytic Myophage Abp53 Encodes Several Proteins Similar to Those Encoded by Host Acinetobacter baumannii and Phage phiKO2 ▿ †

    PubMed Central

    Lee, Chia-Ni; Tseng, Tsai-Tien; Lin, Juey-Wen; Fu, Yung-Chieh; Weng, Shu-Fen; Tseng, Yi-Hsiung

    2011-01-01

    Acinetobacter baumannii is an important Gram-negative opportunistic pathogen causing nosocomial infections. The emergence of multiple-drug-resistant A. baumannii isolates has increased in recent years. Directed toward phage therapy, a lytic phage of A. baumannii, designated Abp53, was isolated from a sputum sample in this study. Abp53 has an isometric head and a contractile tail with tail fibers (belonging to Myoviridae), a latent period of about 10 min, and a burst size of approximately 150 PFU per infected cell. Abp53 could completely lyse 27% of the A. baumannii isolates tested, which were all multiple drug resistant, but not other bacteria. Mg2+ enhanced the adsorption and productivity of, and host lysis by, Abp53. Twenty Abp53 virion proteins were visualized in SDS-polyacrylamide gel electrophoresis, with a 47-kDa protein being the predicted major capsid protein. Abp53 has a double-stranded DNA genome of 95 kb. Sequence analyses of a 10-kb region revealed 8 open reading frames. Five of the encoded proteins, including 3 tail components and 2 hypothetical proteins, were similar to proteins encoded by A. baumannii strain ACICU. ORF1176 (one of the tail components, 1,176 amino acids [aa]), which is also similar to tail protein gp21 of Klebsiella phage phiKO2, contained repeated domains similar to those within the ACICU_02717 protein of A. baumannii ACICU and gp21. These findings suggest a common ancestry and horizontal gene transfer during evolution. As phages can expand the host range by domain duplication in tail fiber proteins, repeated domains in ORF1176 might have a similar significance in Abp53. PMID:21821767

  3. A Drosophila gene encoding a protein resembling the human. beta. -amyloid protein precursor

    SciTech Connect

    Rosen, D.R.; Martin-Morris, L.; Luo, L.; White, K. )

    1989-04-01

    The authors have isolated genomic and cDNA clones for a Drosophila gene resembling the human {beta}-amyloid precursor protein (APP). This gene produces a nervous system-enriched 6.5-kilobase transcript. Sequencing of cDNAs derived from the 6.5-kilobase transcript predicts an 886-amino acid polypeptide. This polypeptide contains a putative transmembrane domain and exhibits strong sequence similarity to cytoplasmic and extracellular regions of the human {beta}-amyloid precursor protein. There is a high probability that this Drosophila gene corresponds to the essential Drosophila locus vnd, a gene required for embryonic nervous system development.

  4. An essential yeast gene with homology to the exonuclease-encoding XRN1/KEM1 gene also encodes a protein with exoribonuclease activity

    SciTech Connect

    Kenna, M.; Douglas, M.G. ); Stevens, A. ); McCammon, M. )

    1993-01-01

    This is a study of a temperature-sensitive (ts) mutant from Saccharomyces cerevisiae which was obtained in a screen for mutants reduced in the synthesis of binding of a hybrid protein which competes for the transport of protein precursors into mitochondria. Examination of this mutant lead to the characterization of a gene with significant primary sequence homology to a previously identified gene, XRN1 or KEM1. Often called XRN1/KEM1, it encodes a protein of 175kDa which appears to have a multitude of properties, including involvement in recombination, RNA processing and turnover, involvement in recombination, RNA processing and turnover, microtubule function, karyogamy and DNA replication. The related gene describes further characterization of the HKE1/RAT1 gene and an hkal mutant and shows that p116 is a protein having 5[prime]-->3[prime] exoribonuclease activity, a major activity of the product of the related XRN1/KEM1 gene.

  5. The Agrobacterium rhizogenes GALLS Gene Encodes Two Secreted Proteins Required for Genetic Transformation of Plants▿

    PubMed Central

    Hodges, Larry D.; Lee, Lan-Ying; McNett, Henry; Gelvin, Stanton B.; Ream, Walt

    2009-01-01

    Agrobacterium tumefaciens and Agrobacterium rhizogenes are related pathogens that cause crown gall and hairy root diseases, which result from integration and expression of bacterial genes in the plant genome. Single-stranded DNA (T strands) and virulence proteins are translocated into plant cells by a type IV secretion system. VirD2 nicks a specific DNA sequence, attaches to the 5′ end, and pilots the DNA into plant cells. A. tumefaciens translocates single-stranded DNA-binding protein VirE2 into plant cells where it likely binds T strands and may aid in targeting them into the nucleus. Although some A. rhizogenes strains lack VirE2, they transfer T strands efficiently due to the GALLS gene, which complements an A. tumefaciens virE2 mutant for tumor formation. Unlike VirE2, full-length GALLS (GALLS-FL) contains ATP-binding and helicase motifs similar to those in TraA, a strand transferase involved in conjugation. GALLS-FL and VirE2 contain nuclear localization signals (NLS) and secretion signals. Mutations in any of these domains abolish the ability of the GALLS gene to substitute for virE2. Here, we show that the GALLS gene encodes two proteins from one open reading frame: GALLS-FL and a protein comprised of the C-terminal domain, which initiates at an internal in-frame start codon. On some hosts, both GALLS proteins were required to substitute for VirE2. GALLS-FL tagged with yellow fluorescent protein localized to the nucleus of tobacco cells in an NLS-dependent manner. In plant cells, the GALLS proteins interacted with themselves, VirD2, and each other. VirD2 interacted with GALLS-FL and localized inside the nucleus, where its predicted helicase activity may pull T strands into the nucleus. PMID:18952790

  6. Chimeras taking shape: potential functions of proteins encoded by chimeric RNA transcripts.

    PubMed

    Frenkel-Morgenstern, Milana; Lacroix, Vincent; Ezkurdia, Iakes; Levin, Yishai; Gabashvili, Alexandra; Prilusky, Jaime; Del Pozo, Angela; Tress, Michael; Johnson, Rory; Guigo, Roderic; Valencia, Alfonso

    2012-07-01

    Chimeric RNAs comprise exons from two or more different genes and have the potential to encode novel proteins that alter cellular phenotypes. To date, numerous putative chimeric transcripts have been identified among the ESTs isolated from several organisms and using high throughput RNA sequencing. The few corresponding protein products that have been characterized mostly result from chromosomal translocations and are associated with cancer. Here, we systematically establish that some of the putative chimeric transcripts are genuinely expressed in human cells. Using high throughput RNA sequencing, mass spectrometry experimental data, and functional annotation, we studied 7424 putative human chimeric RNAs. We confirmed the expression of 175 chimeric RNAs in 16 human tissues, with an abundance varying from 0.06 to 17 RPKM (Reads Per Kilobase per Million mapped reads). We show that these chimeric RNAs are significantly more tissue-specific than non-chimeric transcripts. Moreover, we present evidence that chimeras tend to incorporate highly expressed genes. Despite the low expression level of most chimeric RNAs, we show that 12 novel chimeras are translated into proteins detectable in multiple shotgun mass spectrometry experiments. Furthermore, we confirm the expression of three novel chimeric proteins using targeted mass spectrometry. Finally, based on our functional annotation of exon organization and preserved domains, we discuss the potential features of chimeric proteins with illustrative examples and suggest that chimeras significantly exploit signal peptides and transmembrane domains, which can alter the cellular localization of cognate proteins. Taken together, these findings establish that some chimeric RNAs are translated into potentially functional proteins in humans. PMID:22588898

  7. Solution Structure of Ribosomal Protein L40E, a Unique C4 Zinc Finger Protein Encoded by Archaeon Sulfolobus Solfataricus

    SciTech Connect

    Wu, Bin; Lukin, Jonathan A.; Yee, Adelinda; Lemak, Alexander; Semesi, Anthony; Ramelot, Theresa A.; Kennedy, Michael A.; Arrowsmith, Cheryl H.

    2008-01-31

    The ribosomal protein L40E from archaeon Sulfolobus solfataricus is a component of the 50S ribosomal subunit. L40E is a 56-residue, highly basic protein that contains a C4 zinc finger motif, CRKC_X10_CRRC. Homologs are found in both archaea and eukaryotes but are not present in bacteria. Eukaryotic genomes encode L40E as a ubiquitin-fusion protein. L40E was absent from the crystal structure of euryarchaeota 50S ribosomal subunit. Here we report the three-dimensional solution structure of L40E by NMR spectroscopy. The structure of L40E is a three-stranded b-sheet with a simple b2b1b3 topology. There are two unique characteristics revealed by the structure. First, a large and ordered b2–b3 loop twists to pack across the one side of the protein. L40E contains a buried polar cluster comprising Lys19, Lys20, Cys22, Asn29, and Cys36. Second, the surface of L40E is almost entirely positively charged. Ten conserved basic residues are positioned on the two sides of the surface. It is likely that binding of zinc is essential in stabilizing the tertiary structure of L40E to act as a scaffold to create a broad positively charged surface for RNA and/or protein recognition. A portion of this work was performed in the Environmental Molecular Sciences Facility, a DOE national scientific user facility.

  8. Cancer cells express aberrant DNMT3B transcripts encoding truncated proteins

    PubMed Central

    Ostler, KR; Davis, EM; Payne, SL; Gosalia, BB; Expósito-Céspedes, J; Le Beau, MM; Godley, LA

    2008-01-01

    Cancer cells display an altered distribution of DNA methylation relative to normal cells. Certain tumor suppressor gene promoters are hypermethylated and transcriptionally inactivated, whereas repetitive DNA is hypomethylated and transcriptionally active. Little is understood about how the abnormal DNA methylation patterns of cancer cells are established and maintained. Here, we identify over 20 DNMT3B transcripts from many cancer cell lines and primary acute leukemia cells that contain aberrant splicing at the 5′ end of the gene, encoding truncated proteins lacking the C-terminal catalytic domain. Many of these aberrant transcripts retain intron sequences. Although the aberrant transcripts represent a minority of the DNMT3B transcripts present, Western blot analysis demonstrates truncated DNMT3B isoforms in the nuclear protein extracts of cancer cells. To test if expression of a truncated DNMT3B protein could alter the DNA methylation patterns within cells, we expressed DNMT3B7, the most frequently expressed aberrant transcript, in 293 cells. DNMT3B7-expressing 293 cells have altered gene expression as identified by microarray analysis. Some of these changes in gene expression correlate with altered DNA methylation of corresponding CpG islands. These results suggest that truncated DNMT3B proteins could play a role in the abnormal distribution of DNA methylation found in cancer cells. PMID:17353906

  9. The dyf-3 gene encodes a novel protein required for sensory cilium formation in Caenorhabditis elegans.

    PubMed

    Murayama, Takashi; Toh, Yoshihiro; Ohshima, Yasumi; Koga, Makoto

    2005-02-25

    Ciliated neurons in animals are important for the reception of environmental stimuli. To understand the mechanism of cilium morphogenesis in Caenorhabditis elegans, we analyzed dyf-3 mutants that are defective in uptake of a fluorescent dye and abnormal in sensory cilium structure. Expression of green fluorescent protein in sensory neurons of a dyf-3 mutant revealed that the mutant has stunted cilia and abnormal posterior projections in some sensory neurons. The dyf-3 gene encodes three proteins with different N-terminals. The largest DYF-3 protein has 404 amino acid residues that are 38% identical with those of a predicted human protein of unknown function. Expression of a functional dyf-3Colon, two colonsgfp fusion gene is detected in 26 chemosensory neurons, including six IL2 neurons, eight pairs of amphid neurons (ASE, ADF, ASG, ASH, ASI, ASJ, ASK and ADL) and two pairs of phasmid neurons (PHA and PHB). Expression of a dyf-3 cDNA in specific neurons of dyf-3 animals indicated that dyf-3 acts cell-autonomously for fluorescent dye uptake. Reduction of dyf-3Colon, two colonsgfp expression in a daf-19 mutant suggests that dyf-3 expression is regulated by DAF-19 transcription factor, and DYF-3 may be involved in the intraflagellar transport system. PMID:15713455

  10. Characterization of KfrA proteins encoded by a plasmid of Paenibacillus popilliae ATCC 14706T

    PubMed Central

    Iiyama, Kazuhiro; Mon, Hiroaki; Mori, Kazuki; Mitsudome, Takumi; Lee, Jae Man; Kusakabe, Takahiro; Tashiro, Kousuke; Asano, Shin-ichiro; Yasunaga-Aoki, Chisa

    2015-01-01

    A scaffold obtained from whole-genome shotgun sequencing of Paenibacillus popilliae ATCC 14706T shares partial homology with plasmids found in other strains of P. popilliae. PCR and sequencing for gap enclosure indicated that the scaffold originated from a 15,929-bp circular DNA. The restriction patterns of a plasmid isolated from P. popilliae ATCC 14706T were identical to those expected from the sequence; thus, this circular DNA was identified as a plasmid of ATCC 14706T and designated pPOP15.9. The plasmid encodes 17 putative open reading frames. Orfs 1, 5, 7, 8, and 9 are homologous to Orfs 11, 12, 15, 16, and 17, respectively. Orf1 and Orf11 are annotated as replication initiation proteins. Orf8 and Orf16 are homologs of KfrA, a plasmid-stabilizing protein in Gram-negative bacteria. Recombinant Orf8 and Orf16 proteins were assessed for the properties of KfrA. Indeed, they formed multimers and bound to inverted repeat sequences in upstream regions of both orf8 and orf16. A phylogenetic tree based on amino acid sequences of Orf8, Orf16 and Kfr proteins did not correlate with species lineage. PMID:25853059

  11. Homology probing: identification of cDNA clones encoding members of the protein-serine kinase family

    SciTech Connect

    Hanks, S.K.

    1987-01-01

    Mixed /sup 32/P-labeled oligonucleotide probes were used to screen a HeLa cDNA library for clones encoding amino acid contiguities whose conservation is characteristic of the protein-serine kinase family. Eighty thousand clones were screened, from which 19 were identified as showing strong hybridization to two distinct probes. Four clones were chosen for characterization by partial DNA sequence analysis and 3 of these were found to encode amino acid sequences typical of protein-serine kinases. One deduced amino acid sequence shares 72% identify with rabbit skeletal muscle phosphorylase kinase ..gamma..-subunit, while another is closely related to the yeast protein-serine kinases CDC2 in Schizosaccharomyces pombe and CDC28 in Saccharomyces cerevisiae. This screening approach should have applications in the identification of clones encoding previously unknown or poorly characterized members of other protein families.

  12. Albino Leaf1 That Encodes the Sole Octotricopeptide Repeat Protein Is Responsible for Chloroplast Development.

    PubMed

    Zhang, Zemin; Tan, Jianjie; Shi, Zhenying; Xie, Qingjun; Xing, Yi; Liu, Changhong; Chen, Qiaoling; Zhu, Haitao; Wang, Jiang; Zhang, Jingliu; Zhang, Guiquan

    2016-06-01

    Chloroplast, the photosynthetic organelle in plants, plays a crucial role in plant development and growth through manipulating the capacity of photosynthesis. However, the regulatory mechanism of chloroplast development still remains elusive. Here, we characterized a mutant with defective chloroplasts in rice (Oryza sativa), termed albino leaf1 (al1), which exhibits a distinct albino phenotype in leaves, eventually leading to al1 seedling lethality. Electronic microscopy observation demonstrated that the number of thylakoids was reduced and the structure of thylakoids was disrupted in the al1 mutant during rice development, which eventually led to the breakdown of chloroplast. Molecular cloning revealed that AL1 encodes the sole octotricopeptide repeat protein (RAP) in rice. Genetic complementation of Arabidopsis (Arabidopsis thaliana) rap mutants indicated that the AL1 protein is a functional RAP. Further analysis illustrated that three transcript variants were present in the AL1 gene, and the altered splices occurred at the 3' untranslated region of the AL1 transcript. In addition, our results also indicate that disruption of the AL1 gene results in an altered expression of chloroplast-associated genes. Consistently, proteomic analysis demonstrated that the abundance of photosynthesis-associated proteins is altered significantly, as is that of a group of metabolism-associated proteins. More specifically, we found that the loss of AL1 resulted in altered abundances of ribosomal proteins, suggesting that RAP likely also regulates the homeostasis of ribosomal proteins in rice in addition to the ribosomal RNA. Taken together, we propose that AL1, particularly the AL1a and AL1c isoforms, plays an essential role in chloroplast development in rice. PMID:27208287

  13. The hmc operon of Desulfovibrio vulgaris subsp. vulgaris Hildenborough encodes a potential transmembrane redox protein complex.

    PubMed Central

    Rossi, M; Pollock, W B; Reij, M W; Keon, R G; Fu, R; Voordouw, G

    1993-01-01

    The nucleotide sequence of the hmc operon from Desulfovibrio vulgaris subsp. vulgaris Hildenborough indicated the presence of eight open reading frames, encoding proteins Orf1 to Orf6, Rrf1, and Rrf2. Orf1 is the periplasmic, high-molecular-weight cytochrome (Hmc) containing 16 c-type hemes and described before (W. B. R. Pollock, M. Loutfi, M. Bruschi, B. J. Rapp-Giles, J. D. Wall, and G. Voordouw, J. Bacteriol. 173:220-228, 1991). Orf2 is a transmembrane redox protein with four iron-sulfur clusters, as indicated by its similarity to DmsB from Escherichia coli. Orf3, Orf4, and Orf5 are all highly hydrophobic, integral membrane proteins with similarities to subunits of NADH dehydrogenase or cytochrome c reductase. Orf6 is a cytoplasmic redox protein containing two iron-sulfur clusters, as indicated by its similarity to the ferredoxin domain of [Fe] hydrogenase from Desulfovibrio species. Rrf1 belongs to the family of response regulator proteins, while the function of Rrf2 cannot be derived from the gene sequence. The expression of individual genes in E. coli with the T7 system confirmed the open reading frames for Orf2, Orf6, and Rrf1. Deletion of 0.4 kb upstream from orf1 abolished the expression of Hmc in D. desulfuricans G200, indicating this region to contain the hmc operon promoter. The expression of two truncated hmc genes in D. desulfuricans G200 resulted in stable periplasmic c-type cytochromes, confirming the domain structure of Hmc. We propose that Hmc and Orf2 to Orf6 form a transmembrane protein complex that allows electron flow from the periplasmic hydrogenases to the cytoplasmic enzymes that catalyze the reduction of sulfate. The domain structure of Hmc may be required to allow interaction with multiple hydrogenases. Images PMID:8335628

  14. TOM1, an Arabidopsis gene required for efficient multiplication of a tobamovirus, encodes a putative transmembrane protein.

    PubMed

    Yamanaka, T; Ohta, T; Takahashi, M; Meshi, T; Schmidt, R; Dean, C; Naito, S; Ishikawa, M

    2000-08-29

    Host-encoded factors play an important role in virus multiplication, acting in concert with virus-encoded factors. However, information regarding the host factors involved in this process is limited. Here we report the map-based cloning of an Arabidopsis thaliana gene, TOM1, which is necessary for the efficient multiplication of tobamoviruses, positive-strand RNA viruses infecting a wide variety of plants. The TOM1 mRNA is suggested to encode a 291-aa polypeptide that is predicted to be a multipass transmembrane protein. The Sos recruitment assay supported the hypothesis that TOM1 is associated with membranes, and in addition, that TOM1 interacts with the helicase domain of tobamovirus-encoded replication proteins. Taken into account that the tobamovirus replication complex is associated with membranes, we propose that TOM1 participates in the in vivo formation of the replication complex by serving as a membrane anchor. PMID:10944200

  15. Locus heterogeneity disease genes encode proteins with high interconnectivity in the human protein interaction network

    PubMed Central

    Keith, Benjamin P.; Robertson, David L.; Hentges, Kathryn E.

    2014-01-01

    Mutations in genes potentially lead to a number of genetic diseases with differing severity. These disease genes have been the focus of research in recent years showing that the disease gene population as a whole is not homogeneous, and can be categorized according to their interactions. Locus heterogeneity describes a single disorder caused by mutations in different genes each acting individually to cause the same disease. Using datasets of experimentally derived human disease genes and protein interactions, we created a protein interaction network to investigate the relationships between the products of genes associated with a disease displaying locus heterogeneity, and use network parameters to suggest properties that distinguish these disease genes from the overall disease gene population. Through the manual curation of known causative genes of 100 diseases displaying locus heterogeneity and 397 single-gene Mendelian disorders, we use network parameters to show that our locus heterogeneity network displays distinct properties from the global disease network and a Mendelian network. Using the global human proteome, through random simulation of the network we show that heterogeneous genes display significant interconnectivity. Further topological analysis of this network revealed clustering of locus heterogeneity genes that cause identical disorders, indicating that these disease genes are involved in similar biological processes. We then use this information to suggest additional genes that may contribute to diseases with locus heterogeneity. PMID:25538735

  16. Cloning of apg-2 encoding a novel member of heat shock protein 110 family.

    PubMed

    Kaneko, Y; Kimura, T; Kishishita, M; Noda, Y; Fujita, J

    1997-04-11

    Chinese hamster heat shock protein 110-encoding gene (hsp110), mouse apg-1 and human hsp70RY are structurally related genes, with the first two encoding about 110-kDa HSPs [Yoon et al. (1995) J. Biol. Chem. 270, 15725-15733; Kaneko et al. (1997) J. Biol. Chem., in press; Fathallah et al. (1993) J. Immunol. 151, 810-813]. Using apg-1 cDNA as a probe, we isolated a novel cDNA, apg-2 from a mouse testis cDNA library, which was highly homologous to human hsp70RY. However, the predicted amino acid (aa) sequence of APG-2 was longer (841 aa) than that of HSP70RY (701 aa) and comparable to those of HSP110 and APG-1. Northern blot analysis revealed that the expression of apg-2 transcripts was ubiquitous in various mouse tissues, and most abundant in the testis and ovary. While induction of hsp70 transcripts was observed in mouse TAMA26 Sertoli cells and NIH/3T3 fibroblasts on temperature shift from 37 degrees C to 42 degrees C (traditional heat shock) or from 32 degrees C to 39 degrees C, apg-2 transcripts were not induced under either condition. These results suggest that apg-2 is an isoform of mouse homolog of hsp70RY, but that it belongs to the hsp110 family instead of hsp70 family, and that it plays a role under non-stress conditions. PMID:9161406

  17. Comparative sequence analysis of double stranded RNA binding protein encoding gene of parapoxviruses from Indian camels.

    PubMed

    Nagarajan, G; Swami, Shelesh Kumar; Dahiya, Shyam Singh; Sivakumar, G; Tuteja, F C; Narnaware, S D; Mehta, S C; Singh, Raghvendar; Patil, N V

    2014-03-01

    The dsRNA binding protein (RBP) encoding gene of parapoxviruses (PPVs) from the Dromedary camels, inhabitating different geographical region of Rajasthan, India were amplified by polymerase chain reaction using the primers of pseudocowpoxvirus (PCPV) from Finnish reindeer and cloned into pGEM-T for sequence analysis. Analysis of RBP encoding gene revealed that PPV DNA from Bikaner shared 98.3% and 76.6% sequence identity at the amino acid level, with Pali and Udaipur PPV DNA, respectively. Reference strains of Bovine papular stomatitis virus (BPSV) and PCPV (reindeer PCPV and human PCPV) shared 52.8% and 86.9% amino acid identity with RBP gene of camel PPVs from Bikaner, respectively. But different strains of orf virus (ORFV) from different geographical areas of the world shared 69.5-71.7% amino acid identity with RBP gene of camel PPVs from Bikaner. These findings indicate that the camel PPVs described are closely related to bovine PPV (PCPV) in comparison to caprine and ovine PPV (ORFV). PMID:25685494

  18. Ring finger protein 10 is a novel synaptonuclear messenger encoding activation of NMDA receptors in hippocampus

    PubMed Central

    Dinamarca, Margarita C; Guzzetti, Francesca; Karpova, Anna; Lim, Dmitry; Mitro, Nico; Musardo, Stefano; Mellone, Manuela; Marcello, Elena; Stanic, Jennifer; Samaddar, Tanmoy; Burguière, Adeline; Caldarelli, Antonio; Genazzani, Armando A; Perroy, Julie; Fagni, Laurent; Canonico, Pier Luigi; Kreutz, Michael R; Gardoni, Fabrizio; Luca, Monica Di

    2016-01-01

    Synapses and nuclei are connected by bidirectional communication mechanisms that enable information transfer encoded by macromolecules. Here, we identified RNF10 as a novel synaptonuclear protein messenger. RNF10 is activated by calcium signals at the postsynaptic compartment and elicits discrete changes at the transcriptional level. RNF10 is enriched at the excitatory synapse where it associates with the GluN2A subunit of NMDA receptors (NMDARs). Activation of synaptic GluN2A-containing NMDARs and induction of long term potentiation (LTP) lead to the translocation of RNF10 from dendritic segments and dendritic spines to the nucleus. In particular, we provide evidence for importin-dependent long-distance transport from synapto-dendritic compartments to the nucleus. Notably, RNF10 silencing prevents the maintenance of LTP as well as LTP-dependent structural modifications of dendritic spines. DOI: http://dx.doi.org/10.7554/eLife.12430.001 PMID:26977767

  19. Rice ABERRANT PANICLE ORGANIZATION 1, encoding an F-box protein, regulates meristem fate.

    PubMed

    Ikeda, Kyoko; Ito, Momoyo; Nagasawa, Nobuhiro; Kyozuka, Junko; Nagato, Yasuo

    2007-09-01

    Inflorescence architecture is one of the most important agronomical traits. Characterization of rice aberrant panicle organization 1 (apo1) mutants revealed that APO1 positively controls spikelet number by suppressing the precocious conversion of inflorescence meristems to spikelet meristems. In addition, APO1 is associated with the regulation of the plastchron, floral organ identity, and floral determinacy. Phenotypic analyses of apo1 and floral homeotic double mutants demonstrate that APO1 positively regulates class-C floral homeotic genes, but not class-B genes. Molecular studies revealed that APO1 encodes an F-box protein, an ortholog of Arabidopsis UNUSUAL FLORAL ORGAN (UFO), which is a positive regulator of class-B genes. Overexpression of APO1 caused an increase in inflorescence branches and spikelets. As the mutant inflorescences and flowers differed considerably between apo1 and ufo, the functions of APO1 and UFO appear to have diverged during evolution. PMID:17666027

  20. The Drosophila melanogaster developmental gene g1 encodes a variant zinc-finger-motif protein.

    PubMed

    Bouchard, M L; Côté, S

    1993-03-30

    In Drosophila melanogaster, the mechanisms involved in the pattern formation of complex internal organs are still largely unknown. However, the identity of the molecular determinants that control the development of these specific tissues is emerging from the combined use of genetic and molecular approaches. We have cloned a gene that is expressed in the mesoderm, one of the fundamental embryonic germ layers which gives rise to internal structures, such as the musculature. Here, we describe the molecular characterization of this gene, designated as g1. The nucleotide (nt) sequence of its cDNA shows an open reading frame of 852 nt, which encodes a 32-kDa protein with two putative zinc fingers, and a serine/glutamine/proline-rich region. These features indicate a functional role for g1, which remains to be elucidated, in regulating gene expression during mesoderm formation. PMID:8462875

  1. Identification of pTiC58 plasmid-encoded proteins for virulence in Agrobacterium tumefaciens.

    PubMed Central

    Hagiya, M; Close, T J; Tait, R C; Kado, C I

    1985-01-01

    Analyses were made of the host-dependent-variation (hdv) locus of the virulence (vir) region of the pTiC58 plasmid of Agrobacterium tumefaciens. The hdv locus is comprised of at least four genes that encode polypeptides of 13, 15, 29, and 28 kDa. Insertion of transposon Tn5 in the first gene abolishes the expression of all four genes in vitro and in vivo. Nucleotide sequence analysis of the hdv locus revealed four open reading frames tandemly arranged with spacer sequences having no promoter-like sequences and lacking the ability to bind A. tumefaciens RNA polymerase. These studies suggest that the hdv locus is comprised of at least four genes arranged in an operon in the vir region. The protein products of these genes are likely to function in some aspect of the host-range determination of A. tumefaciens. Images PMID:2986128

  2. mus304 encodes a novel DNA damage checkpoint protein required during Drosophila development

    PubMed Central

    Brodsky, Michael H.; Sekelsky, Jeff J.; Tsang, Garson; Hawley, R. Scott; Rubin, Gerald M.

    2000-01-01

    Checkpoints block cell cycle progression in eukaryotic cells exposed to DNA damaging agents. We show that several Drosophila homologs of checkpoint genes, mei-41, grapes, and 14-3-3ε, regulate a DNA damage checkpoint in the developing eye. We have used this assay to show that the mutagen-sensitive gene mus304 is also required for this checkpoint. mus304 encodes a novel coiled-coil domain protein, which is targeted to the cytoplasm. Similar to mei-41, mus304 is required for chromosome break repair and for genomic stability. mus304 animals also exhibit three developmental defects, abnormal bristle morphology, decreased meiotic recombination, and arrested embryonic development. We suggest that these phenotypes reflect distinct developmental consequences of a single underlying checkpoint defect. Similar mechanisms may account for the puzzling array of symptoms observed in humans with mutations in the ATM tumor suppressor gene. PMID:10733527

  3. Genetically Encoded Fragment-Based Discovery of Glycopeptide Ligands for Carbohydrate-Binding Proteins

    DOE PAGESBeta

    Ng, Simon; Lin, Edith; Kitov, Pavel I.; Tjhung, Katrina F.; Gerlits, Oksana O.; Deng, Lu; Kasper, Brian; Sood, Amika; Paschal, Beth M.; Zhang, Ping; et al

    2015-04-10

    Here we describe an approach to accelerate the search for competitive inhibitors for carbohydrate-recognition domains (CRDs). Genetically encoded fragment-based-discovery (GE-FBD) uses selection of phagedisplayed glycopeptides to dock a glycan fragment at the CRD and guide selection of Synergistic peptide motifs adjacent to the CRD. Starting from concanavalin A (ConA), a mannose (Man)-binding protein, as a bait, we narrowed a library of 108 glycopeptides to 86 leads that share a consensus motif, Man-WYD. Validation of synthetic leads yielded Man-WYDLF that exhibited 40 50-fold enhancement in affinity over methyl α-D-mannopyranoside (MeMan). Lectin array Suggested specificity: Man-WYD derivative bound only to 3 outmore » of 17 proteins-ConA, LcH, and PSA-that bind to Man. An X-ray structure of ConA.:Man-WYD proved that the trimannoside core and Man-WYD exhibit identical CRD docking; but their extra-CRD binding modes are significantly. different. Still, they have comparable affinity and selectivity for various Man-binding proteins. The intriguing observation provides new insight into functional mimicry :of carbohydrates by peptide ligands. GE-FBD may provide an alternative to rapidly search for competitive inhibitors for lectins.« less

  4. Genetically Encoded Fragment-Based Discovery of Glycopeptide Ligands for Carbohydrate-Binding Proteins

    SciTech Connect

    Ng, Simon; Lin, Edith; Kitov, Pavel I.; Tjhung, Katrina F.; Gerlits, Oksana O.; Deng, Lu; Kasper, Brian; Sood, Amika; Paschal, Beth M.; Zhang, Ping; Ling, Chang-Chun; Klassen, John S.; Noren, Christopher J.; Mahal, Lara K.; Woods, Robert J.; Coates, Leighton; Derda, Ratmir

    2015-04-10

    Here we describe an approach to accelerate the search for competitive inhibitors for carbohydrate-recognition domains (CRDs). Genetically encoded fragment-based-discovery (GE-FBD) uses selection of phagedisplayed glycopeptides to dock a glycan fragment at the CRD and guide selection of Synergistic peptide motifs adjacent to the CRD. Starting from concanavalin A (ConA), a mannose (Man)-binding protein, as a bait, we narrowed a library of 108 glycopeptides to 86 leads that share a consensus motif, Man-WYD. Validation of synthetic leads yielded Man-WYDLF that exhibited 40 50-fold enhancement in affinity over methyl α-D-mannopyranoside (MeMan). Lectin array Suggested specificity: Man-WYD derivative bound only to 3 out of 17 proteins-ConA, LcH, and PSA-that bind to Man. An X-ray structure of ConA.:Man-WYD proved that the trimannoside core and Man-WYD exhibit identical CRD docking; but their extra-CRD binding modes are significantly. different. Still, they have comparable affinity and selectivity for various Man-binding proteins. The intriguing observation provides new insight into functional mimicry :of carbohydrates by peptide ligands. GE-FBD may provide an alternative to rapidly search for competitive inhibitors for lectins.

  5. Isolation and characterization of a gene encoding an ethylene responsive factor protein from Ceratoides arborescens.

    PubMed

    Dong, Jie; Wang, Xuemin; Wang, Kang; Wang, Zan; Gao, Hongwen

    2012-02-01

    Ethylene responsive factor (ERF) proteins play important roles in plant growth and development and regulate biotic and abiotic stress responses. In this study, a full length mRNA encoding a novel ERF-type transcription factor namely Ceratoides arborescens ERF protein (CeERF) was isolated from C. arborescens. The deduced amino acid of CeERF had a conserved APETALA2/ERF (AP2/ERF) domain which specifically binds to cis-acting elements GCC box. Under normal conditions, the expression level of CeERF was highest in leaves and lowest in roots. CeERF expression was induced by 20% PEG in a time-dependent pattern and peaked at 8 h. CeERF also acts in salt- and hormones-induced stresses. Transient expression analysis in onion epidermal cells indicated that CeERF protein localized to nucleus. Overexpression of CeERF in transgenic tobacco plants resulted in higher tolerance to abiotic stresses than in control plants. These results suggested that CeERF might play a role in abiotic stress signal transduction and that overexpression of CeERF might serve as a feasible approach to enhance resistance in forage, even crop. PMID:21603850

  6. A prophage-encoded actin-like protein required for efficient viral DNA replication in bacteria.

    PubMed

    Donovan, Catriona; Heyer, Antonia; Pfeifer, Eugen; Polen, Tino; Wittmann, Anja; Krämer, Reinhard; Frunzke, Julia; Bramkamp, Marc

    2015-05-26

    In host cells, viral replication is localized at specific subcellular sites. Viruses that infect eukaryotic and prokaryotic cells often use host-derived cytoskeletal structures, such as the actin skeleton, for intracellular positioning. Here, we describe that a prophage, CGP3, integrated into the genome of Corynebacterium glutamicum encodes an actin-like protein, AlpC. Biochemical characterization confirms that AlpC is a bona fide actin-like protein and cell biological analysis shows that AlpC forms filamentous structures upon prophage induction. The co-transcribed adaptor protein, AlpA, binds to a consensus sequence in the upstream promoter region of the alpAC operon and also interacts with AlpC, thus connecting circular phage DNA to the actin-like filaments. Transcriptome analysis revealed that alpA and alpC are among the early induced genes upon excision of the CGP3 prophage. Furthermore, qPCR analysis of mutant strains revealed that both AlpA and AlpC are required for efficient phage replication. Altogether, these data emphasize that AlpAC are crucial for the spatio-temporal organization of efficient viral replication. This is remarkably similar to actin-assisted membrane localization of eukaryotic viruses that use the actin cytoskeleton to concentrate virus particles at the egress sites and provides a link of evolutionary conserved interactions between intracellular virus transport and actin. PMID:25916847

  7. Characterization of Durham virus, a novel rhabdovirus that encodes both a C and SH protein

    PubMed Central

    Allison, A. B.; Palacios, G.; Rosa, A. Travassos da; Popov, V. L.; Lu, L.; Xiao, S. Y.; DeToy, K.; Briese, T.; Lipkin, W. Ian; Keel, M. K.; Stallknecht, D. E.; Bishop, G. R.; Tesh, R. B.

    2010-01-01

    The family Rhabdoviridae is a diverse group of non-segmented, negative-sense RNA viruses that are distributed worldwide and infect a wide range of hosts including vertebrates, invertebrates, and plants. Of the 114 currently recognized vertebrate rhabdoviruses, relatively few have been well characterized at both the antigenic and genetic level; hence, the phylogenetic relationships between many of the vertebrate rhabdoviruses remain unknown. The present report describes a novel rhabdovirus isolated from the brain of a moribund American coot (Fulica americana) that exhibited neurological signs when found in Durham County, North Carolina, in 2005. Antigenic characterization of the virus revealed that it was serologically unrelated to 68 other known vertebrate rhabdoviruses. Genomic sequencing of the virus indicated that it shared the highest identity to Tupaia rhabdovirus (TUPV), and as only previously observed in TUPV, the genome encoded a putative C protein in an overlapping open reading frame (ORF) of the phosphoprotein gene and a small hydrophobic protein located in a novel ORF between the matrix and glycoprotein genes. Phylogenetic analysis of partial amino acid sequences of the nucleoprotein and polymerase proteins indicated that, in addition to TUPV, the virus was most closely related to avian and small mammal rhabdoviruses from Africa and North America. In this report, we present the morphological, pathological, antigenic, and genetic characterization of the new virus, tentatively named Durham virus (DURV), and discuss its potential evolutionary relationship to other vertebrate rhabdoviruses. PMID:20863863

  8. Direct control of type IIA topoisomerase activity by a chromosomally encoded regulatory protein

    PubMed Central

    Vos, Seychelle M.; Lyubimov, Artem Y.; Hershey, David M.; Schoeffler, Allyn J.; Sengupta, Sugopa; Nagaraja, Valakunja; Berger, James M.

    2014-01-01

    Precise control of supercoiling homeostasis is critical to DNA-dependent processes such as gene expression, replication, and damage response. Topoisomerases are central regulators of DNA supercoiling commonly thought to act independently in the recognition and modulation of chromosome superstructure; however, recent evidence has indicated that cells tightly regulate topoisomerase activity to support chromosome dynamics, transcriptional response, and replicative events. How topoisomerase control is executed and linked to the internal status of a cell is poorly understood. To investigate these connections, we determined the structure of Escherichia coli gyrase, a type IIA topoisomerase bound to YacG, a recently identified chromosomally encoded inhibitor protein. Phylogenetic analyses indicate that YacG is frequently associated with coenzyme A (CoA) production enzymes, linking the protein to metabolism and stress. The structure, along with supporting solution studies, shows that YacG represses gyrase by sterically occluding the principal DNA-binding site of the enzyme. Unexpectedly, YacG acts by both engaging two spatially segregated regions associated with small-molecule inhibitor interactions (fluoroquinolone antibiotics and the newly reported antagonist GSK299423) and remodeling the gyrase holoenzyme into an inactive, ATP-trapped configuration. This study establishes a new mechanism for the protein-based control of topoisomerases, an approach that may be used to alter supercoiling levels for responding to changes in cellular state. PMID:24990966

  9. Ectromelia virus encodes a family of Ankyrin/F-box proteins that regulate NFκB.

    PubMed

    Burles, Kristin; van Buuren, Nicholas; Barry, Michele

    2014-11-01

    A notable feature of poxviruses is their ability to inhibit the antiviral response, including the nuclear factor kappa B (NFκB) pathway. NFκB is a transcription factor that is sequestered in the cytoplasm until cell stimulation, and relies on the SCF (Skp1, culllin-1, F-box) ubiquitin ligase to target its inhibitor, IκBα, for degradation. IκBα is recruited to the SCF by the F-box domain-containing protein βTrCP. Here, we show that ectromelia virus, the causative agent of mousepox, encodes four F-box-containing proteins, EVM002, EVM005, EVM154, and EVM165, all of which contain Ankyrin (Ank) domains. The Ank/F-box proteins inhibit NFκB nuclear translocation, and this inhibition is dependent on the F-box domain. We also demonstrate that EVM002, EVM005, EVM154, and EVM165 prevent IκBα degradation, suggesting that they target the SCF. This study identifies a new mechanism by which ectromelia virus inhibits NFκB. PMID:25240225

  10. KOJAK encodes a cellulose synthase-like protein required for root hair cell morphogenesis in Arabidopsis

    PubMed Central

    Favery, Bruno; Ryan, Eoin; Foreman, Julia; Linstead, Paul; Boudonck, Kurt; Steer, Martin; Shaw, Peter; Dolan, Liam

    2001-01-01

    The cell wall is an important determinant of plant cell form. Here we define a class of Arabidopsis root hair mutants with defective cell walls. Plants homozygous for kojak (kjk) mutations initiate root hairs that rupture at their tip soon after initiation. The KJK gene was isolated by positional cloning, and its identity was confirmed by the molecular complementation of the Kjk− phenotype and the sequence of three kjk mutant alleles. KOJAK encodes a cellulose synthase-like protein, AtCSLD3. KOJAK/AtCSLD3 is the first member of this subfamily of proteins to be shown to have a function in cell growth. Subcellular localization of the KOJAK/AtCSLD3 protein using a GFP fusion shows that KOJAK/AtCSLD3 is located on the endoplasmic reticulum, indicating that KOJAK/AtCSLD3 is required for the synthesis of a noncellulosic wall polysaccharide. Consistent with the cell specific defect in the roots of kjk mutants, KOJAK/AtCSDL3 is preferentially expressed in hair cells of the epidermis. The Kjk− phenotype and the pattern of KOJAK/AtCSLD3 expression suggest that this gene acts early in the process of root hair outgrowth. These results suggest that KOJAK/AtCSLD3 is involved in the biosynthesis of β-glucan-containing polysaccharides that are required during root hair elongation. PMID:11156607

  11. Cloning and functional expression of a complementary DNA encoding a mammalian nucleoside transport protein.

    PubMed

    Huang, Q Q; Yao, S Y; Ritzel, M W; Paterson, A R; Cass, C E; Young, J D

    1994-07-01

    Expression screening in Xenopus oocytes was used to isolate a cDNA from rat jejunal epithelium encoding a Na(+)-dependent nucleoside transport protein (named cNT1). The cDNA sequence of cNT1 predicts a protein of 648 amino acids (relative molecular mass 71,000) with 14 potential transmembrane domains. Data base searches indicate significant sequence similarity to the NUPC proton/nucleoside symporter of Escherichia coli. There is no sequence similarity between cNT1 and proteins of mammalian origin. Functionally, cNT1 exhibited the transport characteristics of the nucleoside transport system cit (selective for pyrimidine nucleosides and adenosine) and accepted both 3'-azido-3'-deoxythymidine (AZT) and 2',3'-dideoxycytidine (ddC) as permeants (Km = 0.49 and 0.51 mM, respectively). The demonstration of transport of AZT by cNT1 expressed in Xenopus oocytes provides the first direct evidence that AZT enters cells by transporter-mediated processes, as well as by passive diffusion. Consistent with the tissue distribution of system cit transport activity, transcripts for cNT1 were detected in kidney as well as jejunum. cNT1 therefore belongs to a potential new gene family and may be involved in the intestinal absorption and renal handling of pyrimidine nucleoside analogs used to treat acquired immunodeficiency syndrome (AIDS). PMID:8027026

  12. Monoclonal antibody against a putative myristoylated membrane protein encoded by grouper iridovirus 59L gene.

    PubMed

    Chen, Zhi-Yu; Chiou, Pinwen Peter; Liou, Chian-Jiun; Lai, Yu-Shen

    2015-04-01

    Groupers (Epinephelus spp.) are economically important fish species worldwide, and ranaviruses are major viral pathogens causing heavy economic losses in grouper aquaculture. In this study, the 59L gene of grouper iridovirus (GIV-59L) was cloned and characterized. This gene is 1521 bp and encodes a protein of 506 amino acids with a predicted molecular mass of 53.9 kDa. Interestingly, GIV-59L and its homologs are found in all genera of the family Iridoviridae. A mouse monoclonal antibody specific for the C-terminal domain (amino acid positions 254-506) of the GIV-59L protein, GIV-59L(760-1518)-MAb-21, was produced and proved to be well suited for use in a number of GIV immunoassays. RT-PCR, Western blotting, and cycloheximide and cytosine arabinoside drug inhibition analyses indicated that GIV-59L is a viral late gene in GIV-infected grouper kidney cells. Immunofluorescence analysis revealed that GIV-59L protein mainly accumulates in the cytoplasm of infected cells and is finally packed into a whole virus particle. The GIV-59L(760-1518)-MAb-21 characterized in this study could have widespread application in GIV immunodiagnostics and other research on GIV. In addition, the results presented here offer important insights into the pathogenesis of GIV. PMID:25850399

  13. A Member of the Notch Group of Interacting Loci, Deltex Encodes a Cytoplasmic Basic Protein

    PubMed Central

    Busseau, I.; Diederich, R. J.; Xu, T.; Artavanis-Tsakonas, S.

    1994-01-01

    Prior genetic studies have suggested a functional relationship between the product of the deltex gene and those of three of the so-called ``neurogenic'' loci, Notch, Delta and mastermind. To gain further insight into this relationship, we have proceeded with a molecular characterization of deltex. We report that deltex encodes a maternally and zygotically expressed transcript that conceptually translates to a basic protein of novel sequence. Immunolocalization of the protein reveals an apparently ubiquitous distribution in embryonic and imaginal tissues. Because our detection methods also reveal a very low level of protein accumulation within the cytoplasm of cells, we have used transgenic flies to confirm this observation by ectopically expressing deltex under the control of a heat shock gene promoter. The resulting overexpression rescues deltex mutant defects but does not produce any obvious phenotypic abnormalities in otherwise wild-type flies. Finally, we examine genetically several Supressor of deltex mutations for evidence of functional integration with deltex and other neurogenic genes. We demonstrate that in addition to suppressing all adult morphological defects of deltex alleles, these suppressors also are capable of suppressing most synergistic effects involving deltex and Notch, Delta and mastermind. PMID:8150285

  14. A prophage-encoded actin-like protein required for efficient viral DNA replication in bacteria

    PubMed Central

    Donovan, Catriona; Heyer, Antonia; Pfeifer, Eugen; Polen, Tino; Wittmann, Anja; Krämer, Reinhard; Frunzke, Julia; Bramkamp, Marc

    2015-01-01

    In host cells, viral replication is localized at specific subcellular sites. Viruses that infect eukaryotic and prokaryotic cells often use host-derived cytoskeletal structures, such as the actin skeleton, for intracellular positioning. Here, we describe that a prophage, CGP3, integrated into the genome of Corynebacterium glutamicum encodes an actin-like protein, AlpC. Biochemical characterization confirms that AlpC is a bona fide actin-like protein and cell biological analysis shows that AlpC forms filamentous structures upon prophage induction. The co-transcribed adaptor protein, AlpA, binds to a consensus sequence in the upstream promoter region of the alpAC operon and also interacts with AlpC, thus connecting circular phage DNA to the actin-like filaments. Transcriptome analysis revealed that alpA and alpC are among the early induced genes upon excision of the CGP3 prophage. Furthermore, qPCR analysis of mutant strains revealed that both AlpA and AlpC are required for efficient phage replication. Altogether, these data emphasize that AlpAC are crucial for the spatio-temporal organization of efficient viral replication. This is remarkably similar to actin-assisted membrane localization of eukaryotic viruses that use the actin cytoskeleton to concentrate virus particles at the egress sites and provides a link of evolutionary conserved interactions between intracellular virus transport and actin. PMID:25916847

  15. spn-F encodes a novel protein that affects oocyte patterning and bristle morphology in Drosophila.

    PubMed

    Abdu, Uri; Bar, Dikla; Schüpbach, Trudi

    2006-04-01

    The anteroposterior and dorsoventral axes of the Drosophila embryo are established during oogenesis through the activities of Gurken (Grk), a Tgfalpha-like protein, and the Epidermal growth factor receptor (Egfr). spn-F mutant females produce ventralized eggs similar to the phenotype produced by mutations in the grk-Egfr pathway. We found that the ventralization of the eggshell in spn-F mutants is due to defects in the localization and translation of grk mRNA during mid-oogenesis. Analysis of the microtubule network revealed defects in the organization of the microtubules around the oocyte nucleus. In addition, spn-F mutants have defective bristles. We cloned spn-F and found that it encodes a novel coiled-coil protein that localizes to the minus end of microtubules in the oocyte, and this localization requires the microtubule network and a Dynein heavy chain gene. We also show that Spn-F interacts directly with the Dynein light chain Ddlc-1. Our results show that we have identified a novel protein that affects oocyte axis determination and the organization of microtubules during Drosophila oogenesis. PMID:16540510

  16. Systematic and searchable classification of cytochrome P450 proteins encoded by fungal and oomycete genomes

    PubMed Central

    2012-01-01

    Background Cytochrome P450 proteins (CYPs) play diverse and pivotal roles in fungal metabolism and adaptation to specific ecological niches. Fungal genomes encode extremely variable “CYPomes” ranging from one to more than 300 CYPs. Despite the rapid growth of sequenced fungal and oomycete genomes and the resulting influx of predicted CYPs, the vast majority of CYPs remain functionally uncharacterized. To facilitate the curation and functional and evolutionary studies of CYPs, we previously developed Fungal Cytochrome P450 Database (FCPD), which included CYPs from 70 fungal and oomycete species. Here we present a new version of FCPD (1.2) with more data and an improved classification scheme. Results The new database contains 22,940 CYPs from 213 species divided into 2,579 clusters and 115 clans. By optimizing the clustering pipeline, we were able to uncover 36 novel clans and to assign 153 orphan CYP families to specific clans. To augment their functional annotation, CYP clusters were mapped to David Nelson’s P450 databases, which archive a total of 12,500 manually curated CYPs. Additionally, over 150 clusters were functionally classified based on sequence similarity to experimentally characterized CYPs. Comparative analysis of fungal and oomycete CYPomes revealed cases of both extreme expansion and contraction. The most dramatic expansions in fungi were observed in clans CYP58 and CYP68 (Pezizomycotina), clans CYP5150 and CYP63 (Agaricomycotina), and family CYP509 (Mucoromycotina). Although much of the extraordinary diversity of the pan-fungal CYPome can be attributed to gene duplication and adaptive divergence, our analysis also suggests a few potential horizontal gene transfer events. Updated families and clans can be accessed through the new version of the FCPD database. Conclusions FCPD version 1.2 provides a systematic and searchable catalogue of 9,550 fungal CYP sequences (292 families) encoded by 108 fungal species and 147 CYP sequences (9 families

  17. Mechanistic Studies of the Genetically Encoded Fluorescent Protein Voltage Probe ArcLight

    PubMed Central

    Han, Zhou; Jin, Lei; Chen, Fuyi; Loturco, Joseph J.; Cohen, Lawrence B.; Bondar, Alexey; Lazar, Josef; Pieribone, Vincent A.

    2014-01-01

    ArcLight, a genetically encoded fluorescent protein voltage probe with a large ΔF/ΔV, is a fusion between the voltage sensing domain of the Ciona instestinalis voltage sensitive phosphatase and super ecliptic pHluorin carrying a single mutation (A227D in the fluorescent protein). Without this mutation the probe produces only a very small change in fluorescence in response to voltage deflections (∼1%). The large signal afforded by this mutation allows optical detection of action potentials and sub-threshold electrical events in single-trials in vitro and in vivo. However, it is unclear how this single mutation produces a probe with such a large modulation of its fluorescence output with changes in membrane potential. In this study, we identified which residues in super ecliptic pHluorin (vs eGFP) are critical for the ArcLight response, as a similarly constructed probe based on eGFP also exhibits large response amplitude if it carries these critical residues. We found that D147 is responsible for determining the pH sensitivity of the fluorescent protein used in these probes but by itself does not result in a voltage probe with a large signal. We also provide evidence that the voltage dependent signal of ArcLight is not simply sensing environmental pH changes. A two-photon polarization microscopy study showed that ArcLight's response to changes in membrane potential includes a reorientation of the super ecliptic pHluorin. We also explored different changes including modification of linker length, deletion of non-essential amino acids in the super ecliptic pHluorin, adding a farnesylation site, using tandem fluorescent proteins and other pH sensitive fluorescent proteins. PMID:25419571

  18. The RCN1-encoded A subunit of protein phosphatase 2A increases phosphatase activity in vivo

    NASA Technical Reports Server (NTRS)

    Deruere, J.; Jackson, K.; Garbers, C.; Soll, D.; Delong, A.; Evans, M. L. (Principal Investigator)

    1999-01-01

    Protein phosphatase 2A (PP2A), a heterotrimeric serine/threonine-specific protein phosphatase, comprises a catalytic C subunit and two distinct regulatory subunits, A and B. The RCN1 gene encodes one of three A regulatory subunits in Arabidopsis thaliana. A T-DNA insertion mutation at this locus impairs root curling, seedling organ elongation and apical hypocotyl hook formation. We have used in vivo and in vitro assays to gauge the impact of the rcn1 mutation on PP2A activity in seedlings. PP2A activity is decreased in extracts from rcn1 mutant seedlings, and this decrease is not due to a reduction in catalytic subunit expression. Roots of mutant seedlings exhibit increased sensitivity to the phosphatase inhibitors okadaic acid and cantharidin in organ elongation assays. Shoots of dark-grown, but not light-grown seedlings also show increased inhibitor sensitivity. Furthermore, cantharidin treatment of wild-type seedlings mimics the rcn1 defect in root curling, root waving and hypocotyl hook formation assays. In roots of wild-type seedlings, RCN1 mRNA is expressed at high levels in root tips, and accumulates to lower levels in the pericycle and lateral root primordia. In shoots, RCN1 is expressed in the apical hook and the basal, rapidly elongating cells in etiolated hypocotyls, and in the shoot meristem and leaf primordia of light-grown seedlings. Our results show that the wild-type RCN1-encoded A subunit functions as a positive regulator of the PP2A holoenzyme, increasing activity towards substrates involved in organ elongation and differential cell elongation responses such as root curling.

  19. Flower-enhanced expression of a nuclear-encoded mitochondrial respiratory protein is associated with changes in mitochondrion number.

    PubMed Central

    Huang, J; Struck, F; Matzinger, D F; Levings, C S

    1994-01-01

    The mitochondrial Rieske iron-sulfur protein is an obligatory component of the respiratory electron transport chain that is encoded by a single-copy gene in mammals and fungi. In contrast, this protein is encoded by a small gene family in dicotyledonous tobacco and monocotyledonous maize. We cloned four cDNAs from tobacco that encode the mitochondrial Rieske iron-sulfur protein. These clones, along with a previously isolated cDNA, represent five independent members of the gene family that can be divided into three subfamilies. All of these genes were derived from the two progenitor species and were expressed in amphidiploid tobacco. The proteins encoded by these five genes are probably functional because they all contain the universally conserved hexyl peptides necessary for the 2Fe-2S cluster formation. The expression of the Rieske protein gene family is differentially regulated; a 6- to 11-fold higher level of steady state transcripts was found in flowers than in leaves, stems, and roots. Members of at least two subfamilies were preferentially expressed in flowers, indicating that they share a common cis-regulatory element(s), which can respond to a flower-specific signal(s). Although approximately 10 times more transcripts occurred in flowers than in leaves, flower and leaf mitochondria contained a similar amount of the Rieske protein. Flowers, however, contained seven times more Rieske proteins than leaves. These results indicated an increase in mitochondrion number in flowers. High-energy demands during anther development might bring about an increase in mitochondrion numbers in flowers and the flower-enhanced expression of the Rieske protein gene family. Our results suggested that nuclear genes encoding mitochondrial respiratory proteins could sense and respond to changes in energy metabolism and/or changes in mitochondrion numbers. PMID:8180500

  20. Genes encoding novel secreted and transmembrane proteins are temporally and spatially regulated during Drosophila melanogaster embryogenesis

    PubMed Central

    Zúñiga, Alejandro; Hödar, Christian; Hanna, Patricia; Ibáñez, Freddy; Moreno, Pablo; Pulgar, Rodrigo; Pastenes, Luis; González, Mauricio; Cambiazo, Verónica

    2009-01-01

    Background Morphogenetic events that shape the Drosophila melanogaster embryo are tightly controlled by a genetic program in which specific sets of genes are up-regulated. We used a suppressive subtractive hybridization procedure to identify a group of developmentally regulated genes during early stages of D. melanogaster embryogenesis. We studied the spatiotemporal activity of these genes in five different intervals covering 12 stages of embryogenesis. Results Microarrays were constructed to confirm induction of expression and to determine the temporal profile of isolated subtracted cDNAs during embryo development. We identified a set of 118 genes whose expression levels increased significantly in at least one developmental interval compared with a reference interval. Of these genes, 53% had a phenotype and/or molecular function reported in the literature, whereas 47% were essentially uncharacterized. Clustering analysis revealed demarcated transcript groups with maximum gene activity at distinct developmental intervals. In situ hybridization assays were carried out on 23 uncharacterized genes, 15 of which proved to have spatiotemporally restricted expression patterns. Among these 15 uncharacterized genes, 13 were found to encode putative secreted and transmembrane proteins. For three of them we validated our protein sequence predictions by expressing their cDNAs in Drosophila S2R+ cells and analyzed the subcellular distribution of recombinant proteins. We then focused on the functional characterization of the gene CG6234. Inhibition of CG6234 by RNA interference resulted in morphological defects in embryos, suggesting the involvement of this gene in germ band retraction. Conclusion Our data have yielded a list of developmentally regulated D. melanogaster genes and their expression profiles during embryogenesis and provide new information on the spatiotemporal expression patterns of several uncharacterized genes. In particular, we recovered a substantial number of

  1. Trypanosoma rangeli and Trypanosoma cruzi: molecular characterization of genes encoding putative calcium-binding proteins, highly conserved in trypanosomatids.

    PubMed

    Porcel, B M; Bontempi, E J; Henriksson, J; Rydåker, M; Aslund, L; Segura, E L; Pettersson, U; Ruiz, A M

    1996-12-01

    Genes encoding a 29-kDa flagellar calcium-binding protein (F29) in Trypanosoma cruzi, strongly homologous to EF-hand calcium-binding protein-encoding genes previously reported in this parasite, were isolated by immunoscreening. F29 is encoded by a number of very similar genes, highly conserved among different T. cruzi isolates. The genes are located on a pair of homologous chromosomes, arranged in one or two clusters of tandem repeats. PCR amplification of Trypanosoma rangeli genomic DNA, using primers derived from the T. cruzi F29 sequence made it possible to isolate the homologous gene in T. rangeli, encoding a 23-kDa protein called TrCaBP. Gene sequence comparisons showed homology to EF-hand calcium-binding proteins from T. cruzi (82.8%), Trypanosoma brucei brucei (60.2%), and Entamoeba histolytica (28.4%). Northern blot analysis revealed that the TrCaBP gene is expressed in T. rangeli as a polyadenylated transcript. The TrCaBP-encoding genes are present in at least 20 copies per cell, organized in tandem arrays, on large T. rangeli chromosomes in some isolates and on two smaller ones in others. This gene, however, seems to be absent from Leishmania. PMID:8948328

  2. ‘Ca. Liberibacter asiaticus’ Proteins Orthologous with pSymA-Encoded Proteins of Sinorhizobium meliloti: Hypothetical Roles in Plant Host Interaction

    PubMed Central

    Kuykendall, L. David; Shao, Jonathan Y.; Hartung, John S.

    2012-01-01

    Sinorhizobium meliloti strain 1021, a nitrogen-fixing, root-nodulating bacterial microsymbiont of alfalfa, has a 3.5 Mbp circular chromosome and two megaplasmids including 1.3 Mbp pSymA carrying nonessential ‘accessory’ genes for nitrogen fixation (nif), nodulation and host specificity (nod). A related bacterium, psyllid-vectored ‘Ca. Liberibacter asiaticus,’ is an obligate phytopathogen with a reduced genome that was previously analyzed for genes orthologous to genes on the S. meliloti circular chromosome. In general, proteins encoded by pSymA genes are more similar in sequence alignment to those encoded by S. meliloti chromosomal orthologs than to orthologous proteins encoded by genes carried on the ‘Ca. Liberibacter asiaticus’ genome. Only two ‘Ca. Liberibacter asiaticus’ proteins were identified as having orthologous proteins encoded on pSymA but not also encoded on the chromosome of S. meliloti. These two orthologous gene pairs encode a Na+/K+ antiporter (shared with intracellular pathogens of the family Bartonellacea) and a Co++, Zn++ and Cd++ cation efflux protein that is shared with the phytopathogen Agrobacterium. Another shared protein, a redox-regulated K+ efflux pump may regulate cytoplasmic pH and homeostasis. The pSymA and ‘Ca. Liberibacter asiaticus’ orthologs of the latter protein are more highly similar in amino acid alignment compared with the alignment of the pSymA-encoded protein with its S. meliloti chromosomal homolog. About 182 pSymA encoded proteins have sequence similarity (≤E-10) with ‘Ca. Liberibacter asiaticus’ proteins, often present as multiple orthologs of single ‘Ca. Liberibacter asiaticus’ proteins. These proteins are involved with amino acid uptake, cell surface structure, chaperonins, electron transport, export of bioactive molecules, cellular homeostasis, regulation of gene expression, signal transduction and synthesis of amino acids and metabolic cofactors. The presence of multiple orthologs defies

  3. Rational design of an efficient, genetically encodable, protein-encased singlet oxygen photosensitizer.

    PubMed

    Westberg, Michael; Holmegaard, Lotte; Pimenta, Frederico M; Etzerodt, Michael; Ogilby, Peter R

    2015-02-01

    Singlet oxygen, O(2)(a(1)Δ(g)), plays a key role in many processes of cell signaling. Limitations in mechanistic studies of such processes are generally associated with the difficulty of controlling the amount and location of O(2)(a(1)Δ(g)) production in or on a cell. As such, there is great need for a system that (a) selectively produces O(2)(a(1)Δ(g)) in appreciable and accurately quantifiable yields and (b) can be localized in a specific place at the suborganelle level. A genetically encodable, protein-encased photosensitizer is one way to achieve this goal. Through a systematic and rational approach involving mutations to a LOV2 protein that binds the chromophore flavin mononucleotide (FMN), we have developed a promising photosensitizer that overcomes many of the problems that affect related systems currently in use. Specifically, by decreasing the extent of hydrogen bonding between FMN and a specific amino acid residue in the local protein environment, we decrease the susceptibility of FMN to undesired photoinitiated electron-transfer reactions that kinetically compete with O(2)(a(1)Δ(g)) production. As a consequence, our protein-encased FMN system produces O(2)(a(1)Δ(g)) with the uniquely large quantum efficiency of 0.25 ± 0.03. We have also quantified other key photophysical parameters that characterize this sensitizer system, including unprecedented H(2)O/D(2)O solvent isotope effects on the O(2)(a(1)Δ(g)) formation kinetics and yields. As such, our results facilitate future systematic developments in this field. PMID:25575190

  4. ERCC4 (XPF) encodes a human nucleotide excision repair protein with eukaryotic recombination homologs.

    PubMed

    Brookman, K W; Lamerdin, J E; Thelen, M P; Hwang, M; Reardon, J T; Sancar, A; Zhou, Z Q; Walter, C A; Parris, C N; Thompson, L H

    1996-11-01

    ERCC4 is an essential human gene in the nucleotide excision repair (NER) pathway, which is responsible for removing UV-C photoproducts and bulky adducts from DNA. Among the NER genes, ERCC4 and ERCC1 are also uniquely involved in removing DNA interstrand cross-linking damage. The ERCC1-ERCC4 heterodimer, like the homologous Rad10-Rad1 complex, was recently found to possess an endonucleolytic activity that incises on the 5' side of damage. The ERCC4 gene, assigned to chromosome 16p13.1-p13.2, was previously isolated by using a chromosome 16 cosmid library. It corrects the defect in Chinese hamster ovary (CHO) mutants of NER complementation group 4 and is implicated in complementation group F of the human disorder xeroderma pigmentosum. We describe the ERCC4 gene structure and functional cDNA sequence encoding a 916-amino-acid protein (104 kDa), which has substantial homology with the eukaryotic DNA repair and recombination proteins MEI-9 (Drosophila melanogaster), Rad16 (Schizosaccharomyces pombe), and Rad1 (Saccharomyces cerevisiae). ERCC4 cDNA efficiently corrected mutants in rodent NER complementation groups 4 and 11, showing the equivalence of these groups, and ERCC4 protein levels were reduced in mutants of both groups. In cells of an XP-F patient, the ERCC4 protein level was reduced to less than 5%, consistent with XPF being the ERCC4 gene. The considerable identity (40%) between ERCC4 and MEI-9 suggests a possible involvement of ERCC4 in meiosis. In baboon tissues, ERCC4 was expressed weakly and was not significantly higher in testis than in nonmeiotic tissues. PMID:8887684

  5. Genes Encoding Cher-TPR Fusion Proteins Are Predominantly Found in Gene Clusters Encoding Chemosensory Pathways with Alternative Cellular Functions

    PubMed Central

    Rico-Jiménez, Miriam; Alfonso, Carlos; Krell, Tino

    2012-01-01

    Chemosensory pathways correspond to major signal transduction mechanisms and can be classified into the functional families flagellum-mediated taxis, type four pili-mediated taxis or pathways with alternative cellular functions (ACF). CheR methyltransferases are core enzymes in all of these families. CheR proteins fused to tetratricopeptide repeat (TPR) domains have been reported and we present an analysis of this uncharacterized family. We show that CheR-TPRs are widely distributed in GRAM-negative but almost absent from GRAM-positive bacteria. Most strains contain a single CheR-TPR and its abundance does not correlate with the number of chemoreceptors. The TPR domain fused to CheR is comparatively short and frequently composed of 2 repeats. The majority of CheR-TPR genes were found in gene clusters that harbor multidomain response regulators in which the REC domain is fused to different output domains like HK, GGDEF, EAL, HPT, AAA, PAS, GAF, additional REC, HTH, phosphatase or combinations thereof. The response regulator architectures coincide with those reported for the ACF family of pathways. Since the presence of multidomain response regulators is a distinctive feature of this pathway family, we conclude that CheR-TPR proteins form part of ACF type pathways. The diversity of response regulator output domains suggests that the ACF pathways form a superfamily which regroups many different regulatory mechanisms, in which all CheR-TPR proteins appear to participate. In the second part we characterize WspC of Pseudomonas putida, a representative example of CheR-TPR. The affinities of WspC-Pp for S-adenosylmethionine and S-adenosylhomocysteine were comparable to those of prototypal CheR, indicating that WspC-Pp activity is in analogy to prototypal CheRs controlled by product feed-back inhibition. The removal of the TPR domain did not impact significantly on the binding constants and consequently not on the product feed-back inhibition. WspC-Pp was found to be

  6. Hypoxia-inducible genes encoding small EF-hand proteins in rice and tomato.

    PubMed

    Otsuka, Chie; Minami, Ikuko; Oda, Kenji

    2010-01-01

    Rice has evolved metabolic and morphological adaptations to low-oxygen stress to grow in submerged paddy fields. To characterize the molecular components that mediate the response to hypoxia in rice, we identified low-oxygen stress early response genes by microarray analysis. Among the highly responsive genes, five genes, OsHREF1 to OsHREF5, shared strong homology. They encoded small proteins harboring two EF-hands, typical Ca(2+)-binding motifs. Homologous genes were found in many land plants, including SlHREF in tomato, which is also strongly induced by hypoxia. SlHREF induction was detected in both roots and shoots of tomato plants under hypoxia. With the exception of OsHREF5, OsHREF expression was unaffected by drought, salinity, cold, or osmotic stress. Fluorescent signals of green fluorescent protein-fused OsHREFs were detected in the cytosol and nucleus. Ruthenium red, an inhibitor of intracellular Ca(2+) release, repressed induction of OsHREF1-4 under hypoxia. The HREFs may be related to the Ca(2+) response to hypoxia. PMID:21150100

  7. uninflatable encodes a novel ectodermal apical surface protein required for tracheal inflation in Drosophila

    PubMed Central

    Zhang, Liang; Ward, Robert E.

    2009-01-01

    The tracheal system of Drosophila melanogaster has proven to be an excellent model system for studying the development of branched tubular organs. Mechanisms regulating the patterning and initial maturation of the tracheal system have been largely worked out, yet important questions remain regarding how the mature tubes inflate with air at the end of embryogenesis, and how the tracheal system grows in response to the oxygen needs of a developing larva that increases nearly 1000-fold in volume over a four day period. Here we describe the cloning and characterization of uninflatable (uif), a gene that encodes a large transmembrane protein containing carbohydrate binding and cell signaling motifs in its extracellular domain. Uif is highly conserved in insect species, but does not appear to have a true ortholog in vertebrate species. uif is expressed zygotically beginning in stage 5 embryos, and Uif protein localizes to the apical plasma membrane in all ectodermally derived epithelia, most notably in the tracheal system. uif mutant animals show defects in tracheal inflation at the end of embryogenesis, and die primarily as larvae. Tracheal tubes in mutant larvae are often crushed or twisted, although tracheal patterning and maturation appear normal during embryogenesis. uif mutants larvae also show defects in tracheal growth and molting of their tracheal cuticle. PMID:19818339

  8. Developmental expression of tobacco pistil-specific genes encoding novel extensin-like proteins.

    PubMed Central

    Goldman, M H; Pezzotti, M; Seurinck, J; Mariani, C

    1992-01-01

    We have sought to identify pistil-specific genes that can be used as molecular markers to study pistil development. For this purpose, a cDNA library was constructed from poly(A)+ RNA extracted from tobacco stigmas and styles at different developmental stages. Differential screening of this library led to the isolation of cDNA clones that correspond to genes preferentially or specifically expressed in the pistil. Seven of these cDNA clones encode proteins containing repetitions of the pentapeptide Ser-Pro4, which is a typical motif found in extensins. Unlike extensin genes, the extensin-like genes described here are not induced under stress conditions. RNA gel blot hybridizations demonstrated the organ-specific expression of the extensin-like genes and their temporal regulation during pistil development. After pollination, the transcript levels of the pistil-specific extensin-like genes change relative to levels in unpollinated pistils. In situ hybridization experiments showed that at least one of these pistil-specific genes is specifically expressed in cells of the transmitting tissue. The possible roles of the extensin-like proteins in pistils are discussed. PMID:1392607

  9. tassel-less1 encodes a boron channel protein required for inflorescence development in maize.

    PubMed

    Leonard, April; Holloway, Beth; Guo, Mei; Rupe, Mary; Yu, GongXin; Beatty, Mary; Zastrow-Hayes, Gina; Meeley, Robert; Llaca, Victor; Butler, Karlene; Stefani, Tony; Jaqueth, Jennifer; Li, Bailin

    2014-06-01

    tassel-less1 (tls1) is a classical maize (Zea mays) inflorescence mutant. Homozygous mutant plants have no tassels or very small tassels, and ear development is also impaired. Using a positional cloning approach, ZmNIP3;1 (a NOD26-like intrinsic protein) was identified as the candidate gene for tls1. The ZmNIP3;1 gene is completely deleted in the tls1 mutant genome. Two Mutator-insertional TUSC alleles of ZmNIP3;1 exhibited tls1-like phenotypes, and allelism tests confirmed that the tls1 gene encodes ZmNIP3;1. Transgenic plants with an RNA interference (RNAi) construct to down-regulate ZmNIP3;1 also showed tls1-like phenotypes, further demonstrating that TLS1 is ZmNIP3;1. Sequence analysis suggests that ZmNIP3;1 is a boron channel protein. Foliar application of boron could rescue the tls1 phenotypes and restore the normal tassel and ear development. Gene expression analysis indicated that in comparison with that of the wild type or tls1 plants treated with boron, the transition from the vegetative to reproductive phase or the development of the floral meristem is impaired in the shoot apical meristem of the tls1 mutant plants. It is concluded that the tls1 mutant phenotypes are caused by impaired boron transport, and boron is essential for inflorescence development in maize. PMID:24685595

  10. Comprehensive Analysis of Transport Proteins Encoded Within the Genome of Bdellovibrio bacteriovorus

    PubMed Central

    Barabote, Ravi D.; Rendulic, Snjezana; Schuster, Stephan C.; Saier, Milton H.

    2012-01-01

    Bdellovibrio bacteriovorus is a bacterial parasite with an unusual lifestyle. It grows and reproduces in the periplasm of a host prey bacterium. The complete genome sequence of B. bacteriovorus has recently been reported. We have reanalyzed the transport proteins encoded within the B. bacteriovorus genome according to the current content of the transporter classification database (TCDB). A comprehensive analysis is given on the types and numbers of transport systems that B. bacteriovorus has. In this regard, the potential protein secretory capabilities of at least 4 types of inner membrane secretion systems and 5 types for outer membrane secretion are described. Surprisingly, B. bacteriovorus has a disproportionate percentage of cytoplasmic membrane channels and outer membrane porins. It has far more TonB/ExbBD-type systems and MotAB-type systems for energizing outer membrane transport and motility than does E. coli. Analysis of probable substrate specificities of its transporters provides clues to its metabolic preferences. Interesting examples of gene fusions and of potentially overlapping genes were also noted. Our analyses provide a comprehensive, detailed appreciation of the transport capabilities of B. bacteriovorus. They should serve as a guide for functional experimental analyses. PMID:17706914

  11. A mutation in PNPT1, encoding mitochondrial-RNA-import protein PNPase, causes hereditary hearing loss.

    PubMed

    von Ameln, Simon; Wang, Geng; Boulouiz, Redouane; Rutherford, Mark A; Smith, Geoffrey M; Li, Yun; Pogoda, Hans-Martin; Nürnberg, Gudrun; Stiller, Barbara; Volk, Alexander E; Borck, Guntram; Hong, Jason S; Goodyear, Richard J; Abidi, Omar; Nürnberg, Peter; Hofmann, Kay; Richardson, Guy P; Hammerschmidt, Matthias; Moser, Tobias; Wollnik, Bernd; Koehler, Carla M; Teitell, Michael A; Barakat, Abdelhamid; Kubisch, Christian

    2012-11-01

    A subset of nuclear-encoded RNAs has to be imported into mitochondria for the proper replication and transcription of the mitochondrial genome and, hence, for proper mitochondrial function. Polynucleotide phosphorylase (PNPase or PNPT1) is one of the very few components known to be involved in this poorly characterized process in mammals. At the organismal level, however, the effect of PNPase dysfunction and impaired mitochondrial RNA import are unknown. By positional cloning, we identified a homozygous PNPT1 missense mutation (c.1424A>G predicting the protein substitution p.Glu475Gly) of a highly conserved PNPase residue within the second RNase-PH domain in a family affected by autosomal-recessive nonsyndromic hearing impairment. In vitro analyses in bacteria, yeast, and mammalian cells showed that the identified mutation results in a hypofunctional protein leading to disturbed PNPase trimerization and impaired mitochondrial RNA import. Immunohistochemistry revealed strong PNPase staining in the murine cochlea, including the sensory hair cells and the auditory ganglion neurons. In summary, we show that a component of the mitochondrial RNA-import machinery is specifically required for auditory function. PMID:23084290

  12. TANG1, Encoding a Symplekin_C Domain-Contained Protein, Influences Sugar Responses in Arabidopsis.

    PubMed

    Zheng, Leiying; Shang, Li; Chen, Xing; Zhang, Limin; Xia, Yan; Smith, Caroline; Bevan, Michael W; Li, Yunhai; Jing, Hai-Chun

    2015-07-01

    Sugars not only serve as energy and cellular carbon skeleton but also function as signaling molecules regulating growth and development in plants. Understanding the molecular mechanisms in sugar signaling pathways will provide more information for improving plant growth and development. Here, we describe a sugar-hypersensitive recessive mutant, tang1. Light-grown tang1 mutants have short roots and increased starch and anthocyanin contents when grown on high-sugar concentration medium. Dark-grown tang1 plants exhibit sugar-hypersensitive hypocotyl elongation and enhanced dark development. The tang1 mutants also show an enhanced response to abscisic acid but reduced response to ethylene. Thus, tang1 displays a range of alterations in sugar signaling-related responses. The TANG1 gene was isolated by a map-based cloning approach and encodes a previously uncharacterized unique protein with a predicted Symplekin tight-junction protein C terminus. Expression analysis indicates that TANG1 is ubiquitously expressed at moderate levels in different organs and throughout the Arabidopsis (Arabidopsis thaliana) life cycle; however, its expression is not affected by high-sugar treatment. Genetic analysis shows that PRL1 and TANG1 have additive effects on sugar-related responses. Furthermore, the mutation of TANG1 does not affect the expression of genes involved in known sugar signaling pathways. Taken together, these results suggest that TANG1, a unique gene, plays an important role in sugar responses in Arabidopsis. PMID:26002908

  13. The protein encoded by the rolB plant oncogene hydrolyses indole glucosides.

    PubMed Central

    Estruch, J J; Schell, J; Spena, A

    1991-01-01

    The rolB gene of Agrobacterium rhizogenes, whose expression stimulates the formation of roots by transformed plant tissues and other growth alterations in transgenic plants, codes for a beta-glucosidase able to hydrolyse indole-beta-glucosides. Indeed, we show that extracts of bacteria and/or plant tissue expressing the rolB protein hydrolyse indoxyl-beta-glucoside (plant indican). Because of the structural similarity between indoxyl-beta-glucoside and indole-3-acetyl-beta-glucoside (IAA-beta-glucoside), we propose that the physiological and developmental alterations in transgenic plants expressing the rolB gene could be the result of an increased intracellular auxin activity caused by the release of active auxins from inactive beta-glucosides. Thus two of the oncogenes carried by the T-DNA of the plant pathogen Agrobacterium rhizogenes (rolB and rolC) perturb plant growth and development by coding for beta-glucosidases with distinct specificities. Whereas the rolC beta-glucosidase releases cytokinins from their glucoside conjugates, the rolB encoded protein hydrolyses indole-beta-glucosides. The combined action of these two genes therefore is expected to modulate the intracellular concentration of two of the main growth factors active in plants. Images PMID:1915286

  14. A Gene Mutated in Nephronophthisis and Retinitis Pigmentosa Encodes a Novel Protein, Nephroretinin, Conserved in Evolution

    PubMed Central

    Otto, Edgar; Hoefele, Julia; Ruf, Rainer; Mueller, Adelheid M.; Hiller, Karl S.; Wolf, Matthias T. F.; Schuermann, Maria J.; Becker, Achim; Birkenhäger, Ralf; Sudbrak, Ralf; Hennies, Hans C.; Nürnberg, Peter; Hildebrandt, Friedhelm

    2002-01-01

    Nephronophthisis (NPHP) comprises a group of autosomal recessive cystic kidney diseases, which constitute the most frequent genetic cause for end-stage renal failure in children and young adults. The most prominent histologic feature of NPHP consists of development of renal fibrosis, which, in chronic renal failure of any origin, represents the pathogenic event correlated most strongly to loss of renal function. Four gene loci for NPHP have been mapped to chromosomes 2q13 (NPHP1), 9q22 (NPHP2), 3q22 (NPHP3), and 1p36 (NPHP4). At all four loci, linkage has also been demonstrated in families with the association of NPHP and retinitis pigmentosa, known as “Senior-Løken syndrome” (SLS). Identification of the gene for NPHP type 1 had revealed nephrocystin as a novel docking protein, providing new insights into mechanisms of cell-cell and cell-matrix signaling. We here report identification of the gene (NPHP4) causing NPHP type 4, by use of high-resolution haplotype analysis and by demonstration of nine likely loss-of-function mutations in six affected families. NPHP4 encodes a novel protein, nephroretinin, that is conserved in evolution—for example, in the nematode Caenorhabditis elegans. In addition, we demonstrate two loss-of-function mutations of NPHP4 in patients from two families with SLS. Thus, we have identified a novel gene with critical roles in renal tissue architecture and ophthalmic function. PMID:12205563

  15. Genomics and Physiology of a Marine Flavobacterium Encoding a Proteorhodopsin and a Xanthorhodopsin-Like Protein

    PubMed Central

    Riedel, Thomas; Gómez-Consarnau, Laura; Tomasch, Jürgen; Martin, Madeleine; Jarek, Michael; González, José M.; Spring, Stefan; Rohlfs, Meike; Brinkhoff, Thorsten; Cypionka, Heribert; Göker, Markus; Fiebig, Anne; Klein, Johannes; Goesmann, Alexander; Fuhrman, Jed A.; Wagner-Döbler, Irene

    2013-01-01

    Proteorhodopsin (PR) photoheterotrophy in the marine flavobacterium Dokdonia sp. PRO95 has previously been investigated, showing no growth stimulation in the light at intermediate carbon concentrations. Here we report the genome sequence of strain PRO95 and compare it to two other PR encoding Dokdonia genomes: that of strain 4H-3-7-5 which shows the most similar genome, and that of strain MED134 which grows better in the light under oligotrophic conditions. Our genome analysis revealed that the PRO95 genome as well as the 4H-3-7-5 genome encode a protein related to xanthorhodopsins. The genomic environment and phylogenetic distribution of this gene suggest that it may have frequently been recruited by lateral gene transfer. Expression analyses by RT-PCR and direct mRNA-sequencing showed that both rhodopsins and the complete β-carotene pathway necessary for retinal production are transcribed in PRO95. Proton translocation measurements showed enhanced proton pump activity in response to light, supporting that one or both rhodopsins are functional. Genomic information and carbon source respiration data were used to develop a defined cultivation medium for PRO95, but reproducible growth always required small amounts of yeast extract. Although PRO95 contains and expresses two rhodopsin genes, light did not stimulate its growth as determined by cell numbers in a nutrient poor seawater medium that mimics its natural environment, confirming previous experiments at intermediate carbon concentrations. Starvation or stress conditions might be needed to observe the physiological effect of light induced energy acquisition. PMID:23526944

  16. Genomics and physiology of a marine flavobacterium encoding a proteorhodopsin and a xanthorhodopsin-like protein.

    PubMed

    Riedel, Thomas; Gómez-Consarnau, Laura; Tomasch, Jürgen; Martin, Madeleine; Jarek, Michael; González, José M; Spring, Stefan; Rohlfs, Meike; Brinkhoff, Thorsten; Cypionka, Heribert; Göker, Markus; Fiebig, Anne; Klein, Johannes; Goesmann, Alexander; Fuhrman, Jed A; Wagner-Döbler, Irene

    2013-01-01

    Proteorhodopsin (PR) photoheterotrophy in the marine flavobacterium Dokdonia sp. PRO95 has previously been investigated, showing no growth stimulation in the light at intermediate carbon concentrations. Here we report the genome sequence of strain PRO95 and compare it to two other PR encoding Dokdonia genomes: that of strain 4H-3-7-5 which shows the most similar genome, and that of strain MED134 which grows better in the light under oligotrophic conditions. Our genome analysis revealed that the PRO95 genome as well as the 4H-3-7-5 genome encode a protein related to xanthorhodopsins. The genomic environment and phylogenetic distribution of this gene suggest that it may have frequently been recruited by lateral gene transfer. Expression analyses by RT-PCR and direct mRNA-sequencing showed that both rhodopsins and the complete β-carotene pathway necessary for retinal production are transcribed in PRO95. Proton translocation measurements showed enhanced proton pump activity in response to light, supporting that one or both rhodopsins are functional. Genomic information and carbon source respiration data were used to develop a defined cultivation medium for PRO95, but reproducible growth always required small amounts of yeast extract. Although PRO95 contains and expresses two rhodopsin genes, light did not stimulate its growth as determined by cell numbers in a nutrient poor seawater medium that mimics its natural environment, confirming previous experiments at intermediate carbon concentrations. Starvation or stress conditions might be needed to observe the physiological effect of light induced energy acquisition. PMID:23526944

  17. Non-human lnc-DC orthologs encode Wdnm1-like protein

    PubMed Central

    Dijkstra, Johannes M.; Ballingall, Keith T.

    2014-01-01

    In a recent publication in Science, Wang et al. found a long noncoding RNA (lncRNA) expressed in human dendritic cells (DC), which they designated lnc-DC. Based on lentivirus-mediated RNA interference (RNAi) experiments in human and murine systems, they concluded that lnc-DC is important in differentiation of monocytes into DC. However, Wang et al. did not mention that their so-called “mouse lnc-DC ortholog” gene was already designated “ Wdnm1-like” and is known to encode a small secreted protein.  We found that incapacitation of the Wdnm1-like open reading frame (ORF) is very rare among mammals, with all investigated primates except for hominids having an intact ORF. The null-hypothesis by Wang et al. therefore should have been that the human lnc-DC transcript might only represent a non-functional relatively young evolutionary remnant of a protein coding locus.  Whether this null-hypothesis can be rejected by the experimental data presented by Wang et al. depends in part on the possible off-target (immunogenic or otherwise) effects of their RNAi procedures, which were not exhaustive in regard to the number of analyzed RNAi sequences and control sequences.  If, however, the conclusions by Wang et al. on their human model are correct, and they may be, current knowledge regarding the Wdnm1-like locus suggests an intriguing combination of different functions mediated by transcript and protein in the maturation of several cell types at some point in evolution. We feel that the article by Wang et al. tends to be misleading without the discussion presented here. PMID:25309733

  18. The fatty acid desaturase 3 gene encodes for different FADS3 protein isoforms in mammalian tissues

    PubMed Central

    Pédrono, Frédérique; Blanchard, Hélène; Kloareg, Maela; D'andréa, Sabine; Daval, Stéphanie; Rioux, Vincent; Legrand, Philippe

    2010-01-01

    In 2000, Marquardt et al. (A. Marquardt, H. Stöhr, K. White, and B. H. F. Weber. 2000. cDNA cloning, genomic structure, and chromosomal localization of three members of the human fatty acid desaturase family. Genomics. 66: 176–183.) described the genomic structure of the fatty acid desaturase (FADS) cluster in humans. This cluster includes the FADS1 and FADS2 genes encoding, respectively, for the Δ5- and Δ6-desaturases involved in polyunsaturated fatty acid biosynthesis. A third gene, named FADS3, has recently been identified but no functional role has yet been attributed to the putative FADS3 protein. In this study, we investigated the FADS3 occurrence in rat tissues by using two specific polyclonal antibodies directed against the N-terminal and C-terminal ends of rat FADS3. Our results showed three potential protein isoforms of FADS3 (75 kDa, 51 kDa, and 37 kDa) present in a tissue-dependent manner. The occurrence of these FADS3 isoforms did not depend on the mRNA level determined by real-time PCR. In parallel, mouse tissues were also tested and showed the same three FADS3 isoforms but with a different tissue distribution. Finally, we reported the existence of FADS3 in human cells and tissues but different new isoforms were identified. To conclude, we showed in this study that FADS3 does exist under multiple protein isoforms depending on the mammalian tissues. These results will help further investigations to determine the physiological function of FADS3. PMID:19752397

  19. Identification of a Gene in Staphylococcus xylosus Encoding a Novel Glucose Uptake Protein

    PubMed Central

    Fiegler, Heike; Bassias, Joannis; Jankovic, Ivana; Brückner, Reinhold

    1999-01-01

    By transposon Tn917 mutagenesis, two mutants of Staphylococcus xylosus were isolated that showed higher levels of β-galactosidase activity in the presence of glucose than the wild type. Both transposons integrated in a gene, designated glcU, encoding a protein involved in glucose uptake in S. xylosus, which is followed by a glucose dehydrogenase gene (gdh). Glucose-mediated repression of β-galactosidase, α-glucosidase, and β-glucuronidase activities was partially relieved in the mutant strains, while repression by sucrose or fructose remained as strong as in the wild type. In addition to the pleiotropic regulatory effect, integration of the transposons into glcU reduced glucose dehydrogenase activity, suggesting cotranscription of glcU and gdh. Insertional inactivation of the gdh gene and deletion of the glcU gene without affecting gdh expression showed that loss of GlcU function is exclusively responsible for the regulatory defect. Reduced glucose repression is most likely the consequence of impaired glucose uptake in the glcU mutant strains. With cloned glcU, an Escherichia coli mutant deficient in glucose transport could grow with glucose as sole carbon source, provided a functional glucose kinase was present. Therefore, glucose is internalized by glcU in nonphosphorylated form. A gene from Bacillus subtilis, ycxE, that is homologous to glcU, could substitute for glcU in the E. coli glucose growth experiments and restored glucose repression in the S. xylosus glcU mutants. Three more proteins with high levels of similarity to GlcU and YcxE are currently in the databases. It appears that these proteins constitute a novel family whose members are involved in bacterial transport processes. GlcU and YcxE are the first examples whose specificity, glucose, has been determined. PMID:10438764

  20. Both Homo and Heterodimers of Marek's Disease Virus Encoded Meq Protein Contribute to Transformation of Lymphocytes in Chickens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Marek’s disease virus (MDV) elicits T-cell lymphomas in chickens. The MDV genome encodes an oncoprotein, Meq, with similarity to the Jun/Fos family of proteins. Similar to Jun, the leucine zipper region of Meq allows the formation of homo- and heterodimers. We have previously shown that Meq homodime...

  1. Influence of energy supply on expression of genes encoding for lipogenic enzymes and regulatory proteins in growing beef steers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Forty crossbred beef steers were used to determine the effects metabolizable energy (ME) intake and of site and complexity of carbohydrate (CHO) infusion on expression of genes encoding lipogenic enzymes and regulatory proteins in subcutaneous (SC), mesenteric (MES) and omental (OM) adipose. Treatm...

  2. Red-shifted fluorescent proteins mPlum and mRaspberry and polynucleotides encoding the same

    DOEpatents

    Tsien, Roger Y.; Wang, Lei

    2008-07-01

    Methods using somatic hypermutation (SHM) for producing polypeptide and nucleic acid variants, and nucleic acids encoding such polypeptide variants are disclosed. Such variants may have desired properties. Also disclosed are novel polypeptides, such as improved fluorescent proteins, produced by the novel methods, and nucleic acids, vectors, and host cells comprising such vectors.

  3. Genes Encoding the PR-4 Protein Wheatwin Are Developmentally Regulated in Wheat Grains and Respond to High Temperatures During Grainfill

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Sequences encoding three wheatwins, including a novel protein not identified previously, were found among expressed sequence tags (ESTs) from grains from the US bread wheat Butte 86 and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was used to detect transcripts specific for...

  4. Identification and characterization of a multigene family encoding germin-like proteins in cultivated peanut (Arachis hypogaea L.)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Germin-like proteins (GLPs) play diversified roles in plant development and defense response. Here, we identified 36 ESTs encoding GLPs from peanut (Arachis hypogaea L.). After assembly, these ESTs were integrated into eight unigenes, named AhGLP1 to AhGLP8, of which, three (AhGLP1-3) were comprised...

  5. Expression of the Immediate-Early Gene-Encoded Protein Egr-1 ("zif268") during in Vitro Classical Conditioning

    ERIC Educational Resources Information Center

    Mokin, Maxim; Keifer, Joyce

    2005-01-01

    Expression of the immediate-early genes (IEGs) has been shown to be induced by activity-dependent synaptic plasticity or behavioral training and is thought to play an important role in long-term memory. In the present study, we examined the induction and expression of the IEG-encoded protein Egr-1 during an in vitro neural correlate of eyeblink…

  6. Cloning and expression analysis of mouse Cclp1, a new gene encoding a coiled-coil-like protein.

    PubMed

    Noben-Trauth, K; Naggert, J K; Nishina, P M

    1997-05-30

    Here we describe the nucleotide sequence and expression pattern of a novel gene termed Coiled-coil-like protein 1 (Cclp1). A 2646bp open reading frame encodes a 882 amino acid protein with a predicted coiled-coil domain at the amino terminus. Cclp1 is expressed in a variety of adult tissues and during different stages of embryogenesis. The broad expression pattern suggests a general cellular function of CCLP1. PMID:9199242

  7. Translational Control of Host Gene Expression by a Cys-Motif Protein Encoded in a Bracovirus.

    PubMed

    Kim, Eunseong; Kim, Yonggyun

    2016-01-01

    Translational control is a strategy that various viruses use to manipulate their hosts to suppress acute antiviral response. Polydnaviruses, a group of insect double-stranded DNA viruses symbiotic to some endoparasitoid wasps, are divided into two genera: ichnovirus (IV) and bracovirus (BV). In IV, some Cys-motif genes are known as host translation-inhibitory factors (HTIF). The genome of endoparasitoid wasp Cotesia plutellae contains a Cys-motif gene (Cp-TSP13) homologous to an HTIF known as teratocyte-secretory protein 14 (TSP14) of Microplitis croceipes. Cp-TSP13 consists of 129 amino acid residues with a predicted molecular weight of 13.987 kDa and pI value of 7.928. Genomic DNA region encoding its open reading frame has three introns. Cp-TSP13 possesses six conserved cysteine residues as other Cys-motif genes functioning as HTIF. Cp-TSP13 was expressed in Plutella xylostella larvae parasitized by C. plutellae. C. plutellae bracovirus (CpBV) was purified and injected into non-parasitized P. xylostella that expressed Cp-TSP13. Cp-TSP13 was cloned into a eukaryotic expression vector and used to infect Sf9 cells to transiently express Cp-TSP13. The synthesized Cp-TSP13 protein was detected in culture broth. An overlaying experiment showed that the purified Cp-TSP13 entered hemocytes. It was localized in the cytosol. Recombinant Cp-TSP13 significantly inhibited protein synthesis of secretory proteins when it was added to in vitro cultured fat body. In addition, the recombinant Cp-TSP13 directly inhibited the translation of fat body mRNAs in in vitro translation assay using rabbit reticulocyte lysate. Moreover, the recombinant Cp-TSP13 significantly suppressed cellular immune responses by inhibiting hemocyte-spreading behavior. It also exhibited significant insecticidal activities by both injection and feeding routes. These results indicate that Cp-TSP13 is a viral HTIF. PMID:27598941

  8. Nuclear factor-κB regulates the expression of multiple genes encoding liver transport proteins.

    PubMed

    Balasubramaniyan, Natarajan; Ananthanarayanan, Meenakshisundaram; Suchy, Frederick J

    2016-04-15

    In this study we identified the mechanisms underlying the inhibitory effects of NF-κB on the expression of genes encoding multiple liver transport proteins. Well-conserved NF-κB binding sites were found in the promoters of farnesoid X receptor (FXR)-target genes. An electromobility shift assay (EMSA) demonstrated the specific interaction between the NF-κB p65 protein and a (32)P-labeled BSEP NF-κB response element (NF-κBE). Chromatin immunoprecipitation (ChIP) analysis confirmed binding of NF-κB p65 to the BSEP locus but not the FXRE in vitro. NF-κB p65 overexpression in Huh-7 cells markedly repressed FXR/RXR transactivation of the BSEP, ABCG5/G8, MRP2, and FXR promoters, which was totally reversed by expression of the IκBα super-repressor. NF-κB interacted directly with FXR on coimmunoprecipitation, suggesting another level for the inhibitory effects of NF-κB on FXR-target genes. In vivo ChIP analysis with liver nuclei obtained from mice after 3 days of common bile duct ligation (BDL) or 6 h post-lipopolysaccharide (LPS) injection showed a markedly increased recruitment of NF-κB p65 to the Bsep promoter compared with controls. There was also increased recruitment of the corepressor silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) and histone deacetylase (HDAC)3 and HDAC2 to the NF-κB sites. We also found that NF-κB p65 was recruited to NF-κB binding sites in the promoters of organic solute transporter, OSTα and OSTβ, and unexpectedly activated rather than repressed gene expression. In mouse liver after BDL NF-κB recruitment to Ostα and Ostβ promoters was associated with increased binding of the potent coactivator cAMP response element binding protein (CREB)-binding protein (CBP)/p300 to the NF-κBE and depletion of CBP/p300 at the FXR element. Overall, these studies demonstrate a novel role for NF-κB in adaptation to obstructive and LPS-induced cholestasis acting through recruitment to specific NF-κB binding sites in

  9. Enhancement of initial equivalency for protein structure alignment based on encoded local structures.

    PubMed

    Hung, Kenneth; Wang, Jui-Chih; Chen, Cheng-Wei; Chuang, Cheng-Long; Tsai, Kun-Nan; Chen, Chung-Ming

    2012-11-01

    Most alignment algorithms find an initial equivalent residue pair followed by an iterative optimization process to explore better near-optimal alignments in the surrounding solution space of the initial alignment. It plays a decisive role in determining the alignment quality since a poor initial alignment may make the final alignment trapped in an undesirable local optimum even with an iterative optimization. We proposed a vector-based alignment algorithm with a new initial alignment approach accounting for local structure features called MIRAGE-align. The new idea is to enhance the quality of the initial alignment based on encoded local structural alphabets to identify the protein structure pair whose sequence identity falls in or below twilight zone. The statistical analysis of alignment quality based on Match Index (MI) and computation time demonstrated that MIRAGE-align algorithm outperformed four previously published algorithms, i.e., the residue-based algorithm (CE), the vector-based algorithm (SSM), TM-align, and Fr-TM-align. MIRAGE-align yields a better estimate of initial solution to enhance the quality of initial alignment and enable the employment of a non-iterative optimization process to achieve a better alignment. PMID:22717522

  10. Mutations in STX1B, encoding a presynaptic protein, cause fever-associated epilepsy syndromes.

    PubMed

    Schubert, Julian; Siekierska, Aleksandra; Langlois, Mélanie; May, Patrick; Huneau, Clément; Becker, Felicitas; Muhle, Hiltrud; Suls, Arvid; Lemke, Johannes R; de Kovel, Carolien G F; Thiele, Holger; Konrad, Kathryn; Kawalia, Amit; Toliat, Mohammad R; Sander, Thomas; Rüschendorf, Franz; Caliebe, Almuth; Nagel, Inga; Kohl, Bernard; Kecskés, Angela; Jacmin, Maxime; Hardies, Katia; Weckhuysen, Sarah; Riesch, Erik; Dorn, Thomas; Brilstra, Eva H; Baulac, Stephanie; Møller, Rikke S; Hjalgrim, Helle; Koeleman, Bobby P C; Jurkat-Rott, Karin; Lehman-Horn, Frank; Roach, Jared C; Glusman, Gustavo; Hood, Leroy; Galas, David J; Martin, Benoit; de Witte, Peter A M; Biskup, Saskia; De Jonghe, Peter; Helbig, Ingo; Balling, Rudi; Nürnberg, Peter; Crawford, Alexander D; Esguerra, Camila V; Weber, Yvonne G; Lerche, Holger

    2014-12-01

    Febrile seizures affect 2-4% of all children and have a strong genetic component. Recurrent mutations in three main genes (SCN1A, SCN1B and GABRG2) have been identified that cause febrile seizures with or without epilepsy. Here we report the identification of mutations in STX1B, encoding syntaxin-1B, that are associated with both febrile seizures and epilepsy. Whole-exome sequencing in independent large pedigrees identified cosegregating STX1B mutations predicted to cause an early truncation or an in-frame insertion or deletion. Three additional nonsense or missense mutations and a de novo microdeletion encompassing STX1B were then identified in 449 familial or sporadic cases. Video and local field potential analyses of zebrafish larvae with antisense knockdown of stx1b showed seizure-like behavior and epileptiform discharges that were highly sensitive to increased temperature. Wild-type human syntaxin-1B but not a mutated protein rescued the effects of stx1b knockdown in zebrafish. Our results thus implicate STX1B and the presynaptic release machinery in fever-associated epilepsy syndromes. PMID:25362483

  11. Comparative analyses of transport proteins encoded within the genomes of Mycobacterium tuberculosis and Mycobacterium leprae

    PubMed Central

    Youm, Jiwon; Saier, Milton H.

    2012-01-01

    The co-emergence of multidrug resistant pathogenic bacterial strains and the HIV pandemic has made tuberculosis a leading public health threat. The causative agent is Mycobacterium tuberculosis (Mtu), a facultative intracellular parasite. Mycobacterium leprae (Mle), a related organism that causes leprosy, is an obligate intracellular parasite. Given that different transporters are required for bacterial growth and persistence under a variety of growth conditions, we conducted comparative analyses of transport proteins encoded within the genomes of these two organisms. A minimal set of genes required for intracellular and extracellular life were identified. Drug efflux systems utilizing primary active transport mechanisms have been preferentially retained in Mle and still others preferentially lost. Transporters associated with environmental adaptation found in Mtu were mostly lost in Mle. These findings provide starting points for experimental studies that may elucidate the dependencies of pathogenesis on transport for these two pathogenic mycobacteria. They also lead to suggestions regarding transporters that function in intra- versus extra-cellular growth. PMID:22179038

  12. Systematic Global Analysis of Genes Encoding Protein Phosphatases in Aspergillus fumigatus

    PubMed Central

    Winkelströter, Lizziane K.; Dolan, Stephen K.; Fernanda dos Reis, Thaila; Bom, Vinícius Leite Pedro; Alves de Castro, Patrícia; Hagiwara, Daisuke; Alowni, Raneem; Jones, Gary W.; Doyle, Sean; Brown, Neil Andrew; Goldman, Gustavo H.

    2015-01-01

    Aspergillus fumigatus is a fungal pathogen that causes several invasive and noninvasive diseases named aspergillosis. This disease is generally regarded as multifactorial, considering that several pathogenicity determinants are present during the establishment of this illness. It is necessary to obtain an increased knowledge of how, and which, A. fumigatus signal transduction pathways are engaged in the regulation of these processes. Protein phosphatases are essential to several signal transduction pathways. We identified 32 phosphatase catalytic subunit-encoding genes in A. fumigatus, of which we were able to construct 24 viable deletion mutants. The role of nine phosphatase mutants in the HOG (high osmolarity glycerol response) pathway was evaluated by measuring phosphorylation of the p38 MAPK (SakA) and expression of osmo-dependent genes. We were also able to identify 11 phosphatases involved in iron assimilation, six that are related to gliotoxin resistance, and three implicated in gliotoxin production. These results present the creation of a fundamental resource for the study of signaling in A. fumigatus and its implications in the regulation of pathogenicity determinants and virulence in this important pathogen. PMID:25943523

  13. Leafy head2, which encodes a putative RNA-binding protein, regulates shoot development of rice.

    PubMed

    Xiong, Guo Sheng; Hu, Xing Ming; Jiao, Yong Qing; Yu, Yan Chun; Chu, Cheng Cai; Li, Jia Yang; Qian, Qian; Wang, Yong Hong

    2006-03-01

    During vegetative development, higher plants continuously form new leaves in regular spatial and temporal patterns. Mutants with abnormal leaf developmental patterns not only provide a great insight into understanding the regulatory mechanism of plant architecture, but also enrich the ways to its modification by which crop yield could be improved. Here, we reported the characterization of the rice leafy-head2 (lhd2) mutant that exhibits shortened plastochron, dwarfism, reduced tiller number, and failure of phase transition from vegetative to reproductive growth. Anatomical and histological study revealed that the rapid emergence of leaves in lhd2 was resulted from the rapid initiation of leaf primordia whereas the reduced tiller number was a consequence of the suppression of the tiller bud outgrowth. The molecular and genetic analysis showed that LHD2 encodes a putative RNA binding protein with 67% similarity to maize TE1. Comparison of genome-scale expression profiles between wild-type and lhd2 plants suggested that LHD2 may regulate rice shoot development through KNOX and hormone-related genes. The similar phenotypes caused by LHD2 mutation and the conserved expression pattern of LHD2 indicated a conserved mechanism in controlling the temporal leaf initiation in grass. PMID:16541125

  14. Multiplexed visualization of dynamic signaling networks using genetically encoded fluorescent protein-based biosensors

    PubMed Central

    Depry, Charlene; Mehta, Sohum; Zhang, Jin

    2012-01-01

    Cells rely on a complex, interconnected network of signaling pathways to sense and interpret changes in their extracellular environment. The development of genetically encoded fluorescent protein (FP)-based biosensors has made it possible for researchers to directly observe and characterize the spatiotemporal dynamics of these intracellular signaling pathways in living cells. However, detailed information regarding the precise temporal and spatial relationships between intersecting pathways is often lost when individual signaling events are monitored in isolation. As the development of biosensor technology continues to advance, it is becoming increasingly feasible to image multiple FP-based biosensors concurrently, permitting greater insights into the intricate coordination of intracellular signaling networks by enabling parallel monitoring of distinct signaling events within the same cell. In this review, we discuss several strategies for multiplexed imaging of FP-based biosensors, while also underscoring some of the challenges associated with these techniques and highlighting additional avenues that could lead to further improvements in parallel monitoring of intracellular signaling events. PMID:23138230

  15. Systematic Global Analysis of Genes Encoding Protein Phosphatases in Aspergillus fumigatus.

    PubMed

    Winkelströter, Lizziane K; Dolan, Stephen K; Fernanda Dos Reis, Thaila; Bom, Vinícius Leite Pedro; Alves de Castro, Patrícia; Hagiwara, Daisuke; Alowni, Raneem; Jones, Gary W; Doyle, Sean; Brown, Neil Andrew; Goldman, Gustavo H

    2015-07-01

    Aspergillus fumigatus is a fungal pathogen that causes several invasive and noninvasive diseases named aspergillosis. This disease is generally regarded as multifactorial, considering that several pathogenicity determinants are present during the establishment of this illness. It is necessary to obtain an increased knowledge of how, and which, A. fumigatus signal transduction pathways are engaged in the regulation of these processes. Protein phosphatases are essential to several signal transduction pathways. We identified 32 phosphatase catalytic subunit-encoding genes in A. fumigatus, of which we were able to construct 24 viable deletion mutants. The role of nine phosphatase mutants in the HOG (high osmolarity glycerol response) pathway was evaluated by measuring phosphorylation of the p38 MAPK (SakA) and expression of osmo-dependent genes. We were also able to identify 11 phosphatases involved in iron assimilation, six that are related to gliotoxin resistance, and three implicated in gliotoxin production. These results present the creation of a fundamental resource for the study of signaling in A. fumigatus and its implications in the regulation of pathogenicity determinants and virulence in this important pathogen. PMID:25943523

  16. Relationships between protein-encoding gene abundance and corresponding process are commonly assumed yet rarely observed.

    PubMed

    Rocca, Jennifer D; Hall, Edward K; Lennon, Jay T; Evans, Sarah E; Waldrop, Mark P; Cotner, James B; Nemergut, Diana R; Graham, Emily B; Wallenstein, Matthew D

    2015-08-01

    For any enzyme-catalyzed reaction to occur, the corresponding protein-encoding genes and transcripts are necessary prerequisites. Thus, a positive relationship between the abundance of gene or transcripts and corresponding process rates is often assumed. To test this assumption, we conducted a meta-analysis of the relationships between gene and/or transcript abundances and corresponding process rates. We identified 415 studies that quantified the abundance of genes or transcripts for enzymes involved in carbon or nitrogen cycling. However, in only 59 of these manuscripts did the authors report both gene or transcript abundance and rates of the appropriate process. We found that within studies there was a significant but weak positive relationship between gene abundance and the corresponding process. Correlations were not strengthened by accounting for habitat type, differences among genes or reaction products versus reactants, suggesting that other ecological and methodological factors may affect the strength of this relationship. Our findings highlight the need for fundamental research on the factors that control transcription, translation and enzyme function in natural systems to better link genomic and transcriptomic data to ecosystem processes. PMID:25535936

  17. Transient expression of protein tyrosine phosphatases encoded in Cotesia plutellae bracovirus inhibits insect cellular immune responses

    NASA Astrophysics Data System (ADS)

    Ibrahim, Ahmed M. A.; Kim, Yonggyun

    2008-01-01

    Several immunosuppressive factors are associated with parasitism of an endoparasitoid wasp, Cotesia plutellae, on the diamondback moth, Plutella xylostella. C. plutellae bracovirus (CpBV) encodes a large number of putative protein tyrosine phosphatases (PTPs), which may play a role in inhibiting host cellular immunity. To address this inhibitory hypothesis of CpBV-PTPs, we performed transient expression of individual CpBV-PTPs in hemocytes of the beet armyworm, Spodoptera exigua, and analyzed their cellular immune responses. Two different forms of CpBV-PTPs were chosen and cloned into a eukaryotic expression vector under the control of the p10 promoter of baculovirus: one with the normal cysteine active site (CpBV-PTP1) and the other with a mutated active site (CpBV-PTP5). The hemocytes transfected with CpBV-PTP1 significantly increased in PTP activity compared to control hemocytes, but those with CpBV-PTP5 exhibited a significant decrease in the PTP activity. All transfected hemocytes exhibited a significant reduction in both cell spreading and encapsulation activities compared to control hemocytes. Co-transfection of CpBV-PTP1 together with its double-stranded RNA reduced the messenger RNA (mRNA) level of CpBV-PTP1 and resulted in recovery of both hemocyte behaviors. This is the first report demonstrating that the polydnaviral PTPs can manipulate PTP activity of the hemocytes to interrupt cellular immune responses.

  18. The HTLV-1-encoded protein HBZ directly inhibits the acetyl transferase activity of p300/CBP

    PubMed Central

    Wurm, Torsten; Wright, Diana G.; Polakowski, Nicholas; Mesnard, Jean-Michel; Lemasson, Isabelle

    2012-01-01

    The homologous cellular coactivators p300 and CBP contain intrinsic lysine acetyl transferase (termed HAT) activity. This activity is responsible for acetylation of several sites on the histones as well as modification of transcription factors. In a previous study, we found that HBZ, encoded by the Human T-cell Leukemia Virus type 1 (HTLV-1), binds to multiple domains of p300/CBP, including the HAT domain. In this study, we found that HBZ inhibits the HAT activity of p300/CBP through the bZIP domain of the viral protein. This effect correlated with a reduction of H3K18 acetylation, a specific target of p300/CBP, in cells expressing HBZ. Interestingly, lower levels of H3K18 acetylation were detected in HTLV-1 infected cells compared to non-infected cells. The inhibitory effect of HBZ was not limited to histones, as HBZ also inhibited acetylation of the NF-κB subunit, p65, and the tumor suppressor, p53. Recent studies reported that mutations in the HAT domain of p300/CBP that cause a defect in acetylation are found in certain types of leukemia. These observations suggest that inhibition of the HAT activity by HBZ is important for the development of adult T-cell leukemia associated with HTLV-1 infection. PMID:22434882

  19. Relationships between protein-encoding gene abundance and corresponding process are commonly assumed yet rarely observed

    PubMed Central

    Rocca, Jennifer D; Hall, Edward K; Lennon, Jay T; Evans, Sarah E; Waldrop, Mark P; Cotner, James B; Nemergut, Diana R; Graham, Emily B; Wallenstein, Matthew D

    2015-01-01

    For any enzyme-catalyzed reaction to occur, the corresponding protein-encoding genes and transcripts are necessary prerequisites. Thus, a positive relationship between the abundance of gene or transcripts and corresponding process rates is often assumed. To test this assumption, we conducted a meta-analysis of the relationships between gene and/or transcript abundances and corresponding process rates. We identified 415 studies that quantified the abundance of genes or transcripts for enzymes involved in carbon or nitrogen cycling. However, in only 59 of these manuscripts did the authors report both gene or transcript abundance and rates of the appropriate process. We found that within studies there was a significant but weak positive relationship between gene abundance and the corresponding process. Correlations were not strengthened by accounting for habitat type, differences among genes or reaction products versus reactants, suggesting that other ecological and methodological factors may affect the strength of this relationship. Our findings highlight the need for fundamental research on the factors that control transcription, translation and enzyme function in natural systems to better link genomic and transcriptomic data to ecosystem processes. PMID:25535936

  20. Mutations in OTOGL, Encoding the Inner Ear Protein Otogelin-like, Cause Moderate Sensorineural Hearing Loss

    PubMed Central

    Yariz, Kemal O.; Duman, Duygu; Seco, Celia Zazo; Dallman, Julia; Huang, Mingqian; Peters, Theo A.; Sirmaci, Asli; Lu, Na; Schraders, Margit; Skromne, Isaac; Oostrik, Jaap; Diaz-Horta, Oscar; Young, Juan I.; Tokgoz-Yilmaz, Suna; Konukseven, Ozlem; Shahin, Hashem; Hetterschijt, Lisette; Kanaan, Moien; Oonk, Anne M.M.; Edwards, Yvonne J.K.; Li, Huawei; Atalay, Semra; Blanton, Susan; DeSmidt, Alexandra A.; Liu, Xue-Zhong; Pennings, Ronald J.E.; Lu, Zhongmin; Chen, Zheng-Yi; Kremer, Hannie; Tekin, Mustafa

    2012-01-01

    Hereditary hearing loss is characterized by a high degree of genetic heterogeneity. Here we present OTOGL mutations, a homozygous one base pair deletion (c.1430 delT) causing a frameshift (p.Val477Glufs∗25) in a large consanguineous family and two compound heterozygous mutations, c.547C>T (p.Arg183∗) and c.5238+5G>A, in a nonconsanguineous family with moderate nonsyndromic sensorineural hearing loss. OTOGL maps to the DFNB84 locus at 12q21.31 and encodes otogelin-like, which has structural similarities to the epithelial-secreted mucin protein family. We demonstrate that Otogl is expressed in the inner ear of vertebrates with a transcription level that is high in embryonic, lower in neonatal, and much lower in adult stages. Otogelin-like is localized to the acellular membranes of the cochlea and the vestibular system and to a variety of inner ear cells located underneath these membranes. Knocking down of otogl with morpholinos in zebrafish leads to sensorineural hearing loss and anatomical changes in the inner ear, supporting that otogelin-like is essential for normal inner ear function. We propose that OTOGL mutations affect the production and/or function of acellular structures of the inner ear, which ultimately leads to sensorineural hearing loss. PMID:23122586

  1. Identification and analysis of a Saccharomyces cerevisiae copper homeostasis gene encoding a homeodomain protein.

    PubMed Central

    Knight, S A; Tamai, K T; Kosman, D J; Thiele, D J

    1994-01-01

    Yeast metallothionein, encoded by the CUP1 gene, and its copper-dependent transcriptional activator ACE1 play a key role in mediating copper resistance in Saccharomyces cerevisiae. Using an ethyl methanesulfonate mutant of a yeast strain in which CUP1 and ACE1 were deleted, we isolated a gene, designated CUP9, which permits yeast cells to grow at high concentrations of environmental copper, most notably when lactate is the sole carbon source. Disruption of CUP9, which is located on chromosome XVI, caused a loss of copper resistance in strains which possessed CUP1 and ACE1, as well as in the cup1 ace1 deletion strain. Measurement of intracellular copper levels of the wild-type and cup9-1 mutant demonstrated that total intracellular copper concentrations were unaffected by CUP9. CUP9 mRNA levels were, however, down regulated by copper when yeast cells were grown with glucose but not with lactate or glycerol-ethanol as the sole carbon source. This down regulation was independent of the copper metalloregulatory transcription factor ACE1. The DNA sequence of CUP9 predicts an open reading frame of 306 amino acids in which a 55-amino-acid sequence showed 47% identity with the homeobox domain of the human proto-oncogene PBX1, suggesting that CUP9 is a DNA-binding protein which regulates the expression of important copper homeostatic genes. Images PMID:7969120

  2. Heterogenic expression of genes encoding secreted proteins at the periphery of Aspergillus niger colonies.

    PubMed

    Vinck, Arman; de Bekker, Charissa; Ossin, Adam; Ohm, Robin A; de Vries, Ronald P; Wösten, Han A B

    2011-01-01

    Colonization of a substrate by fungi starts with the invasion of exploring hyphae. These hyphae secrete enzymes that degrade the organic material into small molecules that can be taken up by the fungus to serve as nutrients. We previously showed that only part of the exploring hyphae of Aspergillus niger highly express the glucoamylase gene glaA. This was an unexpected finding since all exploring hyphae are exposed to the same environmental conditions. Using GFP as a reporter, we here demonstrate that the acid amylase gene aamA, the α-glucuronidase gene aguA, and the feruloyl esterase gene faeA of A. niger are also subject to heterogenic expression within the exploring mycelium. Coexpression studies using GFP and dTomato as reporters showed that hyphae that highly express one of these genes also highly express the other genes encoding secreted proteins. Moreover, these hyphae also highly express the amylolytic regulatory gene amyR, and the glyceraldehyde-3-phosphate dehydrogenase gene gpdA. In situ hybridization demonstrated that the high expressers are characterized by a high 18S rRNA content. Taken together, it is concluded that two subpopulations of hyphae can be distinguished within the exploring mycelium of A. niger. The experimental data indicate that these subpopulations differ in their transcriptional and translational activity. PMID:20722697

  3. Diverse circular replication-associated protein encoding viruses circulating in invertebrates within a lake ecosystem.

    PubMed

    Dayaram, Anisha; Galatowitsch, Mark L; Argüello-Astorga, Gerardo R; van Bysterveldt, Katherine; Kraberger, Simona; Stainton, Daisy; Harding, Jon S; Roumagnac, Philippe; Martin, Darren P; Lefeuvre, Pierre; Varsani, Arvind

    2016-04-01

    Over the last five years next-generation sequencing has become a cost effective and efficient method for identifying known and unknown microorganisms. Access to this technique has dramatically changed the field of virology, enabling a wide range of environmental viral metagenome studies to be undertaken of organisms and environmental samples from polar to tropical regions. These studies have led to the discovery of hundreds of highly divergent single stranded DNA (ssDNA) virus-like sequences encoding replication-associated proteins. Yet, few studies have explored how viruses might be shared in an ecosystem through feeding relationships. Here we identify 169 circular molecules (160 CRESS DNA molecules, nine circular molecules) recovered from a New Zealand freshwater lake, that we have tentatively classified into 51 putatively novel species and five previously described species (DflaCV-3, -5, -6, -8, -10). The CRESS DNA viruses identified in this study were recovered from molluscs (Echyridella menzeisii, Musculium novaezelandiae, Potamopyrgus antipodarum and Physella acuta) and insect larvae (Procordulia grayi, Xanthocnemis zealandica, and Chironomus zealandicus) collected from Lake Sarah, as well as from the lake water and benthic sediments. Extensive diversity was observed across most CRESS DNA molecules recovered. The putative capsid protein of one viral species was found to be most similar to those of members of the Tombusviridae family, thus expanding the number of known RNA-DNA hybrid viruses in nature. We noted a strong association between the CRESS DNA viruses and circular molecules identified in the water and browser organisms (C. zealandicus, P. antipodarum and P. acuta), and between water sediments and undefended prey species (C. zealandicus). However, we were unable to find any significant correlation of viral assemblages to the potential feeding relationships of the host aquatic invertebrates. PMID:26873065

  4. Mycobacterium tuberculosis efpA encodes an efflux protein of the QacA transporter family.

    PubMed Central

    Doran, J L; Pang, Y; Mdluli, K E; Moran, A J; Victor, T C; Stokes, R W; Mahenthiralingam, E; Kreiswirth, B N; Butt, J L; Baron, G S; Treit, J D; Kerr, V J; Van Helden, P D; Roberts, M C; Nano, F E

    1997-01-01

    The Mycobacterium tuberculosis H37Rv efpA gene encodes a putative efflux protein, EfpA, of 55,670 Da. The deduced EfpA protein was similar in secondary structure to Pur8, MmrA, TcmA, LfrA, EmrB, and other members of the QacA transporter family (QacA TF) which mediate antibiotic and chemical resistance in bacteria and yeast. The predicted EfpA sequence possessed all transporter motifs characteristic of the QacA TF, including those associated with proton-antiport function and the motif considered to be specific to exporters. The 1,590-bp efpA open reading frame was G+C rich (65%), whereas the 40-bp region immediately upstream had an A+T bias (35% G+C). Reverse transcriptase-PCR assays indicated that efpA was expressed in vitro and in situ. Putative promoter sequences were partially overlapped by the A+T-rich region and by a region capable of forming alternative secondary structures indicative of transcriptional regulation in analogous systems. PCR single-stranded conformational polymorphism analysis demonstrated that these upstream flanking sequences and the 231-bp, 5' coding region are highly conserved among both drug-sensitive and multiply-drug-resistant isolates of M. tuberculosis. The efpA gene was present in the slow-growing human pathogens M. tuberculosis, Mycobacterium leprae, and Mycobacterium bovis and in the opportunistic human pathogens Mycobacterium avium and Mycobacterium intracellular. However, efpA was not present in 17 other opportunistically pathogenic or nonpathogenic mycobacterial species. PMID:9008277

  5. A human papilloma virus type 11 transcript encoding an E1--E4 protein.

    PubMed

    Nasseri, M; Hirochika, R; Broker, T R; Chow, L T

    1987-08-01

    The human papilloma virus (HPV) associated with a genital wart (condyloma acuminatum) was determined to be type 11. The majority of the viral DNA molecules were monomeric circles present in the cells at high copy number, as demonstrated by one- and two-dimensional agarose gell electrophoretic separation followed by Southern blot analysis. A cDNA library in phage lambda gt11 was constructed from poly(A)-selected mRNA recovered from the tissue. Recombinant clones corresponding to the most abundant 1.2-kb viral mRNA species detected by Northern blot hybridization and by electron microscopic analysis of R loops were isolated and their nucleotide sequence was determined. Comparison to the prototype HPV-11 DNA sequence revealed that this message consisted of two exons. The promotor-proximal exon spanned nucleotides 716 through 847 and the distal exon included nucleotides 3325 through 4390 or 4392. The mRNAs were alternatively polyadenylated after either of these latter two sites, in both cases following a G and preceding a U residue. Fourteen or sixteen bases upstream from the poly(A) was the hexanucleotide AGUAAA, which apparently serves as the signal for cleavage and polyadenylation of the nascent message. The splice donor and acceptor sites conformed to the usual /GU. . .AG/pattern. The exons joined open reading frame (ORF) E1, which contributed the initiation codon and four additional triplets, to ORF E4, which specified 85 amino acids to encode a protein of 10,022 Da. The cDNA also contained the ORFs E5a and E5b toward the 3' end. The complete sequence of the cDNA revealed three single-base changes from the prototype HPV-11, two resulting in altered amino acids in E4. Neither affects the coding potential of the overlapping E2 ORF. The function of the E1--E4 protein is unknown. PMID:2887066

  6. Expression of Epstein-Barr virus-encoded proteins in nasopharyngeal carcinoma.

    PubMed

    Fåhraeus, R; Fu, H L; Ernberg, I; Finke, J; Rowe, M; Klein, G; Falk, K; Nilsson, E; Yadav, M; Busson, P

    1988-09-15

    Expression of the Epstein-Barr virus (EBV) encoded nuclear antigens (EBNA 1 to 6) and membrane-associated protein (LMP) was investigated by immunoblotting in 83 nasopharyngeal carcinoma (NPC) biopsies and 25 other tumor and normal tissue specimens from the head and neck region. Fifty-eight of the 83 NPC biopsies were large enough to yield parallel data on virus DNA and viral expression. All 16 cases of clinically diagnosed and histologically confirmed NPCs from North Africa contained EBV DNA and expressed EBNA-1. Of 31 clinically diagnosed NPCs from China, 29 contained EBV DNA and 25 of these expressed EBNA-1. One control tissue biopsy from the oropharynx of NPC patients contained EBV DNA, but none expressed EBNA-1. The latent membrane protein (LMP) was detected in 22/31 of the Chinese and in 10/16 of the North African NPC biopsies. None of the NPC biopsies or control tissues expressed detectable amounts of EBNA 2 or any of the other 4 nuclear antigens which are invariably expressed in EBV-transformed B cells. A smaller number of tumors from Malaysia and East Africa exhibited a similar pattern of expression. EBV was rescued from a nude-mouse-passaged North African NPC tumor by co-cultivation of the tumor cells with umbilical cord blood lymphocytes. The tumor expressed EBNA 1 and LMP, but not EBNA 2 or the other 4 EBNAs. The resulting LCLs expressed all 6 nuclear antigens, EBNA 1 to 6 and LMP. Our data suggest that expression of the EBV genome is regulated in a tissue-specific fashion. PMID:2843473

  7. Small gene family encoding an eggshell (chorion) protein of the human parasite Schistosoma mansoni

    SciTech Connect

    Bobek, L.A.; Rekosh, D.M.; Lo Verde, P.T.

    1988-08-01

    The authors isolated six independent genomic clones encoding schistosome chorion or eggshell proteins from a Schistosoma mansoni genomic library. A linkage map of five of the clones spanning 35 kilobase pairs (kbp) of the S. mansoni genome was constructed. The region contained two eggshell protein genes closely linked, separated by 7.5 kbp of intergenic DNA. The two genes of the cluster were arranged in the same orientation, that is, they were transcribed from the same strand. The sixth clone probably represents a third copy of the eggshell gene that is not contained within the 35-kbp region. The 5- end of the mRNA transcribed from these genes was defined by primer extension directly off the RNA. The ATCAT cap site sequence was homologous to a silkmoth chorion PuTCATT cap site sequence, where Pu indicates any purine. DNA sequence analysis showed that there were no introns in these genes. The DNA sequences of the three genes were very homologous to each other and to a cDNA clone, pSMf61-46, differing only in three or four nucleotices. A multiple TATA box was located at positions -23 to -31, and a CAAAT sequence was located at -52 upstream of the eggshell transcription unit. Comparison of sequences in regions further upstream with silkmoth and Drosophila sequences revealed very short elements that were shared. One such element, TCACGT, recently shown to be an essential cis-regulatory element for silkmoth chorion gene promoter function, was found at a similar position in all three organisms.

  8. Site-specific chemical modification of recombinant proteins produced in mammalian cells by using the genetically encoded aldehyde tag.

    PubMed

    Wu, Peng; Shui, Wenqing; Carlson, Brian L; Hu, Nancy; Rabuka, David; Lee, Julia; Bertozzi, Carolyn R

    2009-03-01

    The properties of therapeutic proteins can be enhanced by chemical modification. Methods for site-specific protein conjugation are critical to such efforts. Here, we demonstrate that recombinant proteins expressed in mammalian cells can be site-specifically modified by using a genetically encoded aldehyde tag. We introduced the peptide sequence recognized by the endoplasmic reticulum (ER)-resident formylglycine generating enzyme (FGE), which can be as short as 6 residues, into heterologous proteins expressed in mammalian cells. Cotranslational modification of the proteins by FGE produced products bearing a unique aldehyde group. Proteins bearing this "aldehyde tag" were chemically modified by selective reaction with hydrazide- or aminooxy-functionalized reagents. We applied the technique to site-specific modification of monoclonal antibodies, the fastest growing class of biopharmaceuticals, as well as membrane-associated and cytosolic proteins expressed in mammalian cells. PMID:19202059

  9. Ribosomal protein L10 is encoded in the mitochondrial genome of many land plants and green algae

    PubMed Central

    2009-01-01

    Background The mitochondrial genomes of plants generally encode 30-40 identified protein-coding genes and a large number of lineage-specific ORFs. The lack of wide conservation for most ORFs suggests they are unlikely to be functional. However, an ORF, termed orf-bryo1, was recently found to be conserved among bryophytes suggesting that it might indeed encode a functional mitochondrial protein. Results From a broad survey of land plants, we have found that the orf-bryo1 gene is also conserved in the mitochondria of vascular plants and charophycean green algae. This gene is actively transcribed and RNA edited in many flowering plants. Comparative sequence analysis and distribution of editing suggests that it encodes ribosomal protein L10 of the large subunit of the ribosome. In several lineages, such as crucifers and grasses, where the rpl10 gene has been lost from the mitochondrion, we suggest that a copy of the nucleus-encoded chloroplast-derived rpl10 gene may serve as a functional replacement. Conclusion Despite the fact that there are now over 20 mitochondrial genome sequences for land plants and green algae, this gene has remained unidentified and largely undetected until now because of the unlikely coincidence that most of the earlier sequences were from the few lineages that lack the intact gene. These results illustrate the power of comparative sequencing to identify novel genomic features. PMID:19917118

  10. Identification and characterization of the gltK gene encoding a membrane-associated glucose transport protein of pseudomonas aeruginosa.

    PubMed

    Adewoye, L O; Worobec, E A

    2000-08-01

    The Pseudomonas aeruginosa oprB gene encodes the carbohydrate-selective OprB porin, which translocates substrate molecules across the outer membrane to the periplasmic glucose-binding protein. We identified and cloned two open reading frames (ORFs) flanking the oprB gene but are not in operonic arrangement with the oprB gene. The downstream ORF encodes a putative polypeptide homologous to members of a family of transcriptional repressors, whereas the oprB gene is preceded by an ORF encoding a putative product, which exhibits strong homology to several carbohydrate transport ATP-binding cassette (ABC) proteins. The genomic copy of the upstream ORF was mutagenized by homologous recombination. Analysis of the deletion mutant in comparison with the wild type revealed a significant reduction in [14C] glucose transport activity in the mutant strain, suggesting that this ORF likely encodes the inner membrane component of the glucose ABC transporter. It is thus designated gltK gene to reflect its homology to the Pseudomona fluorescens mtlK and its involvement in the high-affinity glucose transport system. Multiple alignment analysis revealed that the P. aeruginosa gltK gene product is a member of the MalK subfamily of ABC proteins. PMID:10940570