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Sample records for 51-kda subunit maps

  1. The neuronal nicotinic acetylcholine receptor {alpha}7 subunit gene: Cloning, mapping, structure, and targeting in mouse

    SciTech Connect

    Orr-Urtreger, A.; Baldini, A.; Beaudet, A.L.

    1994-09-01

    The neuronal nicotinic acetylcholine receptor {alpha}7 subunit is a member of a family of ligand-gated ion channels, and is the only subunit know to bind {alpha}-bungarotoxin in mammalian brain. {alpha}-Bungarotoxin binding sites are known to be more abundant in the hippocampus of mouse strains that are particularly sensitive to nicotine-induced seizures. The {alpha}7 receptor is highly permeable to calcium, which could suggest a role in synaptic plasticity in the nervous system. Auditory gating deficiency, an abnormal response to a second auditory stimulus, is characteristic of schizophrenia. Mouse strains that exhibit a similar gating deficit have reduced hippocampal expression of the {alpha}7 subunit. We have cloned and sequenced the full length cDNA for the mouse {alpha}7 gene (Acra-7) and characterized its gene structure. The murine {alpha}7 shares amino acid identity of 99% and 93% with the rat and human {alpha}7 subunits, respectively. Using an interspecies backcross panel, the murine gene was mapped to chromosome 7 near the p locus, a region syntenic with human chromosome 15; the human gene (CHRNA7) was confirmed to map to 15q13-q14 by FISH. To generate a mouse {alpha}7 mutant by homologous recombination, we have constructed a replacement vector which will delete transmembrane domains II-IV and the cytoplasmic domain from the gene product. Recombinant embryonic stem (ES) cell clones were selected and used to develop mouse chimeras that are currently being bred to obtain germline transmission.

  2. Cytochrome oxidase subunit V gene of Neurospora crassa: DNA sequences, chromosomal mapping, and evidence that the cya-4 locus specifies the structural gene for subunit V.

    PubMed Central

    Sachs, M S; Bertrand, H; Metzenberg, R L; RajBhandary, U L

    1989-01-01

    The sequences of cDNA and genomic DNA clones for Neurospora cytochrome oxidase subunit V show that the protein is synthesized as a 171-amino-acid precursor containing a 27-amino-acid N-terminal extension. The subunit V protein sequence is 34% identical to that of Saccharomyces cerevisiae subunit V; these proteins, as well as the corresponding bovine subunit, subunit IV, contain a single hydrophobic domain which most likely spans the inner mitochondrial membrane. The Neurospora crassa subunit V gene (cox5) contains two introns, 398 and 68 nucleotides long, which share the conserved intron boundaries 5'GTRNGT...CAG3' and the internal consensus sequence ACTRACA. Two short sequences, YGCCAG and YCCGTTY, are repeated four times each in the cox5 gene upstream of the mRNA 5' termini. The cox5 mRNA 5' ends are heterogeneous, with the major mRNA 5' end located 144 to 147 nucleotides upstream from the translational start site. The mRNA contains a 3'-untranslated region of 186 to 187 nucleotides. Using restriction-fragment-length polymorphism, we mapped the cox5 gene to linkage group IIR, close to the arg-5 locus. Since one of the mutations causing cytochrome oxidase deficiency in N. crassa, cya-4-23, also maps there, we transformed the cya-4-23 strain with the wild-type cox5 gene. In contrast to cya-4-23 cells, which grow slowly, cox5 transformants grew quickly, contained cytochrome oxidase, and had 8- to 11-fold-higher levels of subunit V in their mitochondria. These data suggest (i) that the cya-4 locus in N. crassa specifies structural information for cytochrome oxidase subunit V and (ii) that, in N. crassa, as in S. cerevisiae, deficiencies in the production of nuclearly encoded cytochrome oxidase subunits result in deficiency in cytochrome oxidase activity. Finally, we show that the lower levels of subunit V in cya-4-23 cells are most likely due to substantially reduced levels of translatable subunit V mRNA. Images PMID:2540423

  3. Mapping of a conformational epitope on the cashew allergen Ana o 2: a discontinuous large subunit epitope dependent upon homologous or heterologous small subunit association.

    PubMed

    Xia, Lixin; Willison, LeAnna N; Porter, Lauren; Robotham, Jason M; Teuber, Suzanne S; Sathe, Shridhar K; Roux, Kenneth H

    2010-05-01

    The 11S globulins are members of the cupin protein superfamily and represent an important class of tree nut allergens for which a number of linear epitopes have been mapped. However, specific conformational epitopes for these allergens have yet to be described. We have recently reported a cashew Ana o 2 conformational epitope defined by murine mAb 2B5 and competitively inhibited by a subset of patient IgE antibodies. The 2B5 epitope appears to reside on the large (acidic) subunit, is dependent upon small (basic) subunit association for expression, and is highly susceptible to denaturation. Here we fine map the epitope using a combination of recombinant chimeric cashew Ana o 2-soybean Gly m 6 chimeras, deletion and point mutations, molecular modeling, and electron microscopy of 2B5-Ana o 2 immune complexes. Key residues appear confined to a 24 amino acid segment near the N-terminus of the large subunit peptide, a portion of which makes direct contact with the small subunit. These data provide an explanation for both the small subunit dependence and the structurally labile nature of the epitope.

  4. Mapping of the alpha-bungarotoxin binding site within the alpha subunit of the acetylcholine receptor.

    PubMed Central

    Neumann, D; Barchan, D; Safran, A; Gershoni, J M; Fuchs, S

    1986-01-01

    Synthetic peptides and their respective antibodies have been used in order to map the alpha-bungarotoxin binding site within the alpha subunit of the acetylcholine receptor. By using antibodies to a synthetic peptide corresponding to residues 169-181 of the alpha subunit, we demonstrate that this sequence is included within the 18-kDa toxin binding fragment previously reported. Furthermore, the 18-kDa fragment was also found to bind a monoclonal antibody (5.5) directed against the cholinergic binding site. Sequential proteolysis of the acetylcholine receptor with trypsin, prior to Staphylococcus aureus V8 protease digestion, resulted in a 15-kDa toxin binding fragment that is included within the 18-kDa fragment but is shorter than it only at its carboxyl terminus. This 15-kDa fragment therefore initiates beyond Asp-152 and terminates in the region of Arg-313/Lys-314. In addition, experiments are reported that indicate that in the intact acetylcholine receptor, Cys-128 and/or Cys-142 are not crosslinked by disulfide bridges with any of the cysteines (at positions 192, 193, and 222) that reside in the 15-kDa toxin binding fragment. Finally, the synthetic dodecapeptide Lys-His-Trp-Val-Tyr-Tyr-Thr-Cys-Cys-Pro-Asp-Thr, which is present in the 15-kDa fragment (corresponding to residues 185-196 of the alpha subunit) was shown to bind alpha-bungarotoxin directly. This binding was completely inhibited by competition with d-tubocurarine. Images PMID:3458258

  5. Mapping of the alpha-bungarotoxin binding site within the alpha subunit of the acetylcholine receptor.

    PubMed

    Neumann, D; Barchan, D; Safran, A; Gershoni, J M; Fuchs, S

    1986-05-01

    Synthetic peptides and their respective antibodies have been used in order to map the alpha-bungarotoxin binding site within the alpha subunit of the acetylcholine receptor. By using antibodies to a synthetic peptide corresponding to residues 169-181 of the alpha subunit, we demonstrate that this sequence is included within the 18-kDa toxin binding fragment previously reported. Furthermore, the 18-kDa fragment was also found to bind a monoclonal antibody (5.5) directed against the cholinergic binding site. Sequential proteolysis of the acetylcholine receptor with trypsin, prior to Staphylococcus aureus V8 protease digestion, resulted in a 15-kDa toxin binding fragment that is included within the 18-kDa fragment but is shorter than it only at its carboxyl terminus. This 15-kDa fragment therefore initiates beyond Asp-152 and terminates in the region of Arg-313/Lys-314. In addition, experiments are reported that indicate that in the intact acetylcholine receptor, Cys-128 and/or Cys-142 are not crosslinked by disulfide bridges with any of the cysteines (at positions 192, 193, and 222) that reside in the 15-kDa toxin binding fragment. Finally, the synthetic dodecapeptide Lys-His-Trp-Val-Tyr-Tyr-Thr-Cys-Cys-Pro-Asp-Thr, which is present in the 15-kDa fragment (corresponding to residues 185-196 of the alpha subunit) was shown to bind alpha-bungarotoxin directly. This binding was completely inhibited by competition with d-tubocurarine.

  6. Mapping the subunit interface of ribonucleotide reductase (RNR) using photo cross-linking.

    PubMed

    Hassan, A Quamrul; Stubbe, JoAnne

    2008-11-15

    Escherichia coli ribonucleotide reductase (RNR) catalyzes the conversion of nucleoside 5'-diphosphates to deoxynucleoside 5'-diphosphates and is a 1:1 complex of two homodimeric subunits: alpha2 and beta2. As a first step towards mapping the subunit interface, beta2 (V365C) was labeled with [(14)C]-benzophenone (BP) iodoacetamide. The resulting [(14)C]-BP-beta2 (V365C) was complexed with alpha2 and irradiated at 365nm for 30min at 4 degrees C. The cross-linked mixture was purified by anion exchange chromatography and digested with trypsin. The peptides were purified by reverse phase chromatography, identified by scintillation counting and analyzed by Edman sequencing. Three [(14)C]-labeled peptides were identified: two contained a peptide in beta to which the BP was attached. The third contained the same beta peptide and a peptide in alpha found in its alphaD helix. These results provide direct support for the proposed docking model of alpha2beta2.

  7. Temperature-dependent instability of the cTnI subunit in NIST SRM2921 characterized by tryptic peptide mapping.

    PubMed

    van der Burgt, Yuri E M; Cobbaert, Christa M; Dalebout, Hans; Smit, Nico; Deelder, André M

    2012-08-01

    In this study temperature-dependent instability of the cTnI subunit of the three-protein complex NIST SRM2921 was demonstrated using a mass spectrometric tryptic peptide mapping approach. The results were compared to the cTnI subunit obtained as a protein standard from Calbiochem with identical amino acid sequence. Both the three-protein complex from NIST as well as the cTnI subunit were incubated at elevated temperatures and then evaluated with respect to the primary sequence. The corresponding peptide maps were analyzed using LC-MS/MS. From a Mascot database search in combination with "semiTrypsin" tolerance it was found that two peptide backbone cleavages had occurred in subunit cTnI in NIST SRM2921 material upon incubation at 37°C, namely between amino acids at 148/149 and 194/195. The Calbiochem standard did not show increased levels of "unexpected" peptides in tryptic peptide maps. One of the two peptide backbone cleavages could also be monitored using a "single-step" MALDI-MS approach, i.e. without the need for peptide separation. The amount of degradation appeared rather constant in replicate temperature-instability experiments. However, for accurate quantification internal labelled standards are needed. Copyright © 2012 Elsevier B.V. All rights reserved.

  8. Fine mapping of sequential neutralization epitopes on the subunit protein VP8 of human rotavirus.

    PubMed Central

    Kovacs-Nolan, Jennifer; Yoo, Dongwan; Mine, Yoshinori

    2003-01-01

    The epitopes of the HRV (human rotavirus), especially those involved in virus neutralization, have not been determined in their entirety, and would have significant implications for HRV vaccine development. In the present study, we report on the epitope mapping and identification of sequential neutralization epitopes, on the Wa strain HRV subunit protein VP8, using synthetic overlapping peptides. Polyclonal antibodies against recombinant Wa VP8 were produced previously in chicken, and purified from egg yolk, which showed neutralizing activity against HRV in vitro. Overlapping VP8 peptide fragments were synthesized and probed with the anti-VP8 antibodies, revealing five sequential epitopes on VP8. Further analysis suggested that three of the five epitopes detected, M1-L10, I55-D66 and L223-P234, were involved in virus neutralization, indicating that sequential epitopes may also be important for the HRV neutralization. The interactions of the antibodies with the five epitopes were characterized by an examination of the critical amino acids involved in antibody binding. Epitopes comprised primarily of hydrophobic amino acid residues, followed by polar and charged residues. The more critical amino acids appeared to be located near the centre of the epitopes, with proline, isoleucine, serine, glutamine and arginine playing an important role in the binding of antibody to the VP8 epitopes. PMID:12901721

  9. The Gα4 G protein subunit interacts with the MAP kinase ERK2 using a D-motif that regulates developmental morphogenesis in Dictyostelium

    PubMed Central

    Nguyen, Hoai-Nghia; Hadwiger, Jeffrey A.

    2009-01-01

    G protein Gα subunits contribute to the specificity of different signal transduction pathways in Dictyostelium discoideum but Gα subunit-effector interactions have not been previously identified. The requirement of the Dictyostelium Gα4 subunit for MAP kinase (MAPK) activation and the identification of a putative MAPK docking site (D-motif) in this subunit suggested a possible interaction between the Gα4 subunit and MAPKs. In vivo association of the Gα4 subunit and ERK2 was demonstrated by pull-down and co-immunoprecipitation assays. Alteration of the D-motif reduced Gα4 subunit-ERK2 interactions but only slightly altered MAPK activation in response to folate. Expression of the Gα4 subunit with the altered D-motif in gα4− cells allowed for slug formation but not the morphogenesis associated with culmination. Expression of this mutant Gα4 subunit was sufficient to rescue chemotactic movement to folate. Alteration of the D-motif also reduced the aggregation defect associated with constitutively active Gα4 subunits. These results suggest Gα4 subunit-MAPK interactions are necessary for developmental morphogenesis but not for chemotaxis to folate. PMID:19765570

  10. Exon junction complex subunits are required to splice Drosophila MAP kinase, a large heterochromatic gene

    PubMed Central

    Roignant, Jean-Yves; Treisman, Jessica E.

    2010-01-01

    Summary The exon junction complex (EJC) is assembled on spliced mRNAs upstream of exon-exon junctions, and can regulate their subsequent translation, localization, or degradation. We isolated mutations in Drosophila mago nashi (mago), which encodes a core EJC subunit, based on their unexpectedly specific effects on photoreceptor differentiation. Loss of Mago prevents Epidermal growth factor receptor signaling, due to a large reduction in MAPK mRNA levels. MAPK expression also requires the EJC subunits Y14 and eIF4AIII, and EJC-associated splicing factors. Mago depletion does not affect the transcription or stability of MAPK mRNA, but alters its splicing pattern. MAPK expression from an exogenous promoter requires Mago only when the template includes introns. MAPK is the primary functional target of mago in eye development; in cultured cells, Mago knockdown disproportionately affects other large genes located in heterochromatin. These data support a nuclear role for EJC components in splicing a specific subset of introns. PMID:20946982

  11. Tryptic mapping and membrane topology of the benzodiazepine receptor alpha-subunit

    SciTech Connect

    Lentes, K.U.; Venter, J.C.

    1986-05-01

    Rat brain membrane benzodiazepine receptors (BZR) were photoaffinity labelled specifically (in presence or absence of 6 ..mu..M clonazepam) with 10 nM /sup 3/H-flunitrazepam (FNZ). Digestion of the FNZ-labelled, membrane-bound BZR with 200 ..mu..g trypsin/mg membrane protein yielded H/sub 2/O-soluble BZR-fragments of molecular mass (M/sub r/) 34, 31, 28, 24, 21, 18, 16, 12, 10 and 7kDa. Because the 34kDa-peptide is the largest fragment containing a FNZ-binding site they conclude that this represents the extracellular domain of the BZR. In the remaining pellet two labelled peptides with M/sub r/ of 44kDa and 28kDa were found that required the use of detergents for their solubilization; they therefore contain the membrane anchoring domain. Digestion of the 0.5% Na-deoxycholate solubilized, intact BZR (M/sub r/ 51kDa) resulted in the same tryptic pattern as the membrane form of the receptor plus two larger fragments of M/sub r/ 45kDa and 40kDa. Arrangement of all tryptic fragments with reference to the FNZ binding site reveals a membrane topology of the BZR alpha-subunit with 67% (34kDa) for the extracellular domain, 21% (11kDa) for the membrane anchoring domain and 12% (6kDa) for a putative cytoplasmic domain. The overlap between some of the labelled fragments suggest that the BZ binding site must be located near the membrane surface of the extracellular domain.

  12. Structural features of the γ subunit of the Escherichia coli F1 ATPase revealed by a 4.4-Å resolution map obtained by x-ray crystallography

    PubMed Central

    Hausrath, Andrew C.; Grüber, Gerhard; Matthews, Brian W.; Capaldi, Roderick A.

    1999-01-01

    The F1 part of the F1FO ATP synthase from Escherichia coli has been crystallized and its structure determined to 4.4-Å resolution by using molecular replacement based on the structure of the beef-heart mitochondrial enzyme. The bacterial F1 consists of five subunits with stoichiometry α3, β3, γ, δ, and ɛ. δ was removed before crystallization. In agreement with the structure of the beef-heart mitochondrial enzyme, although not that from rat liver, the present study suggests that the α and β subunits are arranged in a hexagonal barrel but depart from exact 3-fold symmetry. In the structures of both beef heart and rat-liver mitochondrial F1, less than half of the structure of the γ subunit was seen because of presumed disorder in the crystals. The present electron-density map includes a number of rod-shaped features which appear to correspond to additional α-helical regions within the γ subunit. These suggest that the γ subunit traverses the full length of the stalk that links the F1 and FO parts and makes significant contacts with the c subunit ring of FO. PMID:10570135

  13. Domain mapping of the retinal cyclic GMP phosphodiesterase gamma-subunit. Function of the domains encoded by the three exons of the gamma-subunit gene.

    PubMed

    Takemoto, D J; Hurt, D; Oppert, B; Cunnick, J

    1992-02-01

    Retinal rod-outer-segment phosphodiesterase (PDE) is a heterotetramer consisting of two similar, but not identical, catalytic subunits (alpha and beta) and two identical inhibitory subunits (gamma 2). Previously, we have reported that the site of PDE alpha/beta interaction with PDE gamma is located within residues 54-87 [Cunnick, Hurt, Oppert, Sakamoto & Takemoto (1990) Biochem. J. 271, 721-727]. The site for PDE gamma interaction with transducin alpha (T alpha) was found to encompass residues 24-45 of PDE gamma [Morrison, Cunnick, Oppert & Takemoto (1989) J. Biol. Chem. 264, 11671-11681]. In order to identify binding sites and other functional domains of PDE gamma, the three peptides which are encoded by the three exons of the PDE gamma gene were synthesized chemically. These exons encode for residues 1-49, 50-62 and 63-87 of bovine PDE gamma [Piriev, Purishko, Khramtsov & Lipkin (1990) Dokl. Akad. Nauk. SSSR 315, 229-230]. The peptide encompassing residues 63-87 was inhibitory in a PDE assay, whereas peptides 1-49 and 50-62 had no effect. However, both peptides 1-49 and 63-87 bound to PDE alpha/beta in a solid-phase binding assay. Only peptide 1-49 bound to T alpha.GTP[S] (GTP[S] is guanosine 5'-[gamma-thio]triphosphate). These data confirm that the inhibitory region of PDE gamma is encoded by exon 3 (residues 63-87), whereas a separate binding site for PDE alpha/beta and for T alpha.GTP[S] is encoded by exon 1 (residues 1-49). To study further the structure-function relationship of PDE gamma, this entire protein and two mutants were chemically synthesized. One mutant (-CT) lacked residues 78-87, whereas another replaced tyrosine-84 with glycine (TYR-84). Whereas the synthetic PDE gamma inhibited PDE alpha/beta catalytic activity, the -CT and TVR-84 mutants did not. All three synthetic proteins bound to both PDE alpha/beta and and T alpha.GTP[S]. These data confirm the presence of an alternative binding site on PDE gamma and demonstrate the importance of tyrosine

  14. Mapping of the gene for the p60 subunit of the human chromatin assembly factor (CAF1A) to the Down syndrome region of chromosome 21

    SciTech Connect

    Blouin, J.L.; Gos, A.; Morris, M.A.; Antonarakis, S.E.

    1996-04-15

    Exon trapping was used to clone portions of genes from the Down syndrome critical region (DSCR) of human chromosome 21. One trapped sequence showed complete homology with nucleotide sequence U20980 (GenBank), which corresponds to the gene for the p60 subunit of the human chromatin assembly factor-1 (CAF1A). We mapped this gene to human chromosome 21 by fluorescence in situ hybridization, by the use of somatic cell hybrids, and by hybridization to chromosome 21-specific YACs and cosmids. The CAF1A gene localizes to YACs 745H11 and 230E8 of the Chumakov et al. YAC contig, within the DSCR on 21q22. This CAF1A, which belongs to the WD-motif family of genes and interacts with other polypeptide subunits to promote assembly of histones to replicating DNA, may contribute in a gene dosage-dependent manner to the phenotype of Down syndrome. 22 refs., 1 fig.

  15. Biochemical mapping of interactions within the intraflagellar transport (IFT) B core complex: IFT52 binds directly to four other IFT-B subunits.

    PubMed

    Taschner, Michael; Bhogaraju, Sagar; Vetter, Melanie; Morawetz, Michaela; Lorentzen, Esben

    2011-07-29

    Cilia and flagella are complex structures emanating from the surface of most eukaroytic cells and serve important functions including motility, signaling, and sensory reception. A process called intraflagellar transport (IFT) is of central importance to ciliary assembly and maintenance. The IFT complex is required for this transport and consists of two distinct multisubunit subcomplexes, IFT-A and IFT-B. Despite the importance of the IFT complex, little is known about its overall architecture. This paper presents a biochemical dissection of the molecular interactions within the IFT-B core complex. Two stable subcomplexes consisting of IFT88/70/52/46 and IFT81/74/27/25 were recombinantly co-expressed and purified. We identify a novel interaction between IFT70/52 and map the interaction domains between IFT52 and the other subunits within the IFT88/70/52/46 complex. Additionally, we show that IFT52 binds directly to the IFT81/74/27/25 complex, indicating that it could mediate the interaction between the two subcomplexes. Our data lead to an improved architectural map for the IFT-B core complex with new interactions as well as domain resolution mapping for several subunits.

  16. Functional reorganization of visual cortex maps after ischemic lesions is accompanied by changes in expression of cytoskeletal proteins and NMDA and GABA(A) receptor subunits.

    PubMed

    Zepeda, Angelica; Sengpiel, Frank; Guagnelli, Miguel Angel; Vaca, Luis; Arias, Clorinda

    2004-02-25

    Reorganization of cortical representations after focal visual cortex lesions has been documented. It has been suggested that functional reorganization may rely on cellular mechanisms involving modifications in the excitatory/inhibitory neurotransmission balance and on morphological changes of neurons peripheral to the lesion. We explored functional reorganization of cortical retinotopic maps after a focal ischemic lesion in primary visual cortex of kittens using optical imaging of intrinsic signals. After 1, 2, and 5 weeks postlesion (wPL), we addressed whether functional reorganization correlated in time with changes in the expression of MAP-2, GAP-43, GFAP, GABA(A) receptor subunit alpha1 (GABA(A)alpha1), subunit 1 of the NMDA receptor (NMDAR1), and in neurotransmitter levels at the border of the lesion. Our results show that: (1) retinotopic maps reorganize with time after an ischemic lesion; (2) MAP-2 levels increase gradually from 1wPL to 5wPL; (3) MAP-2 upregulation is associated with an increase in dendritic-like structures surrounding the lesion and a decrease in GFAP-positive cells; (4) GAP-43 levels reach the highest point at 2wPL; (5) NMDAR1 and glutamate contents increase in parallel from 1wPL to 5wPL; (6) GABA(A)alpha1 levels increase from 1wPL to 2wPL but do not change after this time point; and (7) GABA contents remain low from 1wPL to 5wPL. This is a comprehensive study showing for the first time that functional reorganization correlates in time with dendritic sprouting and with changes in the excitatory/inhibitory neurotransmission systems previously proposed to participate in cortical remodeling and suggests mechanisms by which plasticity of cortical representations may occur.

  17. Genetic contribution to iron status: SNPs related to iron deficiency anaemia and fine mapping of CACNA2D3 calcium channel subunit.

    PubMed

    Baeza-Richer, Carlos; Arroyo-Pardo, Eduardo; Blanco-Rojo, Ruth; Toxqui, Laura; Remacha, Angel; Vaquero, M Pilar; López-Parra, Ana M

    2015-12-01

    Numerous studies associate genetic markers with iron- and erythrocyte-related parameters, but few relate them to iron-clinical phenotypes. Novel SNP rs1375515, located in a subunit of the calcium channel gene CACNA2D3, is associated with a higher risk of anaemia. The aim of this study is to further investigate the association of this SNP with iron-related parameters and iron-clinical phenotypes, and to explore the potential role of calcium channel subunit region in iron regulation. Furthermore, we aim to replicate the association of other SNPs reported previously in our population. We tested 45 SNPs selected via systematic review and fine mapping of CACNA2D3 region, with haematological and biochemical traits in 358 women of reproductive age. Multivariate analyses include back-step logistic regression and decision trees. The results replicate the association of SNPs with iron-related traits, and also confirm the protective effect of both A allele of rs1800562 (HFE) and G allele of rs4895441 (HBS1L-MYB). The risk of developing anaemia is increased in reproductive age women carriers of A allele of rs1868505 (CACNA2D3) and/or T allele of rs13194491 (HIST1H2BJ). Association of SNPs from fine mapping with ferritin and serum iron suggests that calcium channels could be a potential pathway for iron uptake in physiological conditions.

  18. Genetic mapping of the LMP2 proteasome subunit gene to the BoLA class IIb region

    SciTech Connect

    Shalhevet, D.; Da, Y.; Beever, J.E.; Eijk, M.J.T. van; Ma, R.; Lewin, H.A.; Gaskins, H.R.

    1995-01-01

    Recent identification of four tightly-linked genes within the class II region of the major histocompatibility complex (MHC) in humans and rodents has led to a better understanding of class I antigen processing mechanisms. Two of these genes, LMP2 and LMP7, encode subunits of a low molecular mass poypeptide (LMP) complex. Several observations suggest that the LMP complex may be the proteolytic system responsible for generating the size-restricted peptides required for MHC class I assembly. For example, the LMP complex is a large cytoplasmic structure that is antigenically and biochemically related to the proteasome, a proteolytic complex that mediates degradation of ubiquitinated substrates. Data regarding proteolytic specificity indicates that the LMP complex may specifically produce nonamers, the appropriate peptide size for class I binding. In addition, similar to all components of the class I assembly process, intra-MHC LMP genes are regulated by IFN{gamma}. 26 refs., 2 figs., 1 tab.

  19. A susceptibility locus for lung cancer maps to nicotinic acetylcholine receptor subunit genes on 15q25.

    PubMed

    Hung, Rayjean J; McKay, James D; Gaborieau, Valerie; Boffetta, Paolo; Hashibe, Mia; Zaridze, David; Mukeria, Anush; Szeszenia-Dabrowska, Neonilia; Lissowska, Jolanta; Rudnai, Peter; Fabianova, Eleonora; Mates, Dana; Bencko, Vladimir; Foretova, Lenka; Janout, Vladimir; Chen, Chu; Goodman, Gary; Field, John K; Liloglou, Triantafillos; Xinarianos, George; Cassidy, Adrian; McLaughlin, John; Liu, Geoffrey; Narod, Steven; Krokan, Hans E; Skorpen, Frank; Elvestad, Maiken Bratt; Hveem, Kristian; Vatten, Lars; Linseisen, Jakob; Clavel-Chapelon, Françoise; Vineis, Paolo; Bueno-de-Mesquita, H Bas; Lund, Eiliv; Martinez, Carmen; Bingham, Sheila; Rasmuson, Torgny; Hainaut, Pierre; Riboli, Elio; Ahrens, Wolfgang; Benhamou, Simone; Lagiou, Pagona; Trichopoulos, Dimitrios; Holcátová, Ivana; Merletti, Franco; Kjaerheim, Kristina; Agudo, Antonio; Macfarlane, Gary; Talamini, Renato; Simonato, Lorenzo; Lowry, Ray; Conway, David I; Znaor, Ariana; Healy, Claire; Zelenika, Diana; Boland, Anne; Delepine, Marc; Foglio, Mario; Lechner, Doris; Matsuda, Fumihiko; Blanche, Helene; Gut, Ivo; Heath, Simon; Lathrop, Mark; Brennan, Paul

    2008-04-03

    Lung cancer is the most common cause of cancer death worldwide, with over one million cases annually. To identify genetic factors that modify disease risk, we conducted a genome-wide association study by analysing 317,139 single-nucleotide polymorphisms in 1,989 lung cancer cases and 2,625 controls from six central European countries. We identified a locus in chromosome region 15q25 that was strongly associated with lung cancer (P = 9 x 10(-10)). This locus was replicated in five separate lung cancer studies comprising an additional 2,513 lung cancer cases and 4,752 controls (P = 5 x 10(-20) overall), and it was found to account for 14% (attributable risk) of lung cancer cases. Statistically similar risks were observed irrespective of smoking status or propensity to smoke tobacco. The association region contains several genes, including three that encode nicotinic acetylcholine receptor subunits (CHRNA5, CHRNA3 and CHRNB4). Such subunits are expressed in neurons and other tissues, in particular alveolar epithelial cells, pulmonary neuroendocrine cells and lung cancer cell lines, and they bind to N'-nitrosonornicotine and potential lung carcinogens. A non-synonymous variant of CHRNA5 that induces an amino acid substitution (D398N) at a highly conserved site in the second intracellular loop of the protein is among the markers with the strongest disease associations. Our results provide compelling evidence of a locus at 15q25 predisposing to lung cancer, and reinforce interest in nicotinic acetylcholine receptors as potential disease candidates and chemopreventative targets.

  20. The human B22 subunit of the NADH-ubiquinone oxidoreductase maps to the region of chromosome 8 involved in Branchio-oto-renal syndrome

    SciTech Connect

    Gu, J.Z.; Lin, Xin; Wells, D.E.

    1996-07-01

    To identify candidate genes for Branchio-oto-renal (BOR) syndrome, we have made use of a set of cosmids that map to 8q13.3, which has previously been shown to be involved in this syndrome. These cosmids were used as genomic clones in the attempts to isolate corresponding cDNAs using a modified hybrid selection technique. cDNAs using a modified hybrid selection technique. cDNAs from the region were identified and used to search for sequence similarity in human or other species. One cDNA clone was found to have 89% sequence similarity to the bovine B22 subunit of NADH-ubiquinone oxidoreductase, a mitochondrial protein in the respiratory electron transport chain. Given the history of other mitochondrial mutations being involved in hearing loss syndromes, this gene should be considered a strong candidate for involvement in BOR.

  1. Complete nucleotide sequence and mRNA-mapping of the large subunit gene of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from Chlamydomonas moewusii.

    PubMed

    Yang, R C; Dove, M; Seligy, V L; Lemieux, C; Turmel, M; Narang, S A

    1986-01-01

    Nucleotide (nt) sequence of the large subunit (LS) gene of ribulose-1,5-bisphosphate carboxylase/oxygenase from the green alga, Chlamydomonas moewusii, and mapping of transcription ends was achieved by two new strategies. The deduced LS sequence of 475 amino acid residues was compared with similar genes from six other species; cyanobacteria, land plants and a related alga (C. reinhardtii). The most conserved regions are the three ribulose bisphosphate binding sites and the CO2 activator site. The nt sequence conservation outside the coding region is limited to only three segments within the 5'-flanking region: a region of tandem repeats, TATAA box and ribosome-binding site. Termination point of transcription is an 'A' residue 3' to the first of two 18-nt inverted repeats, which has the potential to form a stem-loop hairpin structure. The possible role of these potential regulatory features for transcription and translation, and similar structures in other LS genes is presented.

  2. Tracing the path of DNA substrates in active Tetrahymena telomerase holoenzyme complexes: mapping of DNA contact sites in the RNA subunit

    PubMed Central

    Goldin, Svetlana; Kertesz Rosenfeld, Karin; Manor, Haim

    2012-01-01

    Telomerase, the enzyme that extends single-stranded telomeric DNA, consists of an RNA subunit (TER) including a short template sequence, a catalytic protein (TERT) and accessory proteins. We used site-specific UV cross-linking to map the binding sites for DNA primers in TER within active Tetrahymena telomerase holoenzyme complexes. The mapping was performed at single-nucleotide resolution by a novel technique based on RNase H digestion of RNA–DNA hybrids made with overlapping complementary oligodeoxynucleotides. These data allowed tracing of the DNA path through the telomerase complexes from the template to the TERT binding element (TBE) region of TER. TBE is known to bind TERT and to be involved in the template 5′-boundary definition. Based on these findings, we propose that upstream sequences of each growing telomeric DNA chain are involved in regulation of its growth arrest at the 5′-end of the RNA template. The upstream DNA–TBE interaction may also function as an anchor for the subsequent realignment of the 3′-end of the DNA with the 3′-end of the template to enable initiation of synthesis of a new telomeric repeat. PMID:22584626

  3. Involvement of OS-2 MAP kinase in regulation of the large-subunit catalases CAT-1 and CAT-3 in Neurospora crassa.

    PubMed

    Yamashita, Kazuhiro; Shiozawa, Azusa; Banno, Shinpei; Fukumori, Fumiyasu; Ichiishi, Akihiko; Kimura, Makoto; Fujimura, Makoto

    2007-08-01

    Neurospora crassa has four catalase genes--cat-1, cat-2, cat-3, and ctt-1/cat-4. cat-1 and cat-3 encode two fungal-specific large-subunit catalases CAT-1 and CAT-3 normally produced in conidia and growing hyphae, respectively. cat-2 encodes CAT-2 catalase-peroxidase normally produced in conidia. ctt-1 (or cat-4), of which expression was controlled by OS-2 MAP kinase (Noguchi et al., Fungal Genet. Biol. 44, 208-218), encodes a small-subunit catalase with unknown function. To clarify the contribution of OS-2 on the regulation of CAT-1, CAT-2, and CAT-3, we performed quantitative RT-PCR and in-gel catalase activity analyses. When the hyphae were treated with a fungicide (1 mug/ml fludioxonil) or subjected to an osmotic stress (1 M sorbitol), cat-1 was strongly upregulated and CAT-1 was reasonably induced in the wild-type strain. Interestingly, fludioxonil caused not only the CAT-1 induction but also a remarkable CAT-3 decrease in the wild-type hyphae, implying of an abnormal stimulation of asexual differentiation. These responses were not observed in an os-2 mutant hyphae, indicating an involvement of OS-2 in the cat-1 expression; however, os-2 was dispensable for the production of CAT-1 in conidia. In contrast, the expression of cat-2 was significantly induced by heat shock (45 degrees C) and that of cat-3 was moderately stimulated by an oxidative stress (50 microg/ml methyl viologen) in both the wild-type strain and the os-2 mutant, and corresponding enzyme activities were detected after the treatments. Although basal levels of transcription of cat-1 and cat-3 in an os-2 mutant hyphae were a few-fold lower than in the wild-type hyphae, the os-2 mutant exhibited a considerably lower levels of CAT-3 activity than the wild-type strain. These findings suggest that OS-2 MAP kinase regulated the expression of cat-1 and cat-3 transcriptionally, and probably that of cat-3 posttranscriptionally, even though the presence of another regulatory system for each of these two

  4. Mapping of the glutamate-cysteine ligase catalytic subunit gene (GLCLC) to human chromosome 6p12 and mouse chromosome 9D-E and of the regulatory subunit gene (GLCLR) to human chromosome 1p21-p22 and mouse chromosome 3H1-3

    SciTech Connect

    Tsuchiya, K.; Disteche, C.M.; Reid, L.L.

    1995-12-10

    Glutamate-cysteine ligase (EC 6.3.2.2, GLCL), formerly called {gamma}-glutamylcysteine synthetase (GCS), is the rate-limiting enzyme in the de novo synthesis of the antioxidant tripeptide glutathione. GLCL consists of a heavy subunit, which possesses catalytic activity and is the site of glutathione feedback inhibition, and a light subunit, which has a regulatory function. Glutathione is ubiquitous in mammalian tissues and performs a variety of functions, including protection from reactive oxygen species through antioxidant properties; detoxification of xenobiotics, organic peroxides, and heavy metals; and maintenance of sulfhydryl groups of other molecules. Increased intracellular levels of glutathione have also been found in tumor cells resistant to chemotherapeutic agents. Increased expression of GLCL in melphalan-resistant myeloma and prostate carcinoma cells and cisplatinum-resistant ovarian carcinoma cells suggests that this enzyme may be involved in glutathione-associated drug resistance. Moreover, GLCL has been shown to be induced by phenolic antioxidants and heavy metals. Recently, Mulcahy and Gipp have shown that the GLCL catalytic subunit gene (GLCLC) contains a putative antioxidant regulatory element, which may explain the responsiveness of this gene to agents that induce oxidative stress. To further our understanding of GLCL, which is linked to such a wide variety of metabolic and physiological functions through its role in glutathione synthesis, we have mapped both the catalytic and regulatory subunit genes (GLCLC and GLCLR) to human and mouse chromosomes by fluorescence in situ hybridization (FISH). 16 refs., 1 fig.

  5. The genes for nicein/kalinin 125- and 100-kDa subunits, candidates for junctional epidermiolysis bullosa, map to chromosomes 1q32 and 1q25-q31

    SciTech Connect

    Vailly, J.; Ortonne, J.P.; Meneguzzi, G.; Szepetowski, P.; Pedeutour, F. ); Mattei, M.G. ); Burgeson, R. )

    1994-05-01

    Expression of nicein is specifically hampered in the severe form of junctional epidermolysis bullosa (JEB), a recessive genodermatosis characterized by blister formation of integument believed to be due to defects in hemidesmosomes. Nicein genes are therefore the prime candidates for involvement in JEB. To map the gene encoding the 125-kDa subunit of nicein, the authors used the cDNA Kal5.5C coding for the amino-terminal domain of the protein. In situ hybridization was carried out on chromosomes in phytohemagglutinin-stimulated blood lymphocytes of healthy donors. In 100 metaphases examined, 153 silver grains were found associated with chromosomes; 45 (29%) of these were located on chromosome 1, and 33 (73%) of these 45 grains mapped to region 1q32.1-q41 with a maximum in band 1q32. To confirm the regional localization of the genes for nicein subunits of 100 and 125 kDa, fluorescence in situ hybridization was performed on normal lymphocytes from two unrelated normal males and fibroblast cell lines GM00257 (karyotype 46,XX, t(1;2)(1q32;2p23)) and GM004088 (46,XY,t(1;4)(q32;p16)). It was thus confirmed that the genes for nicein 125- and 100-kDa subunits are localized at 1q32 and 1q25-q31, respectively. 9 refs., 1 fig.

  6. A new sodium channel alpha-subunit gene (Scn9a) from Schwann cells maps to the Scn1a, Scn2a, Scn3a cluster of mouse chromosome 2.

    PubMed

    Beckers, M C; Ernst, E; Belcher, S; Howe, J; Levenson, R; Gros, P

    1996-08-15

    We have used a total of 27 AXB/BXA recombinant inbred mouse strains to determine the chromosomal location of a newly identified gene encoding an alpha-subunit isoform of the sodium channel from Schwann cells, Scn9a. Linkage analysis established that Scn9a mapped to the proximal segment of mouse chromosome 2. The segregation of restriction fragment length polymorphisms in 145 progeny from a Mus spretus x C57BL/6J backcross indicates that Scn9a is very tightly linked to Scn1a (gene encoding the type I sodium channel alpha-subunit of the brain) and forms part of a cluster of four Scna genes located on mouse chromosome 2.

  7. High-resolution mapping of the [gamma]-aminobutyric acid receptor subunit [beta]3 and [alpha]5 gene cluster on chromosome 15q11-q13, and localization of breakpoints in two Angelman syndrome patients

    SciTech Connect

    Sinnett, D.; Wagstaff, J.; Woolf, E. Harvard Medical School, Boston, MA ); Glatt, K. ); Kirkness, E.J. )Lalande, M. Harvard Medical School, Boston, MA Howard Hughes Medical Inst., Boston, MA )

    1993-06-01

    The [gamma]-aminobutyric acid (GABA[sub A]) receptors are a family of ligand-gated chloride channels constituting the major inhibitory neurotransmitter receptors in the nervous system. In order to determine the genomic organization of the GABA[sub A] receptor [beta]3 subunit gene (GABRB3) and [alpha]5 subunit gene (GABRA5) in chromosome 15q11-q13, the authors have constructed a high-resolution physical map using the combined techniques of field-inversion gel electrophoresis and phage genomic library screening. This map, which covers nearly 1.0 Mb, shows that GABRB3 and GABRA5 are separated by less than 100 kb and are arranged in a head-to-head configuration. GABRB3 encompasses approximately 250 kb, while GABRA5 is contained within 70 kb. This difference in size is due in large part to an intron of 150 kb within GABRB3. The authors have also identified seven putative CpG islands within a 600-kb interval. Chromosomal rearrangement breakpoints -- in one Angelman syndrome (AS) patient with an unbalanced translocation and in another patient with a submicroscopic deletion -- are located within the large GABRB3 intron. These findings will facilitate chromosomal walking strategies for cloning the regions disrupted by the DNA rearrangements in these AS patients and will be valuable for mapping new genes to the AS chromosomal region. 64 refs., 6 figs., 2 tabs.

  8. The Light Chain 1 Subunit of the Microtubule-Associated Protein 1B (MAP1B) Is Responsible for Tiam1 Binding and Rac1 Activation in Neuronal Cells

    PubMed Central

    Henríquez, Daniel R.; Bodaleo, Felipe J.; Montenegro-Venegas, Carolina; González-Billault, Christian

    2012-01-01

    Microtubule-associated protein 1B (MAP1B) is a neuronal protein involved in the stabilization of microtubules both in the axon and somatodendritic compartments. Acute, genetic inactivation of MAP1B leads to delayed axonal outgrowth, most likely due to changes in the post-translational modification of tubulin subunits, which enhances microtubule polymerization. Furthermore, MAP1B deficiency is accompanied by abnormal actin microfilament polymerization and dramatic changes in the activity of small GTPases controlling the actin cytoskeleton. In this work, we showed that MAP1B interacts with a guanine exchange factor, termed Tiam1, which specifically activates Rac1. These proteins co-segregated in neurons, and interact in both heterologous expression systems and primary neurons. We dissected the molecular domains involved in the MAP1B-Tiam1 interaction, and demonstrated that pleckstrin homology (PH) domains in Tiam1 are responsible for MAP1B binding. Interestingly, only the light chain 1 (LC1) of MAP1B was able to interact with Tiam1. Moreover, it was able to increase the activity of the small GTPase, Rac1. These results suggest that the interaction between Tiam1 and MAP1B, is produced by the binding of LC1 with PH domains in Tiam1. The formation of such a complex impacts on the activation levels of Rac1 confirming a novel function of MAP1B related with the control of small GTPases. These results also support the idea of cross-talk between cytoskeleton compartments inside neuronal cells. PMID:23300879

  9. The light chain 1 subunit of the microtubule-associated protein 1B (MAP1B) is responsible for Tiam1 binding and Rac1 activation in neuronal cells.

    PubMed

    Henríquez, Daniel R; Bodaleo, Felipe J; Montenegro-Venegas, Carolina; González-Billault, Christian

    2012-01-01

    Microtubule-associated protein 1B (MAP1B) is a neuronal protein involved in the stabilization of microtubules both in the axon and somatodendritic compartments. Acute, genetic inactivation of MAP1B leads to delayed axonal outgrowth, most likely due to changes in the post-translational modification of tubulin subunits, which enhances microtubule polymerization. Furthermore, MAP1B deficiency is accompanied by abnormal actin microfilament polymerization and dramatic changes in the activity of small GTPases controlling the actin cytoskeleton. In this work, we showed that MAP1B interacts with a guanine exchange factor, termed Tiam1, which specifically activates Rac1. These proteins co-segregated in neurons, and interact in both heterologous expression systems and primary neurons. We dissected the molecular domains involved in the MAP1B-Tiam1 interaction, and demonstrated that pleckstrin homology (PH) domains in Tiam1 are responsible for MAP1B binding. Interestingly, only the light chain 1 (LC1) of MAP1B was able to interact with Tiam1. Moreover, it was able to increase the activity of the small GTPase, Rac1. These results suggest that the interaction between Tiam1 and MAP1B, is produced by the binding of LC1 with PH domains in Tiam1. The formation of such a complex impacts on the activation levels of Rac1 confirming a novel function of MAP1B related with the control of small GTPases. These results also support the idea of cross-talk between cytoskeleton compartments inside neuronal cells.

  10. Conformation of yeast 18S rRNA. Direct chemical probing of the 5' domain in ribosomal subunits and in deproteinized RNA by reverse transcriptase mapping of dimethyl sulfate-accessible.

    PubMed Central

    Lempereur, L; Nicoloso, M; Riehl, N; Ehresmann, C; Ehresmann, B; Bachellerie, J P

    1985-01-01

    The structure of the 5' domain of yeast 18S rRNA has been probed by dimethyl sulfate (DMS), either in "native" deproteinized molecules or in the 40S ribosomal subunits. DMS-reacted RNA has been used as a template for reverse transcription and a large number of reactive sites, corresponding to all types of bases have been mapped by a primer extension procedure, taking advantage of blocks in cDNA elongation immediately upstream from bases methylated at atom positions involved in the base-pair recognition of the template. Since the same atom positions are protected from DMS in base-paired nucleotides, the secondary structure status of each nucleotide can be directly assessed in this procedure, thus allowing to evaluate the potential contribution of proteins in modulating subunit rRNA conformation. While the DMS probing of deproteinized rRNA confirms a number of helical stems predicted by phylogenetic comparisons, it is remarkable that a few additional base-pairings, while proven by the comparative analysis, appear to require the presence of the bound ribosomal subunit proteins to be stabilized. Images PMID:2417197

  11. Mapping.

    ERIC Educational Resources Information Center

    Kinney, Douglas M.; McIntosh, Willard L.

    1979-01-01

    The area of geological mapping in the United States in 1978 increased greatly over that reported in 1977; state geological maps were added for California, Idaho, Nevada, and Alaska last year. (Author/BB)

  12. Mapping the domain structure of the influenza A virus polymerase acidic protein (PA) and its interaction with the basic protein 1 (PB1) subunit

    SciTech Connect

    Guu, Tom S.Y.; Dong Liping; Wittung-Stafshede, Pernilla; Tao, Yizhi J.

    2008-09-15

    The influenza A virus polymerase consists of three subunits (PA, PB1, and PB2) necessary for viral RNA synthesis. The heterotrimeric polymerase complex forms through PA interacting with PB1 and PB1 interacting with PB2. PA has been shown to play critical roles in the assembly, catalysis, and nuclear localization of the polymerase. To probe the structure of PA, we isolated recombinant PA from insect cells. Limited proteolysis revealed that PA contained two domains connected by a 20-residue linker (residues 257-276). Far-UV circular dichroism established that the two domains folded into a mixed {alpha}/{beta} structure when separately expressed. In vitro pull-down assays showed that neither individually nor cooperatively expressed PA domains, without the linker, could assure PA-PB1 interaction. Protease treatment of PA-PB1 complex indicated that its PA subunit was significantly more stable than free PA, suggesting that the linker is protected and it constitutes an essential component of the PA-PB1 interface.

  13. Subunits of phycoerythrin from Fremyella diplosiphon: chemical and immunochemical characterization.

    PubMed

    Takemoto, J; Bogorad, L

    1975-03-25

    The alpha and beta subunits of the phycobiliprotein, phycoerythrin, isolated from the filamentous blue-green alga, Fremyella diplosiphon, have been separated by chromatography on Bio-Rex 70 ion exchange resin. Analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis shows no detectable cross-contamination of these subunit preparations. The molar extinction coefficients at 552 nm of the alpha and beta subunits in 8 M urea are 25,549 and 48,456, respectively. The amino acid compositions of the subunits are very similar. Molecular weights of the alpha and beta subunits are 19,500 and 21,700, respectively, based on the amino acid composition analyses. Antisera prepared against the alpha subunit reacts with the beta subunit, and vice versa. Tryptic peptide maps reveal that the subunits share share at least eight common tryptic peptides. These results indicate that the phycoerythrin subunits are chemically very similar.

  14. Complexing of the CD-3 subunit by a monoclonal antibody activates a microtubule-associated protein 2 (MAP-2) serine kinase in Jurkat cells.

    PubMed Central

    Hanekom, C; Nel, A; Gittinger, C; Rheeder, A; Landreth, G

    1989-01-01

    Treatment of Jurkat T-cells with anti-CD-3 monoclonal antibodies resulted in the rapid and transient activation of a serine kinase which utilized the microtubule-associated protein, MAP-2, as a substrate in vitro. The kinase was also activated on treatment of Jurkat cells with phytohaemagglutinin, but with a different time course. The activation of the MAP-2 kinase by anti-CD-3 antibodies was dose-dependent, with maximal activity observed at concentrations of greater than 500 ng/ml. Normal human E-rosette-positive T-cells also exhibited induction of MAP-2 kinase activity during anti-CD-3 treatment. The enzyme was optimally active in the presence of 2 mM-Mn2+; lower levels of activity were observed with Mg2+, even at concentrations up to 20 mM. The kinase was partially purified by passage over DE-52 Sephacel with the activity eluting as a single peak at 0.25 M-NaCl. The molecular mass was estimated to be 45 kDa by gel filtration. The activation of the MAP-2 kinase was probably due to phosphorylation of this enzyme as treatment with alkaline phosphatase diminished its activity. These data demonstrate that the stimulation of T-cells through the CD-3 complex results in the activation of a novel serine kinase which may be critically involved in signal transduction in these cells. Images Fig. 1. Fig. 7. Fig. 8. PMID:2552997

  15. EMatch: an efficient method for aligning atomic resolution subunits into intermediate-resolution cryo-EM maps of large macromolecular assemblies

    SciTech Connect

    Dror, Oranit Lasker, Keren; Nussinov, Ruth; Wolfson, Haim

    2007-01-01

    A method for detecting structural homologs of components in an intermediate resolution cryo-EM map and their spatial configuration is presented. Structural analysis of biological machines is essential for inferring their function and mechanism. Nevertheless, owing to their large size and instability, deciphering the atomic structure of macromolecular assemblies is still considered as a challenging task that cannot keep up with the rapid advances in the protein-identification process. In contrast, structural data at lower resolution is becoming more and more available owing to recent advances in cryo-electron microscopy (cryo-EM) techniques. Once a cryo-EM map is acquired, one of the basic questions asked is what are the folds of the components in the assembly and what is their configuration. Here, a novel knowledge-based computational method, named EMatch, towards tackling this task for cryo-EM maps at 6–10 Å resolution is presented. The method recognizes and locates possible atomic resolution structural homologues of protein domains in the assembly. The strengths of EMatch are demonstrated on a cryo-EM map of native GroEL at 6 Å resolution.

  16. Subunit mass analysis for monitoring antibody oxidation

    PubMed Central

    Sokolowska, Izabela; Mo, Jingjie; Dong, Jia; Lewis, Michael J.; Hu, Ping

    2017-01-01

    ABSTRACT Methionine oxidation is a common posttranslational modification (PTM) of monoclonal antibodies (mAbs). Oxidation can reduce the in-vivo half-life, efficacy and stability of the product. Peptide mapping is commonly used to monitor the levels of oxidation, but this is a relatively time-consuming method. A high-throughput, automated subunit mass analysis method was developed to monitor antibody methionine oxidation. In this method, samples were treated with IdeS, EndoS and dithiothreitol to generate three individual IgG subunits (light chain, Fd’ and single chain Fc). These subunits were analyzed by reversed phase-ultra performance liquid chromatography coupled with an online quadrupole time-of-flight mass spectrometer and the levels of oxidation on each subunit were quantitated based on the deconvoluted mass spectra using the UNIFI software. The oxidation results obtained by subunit mass analysis correlated well with the results obtained by peptide mapping. Method qualification demonstrated that this subunit method had excellent repeatability and intermediate precision. In addition, UNIFI software used in this application allows automated data acquisition and processing, which makes this method suitable for high-throughput process monitoring and product characterization. Finally, subunit mass analysis revealed the different patterns of Fc methionine oxidation induced by chemical and photo stress, which makes it attractive for investigating the root cause of oxidation. PMID:28106519

  17. Subunit mass analysis for monitoring antibody oxidation.

    PubMed

    Sokolowska, Izabela; Mo, Jingjie; Dong, Jia; Lewis, Michael J; Hu, Ping

    2017-04-01

    Methionine oxidation is a common posttranslational modification (PTM) of monoclonal antibodies (mAbs). Oxidation can reduce the in-vivo half-life, efficacy and stability of the product. Peptide mapping is commonly used to monitor the levels of oxidation, but this is a relatively time-consuming method. A high-throughput, automated subunit mass analysis method was developed to monitor antibody methionine oxidation. In this method, samples were treated with IdeS, EndoS and dithiothreitol to generate three individual IgG subunits (light chain, Fd' and single chain Fc). These subunits were analyzed by reversed phase-ultra performance liquid chromatography coupled with an online quadrupole time-of-flight mass spectrometer and the levels of oxidation on each subunit were quantitated based on the deconvoluted mass spectra using the UNIFI software. The oxidation results obtained by subunit mass analysis correlated well with the results obtained by peptide mapping. Method qualification demonstrated that this subunit method had excellent repeatability and intermediate precision. In addition, UNIFI software used in this application allows automated data acquisition and processing, which makes this method suitable for high-throughput process monitoring and product characterization. Finally, subunit mass analysis revealed the different patterns of Fc methionine oxidation induced by chemical and photo stress, which makes it attractive for investigating the root cause of oxidation.

  18. MAPS

    Atmospheric Science Data Center

    2014-07-03

    ... Measurement of Air Pollution from Satellites (MAPS) data were collected during Space Shuttle flights in 1981, ... Facts Correlative Data  - CDIAC - Spring & Fall 1994 - Field and Aircraft Campaigns SCAR-B Block:  ...

  19. The gene for human U2 snRNP auxiliary factor small 35-kDa subunit (U2AF1) maps to the progressive myoclonus epilepsy (EPM1) critical region on chromosome 21q22.3

    SciTech Connect

    Lalioti, M.D.; Rossier, C.; Antonarakis, S.E.

    1996-04-15

    We used targeted exon trapping to clone portions of genes from human chromosome 21q22.3. One trapped sequence showed complete homology with the cDNA of human U2AF{sup 35} (M96982; HGM-approved nomenclature U2AF1), which encodes for the small 35-kDa subunit of the U2 snRNP auxiliary factor. Using the U2AF1 cDNA as a probe, we mapped this gene to cosmid Q15D2, a P1, and YAC 350F7 of the Chumakov et al. contig, close to the cystathionine-{beta}-synthase gene (CBS) on 21q22.3. This localization was confirmed by PCR using oligonucleotides from the 3{prime} UTR and by FISH. As U2AF1 associated with a number of different factors during mRNA splicing, overexpression in trisomy 21 individuals could contribute to some Down syndrome phenotypes by interfering with the splicing process. Furthermore, because this gene maps in the critical region for the progressive myoclonus epilepsy I locus (EPM1), mutation analysis will be carried out in patients to evaluate the potential role of U2AF1 as a candidate for EPM1. 24 refs., 1 fig.

  20. Complete modification maps for the cytosolic small and large subunit rRNAs of Euglena gracilis: functional and evolutionary implications of contrasting patterns between the two rRNA components.

    PubMed

    Schnare, Murray N; Gray, Michael W

    2011-10-14

    In the protist Euglena gracilis, the cytosolic small subunit (SSU) rRNA is a single, covalently continuous species typical of most eukaryotes; in contrast, the large subunit (LSU) rRNA is naturally fragmented, comprising 14 separate RNA molecules instead of the bipartite (28S+5.8S) eukaryotic LSU rRNA typically seen. We present extensively revised secondary structure models of the E. gracilis SSU and LSU rRNAs and have mapped the positions of all of the modified nucleosides in these rRNAs (88 in SSU rRNA and 262 in LSU rRNA, with only 3 LSU rRNA modifications incompletely characterized). The relative proportions of ribose-methylated nucleosides and pseudouridine (∼60% and ∼35%, respectively) are closely similar in the two rRNAs; however, whereas the Euglena SSU rRNA has about the same absolute number of modifications as its human counterpart, the Euglena LSU rRNA has twice as many modifications as the corresponding human LSU rRNA. The increased levels of rRNA fragmentation and modification in E. gracilis LSU rRNA are correlated with a 3-fold increase in the level of mispairing in helical regions compared to the human LSU rRNA. In contrast, no comparable increase in mispairing is seen in helical regions of the SSU rRNA compared to its homologs in other eukaryotes. In view of the reported effects of both ribose-methylated nucleoside and pseudouridine residues on RNA structure, these correlations lead us to suggest that increased modification in the LSU rRNA may play a role in stabilizing a 'looser' structure promoted by elevated helical mispairing and a high degree of fragmentation.

  1. Mapping precursor-binding site on TatC subunit of twin arginine-specific protein translocase by site-specific photo cross-linking.

    PubMed

    Zoufaly, Stefan; Fröbel, Julia; Rose, Patrick; Flecken, Tobias; Maurer, Carlo; Moser, Michael; Müller, Matthias

    2012-04-13

    A number of secreted precursor proteins of bacteria, archaea, and plant chloroplasts stand out by a conserved twin arginine-containing sequence motif in their signal peptides. Many of these precursor proteins are secreted in a completely folded conformation by specific twin arginine translocation (Tat) machineries. Tat machineries are high molecular mass complexes consisting of two types of membrane proteins, a hexahelical TatC protein, and usually one or two single-spanning membrane proteins, called TatA and TatB. TatC has previously been shown to be involved in the recognition of twin arginine signal peptides. We have performed an extensive site-specific cross-linking analysis of the Escherichia coli TatC protein under resting and translocating conditions. This strategy allowed us to map the recognition site for twin arginine signal peptides to the cytosolic N-terminal region and first cytosolic loop of TatC. In addition, discrete contact sites between TatC, TatB, and TatA were revealed. We discuss a tentative model of how a twin arginine signal sequence might be accommodated in the Tat translocase.

  2. Mapping Precursor-binding Site on TatC Subunit of Twin Arginine-specific Protein Translocase by Site-specific Photo Cross-linking*

    PubMed Central

    Zoufaly, Stefan; Fröbel, Julia; Rose, Patrick; Flecken, Tobias; Maurer, Carlo; Moser, Michael; Müller, Matthias

    2012-01-01

    A number of secreted precursor proteins of bacteria, archaea, and plant chloroplasts stand out by a conserved twin arginine-containing sequence motif in their signal peptides. Many of these precursor proteins are secreted in a completely folded conformation by specific twin arginine translocation (Tat) machineries. Tat machineries are high molecular mass complexes consisting of two types of membrane proteins, a hexahelical TatC protein, and usually one or two single-spanning membrane proteins, called TatA and TatB. TatC has previously been shown to be involved in the recognition of twin arginine signal peptides. We have performed an extensive site-specific cross-linking analysis of the Escherichia coli TatC protein under resting and translocating conditions. This strategy allowed us to map the recognition site for twin arginine signal peptides to the cytosolic N-terminal region and first cytosolic loop of TatC. In addition, discrete contact sites between TatC, TatB, and TatA were revealed. We discuss a tentative model of how a twin arginine signal sequence might be accommodated in the Tat translocase. PMID:22362773

  3. Sodium channel auxiliary subunits.

    PubMed

    Tseng, Tsai-Tien; McMahon, Allison M; Johnson, Victoria T; Mangubat, Erwin Z; Zahm, Robert J; Pacold, Mary E; Jakobsson, Eric

    2007-01-01

    Voltage-gated ion channels are well known for their functional roles in excitable tissues. Excitable tissues rely on voltage-gated ion channels and their auxiliary subunits to achieve concerted electrical activity in living cells. Auxiliary subunits are also known to provide functional diversity towards the transport and biogenesis properties of the principal subunits. Recent interests in pharmacological properties of these auxiliary subunits have prompted significant amounts of efforts in understanding their physiological roles. Some auxiliary subunits can potentially serve as drug targets for novel analgesics. Three families of sodium channel auxiliary subunits are described here: beta1 and beta3, beta2 and beta4, and temperature-induced paralytic E (TipE). While sodium channel beta-subunits are encoded in many animal genomes, TipE has only been found exclusively in insects. In this review, we present phylogenetic analyses, discuss potential evolutionary origins and functional data available for each of these subunits. For each family, we also correlate the functional specificity with the history of evolution for the individual auxiliary subunits.

  4. Long-Term Potentiation in the CA1 Hippocampus Induced by NR2A Subunit-Containing NMDA Glutamate Receptors Is Mediated by Ras-GRF2/Erk Map Kinase Signaling

    PubMed Central

    Jin, Shan-xue; Feig, Larry A.

    2010-01-01

    Background NMDA-type glutamate receptors (NMDARs) are major contributors to long-term potentiation (LTP), a form of synaptic plasticity implicated in the process of learning and memory. These receptors consist of calcium-permeating NR1 and multiple regulatory NR2 subunits. A majority of studies show that both NR2A and NR2B-containing NMDARs can contribute to LTP, but their unique contributions to this form of synaptic plasticity remain poorly understood. Methodology/Principal Findings In this study, we show that NR2A and NR2B-containing receptors promote LTP differently in the CA1 hippocampus of 1-month old mice, with the NR2A receptors functioning through Ras-GRF2 and its downstream effector, Erk Map kinase, and NR2B receptors functioning independently of these signaling molecules. Conclusions/Significance This study demonstrates that NR2A-, but not NR2B, containing NMDA receptors induce LTP in pyramidal neurons of the CA1 hippocamus from 1 month old mice through Ras-GRF2 and Erk. This difference add new significance to the observation that the relative levels of these NMDAR subtypes is regulated in neurons, such that NR2A-containing receptors become more prominent late in postnatal development, after sensory experience and synaptic activity. PMID:20661302

  5. Further characterization of the ovine lutropin alpha and beta subunits prepared by the salt precipitation method.

    PubMed

    Sairam, M R

    1979-08-01

    The subunits of ovine lutropin prepared by acid dissociation and salt precipitation were characterized by end group analysis, tryptic peptide mapping, SDS gel electrophoresis and biological activity. No evidence of internal peptide cleavage was found in the alpha subunit. The subunits possessed low activity. The alpha and beta subunits recombined effectively to generate a complex that had full receptor binding activity and in vitro biological activity. The recombinants of subunits prepared by countercurrent distribution showed only 50% activity in both assays. The salt precipitation method alpha subunit could be completely reduced and reoxidized in the absence of denaturants. The reoxidized alpha subunit combines with the native beta subunit generating full activity. However, this recombined hormone tends to lose activity with time, suggesting that the reoxidation may not fully restore the native structur of the reduced alpha subunit. The native lutropin alpha subunit effectively combined with follitropin beta subunit generating complete follitropin activity.

  6. Gel-based chemical cross-linking analysis of 20S proteasome subunit-subunit interactions in breast cancer.

    PubMed

    Song, Hai; Xiong, Hua; Che, Jing; Xi, Qing-Song; Huang, Liu; Xiong, Hui-Hua; Zhang, Peng

    2016-08-01

    The ubiquitin-proteasome system plays a pivotal role in breast tumorigenesis by controlling transcription factors, thus promoting cell cycle growth, and degradation of tumor suppressor proteins. However, breast cancer patients have failed to benefit from proteasome inhibitor treatment partially due to proteasome heterogeneity, which is poorly understood in malignant breast neoplasm. Chemical crosslinking is an increasingly important tool for mapping protein three-dimensional structures and proteinprotein interactions. In the present study, two cross-linkers, bis (sulfosuccinimidyl) suberate (BS(3)) and its water-insoluble analog disuccinimidyl suberate (DSS), were used to map the subunit-subunit interactions in 20S proteasome core particle (CP) from MDA-MB-231 cells. Different types of gel electrophoresis technologies were used. In combination with chemical cross-linking and mass spectrometry, we applied these gel electrophoresis technologies to the study of the noncovalent interactions among 20S proteasome subunits. Firstly, the CP subunit isoforms were profiled. Subsequently, using native/SDSPAGE, it was observed that 0.5 mmol/L BS(3) was a relatively optimal cross-linking concentration for CP subunit-subunit interaction study. 2-DE analysis of the cross-linked CP revealed that α1 might preinteract with α2, and α3 might pre-interact with α4. Moreover, there were different subtypes of α1α2 and α3α4 due to proteasome heterogeneity. There was no significant difference in cross-linking pattern for CP subunits between BS(3) and DSS. Taken together, the gel-based characterization in combination with chemical cross-linking could serve as a tool for the study of subunit interactions within a multi-subunit protein complex. The heterogeneity of 20S proteasome subunit observed in breast cancer cells may provide some key information for proteasome inhibition strategy.

  7. Characterization of the mouse PA28 activator complex gene family: complete organizations of the three member genes and a physical map of the approximately 150-kb region containing the alpha- and beta-subunit genes.

    PubMed

    Kohda, K; Ishibashi, T; Shimbara, N; Tanaka, K; Matsuda, Y; Kasahara, M

    1998-05-15

    The proteasome is a multisubunit protease responsible for the generation of peptides loaded onto MHC class I molecules. Recent evidence indicates that binding of an IFN-gamma-inducible PA28 activator complex to the 20S proteasome enhances the generation of class I binding peptides. The alpha- and beta-subunits, which constitute the PA28 activator complex in the form of an (alphabeta)3 heterohexamer, show significant amino acid sequence similarity to a protein, designated Ki or the gamma-subunit, that is capable of binding to the 20S proteasome. In this study, we describe the complete nucleotide sequences of the mouse genes, Psme1, Psme2, and Psme3, coding for the alpha-, beta-, and gamma-subunits, respectively. The overall exon-intron organizations of the three Psme genes are virtually identical, thus providing evidence that they are descended from a single ancestral gene. The promoter regions of the Psme1 and Psme2 genes contain sequence motifs that qualify as IFN-stimulated response elements, consistent with the observation that their expression is induced strongly by IFN-gamma. The Psme1 and Psme2 genes are located approximately 6 kb apart with their 3'-ends pointing toward each other on bands C2 to D1 of mouse chromosome 14, supporting the idea that they emerged by tandem duplication.

  8. Cloning of the cDNA for the human ATP synthase OSCP subunit (ATP5O) by exon trapping and mapping to chromosome 21q22.1-q22.2

    SciTech Connect

    Chen, Haiming; Morris, M.A.; Rossier, C.

    1995-08-10

    Exon trapping was used to clone portions of potential genes from human chromosome 21. One trapped sequence showed striking homology with the bovine and rat ATP synthase OSCP (oligomycin sensitivity conferring protein) subunit. We subsequently cloned the full-length human ATP synthase OSCP cDNA (GDB/HGMW approved name ATP50) from infant brain and muscle libraries and determined its nucleotide and deduced amino acid sequence (EMBL/GenBank Accession No. X83218). The encoded polypeptide contains 213 amino acids, with more than 80% identity to bovine and murine ATPase OSCP subunits and over 35% identity to Saccharomyces cerevisiae and sweet potato sequences. The human ATP5O gene is located at 21q22.1-q22.2, just proximal to D21S17, in YACs 860G11 and 838C7 of the Chumakov et al. YAC contig. The gene is expressed in all human tissues examined, most strongly in muscle and heart. This ATP5O subunit is a key structural component of the stalk of the mitochondrial respiratory chain F{sub 1}F{sub 0}-ATP synthase and as such may contribute in a gene dosage-dependent manner to the phenotype of Down syndrome (trisomy 21). 39 refs., 5 figs.

  9. PA28 subunits of the mouse proteasome: primary structures and chromosomal localization of the genes.

    PubMed

    Kandil, E; Kohda, K; Ishibashi, T; Tanaka, K; Kasahara, M

    1997-01-01

    The 20S proteasome is a multi-subunit protease responsible for the production of peptides presented by major histocompatibility complex (MHC) class I molecules. Recent evidence indicates that an interferon-gamma (IFN-gamma)-inducible PA28 activator complex enhances the generation of class I binding peptides by altering the cleavage pattern of the proteasome. In the present study, we determined the primary structures of the mouse PA28 alpha- and beta-subunits. The deduced amino acid sequences of the alpha- and beta-subunits were 49% identical. We also determined the primary structure of the mouse PA28 gamma-subunit (Ki antigen), a protein of unknown function structurally related to the alpha- and beta-subunits. The amino acid sequence identity of the gamma-subunit to the alpha- and beta-subunits was 40% and 32%, respectively. Interspecific backcross mapping showed that the mouse genes coding for the alpha- and beta-subunits (designated Psme1 and Psme2, respectively) are tightly linked and map close to the Atp5g1 locus on chromosome 14. Thus, unlike the LMP2 and LMP7 subunits, the IFN-gamma-inducible subunits of PA28 are encoded outside the MHC. The gene coding for the gamma-subunit (designated Psme3) was mapped to the vicinity of the Brca1 locus on chromosome 11. A computer search of the DNA databases identified a gamma-subunit-like protein in ticks and Caenorhabditis elegans, the organisms with no adaptive immune system. It appears that the IFN-gamma-inducible alpha- and beta-subunits emerged by gene duplication from a gamma-subunit-like precursor.

  10. Altered 40 S ribosomal subunits in omnipotent suppressors of yeast.

    PubMed

    Eustice, D C; Wakem, L P; Wilhelm, J M; Sherman, F

    1986-03-20

    The five suppressors SUP35, SUP43, SUP44, SUP45 and SUP46, each mapping at a different chromosomal locus in the yeast Saccharomyces cerevisiae, suppress a wide range of mutations, including representatives of all three types of nonsense mutations, UAA, UAG and UGA. We have demonstrated that ribosomes from the four suppressors SUP35, SUP44, SUP45 and SUP46 translate polyuridylate templates in vitro with higher errors than ribosomes from the normal stain, and that this misreading is substantially enhanced by the antibiotic paromomycin. Furthermore, ribosomal subunit mixing experiments established that the 40 S ribosomal subunit, and this subunit only, is responsible for the higher levels of misreading. Thus, the gene products of SUP35, SUP44, SUP45 and SUP46 are components of the 40 S subunit or are enzymes that modify the subunit. In addition, a protein from the 40 S subunit of the SUP35 suppressor has an altered electrophoretic mobility; this protein is distinct from the altered protein previously uncovered in the 40 S subunit of the SUP46 suppressor. In contrast to the ribosomes from the four suppressors SUP35, SUP44, SUP45 and SUP46, the ribosomes from the SUP43 suppressor do not significantly misread polyuridylate templates in vitro, suggesting that this locus may not encode a ribosomal component or that the misreading is highly specific.

  11. Mapping of the {beta}{sub 2} subunit gene (GABRB2) to microdissected human chromosome 5q34-q35 defines a gene cluster for the most abundant GABA{sub A} receptor isoform

    SciTech Connect

    Russek, S.J.; Farb, D.H. |

    1994-10-01

    The {gamma}-aminobutyric acid receptor (GABA{sub A}R) is a multisubunit Cl{sup -} channel that mediates most fast inhibitory synaptic transmission in the central nervous system. Molecular evolution has given rise to many genetic variants of GABA{sub A}R subunits, including {alpha}{sub 1-6}, {beta}{sub 1-4}, {gamma}{sub 1-4}, {sigma}, and {rho}{sub 1-2}, suggesting that an enormous number of combinations of subunits are possible. Here we report that the {beta}{sub 2} gene is located on chromosome 5q34-q35, defining a cluster comprising {alpha}{sub 1}, {beta}{sub 2}, and {gamma}{sub 2} genes that together code for the most abundant GABA{sub A}R isoform. The fact that intron position is conserved in the {beta}{sub 1-3} genes, taken together with the observation that chromosomes 4 and 15 also contain distinct {alpha}-{beta}-{gamma} gene clusters, strongly suggests that an ancestral {alpha}-{beta}-{gamma} cluster was duplicated and translocated to at least two different chromosomes. This organization of GABA{sub A}R gene clusters may have been preserved as linkage provides a mechanism for facilitating coordinate gene expression. 34 refs., 5 figs., 1 tab.

  12. Interactions among rice ORC subunits.

    PubMed

    Tan, Deyong; Lv, Qundan; Chen, Xinai; Shi, Jianghua; Ren, Meiyan; Wu, Ping; Mao, Chuanzao

    2013-08-01

    The origin recognition complex (ORC) is composed of six subunits and plays an important role in DNA replication in all eukaryotes. The ORC subunits OsORC6 as well as the other five ORC subunits in rice were experimentally isolated and sequenced. It indicated that there also exist six ORC subunits in rice. Results of RT-PCR indicated that expression of all the rice ORC genes are no significant difference under 26°C and 34°C. Yeast two hybridization indicated that OsORC2, -3, -5 interact with each other. OsORC5 can then bind OsORC4 to form the OsORC2, -3,-4,-5 core complex. It suggested that the basic interactions have been conserved through evolution. No binding of OsORC1 and OsORC6 with the other subunits were observed. A model of ORC complex in rice is proposed.

  13. Highly conserved small subunit residues influence rubisco large subunit catalysis.

    PubMed

    Genkov, Todor; Spreitzer, Robert J

    2009-10-30

    The chloroplast enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the rate-limiting step of photosynthetic CO(2) fixation. With a deeper understanding of its structure-function relationships and competitive inhibition by O(2), it may be possible to engineer an increase in agricultural productivity and renewable energy. The chloroplast-encoded large subunits form the active site, but the nuclear-encoded small subunits can also influence catalytic efficiency and CO(2)/O(2) specificity. To further define the role of the small subunit in Rubisco function, the 10 most conserved residues in all small subunits were substituted with alanine by transformation of a Chlamydomonas reinhardtii mutant that lacks the small subunit gene family. All the mutant strains were able to grow photosynthetically, indicating that none of the residues is essential for function. Three of the substitutions have little or no effect (S16A, P19A, and E92A), one primarily affects holoenzyme stability (L18A), and the remainder affect catalysis with or without some level of associated structural instability (Y32A, E43A, W73A, L78A, P79A, and F81A). Y32A and E43A cause decreases in CO(2)/O(2) specificity. Based on the x-ray crystal structure of Chlamydomonas Rubisco, all but one (Glu-92) of the conserved residues are in contact with large subunits and cluster near the amino- or carboxyl-terminal ends of large subunit alpha-helix 8, which is a structural element of the alpha/beta-barrel active site. Small subunit residues Glu-43 and Trp-73 identify a possible structural connection between active site alpha-helix 8 and the highly variable small subunit loop between beta-strands A and B, which can also influence Rubisco CO(2)/O(2) specificity.

  14. Neutron scattering and the 30 S ribosomal subunit of E. coli

    SciTech Connect

    Moore, P.B.; Engelman, D.M.; Langer, J.A.; Ramakrishnan, V.R.; Schindler, D.G.; Schoenborn, B.P.; Sillers, I.Y.; Yabuki, S.

    1982-01-01

    This paper reviews the progress made in the study of the internal organization of the 30 S ribosomal subunit of E. coli by neutron scattering since 1975. A map of that particle showing the position of 14 of the subunit's 21 proteins is presented, and the methods currently used for collecting and analyzing such data are discussed. Also discussed is the possibility of extending the interpretation of neutron mapping data beyond the limits practical today. 30 references, 5 figures.

  15. Neutron Scattering and the 30 S Ribosomal Subunit of E. Coli

    DOE R&D Accomplishments Database

    Moore, P. B.; Engelman, D. M.; Langer, J. A.; Ramakrishnan, V. R.; Schindler, D. G.; Schoenborn, B. P.; Sillers, I. Y.; Yabuki, S.

    1982-06-01

    This paper reviews the progress made in the study of the internal organization of the 30 S ribosomal subunit of E. coli by neutron scattering since 1975. A map of that particle showing the position of 14 of the subunit's 21 proteins is presented, and the methods currently used for collecting and analyzing such data are discussed. Also discussed is the possibility of extending the interpretation of neutron mapping data beyond the limits practical today.

  16. Characterization of the interface between gamma and epsilon subunits of Escherichia coli F1-ATPase.

    PubMed

    Tang, C; Capaldi, R A

    1996-02-09

    The interaction faces of the gamma and epsilon subunits in the Escherichia coli F1-ATPase have been explored by a combination of cross-linking and chemical modification experiments using several mutant epsilon subunits as follows: epsilonS10C, epsilonH38C, epsilonT43C, epsilonS65C, epsilonS108C, and epsilonM138C, along with a mutant of the gamma subunit, gammaT106C. The replacement of Ser-10 by a Cys or Met-138 by a Cys reduced the inhibition of ECF1 by the epsilon subunit, while the mutation S65C increased this inhibitory effect. Modification of the Cys at position 10 with N-ethylmaleimide or fluoroscein maleimide further reduced the binding affinity of, and the maximal inhibition by, the epsilon subunit. Similar chemical modification of the Cys at position 43 of the epsilon subunit (in the mutant epsilonT43C) and a Cys at position 106 of the gamma subunit (gammaT106C) also affected the inhibition of ECF1 by the epsilon subunit. The various epsilon subunit mutants were reacted with TFPAM3, and the site(s) of cross-linking within the ECF1 complex was determined. Previous studies have shown cross-linking from the Cys at positions 10 and 38 with the gamma subunit and from a Cys at position 108 to an alpha subunit (Aggeler, R., Chicas-Cruz, K., Cai, S. X., Keana, J. F. W., and Capaldi, R. A. (1992) Biochemistry 31, 2956-2961; Aggeler, R., Weinreich, F., and Capaldi, R. A. (1995) Biochim. Biophys. Acta 1230, 62-68). Here, cross-linking was found from a Cys at position 43 to the gamma subunit and from the Cys at position 138 to a beta subunit. The site of cross-linking from Cys-10 of epsilon to the gamma subunit was localized by peptide mapping to a region of the gamma subunit between residues 222 and 242. Cross-linking from a Cys at position 38 and at position 43 was with the C-terminal part of the gamma subunit, between residues 202 and 286. ECF1 treated with trypsin at pH 7.0 still binds purified epsilon subunit, while enzyme treated with the protease at pH 8.0 does

  17. Characterization of the basic glutathione S-transferase B1 and B2 subunits from human liver.

    PubMed Central

    Stockman, P K; McLellan, L I; Hayes, J D

    1987-01-01

    The basic glutathione S-transferases in human liver are composed of at least two immunochemically distinct polypeptides, designated B1 and B2. These subunits exist as homodimers, but can hybridize to form the B1B2 heterodimer [Stockman, Beckett & Hayes (1985) Biochem. J. 227, 457-465]. Although these basic glutathione S-transferases possess similar catalytic properties, the B2 subunit exhibits significantly greater selenium-independent glutathione peroxidase activity than subunit B1. The use of the ligands haematin, tributyltin acetate and Bromosulphophthalein as inhibitors of 1-chloro-2,4-dinitrobenzene-GSH-conjugating activity clearly discriminate between the B1 and B2 subunits and should help facilitate their identification. Peptide mapping experiments showed that B1 and B2 are structurally distinct, but related, subunits; subunit B1 yielded 43 tryptic peptides, seven of which were unique, whereas subunit B2 yielded 40 tryptic peptides, four of which were unique. PMID:3663118

  18. Localization of yeast RNA polymerase I core subunits by immunoelectron microscopy.

    PubMed Central

    Klinger, C; Huet, J; Song, D; Petersen, G; Riva, M; Bautz, E K; Sentenac, A; Oudet, P; Schultz, P

    1996-01-01

    Immunoelectron microscopy was used to determine the spatial organization of the yeast RNA polymerase I core subunits on a three-dimensional model of the enzyme. Images of antibody-labeled enzymes were compared with the native enzyme to determine the localization of the antibody binding site on the surface of the model. Monoclonal antibodies were used as probes to identify the two largest subunits homologous to the bacterial beta and beta' subunits. The epitopes for the two monoclonal antibodies were mapped using subunit-specific phage display libraries, thus allowing a direct correlation of the structural data with functional information on conserved sequence elements. An epitope close to conserved region C of the beta-like subunit is located at the base of the finger-like domain, whereas a sequence between conserved regions C and D of the beta'-like subunit is located in the apical region of the enzyme. Polyclonal antibodies outlined the alpha-like subunit AC40 and subunit AC19 which were found co-localized also in the apical region of the enzyme. The spatial location of the subunits is correlated with their biological activity and the inhibitory effect of the antibodies. Images PMID:8887555

  19. The ribosomal subunit assembly line

    PubMed Central

    Dlakić, Mensur

    2005-01-01

    Recent proteomic studies in Saccharomyces cerevisiae have identified nearly 200 proteins, other than the structural ribosomal proteins, that participate in the assembly of ribosomal subunits and their transport from the nucleus. In a separate line of research, proteomic studies of mature plant ribosomes have revealed considerable variability in the protein composition of individual ribosomes. PMID:16207363

  20. Rotating proton pumping ATPases: subunit/subunit interactions and thermodynamics.

    PubMed

    Nakanishi-Matsui, Mayumi; Sekiya, Mizuki; Futai, Masamitsu

    2013-03-01

    In this article, we discuss single molecule observation of rotational catalysis by E. coli ATP synthase (F-ATPase) using small gold beads. Studies involving a low viscous drag probe showed the stochastic properties of the enzyme in alternating catalytically active and inhibited states. The importance of subunit interaction between the rotor and the stator, and thermodynamics of the catalysis are also discussed. "Single Molecule Enzymology" is a new trend for understanding enzyme mechanisms in biochemistry and physiology.

  1. The Evolution of the Four Subunits of Voltage-Gated Calcium Channels: Ancient Roots, Increasing Complexity, and Multiple Losses

    PubMed Central

    Moran, Yehu; Zakon, Harold H.

    2014-01-01

    The alpha subunits of voltage-gated calcium channels (Cavs) are large transmembrane proteins responsible for crucial physiological processes in excitable cells. They are assisted by three auxiliary subunits that can modulate their electrical behavior. Little is known about the evolution and roles of the various subunits of Cavs in nonbilaterian animals and in nonanimal lineages. For this reason, we mapped the phyletic distribution of the four channel subunits and reconstructed their phylogeny. Although alpha subunits have deep evolutionary roots as ancient as the split between plants and opistokonths, beta subunits appeared in the last common ancestor of animals and their close-relatives choanoflagellates, gamma subunits are a bilaterian novelty and alpha2/delta subunits appeared in the lineage of Placozoa, Cnidaria, and Bilateria. We note that gene losses were extremely common in the evolution of Cavs, with noticeable losses in multiple clades of subfamilies and also of whole Cav families. As in vertebrates, but not protostomes, Cav channel genes duplicated in Cnidaria. We characterized by in situ hybridization the tissue distribution of alpha subunits in the sea anemone Nematostella vectensis, a nonbilaterian animal possessing all three Cav subfamilies common to Bilateria. We find that some of the alpha subunit subtypes exhibit distinct spatiotemporal expression patterns. Further, all six sea anemone alpha subunit subtypes are conserved in stony corals, which separated from anemones 500 MA. This unexpected conservation together with the expression patterns strongly supports the notion that these subtypes carry unique functional roles. PMID:25146647

  2. Interactions among the protein and RNA subunits of Saccharomyces cerevisiae nuclear RNase P

    PubMed Central

    Houser-Scott, Felicia; Xiao, Shaohua; Millikin, Christopher E.; Zengel, Janice M.; Lindahl, Lasse; Engelke, David R.

    2002-01-01

    Ribonuclease P (RNase P) is a ubiquitous endoribonuclease that cleaves precursor tRNAs to generate mature 5′ termini. Although RNase P from all kingdoms of life have been found to have essential RNA subunits, the number and size of the protein subunits ranges from one small protein in bacteria to at least nine proteins of up to 100 kDa. In Saccharomyces cerevisiae nuclear RNase P, the enzyme is composed of ten subunits: a single RNA and nine essential proteins. The spatial organization of these components within the enzyme is not yet understood. In this study we examine the likely binary protein–protein and protein–RNA subunit interactions by using directed two- and three-hybrid tests in yeast. Only two protein subunits, Pop1p and Pop4p, specifically bind the RNA subunit. Pop4p also interacted with seven of the other eight protein subunits. The remaining protein subunits all showed one or more specific protein–protein interactions with the other integral protein subunits. Of particular interest was the behavior of Rpr2p, the only protein subunit found in RNase P but not in the closely related enzyme, RNase MRP. Rpr2p interacts strongly with itself as well as with Pop4p. Similar interactions with self and Pop4p were also detected for Snm1p, the only unique protein subunit so far identified in RNase MRP. This observation is consistent with Snm1p and Rpr2p serving analogous functions in the two enzymes. This study provides a low-resolution map of the multisubunit architecture of the ribonucleoprotein enzyme, nuclear RNase P from S. cerevisiae. PMID:11880623

  3. Interactions among the protein and RNA subunits of Saccharomyces cerevisiae nuclear RNase P.

    PubMed

    Houser-Scott, Felicia; Xiao, Shaohua; Millikin, Christopher E; Zengel, Janice M; Lindahl, Lasse; Engelke, David R

    2002-03-05

    Ribonuclease P (RNase P) is a ubiquitous endoribonuclease that cleaves precursor tRNAs to generate mature 5' termini. Although RNase P from all kingdoms of life have been found to have essential RNA subunits, the number and size of the protein subunits ranges from one small protein in bacteria to at least nine proteins of up to 100 kDa. In Saccharomyces cerevisiae nuclear RNase P, the enzyme is composed of ten subunits: a single RNA and nine essential proteins. The spatial organization of these components within the enzyme is not yet understood. In this study we examine the likely binary protein-protein and protein-RNA subunit interactions by using directed two- and three-hybrid tests in yeast. Only two protein subunits, Pop1p and Pop4p, specifically bind the RNA subunit. Pop4p also interacted with seven of the other eight protein subunits. The remaining protein subunits all showed one or more specific protein-protein interactions with the other integral protein subunits. Of particular interest was the behavior of Rpr2p, the only protein subunit found in RNase P but not in the closely related enzyme, RNase MRP. Rpr2p interacts strongly with itself as well as with Pop4p. Similar interactions with self and Pop4p were also detected for Snm1p, the only unique protein subunit so far identified in RNase MRP. This observation is consistent with Snm1p and Rpr2p serving analogous functions in the two enzymes. This study provides a low-resolution map of the multisubunit architecture of the ribonucleoprotein enzyme, nuclear RNase P from S. cerevisiae.

  4. Isolation and characterization of the subunits of Phaseolus vulgaris alpha-amylase inhibitor.

    PubMed

    Yamaguchi, H

    1991-11-01

    An alpha-amylase inhibitor (PHA-I) of the white kidney bean (Phaseolus vulgaris) was found to be composed of two kinds of subunits and they were isolated on a size-exclusion column by HPLC under denaturing conditions. The alpha-subunit was free from tryptophan and cysteine and the beta-subunit contained no methionine or cysteine. There was no marked resemblance in tryptic peptide map between these subunit polypeptides. The alpha-subunit contained 28% by weight of carbohydrate, mainly made up of high mannose-type oligosacharides, whereas the sugar moiety of the beta-subunit amounted to 7% by weight and seemed to be predominantly composed of xylomannose-type oligosaccharides. By SDS-PAGE following deglycosylation, the molecular weights of the polypeptides of alpha- and beta-subunits were shown to be 7,800 and 14,000, respectively. These values were consistent with molecular sizes obtained for alpha- and beta-subunits by gel permeation HPLC in 6 M guanidine hydrochloride. The molecular weight of the native PHA-I, 28,800, obtained by gel permeation HPLC under non-denaturing conditions, suggested a heterodimeric structure for PHA-I.

  5. Interaction between G-protein beta and gamma subunit types is selective.

    PubMed Central

    Pronin, A N; Gautam, N

    1992-01-01

    Signal-transducing guanine nucleotide-binding proteins (G proteins) are made up of three subunits, alpha, beta, and gamma. Each of these subunits comprises a family of proteins. The rules for association between members of one family with members of another to form a multimer are not known; it is not clear whether associations are specific or nonspecific. Other than transducin (Gt), the G protein in rod photoreceptors, most purified G proteins contain more than one subtype of beta or gamma subunits. The Gt alpha subunit is associated only with beta 1 and gamma 1. It is not known whether this specificity is due to the differential expression of these subunit types in a cell type or due to intrinsically different affinities between different beta and gamma subunit types. We have used a transfected cell assay system to examine the association of the beta 1, beta 2, and beta 3 proteins with the gamma 1 and gamma 2 proteins. Results show that gamma 1 does not associate with beta 2 and that beta 3 does not associate with gamma 1 or gamma 2. Differences in affinities between types of G protein subunits will impose restrictions on the formation of certain heterotrimers and determine which G protein will be active in a cell. A chimeric molecule of beta 1 and beta 2 was used to broadly map the regions on these subunits that determine specificity of association. Images PMID:1631113

  6. Chromosomal localization of genes encoding guanine nucleotide-binding protein subunits in mouse and human

    SciTech Connect

    Blatt, C.; Eversole-Cire, P.; Cohn, V.H.; Zollman, S.; Fournier, R.E.K.; Mohandas, L.T.; Nesbitt, M.; Lugo, T.; Jones, D.T.; Reed, R.R.; Weiner, L.P.; Sparkes, R.S.; Simon, M.I. )

    1988-10-01

    A variety of genes have been identified that specify the synthesis of the components of guanine nucleotide-binding proteins (G proteins). Eight different guanine nucleotide-binding {alpha}-subunit proteins, two different {beta} subunits, and one {gamma} subunit have been described. Hybridization of cDNA clones with DNA from human-mouse somatic cell hybrids was used to assign many of these genes to human chromosomes. The retinal-specific transducin subunit genes GNAT1 and GNAT2 were on chromosomes 3 and 1; GNAI1, GNAI2, and GNAI3 were assigned to chromosomes 7, 3, and 1, respectively; GNAZ and GNAS were found on chromosomes 22 and 20. The {beta} subunits were also assigned-GNB1 to chromosome 1 and GNB2 to chromosome 7. Restriction fragment length polymorphisms were used to map the homologues of some of these genes in the mouse. GNAT1 and GNAI2 were found to map adjacent to each other on mouse chromosome 9 and GNAT2 was mapped on chromosome 17. The mouse GNB1 gene was assigned to chromosome 19. These mapping assignments will be useful in defining the extend of the G{alpha} gene family and may help in attempts to correlate specific genetic diseases and with genes corresponding to G proteins.

  7. Stoichiometry of δ subunit containing GABAA receptors

    PubMed Central

    Patel, B; Mortensen, M; Smart, T G

    2014-01-01

    Background and Purpose Although the stoichiometry of the major synaptic αβγ subunit-containing GABAA receptors has consensus support for 2α:2β:1γ, a clear view of the stoichiometry of extrasynaptic receptors containing δ subunits has remained elusive. Here we examine the subunit stoichiometry of recombinant α4β3δ receptors using a reporter mutation and a functional electrophysiological approach. Experimental Approach Using site-directed mutagenesis, we inserted a highly characterized 9′ serine to leucine mutation into the second transmembrane (M2) region of α4, β3 and δ subunits that increases receptor sensitivity to GABA. Whole-cell, GABA-activated currents were recorded from HEK-293 cells co-expressing different combinations of wild-type (WT) and/or mutant α4(L297S), β3(L284S) and δ(L288S) subunits. Key Results Recombinant receptors containing one or more mutant subunits showed increased GABA sensitivity relative to WT receptors by approximately fourfold, independent of the subunit class (α, β or δ) carrying the mutation. GABA dose–response curves of cells co-expressing WT subunits with their respective L9′S mutants exhibited multiple components, with the number of discernible components enabling a subunit stoichiometry of 2α, 2β and 1δ to be deduced for α4β3δ receptors. Varying the cDNA transfection ratio by 10-fold had no significant effect on the number of incorporated δ subunits. Conclusions and Implications Subunit stoichiometry is an important determinant of GABAA receptor function and pharmacology, and δ subunit-containing receptors are important mediators of tonic inhibition in several brain regions. Here we demonstrate a preferred subunit stoichiometry for α4β3δ receptors of 2α, 2β and 1δ. PMID:24206220

  8. The Subunit Structure of Benzylsuccinate Synthase†

    PubMed Central

    Li, Lei; Patterson, Dustin P.; Fox, Christel C.; Lin, Brian; Coschigano, Peter W.; Marsh, E. Neil G.

    2010-01-01

    Benzylsuccinate synthase is a member of the glycyl radical family of enzymes. It catalyzes the addition of toluene to fumarate to form benzylsuccinate as the first step in the anaerobic pathway of toluene fermentation. The enzyme comprises three subunits α, β and γ that in Thauera Aromatica T1 strain are encoded by the tutD, tutG and tutF genes respectively. The large α-subunit contains the essential glycine and cysteine residues that are conserved in all glycyl radical enzymes. However, the function of the small β- and γ-subunits has remained unclear. We have over-expressed all three subunits of benzylsuccinate synthase in E. coli, both individually and in combination. Co-expression of the γ-subunit (but not the β-subunit) is essential for efficient expression of the α-subunit. The benzylsuccinate synthase complex lacking the glycyl radical could be purified as an α2β2γ2 hexamer by nickel-affinity chromatography through a ‘His6’ affinity tag engineered onto the C-terminus of the α-subunit. Unexpectedly, BSS was found to contain two iron-sulfur clusters, one associated with the β-subunit and the other with the γ-subunit that appear to be necessary for the structural integrity of the complex. The spectroscopic properties of these clusters suggest that they are most likely [4Fe-4S] clusters. Removal of iron with chelating agents results in dissociation of the complex; similarly a mutant γ-subunit lacking the [4Fe-4S] cluster is unable to stabilize the α-subunit when the proteins are co-expressed. PMID:19159265

  9. Subunit connectivity, assembly determinants, and architecture of the yeast exocyst complex

    PubMed Central

    Heider, Margaret R.; Gu, Mingyu; Duffy, Caroline M.; Mirza, Anne M.; Marcotte, Laura L.; Walls, Alexandra C.; Farrall, Nicholas; Hakhverdyan, Zhanna; Field, Mark C.; Rout, Michael P.; Frost, Adam; Munson, Mary

    2016-01-01

    The exocyst is a hetero-octameric complex proposed to serve as the tethering complex for exocytosis, although it remains poorly understood at the molecular level. Here, we purified endogenous exocyst from Saccharomyces cerevisiae, and show that the purified complexes are stable and consist of all eight subunits with equal stoichiometry. Using a combination of biochemical and auxin-induced degradation experiments in yeast, we mapped the subunit connectivity, identified two stable four-subunit modules within the octamer, and demonstrated that several known exocyst binding partners are not necessary for exocyst assembly and stability. Furthermore, we visualized the structure of the yeast complex using negative stain electron microscopy; our results indicate that exocyst exists predominantly as a stable, octameric complex with an elongated architecture that suggests the subunits are contiguous helical bundles packed together into a bundle of long rods. PMID:26656853

  10. Comparison of the subunit structure of acetylcholine receptors from muscle and electric organ of Electrophorus electricus.

    PubMed

    Gullick, W J; Lindstrom, J M

    1983-08-02

    The acetylcholine receptors of the electric organ and muscle tissues of Electrophorus electricus are composed of alpha, beta, gamma, and delta subunits. Receptor subunits from the two tissues were compared by peptide mapping with monoclonal antibodies, an affinity-labeling reagent, and a lectin to characterize particular peptide fragments. These experiments indicate that the corresponding receptor subunits from the two tissues are extensively homologous or identical throughout their amino acid sequences. Small differences in the electrophoresis of peptide fragments of alpha subunits between the two tissues occurred on fragments which bound labeled lectin. These results suggest that the acetylcholine receptors in electric organ and muscle tissues of Electrophorus differ in structure only by minor posttranslational modifications perhaps involving carbohydrate.

  11. Role of the Rubisco Small Subunit

    SciTech Connect

    Spreitzer, Robert Joseph

    2016-11-05

    Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the rate-limiting step of CO2 fixation in photosynthesis. However, it is a slow enzyme, and O2 competes with CO2 at the active site. Oxygenation initiates the photorespiratory pathway, which also results in the loss of CO2. If carboxylation could be increased or oxygenation decreased, an increase in net CO2 fixation would be realized. Because Rubisco provides the primary means by which carbon enters all life on earth, there is much interest in engineering Rubisco to increase the production of food and renewable energy. Rubisco is located in the chloroplasts of plants, and it is comprised of two subunits. Much is known about the chloroplast-gene-encoded large subunit (rbcL gene), which contains the active site, but much less is known about the role of the nuclear-gene-encoded small subunit in Rubisco function (rbcS gene). Both subunits are coded by multiple genes in plants, which makes genetic engineering difficult. In the eukaryotic, green alga Chlamydomonas reinhardtii, it has been possible to eliminate all the Rubisco genes. These Rubisco-less mutants can be maintained by providing acetate as an alternative carbon source. In this project, focus has been placed on determining whether the small subunit might be a better genetic-engineering target for improving Rubisco. Analysis of a variable-loop structure (βA-βB loop) of the small subunit by genetic selection, directed mutagenesis, and construction of chimeras has shown that the small subunit can influence CO2/O2 specificity. X-ray crystal structures of engineered chimeric-loop enzymes have indicated that additional residues and regions of the small subunit may also contribute to Rubisco function. Structural dynamics of the small-subunit carboxyl terminus was also investigated. Alanine-scanning mutagenesis of the most-conserved small-subunit residues has identified a

  12. 28 CFR 51.6 - Political subunits.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 28 Judicial Administration 2 2010-07-01 2010-07-01 false Political subunits. 51.6 Section 51.6 Judicial Administration DEPARTMENT OF JUSTICE (CONTINUED) PROCEDURES FOR THE ADMINISTRATION OF SECTION 5 OF THE VOTING RIGHTS ACT OF 1965, AS AMENDED General Provisions § 51.6 Political subunits. All...

  13. Spectroscopic properties of Callinectes sapidus hemocyanin subunits

    NASA Astrophysics Data System (ADS)

    Stoeva, Stanka; Dolashka, Pavlina; Bankov, Banko; Voelter, Wolfgang; Salvato, Benedeto; Genov, Nicolay

    1995-10-01

    The two major subunits of the Callinectes sapidus hemocyanin were isolated and characterized by spectroscopic techniques. They consist of 641 and 652 residues, respectively. Circular dichroism spectra showed that the structural integrity of the isolated polypeptide chains is preserved. Tryptophan fluorescence parameters were determined for the hemocyanin aggregates and for the subunits Cs1 and Cs2. The emitting tryptophyl fluorophores in the native hemocyanin are deeply buried in hydrophobic regions and are shielded from the solvent by the quaternary structure of the protein aggregates. In two subunits, obtained after dissociation of the aggregates, these residues become "exposed". It is concluded that the tryptophyl side chains in Cs1 and Cs2 are located in subunit interfaces (contact regions) in a negatively charged environment when the polypeptide chains are aggregated. Most probably they participate in hydrophobic protein-protein interactions. The environment of these fluorophores is more negatively charged after the dissociation of the aggregates to subunits.

  14. Structural Characterization of Tip20p and Dsl1p, Subunits of the Dsl1p Vesicle Tethering Complex

    SciTech Connect

    Tripathi, A.; Ren, Y; Jeffrey, P; Hughson, F

    2009-01-01

    Multisubunit tethering complexes are essential for intracellular trafficking and have been proposed to mediate the initial interaction between vesicles and the membranes with which they fuse. Here we report initial structural characterization of the Dsl1p complex, whose three subunits are essential for trafficking from the Golgi apparatus to the endoplasmic reticulum (ER). Crystal structures reveal that two of the three subunits, Tip20p and Dsl1p, resemble known subunits of the exocyst complex, establishing a structural connection among several multisubunit tethering complexes and implying that many of their subunits are derived from a common progenitor. We show, moreover, that Tip20p and Dsl1p interact directly via N-terminal alpha-helices. Finally, we establish that different Dsl1p complex subunits bind independently to different ER SNARE proteins. Our results map out two alternative protein-interaction networks capable of tethering COPI-coated vesicles, via the Dsl1p complex, to ER membranes.

  15. The herpes zoster subunit vaccine.

    PubMed

    Cunningham, Anthony L

    2016-01-01

    Herpes zoster (HZ) causes severe pain and rash in older people and may be complicated by prolonged pain (postherpetic neuralgia; PHN). HZ results from reactivation of latent varicella-zoster virus (VZV) infection, often associated with age related or other causes of decreased T cell immunity. A concentrated live attenuated vaccine boosts this immunity and provides partial protection against HZ, but this decreases with age and declines over 5-8 years. The new HZ subunit (HZ/su or Shingrix) vaccine combines a key surface VZV glycoprotein (E) with T cell boosting adjuvant (AS01B). It is highly efficacious in protection (97%) against HZ in immunocompetent subjects, with no decline in advancing age and protection maintained for >3 years. Phase I-II trials showed safety and similar immunogenicity in severely immunocompromised patients. Local injection site pain and swelling can be severe in a minority (9.5%) but is transient (2 days). The HZ/su vaccine appears very promising in immunocompetent patients in the ZoE-50 controlled trial. The unblinding of the current ZoE-50 trial and publication of results from the accompanying ZoE-70 trial will reveal more about its mechanism of action and its efficacy against PHN, particularly in subjects >70 years. Phase III trial results in immunocompromised patients are eagerly awaited.

  16. [Co-expression of beta-subunit with other subunits of Qbeta replicase].

    PubMed

    Wang, Dong

    2004-12-01

    In researches involving in vitro protein synthesis and self-replication system, Qbeta replicase is one of the key enzymes, which are demanded for the high availability. Qbeta replicase is a RNA-dependent RNA polymerase of Qbeta coliphage. It consists of four subunits (alpha, beta, gamma, and delta subunit), where the beta-subunit is encoded by the viral genome, while the other three subunits are host proteins normally involved in protein synthesis, namely, ribosomal protein S1 (alpha), elongation factors EF-Tu (gamma) and EF-Ts (delta). To increase the production of the Qbeta replicase holoenzyme, several types of expression vectors, including pKK, pET and others, were employed to produce Qbeta replicase. However, the beta-subunit was almost in the precipitate fraction. Considering that the four subunits of Qbeta replicase holoenzyme are in equivalent molar ratio and the amount of the subunits, ribosomal S1 and EF-Ts, being produced by the host cells is relatively low, co-expression of beta-subunit with the other three subunits was performed to know whether the availability of the host subunits is the contributing factor for the solubility of the Qbeta replicase. pBAD33-rep was constructed by cloning the beta-subunit gene into pBAD 33, a pACYC derivative, and pET21a(+) was employed as expression vector for the three other subunits. Among the different combinations of co-expression experiments, solubility was found to slightly increase by SDS-PAGE analysis when the beta-subunit was co-expressed with EF-Tu-Ts. And the replicase activity assay showed this soluble enzyme is in active form. The expression of beta-subunit was enhanced by decreasing the level of inducer IPTG in co-expression, and more soluble enzyme were obtained.

  17. Isolation and characterization of the subunits of a heat-labile alpha-amylase inhibitor from Phaseolus vulgaris white kidney bean.

    PubMed

    Yamaguchi, H

    1993-02-01

    The heat-labile one of the two alpha-amylase inhibitors of the white kidney bean (Phaseolus vulgaris) was found to be composed of three kinds of subunits, and they were isolated and characterized. The alpha-subunit was free from tryptophan and cysteine and the beta-subunit contained no methionine or cysteine. There was no marked resemblance in tryptic peptide maps between the alpha- and beta-subunit polypeptides. The alpha-subunit contained 30% by weight of carbohydrate, mainly made up of high mannose-oligosaccharides, and the sugar moiety of the beta-subunit amounted 7% and appeared to be predominantly composed of xylomannose-type oligosaccharides. The largest subunit, gamma, was very similar in molecular features to a postulated alpha beta-dimer and its N-terminal sequence coincided with that of the alpha-subunit. The molecular weights of the polypeptides of alpha, beta-, and gamma-subunits were shown to be 7,800, 14,000, and 22,000, respectively, by SDS-PAGE. It seemed likely that the alpha- and beta-subunits are common to both of the inhibitors and that the heat-lability of this inhibitor arises from the gamma-subunit.

  18. Conformational flexibility and subunit arrangement of the modular yeast Spt-Ada-Gcn5 acetyltransferase complex.

    PubMed

    Setiaputra, Dheva; Ross, James D; Lu, Shan; Cheng, Derrick T; Dong, Meng-Qiu; Yip, Calvin K

    2015-04-17

    The Spt-Ada-Gcn5 acetyltransferase (SAGA) complex is a highly conserved, 19-subunit histone acetyltransferase complex that activates transcription through acetylation and deubiquitination of nucleosomal histones in Saccharomyces cerevisiae. Because SAGA has been shown to display conformational variability, we applied gradient fixation to stabilize purified SAGA and systematically analyzed this flexibility using single-particle EM. Our two- and three-dimensional studies show that SAGA adopts three major conformations, and mutations of specific subunits affect the distribution among these. We also located the four functional modules of SAGA using electron microscopy-based labeling and transcriptional activator binding analyses and show that the acetyltransferase module is localized in the most mobile region of the complex. We further comprehensively mapped the subunit interconnectivity of SAGA using cross-linking mass spectrometry, revealing that the Spt and Taf subunits form the structural core of the complex. These results provide the necessary restraints for us to generate a model of the spatial arrangement of all SAGA subunits. According to this model, the chromatin-binding domains of SAGA are all clustered in one face of the complex that is highly flexible. Our results relate information of overall SAGA structure with detailed subunit level interactions, improving our understanding of its architecture and flexibility.

  19. Membrane insertion and topology of the translocon-associated protein (TRAP) gamma subunit.

    PubMed

    Bañó-Polo, Manuel; Martínez-Garay, Carlos A; Grau, Brayan; Martínez-Gil, Luis; Mingarro, Ismael

    2017-05-01

    Translocon-associated protein (TRAP) complex is intimately associated with the ER translocon for the insertion or translocation of newly synthesised proteins in eukaryotic cells. The TRAP complex is comprised of three single-spanning and one multiple-spanning subunits. We have investigated the membrane insertion and topology of the multiple-spanning TRAP-γ subunit by glycosylation mapping and green fluorescent protein fusions both in vitro and in cell cultures. Results demonstrate that TRAP-γ has four transmembrane (TM) segments, an Nt/Ct cytosolic orientation and that the less hydrophobic TM segment inserts efficiently into the membrane only in the cellular context of full-length protein.

  20. Gene targeting of CK2 catalytic subunits

    PubMed Central

    Lou, David Y.; Toselli, Paul; Landesman-Bollag, Esther; Dominguez, Isabel

    2013-01-01

    Protein kinase CK2 is a highly conserved and ubiquitous serine–threonine kinase. It is a tetrameric enzyme that is made up of two regulatory CK2β subunits and two catalytic subunits, either CK2α/CK2α, CK2α/ CK2α′, or CK2α′/CK2α′. Although the two catalytic subunits diverge in their C termini, their enzymatic activities are similar. To identify the specific function of the two catalytic subunits in development, we have deleted them individually from the mouse genome by homologous recombination. We have previously reported that CK2α′is essential for male germ cell development, and we now demonstrate that CK2α has an essential role in embryogenesis, as mice lacking CK2α die in mid-embryogenesis, with cardiac and neural tube defects. PMID:18594950

  1. Anthranilate synthase subunit organization in Chromobacterium violaceum.

    PubMed

    Carminatti, C A; Oliveira, I L; Recouvreux, D O S; Antônio, R V; Porto, L M

    2008-09-16

    Tryptophan is an aromatic amino acid used for protein synthesis and cellular growth. Chromobacterium violaceum ATCC 12472 uses two tryptophan molecules to synthesize violacein, a secondary metabolite of pharmacological interest. The genome analysis of this bacterium revealed that the genes trpA-F and pabA-B encode the enzymes of the tryptophan pathway in which the first reaction is the conversion of chorismate to anthranilate by anthranilate synthase (AS), an enzyme complex. In the present study, the organization and structure of AS protein subunits from C. violaceum were analyzed using bioinformatics tools available on the Web. We showed by calculating molecular masses that AS in C. violaceum is composed of alpha (TrpE) and beta (PabA) subunits. This is in agreement with values determined experimentally. Catalytic and regulatory sites of the AS subunits were identified. The TrpE and PabA subunits contribute to the catalytic site while the TrpE subunit is involved in the allosteric site. Protein models for the TrpE and PabA subunits were built by restraint-based homology modeling using AS enzyme, chains A and B, from Salmonella typhimurium (PDB ID 1I1Q).

  2. Subunit Organisation of In Vitro Reconstituted HOPS and CORVET Multisubunit Membrane Tethering Complexes

    PubMed Central

    Guo, Zhong; Johnston, Wayne; Kovtun, Oleksiy; Mureev, Sergey; Bröcker, Cornelia; Ungermann, Christian; Alexandrov, Kirill

    2013-01-01

    Biochemical and structural analysis of macromolecular protein assemblies remains challenging due to technical difficulties in recombinant expression, engineering and reconstitution of multisubunit complexes. Here we use a recently developed cell-free protein expression system based on the protozoan Leishmania tarentolae to produce in vitro all six subunits of the 600 kDa HOPS and CORVET membrane tethering complexes. We demonstrate that both subcomplexes and the entire HOPS complex can be reconstituted in vitro resulting in a comprehensive subunit interaction map. To our knowledge this is the largest eukaryotic protein complex in vitro reconstituted to date. Using the truncation and interaction analysis, we demonstrate that the complex is assembled through short hydrophobic sequences located in the C-terminus of the individual Vps subunits. Based on this data we propose a model of the HOPS and CORVET complex assembly that reconciles the available biochemical and structural data. PMID:24312556

  3. Protein synthesis by ribosomes with tethered subunits.

    PubMed

    Orelle, Cédric; Carlson, Erik D; Szal, Teresa; Florin, Tanja; Jewett, Michael C; Mankin, Alexander S

    2015-08-06

    The ribosome is a ribonucleoprotein machine responsible for protein synthesis. In all kingdoms of life it is composed of two subunits, each built on its own ribosomal RNA (rRNA) scaffold. The independent but coordinated functions of the subunits, including their ability to associate at initiation, rotate during elongation, and dissociate after protein release, are an established model of protein synthesis. Furthermore, the bipartite nature of the ribosome is presumed to be essential for biogenesis, since dedicated assembly factors keep immature ribosomal subunits apart and prevent them from translation initiation. Free exchange of the subunits limits the development of specialized orthogonal genetic systems that could be evolved for novel functions without interfering with native translation. Here we show that ribosomes with tethered and thus inseparable subunits (termed Ribo-T) are capable of successfully carrying out protein synthesis. By engineering a hybrid rRNA composed of both small and large subunit rRNA sequences, we produced a functional ribosome in which the subunits are covalently linked into a single entity by short RNA linkers. Notably, Ribo-T was not only functional in vitro, but was also able to support the growth of Escherichia coli cells even in the absence of wild-type ribosomes. We used Ribo-T to create the first fully orthogonal ribosome-messenger RNA system, and demonstrate its evolvability by selecting otherwise dominantly lethal rRNA mutations in the peptidyl transferase centre that facilitate the translation of a problematic protein sequence. Ribo-T can be used for exploring poorly understood functions of the ribosome, enabling orthogonal genetic systems, and engineering ribosomes with new functions.

  4. Distinct expression patterns of glycoprotein hormone subunits in the lophotrochozoan Aplysia: implications for the evolution of neuroendocrine systems in animals.

    PubMed

    Heyland, Andreas; Plachetzki, David; Donelly, Evonne; Gunaratne, Dinuka; Bobkova, Yelena; Jacobson, John; Kohn, Andrea B; Moroz, Leonid L

    2012-11-01

    Glycoprotein hormones (GPHs) comprise a group of signaling molecules critical for major metabolic and reproductive functions. In vertebrates they include chorionic gonadotropin, LH, FSH, and TSH. The active hormones are characterized by heterodimerization between a common α and hormone-specific β subunit, which activate leucine-rich repeat-containing G protein coupled receptors. To date, genes referred to as GPHα2 and GPHβ5 have been the only glycoprotein hormone subunits identified in invertebrates, suggesting that other GPHα and GPHβ subunits diversified during vertebrate evolution. Still the functions of GPHα2 and GPHβ5 remain largely unknown for both vertebrates and invertebrates. To further understand the evolution and putative function of these subunits, we cloned and analyzed phylogenetically two glycoprotein subunits, AcaGPHα and AcaGPHβ, from the sea hare Aplysia californica. Model based three-dimensional predictions of AcaGPHβ confirm the presence of a complete cysteine knot, two hairpin loops, and a long loop. As in the human GPHβ5 subunit the seatbelt structure is absent in AcaGPHβ. We also found that AcaGPHα and AcaGPHβ subunits are expressed in larval stages of Aplysia, and we present a detailed expression map of the subunits in the adult central nervous system using in situ hybridizations. Both subunits are expressed in subpopulations of pleural and buccal mechanosensory neurons, suggesting a neuronal modulatory function of these subunits in Aplysia. Furthermore it supports the model of a relatively diffuse neuroendocrine-like system in molluscs, where specific primary sensory neurons release peptides extrasynaptically (paracrine secretion). This is in contrast to vertebrates and insects, in which releasing and stimulating factor from centralized sensory regions of the central nervous system ultimately regulate hormone release in peripheral glands.

  5. Distinct Expression Patterns of Glycoprotein Hormone Subunits in the Lophotrochozoan Aplysia: Implications for the Evolution of Neuroendocrine Systems in Animals

    PubMed Central

    Plachetzki, David; Donelly, Evonne; Gunaratne, Dinuka; Bobkova, Yelena; Jacobson, John; Kohn, Andrea B.; Moroz, Leonid L.

    2012-01-01

    Glycoprotein hormones (GPHs) comprise a group of signaling molecules critical for major metabolic and reproductive functions. In vertebrates they include chorionic gonadotropin, LH, FSH, and TSH. The active hormones are characterized by heterodimerization between a common α and hormone-specific β subunit, which activate leucine-rich repeat-containing G protein coupled receptors. To date, genes referred to as GPHα2 and GPHβ5 have been the only glycoprotein hormone subunits identified in invertebrates, suggesting that other GPHα and GPHβ subunits diversified during vertebrate evolution. Still the functions of GPHα2 and GPHβ5 remain largely unknown for both vertebrates and invertebrates. To further understand the evolution and putative function of these subunits, we cloned and analyzed phylogenetically two glycoprotein subunits, AcaGPHα and AcaGPHβ, from the sea hare Aplysia californica. Model based three-dimensional predictions of AcaGPHβ confirm the presence of a complete cysteine knot, two hairpin loops, and a long loop. As in the human GPHβ5 subunit the seatbelt structure is absent in AcaGPHβ. We also found that AcaGPHα and AcaGPHβ subunits are expressed in larval stages of Aplysia, and we present a detailed expression map of the subunits in the adult central nervous system using in situ hybridizations. Both subunits are expressed in subpopulations of pleural and buccal mechanosensory neurons, suggesting a neuronal modulatory function of these subunits in Aplysia. Furthermore it supports the model of a relatively diffuse neuroendocrine-like system in molluscs, where specific primary sensory neurons release peptides extrasynaptically (paracrine secretion). This is in contrast to vertebrates and insects, in which releasing and stimulating factor from centralized sensory regions of the central nervous system ultimately regulate hormone release in peripheral glands. PMID:22977258

  6. Genetic exploration of interactive domains in RNA polymerase II subunits.

    PubMed Central

    Martin, C; Okamura, S; Young, R

    1990-01-01

    The two large subunits of RNA polymerase II, RPB1 and RPB2, contain regions of extensive homology to the two large subunits of Escherichia coli RNA polymerase. These homologous regions may represent separate protein domains with unique functions. We investigated whether suppressor genetics could provide evidence for interactions between specific segments of RPB1 and RPB2 in Saccharomyces cerevisiae. A plasmid shuffle method was used to screen thoroughly for mutations in RPB2 that suppress a temperature-sensitive mutation, rpb1-1, which is located in region H of RPB1. All six RPB2 mutations that suppress rpb1-1 were clustered in region I of RPB2. The location of these mutations and the observation that they were allele specific for suppression of rpb1-1 suggests an interaction between region H of RPB1 and region I of RPB2. A similar experiment was done to isolate and map mutations in RPB1 that suppress a temperature-sensitive mutation, rpb2-2, which occurs in region I of RPB2. These suppressor mutations were not clustered in a particular region. Thus, fine structure suppressor genetics can provide evidence for interactions between specific segments of two proteins, but the results of this type of analysis can depend on the conditional mutation to be suppressed. Images PMID:2183012

  7. Drosophila laminin: sequence of B2 subunit and expression of all three subunits during embryogenesis

    PubMed Central

    1989-01-01

    In a previous study, we described the cloning of the genes encoding the three subunits of Drosophila laminin, a substrate adhesion molecule, and the cDNA sequence of the B1 subunit (Montell and Goodman, 1988). This analysis revealed the similarity of Drosophila laminin with the mouse and human complexes in subunit composition, domain structure, and amino acid sequence. In this paper, we report the deduced amino acid sequence of the B2 subunit. We then describe the expression and tissue distribution of the three subunits of laminin during Drosophila embryogenesis using both in situ hybridization and immunolocalization techniques, with particular emphasis on its expression in and around the developing nervous system. PMID:2808533

  8. DNA-dependent RNA polymerase subunits encoded within the vaccinia virus genome.

    PubMed Central

    Jones, E V; Puckett, C; Moss, B

    1987-01-01

    Antiserum to a multisubunit DNA-dependent RNA polymerase from vaccinia virions was prepared to carry out genetic studies. This antiserum selectively inhibited the activity of the viral polymerase but had no effect on calf thymus RNA polymerase II. The specificity of the antiserum was further demonstrated by immunoprecipitation of RNA polymerase subunits from dissociated virus particles. The presence in vaccinia virus-infected cells of mRNA that encodes the polymerase subunits was determined by in vitro translation. Immunoprecipitable polypeptides with Mrs of about 135,000, 128,000, 36,000, 34,000, 31,000, 23,000, 21,000, 20,000, and 17,000 were made when early mRNA was added to reticulocyte extracts. The subunits were encoded within the vaccinia virus genome, as demonstrated by translation of early mRNA that hybridized to vaccinia virus DNA. The locations of the subunit genes were determined initially by hybridization of RNA to a series of overlapping 40-kilobase-pair DNA fragments that were cloned in a cosmid vector. Further mapping was achieved with cloned HindIII restriction fragments. Results of these studies indicated that RNA polymerase subunit genes are transcribed early in infection and are distributed within the highly conserved central portion of the poxvirus genome in HindIII fragments E, J, H, D, and A. Images PMID:3033308

  9. Localization of the regulatory particle subunit Sem1 in the 26S proteasome

    SciTech Connect

    Bohn, Stefan; Sakata, Eri; Beck, Florian; Pathare, Ganesh R.; Schnitger, Jérôme; Nágy, Istvan; Baumeister, Wolfgang Förster, Friedrich

    2013-05-31

    Highlights: •26S proteasome subunit Sem1 was mapped using cryo-EM and cross-linking data. •C-terminal helix of Sem1 located near winged helix motif of Rpn7. •N-terminal part of Sem1 tethers Rpn7, Rpn3 and lid helical bundle. •Sem1 binds differently to PCI-domains of proteasome subunit Rpn7 and TREX-2 subunit Thp1. -- Abstract: The ubiquitin–proteasome system is responsible for regulated protein degradation in the cell with the 26S proteasome acting as its executive arm. The molecular architecture of this 2.5 MDa complex has been established recently, with the notable exception of the small acidic subunit Sem1. Here, we localize the C-terminal helix of Sem1 binding to the PCI domain of the subunit Rpn7 using cryo-electron microscopy single particle reconstruction of proteasomes purified from yeast cells with sem1 deletion. The approximate position of the N-terminal region of Sem1 bridging the cleft between Rpn7 and Rpn3 was inferred based on site-specific cross-linking data of the 26S proteasome. Our structural studies indicate that Sem1 can assume different conformations in different contexts, which supports the idea that Sem1 functions as a molecular glue stabilizing the Rpn3/Rpn7 heterodimer.

  10. Diversity of insect nicotinic acetylcholine receptor subunits.

    PubMed

    Jones, Andrew K; Sattelle, David B

    2010-01-01

    Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that mediate fast synaptic transmission in the insect nervous system and are targets of a major group of insecticides, the neonicotinoids. They consist of five subunits arranged around a central ion channeL Since the subunit composition determines the functional and pharmacological properties of the receptor the presence of nAChR families comprising several subunit-encodinggenes provides a molecular basis for broad functional diversity. Analyses of genome sequences have shown that nAChR gene families remain compact in diverse insect species, when compared to their nematode andvertebrate counterparts. Thus, the fruit fly (Drosophila melanogaster), malaria mosquito (Anopheles gambiae), honey bee (Apis mellifera), silk worm (Bombyx mon) and the red flour beetle (Tribolium castaneum) possess 10-12 nAChR genes while human and the nematode Caenorhabditis elegans have 16 and 29 respectively. Although insect nAChRgene families are amongst the smallest known, receptor diversity can be considerably increased by the posttranscriptional processes alternative splicing and mRNA A-to-I editingwhich can potentially generate protein products which far outnumber the nAChR genes. These two processes can also generate species-specific subunit isoforms. In addition, each insect possesses at least one highly divergent nAChR subunit which may perform species-specific functions. Species-specific subunit diversification may offer promising targets for future rational design of insecticides that target specific pest insects while sparing beneficial species.

  11. Allele-specific interactions between the yeast RFC1 and RFC5 genes suggest a basis for RFC subunit-subunit interactions.

    PubMed

    Beckwith, W; McAlear, M A

    2000-11-01

    Replication factor C (RFC) is an essential, multi-subunit ATPase that functions in DNA replication, DNA repair, and DNA metabolism-related checkpoints. In order to investigate how the individual RFC subunits contribute to these functions in vivo, we undertook a genetic analysis of RFC genes from budding yeast. We isolated and characterized mutations in the RFC5 gene that could suppress the cold-sensitive phenotype of rfc1-1 mutants. Analysis of the RFC5 suppressors revealed that they could not suppress the elongated telomere phenotype, the sensitivity to DNA damaging agents, or the mutator phenotype of rfc1-1 mutants. Unlike the checkpoint-defective rfc5-1 mutation, the RFC5 suppressor mutations did not interfere with the methylmethane sulfonate- or hydroxyurea-induced phosphorylation of Rad53p. The Rfc5p suppressor substitutions mapped to amino acid positions in the conserved RFC box motifs IV-VII. Comparisons of the structures of related RFC box-containing proteins suggest that these RFC motifs may function to coordinate interactions between neighboring subunits of multi-subunit ATPases.

  12. Optimized subunit vaccine protects against experimental leishmaniasis

    PubMed Central

    Bertholet, Sylvie; Goto, Yasuyuki; Carter, Lauren; Bhatia, Ajay; Howard, Randall F.; Carter, Darrick; Coler, Rhea N.; Vedvick, Thomas S.; Reed, Steven G.

    2009-01-01

    Development of a protective subunit vaccine against Leishmania spp. depends on antigens and adjuvants that induce appropriate immune responses. We evaluated a second generation polyprotein antigen (Leish-110f) in different adjuvant formulations for immunogenicity and protective efficacy against Leishmania spp. challenges. Vaccine-induced protection was associated with antibody and T cell responses to Leish-110f. CD4 T cells were the source of IFN-γ, TNF, and IL-2 double and triple positive populations. This study establishes the immunogenicity and protective efficacy of the improved Leish-110f subunit vaccine antigen adjuvanted with natural (MPL-SE) or synthetic (EM005) Toll-like receptor 4 agonists. PMID:19786136

  13. Optimized subunit vaccine protects against experimental leishmaniasis.

    PubMed

    Bertholet, Sylvie; Goto, Yasuyuki; Carter, Lauren; Bhatia, Ajay; Howard, Randall F; Carter, Darrick; Coler, Rhea N; Vedvick, Thomas S; Reed, Steven G

    2009-11-23

    Development of a protective subunit vaccine against Leishmania spp. depends on antigens and adjuvants that induce appropriate immune responses. We evaluated a second generation polyprotein antigen (Leish-110f) in different adjuvant formulations for immunogenicity and protective efficacy against Leishmania spp. challenges. Vaccine-induced protection was associated with antibody and T cell responses to Leish-110f. CD4 T cells were the source of IFN-gamma, TNF, and IL-2 double- and triple-positive populations. This study establishes the immunogenicity and protective efficacy of the improved Leish-110f subunit vaccine antigen adjuvanted with natural (MPL-SE) or synthetic (EM005) Toll-like receptor 4 agonists.

  14. Planetary maps

    USGS Publications Warehouse

    ,

    1992-01-01

    An important goal of the USGS planetary mapping program is to systematically map the geology of the Moon, Mars, Venus, and Mercury, and the satellites of the outer planets. These geologic maps are published in the USGS Miscellaneous Investigations (I) Series. Planetary maps on sale at the USGS include shaded-relief maps, topographic maps, geologic maps, and controlled photomosaics. Controlled photomosaics are assembled from two or more photographs or images using a network of points of known latitude and longitude. The images used for most of these planetary maps are electronic images, obtained from orbiting television cameras, various optical-mechanical systems. Photographic film was only used to map Earth's Moon.

  15. Assembly of the 30S ribosomal subunit: positioning ribosomal protein S13 in the S7 assembly branch.

    PubMed

    Grondek, Joel F; Culver, Gloria M

    2004-12-01

    Studies of Escherichia coli 30S ribosomal subunit assembly have revealed a hierarchical and cooperative association of ribosomal proteins with 16S ribosomal RNA; these results have been used to compile an in vitro 30S subunit assembly map. In single protein addition and omission studies, ribosomal protein S13 was shown to be dependent on the prior association of ribosomal protein S20 for binding to the ribonucleoprotein particle. While the overwhelming majority of interactions revealed in the assembly map are consistent with additional data, the dependency of S13 on S20 is not. Structural studies position S13 in the head of the 30S subunit > 100 A away from S20, which resides near the bottom of the body of the 30S subunit. All of the proteins that reside in the head of the 30S subunit, except S13, have been shown to be part of the S7 assembly branch, that is, they all depend on S7 for association with the assembling 30S subunit. Given these observations, the assembly requirements for S13 were investigated using base-specific chemical footprinting and primer extension analysis. These studies reveal that S13 can bind to 16S rRNA in the presence of S7, but not S20. Additionally, interaction between S13 and other members of the S7 assembly branch have been observed. These results link S13 to the 3' major domain family of proteins, and the S7 assembly branch, placing S13 in a new location in the 30S subunit assembly map where its position is in accordance with much biochemical and structural data.

  16. Localization of a gene for a glutamate binding subunit of a NMDA receptor (GRINA) to 8q24

    SciTech Connect

    Lewis, T.B.; DuPont, B.R.; Leach, R.

    1996-02-15

    This article reports on the localization of a gene for a glutamate binding subunit of an N-methyl-D-aspartate (NMDA) receptor, called GRINA, to human chromosome 8q24 using fluorescence in situ hybridization and radiation hybridization mapping. This gene mapped outside the critical region for benign familial neonatal convulsions (BFNC), a rare form of epilepsy; however, GRINA could be the causative genetic factor inducing idiopathic generalized epilepsy. Further studies need to be conducted. 15 refs., 2 figs.

  17. The β-conglycinin deficiency in wild soybean is associated with the tail-to-tail inverted repeat of the α-subunit genes.

    PubMed

    Tsubokura, Yasutaka; Hajika, Makita; Kanamori, Hiroyuki; Xia, Zhengjun; Watanabe, Satoshi; Kaga, Akito; Katayose, Yuichi; Ishimoto, Masao; Harada, Kyuya

    2012-02-01

    β-conglycinin, a major seed protein in soybean, is composed of α, α', and β subunits sharing a high homology among them. Despite its many health benefits, β-conglycinin has a lower amino acid score and lower functional gelling properties compared to glycinin, another major soybean seed protein. In addition, the α, α', and β subunits also contain major allergens. A wild soybean (Glycine soja Sieb et Zucc.) line, 'QT2', lacks all of the β-conglycinin subunits, and the deficiency is controlled by a single dominant gene, Scg-1 (Suppressor of β-conglycinin). This gene was characterized using a soybean cultivar 'Fukuyutaka', 'QY7-25', (its near-isogenic line carrying the Scg-1 gene), and the F₂ population derived from them. The physical map of the Scg-1 region covered by lambda phage genomic clones revealed that the two α-subunit genes, a β-subunit gene, and a pseudo α-subunit gene were closely organized. The two α-subunit genes were arranged in a tail-to-tail orientation, and the genes were separated by 197 bp in Scg-1 compared to 3.3 kb in the normal allele (scg-1). In addition, small RNA was detected in immature seeds of the mutants by northern blot analysis using an RNA probe of the α subunit. These results strongly suggest that β-conglycinin deficiency in QT2 is controlled by post-transcriptional gene silencing through the inverted repeat of the α subunits.

  18. The gamma subunit of transducin is farnesylated.

    PubMed Central

    Lai, R K; Perez-Sala, D; Cañada, F J; Rando, R R

    1990-01-01

    Protein prenylation with farnesyl or geranylgeranyl moieties is an important posttranslational modification that affects the activity of such diverse proteins as the nuclear lamins, the yeast mating factor mata, and the ras oncogene products. In this article, we show that whole retinal cultures incorporate radioactive mevalonic acid into proteins of 23-26 kDa and one of 8 kDa. The former proteins are probably the "small" guanine nucleotide-binding regulatory proteins (G proteins) and the 8-kDa protein is the gamma subunit of the well-studied retinal heterotrimeric G protein (transducin). After deprenylating purified transducin and its subunits with Raney nickel or methyl iodide/base, the adducted prenyl group can be identified as an all-trans-farnesyl moiety covalently linked to a cysteine residue. Thus far, prenylation reactions have been found to occur at cysteine in a carboxyl-terminal consensus CAAX sequence, where C is the cysteine, A is an aliphatic amino acid, and X is undefined. Both the alpha and gamma subunits of transducin have this consensus sequence, but only the gamma subunit is prenylated. Therefore, the CAAX motif is not necessary and sufficient to direct prenylation. Finally, since transducin is the best understood G protein, both structurally and mechanistically, the discovery that it is farnesylated should allow for a quantitative understanding of this post-translational modification. Images PMID:2217200

  19. Binding of the sigma 70 protein to the core subunits of Escherichia coli RNA polymerase, studied by iron-EDTA protein footprinting.

    PubMed Central

    Greiner, D P; Hughes, K A; Gunasekera, A H; Meares, C F

    1996-01-01

    We have used a nonspecific protein cleaving reagent to map the interactions between subunits of the multisubunit enzyme RNA polymerase (Escherichia coli). We developed suitable conditions for using an untethered Fe-EDTA reagent, which does not bind significantly to proteins. Comparison of the cleaved fragments of the subunits from the core enzyme (alpha 2 beta beta') and the holoenzyme (core+sigma 70) shows that absence of the sigma 70 subunit is associated with the appearance of several cleavage sites on the subunits beta (within 10 residues of sequence positions 745, 764, 795, and 812) and beta' (within 10 residues of sequence positions 581, 613, and 728). A cleavage site near beta residue 604 is present in the holoenzyme but absent in the core, demonstrating that a conformational change occurs when sigma 70 binds. No differences are observed for the alpha subunit. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8552678

  20. Interaction between Subunits of Heterodimeric Splicing Factor U2AF Is Essential In Vivo

    PubMed Central

    Rudner, David Z.; Kanaar, Roland; Breger, Kevin S.; Rio, Donald C.

    1998-01-01

    The heterodimeric pre-mRNA splicing factor, U2AF (U2 snRNP auxiliary factor), plays a critical role in 3′ splice site selection. Although the U2AF subunits associate in a tight complex, biochemical experiments designed to address the requirement for both subunits in splicing have yielded conflicting results. We have taken a genetic approach to assess the requirement for the Drosophila U2AF heterodimer in vivo. We developed a novel Escherichia coli copurification assay to map the domain on the Drosophila U2AF large subunit (dU2AF50) that interacts with the Drosophila small subunit (dU2AF38). A 28-amino-acid fragment on dU2AF50 that is both necessary and sufficient for interaction with dU2AF38 was identified. Using the copurification assay, we scanned this 28-amino-acid interaction domain for mutations that abrogate heterodimer formation. A collection of these dU2AF50 point mutants was then tested in vivo for genetic complementation of a recessive lethal dU2AF50 allele. A mutation that completely abolished interaction with dU2AF38 was incapable of complementation, whereas dU2AF50 mutations that did not effect heterodimer formation rescued the recessive lethal dU2AF50 allele. Analysis of heterodimer formation in embryo extracts derived from these interaction mutant lines revealed a perfect correlation between the efficiency of subunit association and the ability to complement the dU2AF50 recessive lethal allele. These data indicate that Drosophila U2AF heterodimer formation is essential for viability in vivo, consistent with a requirement for both subunits in splicing in vitro. PMID:9528748

  1. Involvement of proteasomal subunits zeta and iota in RNA degradation.

    PubMed Central

    Petit, F; Jarrousse, A S; Dahlmann, B; Sobek, A; Hendil, K B; Buri, J; Briand, Y; Schmid, H P

    1997-01-01

    We have identified two distinct subunits of 20 S proteasomes that are associated with RNase activity. Proteasome subunits zeta and iota, eluted from two-dimensional Western blots, hydrolysed tobacco mosaic virus RNA, whereas none of the other subunits degraded this substrate under the same conditions. Additionally, proteasomes were dissociated by 6 M urea, and subunit zeta, containing the highest RNase activity, was isolated by anion-exchange chromatography and gel filtration. Purified subunit zeta migrated as a single spot on two-dimensional PAGE with a molecular mass of approx. 28 kDa. Addition of anti-(subunit zeta) antibodies led to the co-precipitation of this proteasome subunit and nuclease activity. This is the first evidence that proteasomal alpha-type subunits are associated with an enzymic activity, and our results provide further evidence that proteasomes may be involved in cellular RNA metabolism. PMID:9337855

  2. Active Mapping.

    ERIC Educational Resources Information Center

    Day, Dennis

    1994-01-01

    Explains a social studies lesson for third graders that uses KidPix, a computer software graphics program to help students make maps and map keys. Advantages to using the computer versus hand drawing maps are discussed, and an example of map requirements for the lesson is included. (LRW)

  3. Exploring maps

    USGS Publications Warehouse

    ,

    1993-01-01

    Exploring Maps is an interdisciplinary set of materials on mapping for grades 7-12. Students will learn basic mapmaking and map reading skills and will see how maps can answer fundamental geographic questions: "Where am I?" "What else is here?" "Where am I going?"

  4. Contour Mapping

    NASA Technical Reports Server (NTRS)

    1995-01-01

    In the early 1990s, the Ohio State University Center for Mapping, a NASA Center for the Commercial Development of Space (CCDS), developed a system for mobile mapping called the GPSVan. While driving, the users can map an area from the sophisticated mapping van equipped with satellite signal receivers, video cameras and computer systems for collecting and storing mapping data. George J. Igel and Company and the Ohio State University Center for Mapping advanced the technology for use in determining the contours of a construction site. The new system reduces the time required for mapping and staking, and can monitor the amount of soil moved.

  5. Recent Advances in Subunit Vaccine Carriers

    PubMed Central

    Vartak, Abhishek; Sucheck, Steven J.

    2016-01-01

    The lower immunogenicity of synthetic subunit antigens, compared to live attenuated vaccines, is being addressed with improved vaccine carriers. Recent reports indicate that the physio-chemical properties of these carriers can be altered to achieve optimal antigen presentation, endosomal escape, particle bio-distribution, and cellular trafficking. The carriers can be modified with various antigens and ligands for dendritic cells targeting. They can also be modified with adjuvants, either covalently or entrapped in the matrix, to improve cellular and humoral immune responses against the antigen. As a result, these multi-functional carrier systems are being explored for use in active immunotherapy against cancer and infectious diseases. Advancing technology, improved analytical methods, and use of computational methodology have also contributed to the development of subunit vaccine carriers. This review details recent breakthroughs in the design of nano-particulate vaccine carriers, including liposomes, polymeric nanoparticles, and inorganic nanoparticles. PMID:27104575

  6. PKA regulatory subunit expression in tooth development.

    PubMed

    de Sousa, Sílvia Ferreira; Kawasaki, Katsushige; Kawasaki, Maiko; Volponi, Ana Angelova; Gomez, Ricardo Santiago; Gomes, Carolina Cavaliéri; Sharpe, Paul T; Ohazama, Atsushi

    2014-05-01

    Protein kinase A (PKA) plays critical roles in many biological processes including cell proliferation, cell differentiation, cellular metabolism and gene regulation. Mutation in PKA regulatory subunit, PRKAR1A has previously been identified in odontogenic myxomas, but it is unclear whether PKA is involved in tooth development. The aim of the present study was to assess the expression of alpha isoforms of PKA regulatory subunit (Prkar1a and Prkar2a) in mouse and human odontogenesis by in situ hybridization. PRKAR1A and PRKAR2A mRNA transcription was further confirmed in a human deciduous germ by qRT-PCR. Mouse Prkar1a and human PRKAR2A exhibited a dynamic spatio-temporal expression in tooth development, whereas neither human PRKAR1A nor mouse Prkar2a showed their expression in odontogenesis. These isoforms thus showed different expression pattern between human and mouse tooth germs. Copyright © 2014 Elsevier B.V. All rights reserved.

  7. β1-subunit-induced structural rearrangements of the Ca2+- and voltage-activated K+ (BK) channel.

    PubMed

    Castillo, Juan P; Sánchez-Rodríguez, Jorge E; Hyde, H Clark; Zaelzer, Cristian A; Aguayo, Daniel; Sepúlveda, Romina V; Luk, Louis Y P; Kent, Stephen B H; Gonzalez-Nilo, Fernando D; Bezanilla, Francisco; Latorre, Ramón

    2016-06-07

    Large-conductance Ca(2+)- and voltage-activated K(+) (BK) channels are involved in a large variety of physiological processes. Regulatory β-subunits are one of the mechanisms responsible for creating BK channel diversity fundamental to the adequate function of many tissues. However, little is known about the structure of its voltage sensor domain. Here, we present the external architectural details of BK channels using lanthanide-based resonance energy transfer (LRET). We used a genetically encoded lanthanide-binding tag (LBT) to bind terbium as a LRET donor and a fluorophore-labeled iberiotoxin as the LRET acceptor for measurements of distances within the BK channel structure in a living cell. By introducing LBTs in the extracellular region of the α- or β1-subunit, we determined (i) a basic extracellular map of the BK channel, (ii) β1-subunit-induced rearrangements of the voltage sensor in α-subunits, and (iii) the relative position of the β1-subunit within the α/β1-subunit complex.

  8. Structural and functional consequences of succinate dehydrogenase subunit B mutations.

    PubMed

    Kim, E; Rath, E M; Tsang, V H M; Duff, A P; Robinson, B G; Church, W B; Benn, D E; Dwight, T; Clifton-Bligh, R J

    2015-06-01

    Mitochondrial dysfunction, due to mutations of the gene encoding succinate dehydrogenase (SDH), has been implicated in the development of adrenal phaeochromocytomas, sympathetic and parasympathetic paragangliomas, renal cell carcinomas, gastrointestinal stromal tumours and more recently pituitary tumours. Underlying mechanisms behind germline SDH subunit B (SDHB) mutations and their associated risk of disease are not clear. To investigate genotype-phenotype correlation of SDH subunit B (SDHB) variants, a homology model for human SDH was developed from a crystallographic structure. SDHB mutations were mapped, and biochemical effects of these mutations were predicted in silico. Results of structural modelling indicated that many mutations within SDHB are predicted to cause either failure of functional SDHB expression (p.Arg27*, p.Arg90*, c.88delC and c.311delAinsGG), or disruption of the electron path (p.Cys101Tyr, p.Pro197Arg and p.Arg242His). GFP-tagged WT SDHB and mutant SDHB constructs were transfected (HEK293) to determine biological outcomes of these mutants in vitro. According to in silico predictions, specific SDHB mutations resulted in impaired mitochondrial localisation and/or SDH enzymatic activity. These results indicated strong genotype-functional correlation for SDHB variants. This study reveals new insights into the effects of SDHB mutations and the power of structural modelling in predicting biological consequences. We predict that our functional assessment of SDHB mutations will serve to better define specific consequences for SDH activity as well as to provide a much needed assay to distinguish pathogenic mutations from benign variants. © 2015 Society for Endocrinology.

  9. Subunit organization in cytoplasmic dynein subcomplexes

    PubMed Central

    King, Stephen J.; Bonilla, Myriam; Rodgers, Michael E.; Schroer, Trina A.

    2002-01-01

    Because cytoplasmic dynein plays numerous critical roles in eukaryotic cells, determining the subunit composition and the organization and functions of the subunits within dynein are important goals. This has been difficult partly because of accessory polypeptide heterogeneity of dynein populations. The motor domain containing heavy chains of cytoplasmic dynein are associated with multiple intermediate, light intermediate, and light chain accessory polypeptides. We examined the organization of these subunits within cytoplasmic dynein by separating the molecule into two distinct subcomplexes. These subcomplexes were competent to reassemble into a molecule with dynein-like properties. One subcomplex was composed of the dynein heavy and light intermediate chains whereas the other subcomplex was composed of the intermediate and light chains. The intermediate and light chain subcomplex could be further separated into two pools, only one of which contained dynein light chains. The two pools had distinct intermediate chain compositions, suggesting that intermediate chain isoforms have different light chain–binding properties. When the two intermediate chain pools were characterized by analytical velocity sedimentation, at least four molecular components were seen: intermediate chain monomers, intermediate chain dimers, intermediate chain monomers with bound light chains, and a mixture of intermediate chain dimers with assorted bound light chains. These data provide new insights into the compositional heterogeneity and assembly of the cytoplasmic dynein complex and suggest that individual dynein molecules have distinct molecular compositions in vivo. PMID:11967380

  10. DNA sequences, recombinant DNA molecules and processes for producing the A and B subunits of cholera toxin and preparations containing so-obtained subunit or subunits

    SciTech Connect

    Harford, N.; De Wilde, M.

    1987-05-19

    A recombinant DNA molecule is described comprising at least a portion coding for subunits A and B of cholera toxin, or a fragment or derivative of the portion wherein the fragment or derivative codes for a polypeptide have an activity which can induce an immune response to subunit A; can induce an immune response to subunit A and cause epithelial cell penetration and the enzymatic effect leading to net loss of fluid into the gut lumen; can bind to the membrane receptor for the B subunit of cholera toxin; can induce an immune response to subunit B; can induce an immune response to subunit B and bind to the membrane receptor; or has a combination of the activities.

  11. 3D Gel Map of Arabidopsis Complex I

    PubMed Central

    Peters, Katrin; Belt, Katharina; Braun, Hans-Peter

    2013-01-01

    Complex I has a unique structure in plants and includes extra subunits. Here, we present a novel study to define its protein constituents. Mitochondria were isolated from Arabidopsis thaliana cell cultures, leaves, and roots. Subunits of complex I were resolved by 3D blue-native (BN)/SDS/SDS-PAGE and identified by mass spectrometry. Overall, 55 distinct proteins were found, seven of which occur in pairs of isoforms. We present evidence that Arabidopsis complex I consists of 49 distinct types of subunits, 40 of which represent homologs of bovine complex I. The nine other subunits represent special proteins absent in the animal linage of eukaryotes, most prominently a group of subunits related to bacterial gamma-type carbonic anhydrases. A GelMap http://www.gelmap.de/arabidopsis-3d-complex-i/ is presented for promoting future complex I research in Arabidopsis thaliana. PMID:23761796

  12. Models for the a subunits of the Thermus thermophilus V/A-ATPase and Saccharomyces cerevisiae V-ATPase enzymes by cryo-EM and evolutionary covariance

    PubMed Central

    Schep, Daniel G.; Rubinstein, John L.

    2016-01-01

    Rotary ATPases couple ATP synthesis or hydrolysis to proton translocation across a membrane. However, understanding proton translocation has been hampered by a lack of structural information for the membrane-embedded a subunit. The V/A-ATPase from the eubacterium Thermus thermophilus is similar in structure to the eukaryotic V-ATPase but has a simpler subunit composition and functions in vivo to synthesize ATP rather than pump protons. We determined the T. thermophilus V/A-ATPase structure by cryo-EM at 6.4 Å resolution. Evolutionary covariance analysis allowed tracing of the a subunit sequence within the map, providing a complete model of the rotary ATPase. Comparing the membrane-embedded regions of the T. thermophilus V/A-ATPase and eukaryotic V-ATPase from Saccharomyces cerevisiae allowed identification of the α-helices that belong to the a subunit and revealed the existence of previously unknown subunits in the eukaryotic enzyme. Subsequent evolutionary covariance analysis enabled construction of a model of the a subunit in the S. cerevisae V-ATPase that explains numerous biochemical studies of that enzyme. Comparing the two a subunit structures determined here with a structure of the distantly related a subunit from the bovine F-type ATP synthase revealed a conserved pattern of residues, suggesting a common mechanism for proton transport in all rotary ATPases. PMID:26951669

  13. Models for the a subunits of the Thermus thermophilus V/A-ATPase and Saccharomyces cerevisiae V-ATPase enzymes by cryo-EM and evolutionary covariance.

    PubMed

    Schep, Daniel G; Zhao, Jianhua; Rubinstein, John L

    2016-03-22

    Rotary ATPases couple ATP synthesis or hydrolysis to proton translocation across a membrane. However, understanding proton translocation has been hampered by a lack of structural information for the membrane-embedded a subunit. The V/A-ATPase from the eubacterium Thermus thermophilus is similar in structure to the eukaryotic V-ATPase but has a simpler subunit composition and functions in vivo to synthesize ATP rather than pump protons. We determined the T. thermophilus V/A-ATPase structure by cryo-EM at 6.4 Å resolution. Evolutionary covariance analysis allowed tracing of the a subunit sequence within the map, providing a complete model of the rotary ATPase. Comparing the membrane-embedded regions of the T. thermophilus V/A-ATPase and eukaryotic V-ATPase from Saccharomyces cerevisiae allowed identification of the α-helices that belong to the a subunit and revealed the existence of previously unknown subunits in the eukaryotic enzyme. Subsequent evolutionary covariance analysis enabled construction of a model of the a subunit in the S. cerevisae V-ATPase that explains numerous biochemical studies of that enzyme. Comparing the two a subunit structures determined here with a structure of the distantly related a subunit from the bovine F-type ATP synthase revealed a conserved pattern of residues, suggesting a common mechanism for proton transport in all rotary ATPases.

  14. USGS maps

    USGS Publications Warehouse

    ,

    2005-01-01

    Discover a small sample of the millions of maps produced by the U.S. Geological Survey (USGS) in its mission to map the Nation and survey its resources. This booklet gives a brief overview of the types of maps sold and distributed by the USGS through its Earth Science Information Centers (ESIC) and also available from business partners located in most States. The USGS provides a wide variety of maps, from topographic maps showing the geographic relief and thematic maps displaying the geology and water resources of the United States, to special studies of the moon and planets.

  15. Differential accumulation of ribonucleotide reductase subunits in clam oocytes: the large subunit is stored as a polypeptide, the small subunit as untranslated mRNA

    PubMed Central

    1986-01-01

    Within minutes of fertilization of clam oocytes, translation of a set of maternal mRNAs is activated. One of the most abundant of these stored mRNAs encodes the small subunit of ribonucleotide reductase (Standart, N. M., S. J. Bray, E. L. George, T. Hunt, and J. V. Ruderman, 1985, J. Cell Biol., 100:1968-1976). Unfertilized oocytes do not contain any ribonucleotide reductase activity; such activity begins to appear shortly after fertilization. In virtually all organisms, this enzyme is composed of two dissimilar subunits with molecular masses of approximately 44 and 88 kD, both of which are required for activity. This paper reports the identification of the large subunit of clam ribonucleotide reductase isolated by dATP-Sepharose chromatography as a relatively abundant 86-kD polypeptide which is already present in oocytes, and whose level remains constant during early development. The enzyme activity of this large subunit was established in reconstitution assays using the small subunit isolated from embryos by virtue of its binding to the anti-tubulin antibody YL 1/2. Thus the two components of clam ribonucleotide reductase are differentially stored in the oocyte: the small subunit in the form of untranslated mRNA and the large subunit as protein. When fertilization triggers the activation of translation of the maternal mRNA, the newly synthesized small subunit combines with the preformed large subunit to generate active ribonucleotide reductase. PMID:3536960

  16. Prefoldin Subunits Are Protected from Ubiquitin-Proteasome System-mediated Degradation by Forming Complex with Other Constituent Subunits*

    PubMed Central

    Miyazawa, Makoto; Tashiro, Erika; Kitaura, Hirotake; Maita, Hiroshi; Suto, Hiroo; Iguchi-Ariga, Sanae M. M.; Ariga, Hiroyoshi

    2011-01-01

    The molecular chaperone prefoldin (PFD) is a complex comprised of six different subunits, PFD1-PFD6, and delivers newly synthesized unfolded proteins to cytosolic chaperonin TRiC/CCT to facilitate the folding of proteins. PFD subunits also have functions different from the function of the PFD complex. We previously identified MM-1α/PFD5 as a novel c-Myc-binding protein and found that MM-1α suppresses transformation activity of c-Myc. However, it remains unclear how cells regulate protein levels of individual subunits and what mechanisms alter the ratio of their activities between subunits and their complex. In this study, we found that knockdown of one subunit decreased protein levels of other subunits and that transfection of five subunits other than MM-1α into cells increased the level of endogenous MM-1α. We also found that treatment of cells with MG132, a proteasome inhibitor, increased the level of transfected/overexpressed MM-1α but not that of endogenous MM-1α, indicating that overexpressed MM-1α, but not endogenous MM-1α, was degraded by the ubiquitin proteasome system (UPS). Experiments using other PFD subunits showed that the UPS degraded a monomer of PFD subunits, though extents of degradation varied among subunits. Furthermore, the level of one subunit was increased after co-transfection with the respective subunit, indicating that there are specific combinations between subunits to be stabilized. These results suggest mutual regulation of protein levels among PFD subunits and show how individual subunits form the PFD complex without degradation. PMID:21478150

  17. Prefoldin subunits are protected from ubiquitin-proteasome system-mediated degradation by forming complex with other constituent subunits.

    PubMed

    Miyazawa, Makoto; Tashiro, Erika; Kitaura, Hirotake; Maita, Hiroshi; Suto, Hiroo; Iguchi-Ariga, Sanae M M; Ariga, Hiroyoshi

    2011-06-03

    The molecular chaperone prefoldin (PFD) is a complex comprised of six different subunits, PFD1-PFD6, and delivers newly synthesized unfolded proteins to cytosolic chaperonin TRiC/CCT to facilitate the folding of proteins. PFD subunits also have functions different from the function of the PFD complex. We previously identified MM-1α/PFD5 as a novel c-Myc-binding protein and found that MM-1α suppresses transformation activity of c-Myc. However, it remains unclear how cells regulate protein levels of individual subunits and what mechanisms alter the ratio of their activities between subunits and their complex. In this study, we found that knockdown of one subunit decreased protein levels of other subunits and that transfection of five subunits other than MM-1α into cells increased the level of endogenous MM-1α. We also found that treatment of cells with MG132, a proteasome inhibitor, increased the level of transfected/overexpressed MM-1α but not that of endogenous MM-1α, indicating that overexpressed MM-1α, but not endogenous MM-1α, was degraded by the ubiquitin proteasome system (UPS). Experiments using other PFD subunits showed that the UPS degraded a monomer of PFD subunits, though extents of degradation varied among subunits. Furthermore, the level of one subunit was increased after co-transfection with the respective subunit, indicating that there are specific combinations between subunits to be stabilized. These results suggest mutual regulation of protein levels among PFD subunits and show how individual subunits form the PFD complex without degradation.

  18. Inherent conformational flexibility of F1-ATPase α-subunit.

    PubMed

    Hahn-Herrera, Otto; Salcedo, Guillermo; Barril, Xavier; García-Hernández, Enrique

    2016-09-01

    The core of F1-ATPase consists of three catalytic (β) and three noncatalytic (α) subunits, forming a hexameric ring in alternating positions. A wealth of experimental and theoretical data has provided a detailed picture of the complex role played by catalytic subunits. Although major conformational changes have only been seen in β-subunits, it is clear that α-subunits have to respond to these changes in order to be able to transmit information during the rotary mechanism. However, the conformational behavior of α-subunits has not been explored in detail. Here, we have combined unbiased molecular dynamics (MD) simulations and calorimetrically measured thermodynamic signatures to investigate the conformational flexibility of isolated α-subunits, as a step toward deepening our understanding of its function inside the α3β3 ring. The simulations indicate that the open-to-closed conformational transition of the α-subunit is essentially barrierless, which is ideal to accompany and transmit the movement of the catalytic subunits. Calorimetric measurements of the recombinant α-subunit from Geobacillus kaustophilus indicate that the isolated subunit undergoes no significant conformational changes upon nucleotide binding. Simulations confirm that the nucleotide-free and nucleotide-bound subunits show average conformations similar to that observed in the F1 crystal structure, but they reveal an increased conformational flexibility of the isolated α-subunit upon MgATP binding, which might explain the evolutionary conserved capacity of α-subunits to recognize nucleotides with considerable strength. Furthermore, we elucidate the different dependencies that α- and β-subunits show on Mg(II) for recognizing ATP.

  19. RICH MAPS

    EPA Science Inventory

    Michael Goodchild recently gave eight reasons why traditional maps are limited as communication devices, and how interactive internet mapping can overcome these limitations. In the past, many authorities in cartography, from Jenks to Bertin, have emphasized the importance of sim...

  20. RICH MAPS

    EPA Science Inventory

    Michael Goodchild recently gave eight reasons why traditional maps are limited as communication devices, and how interactive internet mapping can overcome these limitations. In the past, many authorities in cartography, from Jenks to Bertin, have emphasized the importance of sim...

  1. Historical Mapping

    USGS Publications Warehouse

    ,

    1999-01-01

    Maps become out of date over time. Maps that are out of date, however, can be useful to historians, attorneys, environmentalists, genealogists, and others interested in researching the background of a particular area. Local historians can compare a series of maps of the same area compiled over a long period of time to learn how the area developed. A succession of such maps can provide a vivid picture of how a place changed over time.

  2. Molecular characterization of the gene encoding the DNA gyrase A subunit of Streptococcus pneumoniae.

    PubMed

    Balas, D; Fernández-Moreira, E; De La Campa, A G

    1998-06-01

    The gene encoding the DNA gyrase A subunit of Streptococcus pneumoniae was cloned and sequenced. The gyrA gene codes for a protein of 822 amino acids homologous to the gyrase A subunit of eubacteria. Translation of the gene in an Escherichia coli expression system revealed a 92-kDa polypeptide. A sequence-directed DNA curvature was identified in the promoter region of gyrA. The bend center was mapped and located between the -35 and -10 regions of the promoter. Primer extension analysis showed that gyrA transcription initiates 6 bp downstream of an extended -10 promoter. The possible implications of the bent DNA region as a regulatory element in the transcription of gyrA are discussed.

  3. Molecular Characterization of the Gene Encoding the DNA Gyrase A Subunit of Streptococcus pneumoniae

    PubMed Central

    Balas, Delia; Fernández-Moreira, Esteban; De La Campa, Adela G.

    1998-01-01

    The gene encoding the DNA gyrase A subunit of Streptococcus pneumoniae was cloned and sequenced. The gyrA gene codes for a protein of 822 amino acids homologous to the gyrase A subunit of eubacteria. Translation of the gene in an Escherichia coli expression system revealed a 92-kDa polypeptide. A sequence-directed DNA curvature was identified in the promoter region of gyrA. The bend center was mapped and located between the −35 and −10 regions of the promoter. Primer extension analysis showed that gyrA transcription initiates 6 bp downstream of an extended −10 promoter. The possible implications of the bent DNA region as a regulatory element in the transcription of gyrA are discussed. PMID:9603872

  4. Exclusion of five subunits of cGMP phosphodiesterase in Leber's congenital amaurosis.

    PubMed

    Perrault, I; Châtelin, S; Nancy, V; Rozet, J M; Gerber, S; Ghazi, I; Souied, E; Dufier, J L; Munnich, A; de Gunzburg, J; Kaplan, J

    1998-03-01

    Leber's congenital amaurosis (LCA) is the earliest and most severe of all inherited retinal dystrophies. Recently, we mapped an LCA gene to chromosome 17p13.1 (LCA1) and ascribed the disease to mutations of the retinal guanylate cyclase (ret GC) gene in a subset of families of North African ancestry. Owing to the genetic heterogeneity of LCA and considering that LCA1 results from an impaired production of cGMP in the retina (with permanent closure of cGMP-gated cation channels), we hypothesized that the activation of the cGMP phosphodiesterase (PDE) could trigger the disease by lowering the intracellular cGMP level in the retina. The rod and cone cGMP-PDE inhibitory subunits were regarded therefore as candidate genes in LCA. Here, we report the exclusion of five rod and cone cGMP-PDE subunits in LCA families unlinked to chromosome 17p13.

  5. Production and characterization of antibody probes directed at constant regions of the alpha and beta subunit of the human T cell receptor.

    PubMed

    Fabbi, M; Acuto, O; Bensussan, A; Poole, C B; Reinherz, E L

    1985-08-01

    To generate antibodies directed at constant regions of the human T cell receptor, purified alpha and beta subunits of a human T cell antigen/major histocompatibility complex receptor from the REX tumor (Ti-REX) were isolated by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and utilized to immunize rabbits. H36 (anti-alpha subunit) and H38 (anti-beta subunit) antisera were strongly reactive with the denatured subunits and also immunoprecipitated the Ti heterodimer from 125I surface-labeled lysates of REX, inducer, suppressor and cytotoxic T cell clones, peripheral T lymphocytes and thymocytes. Moreover, immunodepletion experiments showed that such antisera recognized antigenic determinant(s) shared by all Ti molecules expressed in the thymus. Several observations were made with these anticonstant region antibodies. First, peptide map analysis showed that the T cell receptor molecules recognized by the anti-clonotype and the anti-constant region heteroantisera on a given T cell clone are identical, thus supporting the view that the T cell receptor undergoes allelic exclusion. Second, since the individual antisera were weakly cross-reactive with the other denatured subunit, such subunits probably share conserved sequences. Third, the absence of antisera reactivity with intact cells implies that most of these constant region epitopes must be obscured by associated molecules, perhaps including one or more of the 20-25-kDa T3 subunits. Fourth, the extensive difference in two-dimensional peptide maps of Ti alpha subunits from clones of differing specificities makes it likely that the subunit contributes in a major way to antigen/major histocompatibility complex binding.

  6. [Nose surgical anatomy in six aesthetic subunits].

    PubMed

    Chaput, B; Lauwers, F; Lopez, R; Saboye, J; André, A; Grolleau, J-L; Chavoin, J-P

    2013-04-01

    The nose is a complex entity, combining aesthetic and functional roles. Descriptive anatomy is a fundamental science that it can be difficult to relate directly to our daily surgical activity. Reasoning in terms of aesthetic subunits to decide on his actions appeared to us so obvious. The aim of this paper is to resume the anatomical bases relevant to our daily practice in order to fully apprehend the restorative or cosmetic procedures. We discuss the limits of the systematization of these principles in nasal oncology. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

  7. Structural model of the 50S subunit of E.Coli ribosomes from solution scattering

    SciTech Connect

    Svergun, D.I.; Koch, M.H.J.; Pedersen, J.S.; Serdyuk, I.N.

    1994-12-31

    The application of new methods of small-angle scattering data interpretation to a contrast variation study of the 50S ribosomal subunit of Escherichia coli in solution is described. The X-ray data from contrast variation with sucrose are analyzed in terms of the basic scattering curves from the volume inaccessible to sucrose and from the regions inside this volume occupied mainly by RNA and by proteins. From these curves models of the shape of the 50S and its RNA-rich core are evaluated and positioned so that their difference produces a scattering curve which is in good agreement with the scattering from the protein moiety. Basing on this preliminary model, the X-ray and neutron contrast variation data of the 50S subunit in aqueous solutions are interpreted in the frame of the advanced two-phase model described by the shapes of the 50S subunit and its RNA-rich core taking into account density fluctuations inside the RNA and the protein moiety. The shape of the envelope of the 50S subunit and of the RNA-rich core are evaluated with a resolution of about 40A. The shape of the envelope is in good agreement with the models of the 50S subunit obtained from electron microscopy on isolated particles. The shape of the RNA-rich core correlates well with the model of the entire particle determined by the image reconstruction from ordered sheets indicating that the latter model which is based on the subjective contouring of density maps is heavily biased towards the RNA.

  8. Identification and linkage of the proteasome activator complex PA28 subunit genes in zebrafish.

    PubMed

    Murray, B W; Sültmann, H; Klein, J

    2000-06-01

    PA28 is an activator of the latent 20S proteasome, a large multisubunit complex involved in intracellular proteolysis. Two forms of hexameric PA28 have been identified, PA28-(alphabeta)3 and PA28-(gamma)6, of which the former is of immunological importance. Both the PA28-alpha and PA28-beta subunits are inducible by interferon-gamma (IFN-gamma) and the PA28-(alphabeta)3 complex enhances the ability of the 20S proteasome to produce peptides suited for binding to major histocompatibility complex (Mhc) class I molecules. To identify the homologues of the PA28 subunits in zebrafish we screened a cDNA library and obtained full-length cDNA sequences of the genes PSME1, PSME2 and PSME3 coding for the PA28-alpha, PA28-beta and PA28-gamma subunits, respectively. Phylogenetic analysis indicates the existence of the ancestors of all three genes prior to the divergence of tetrapods and bony fishes. The IFN-gamma-inducible subunits, PA28-alpha and PA28-beta, evolve faster than the presumably older PA28-gamma subunit. Using zebrafish radiation hybrid panels, the genes PSME2 and PSME3 were mapped to linkage group 12 and shown to be separated by a distance of less than 2.4 cM. This observation suggests that an intrachromosomal duplication event created the precursor of the IFN-gamma-inducible genes from a PA28-gamma-like ancestor prior to their recruitment into the Mhc class I peptide presentation pathway.

  9. Structural requirement of the calcium-channel subunit alpha2delta for gabapentin binding.

    PubMed Central

    Wang, M; Offord, J; Oxender, D L; Su, T Z

    1999-01-01

    Gabapentin [Neurontin, 1-(aminomethyl)cyclohexaneacetic acid] is a novel anticonvulsant drug with a high binding affinity for the Ca(2+)-channel subunit alpha(2)delta. In this study, the gabapentin-binding properties of wild-type and mutated porcine brain alpha(2)delta proteins were investigated. Removal of the disulphide bonds between the alpha(2) and the delta subunits did not result in a significant loss of gabapentin binding, suggesting that the disulphide linkage between the two subunits is not required for binding. Singly expressed alpha(2) protein remained membrane associated. However, alpha(2) alone was unable to bind gabapentin, unless the cells were concurrently transfected with the expression vector for delta, suggesting that both alpha(2) and delta are required for gabapentin binding. Using internal deletion mutagenesis, we mapped two regions [amino acid residues 339-365 (DeltaF) and 875-905 (DeltaJ)] within the alpha(2) subunit that are not required for gabapentin binding. Further, deletion of three other individual regions [amino acid residues 206-222 (DeltaD), 516-537 (DeltaH) and 583-603 (DeltaI)] within the alpha(2) subunit disrupted gabapentin binding, suggesting the structural importance of these regions. Using alanine to replace four to six amino acid residues in each of these regions abolished gabapentin binding. These results demonstrate that region D, between the N-terminal end and the first putative transmembrane domain of alpha(2), and regions H and I, between the putative splicing acceptor sites (Gln(511) and Ser(601)), may play important roles in maintaining the structural integrity for gabapentin binding. Further single amino acid replacement mutagenesis within these regions identified Arg(217) as critical for gabapentin binding. PMID:10455017

  10. Amino-terminal truncations of the ribulose-bisphosphate carboxylase small subunit influence catalysis and subunit interactions.

    PubMed Central

    Paul, K; Morell, M K; Andrews, T J

    1993-01-01

    The first 20 residues at the amino terminus of the small subunit of spinach ribulose-1,5-bisphosphate carboxylase form an irregular arm that makes extensive contacts with the large subunit and also with another small subunit (S. Knight, I. Andersson, and C.-I. Brändén [1990] J Mol Biol 215: 113-160). The influence of these contacts on subunit binding and, indirectly, on catalysis was investigated by constructing truncations from the amino terminus of the small subunit of the highly homologous enzyme from Synechococcus PCC 6301 expressed in Escherichia coli. Removal of the first six residues (and thus the region of contact with a neighboring small subunit) affected neither the affinity with which the small subunits bound to the large subunits nor the catalytic properties of the assembled holoenzyme. Extending the truncation to include the first 12 residues (which encroaches into a highly conserved region that interacts with the large subunit) also did not weaken intersubunit binding appreciably, but it reduced the catalytic activity of the holoenzyme nearly 5-fold. Removal of an additional single residue (i.e. removal of a total of 13 residues) weakened intersubunit binding approximately 80-fold. Paradoxically, this partially restored catalytic activity to approximately 40% of that of the wild-type holoenzyme. None of these truncations materially affected the Km values for ribulose-1,5-bisphosphate or CO2. Removal of all 20 residues of the irregular arm (thereby deleting the conserved region of contact with large subunits) totally abolished the small subunit's ability to bind to large subunits to form a stable holoenzyme. However, this truncated small subunit was still synthesized by the E. coli cells. These data are interpreted in terms of the role of the amino-terminal arm of the small subunit in maintaining the structure of the holoenzyme. PMID:8278544

  11. Homologous and unique G protein alpha subunits in the nematode Caenorhabditis elegans.

    PubMed Central

    Lochrie, M A; Mendel, J E; Sternberg, P W; Simon, M I

    1991-01-01

    A cDNA corresponding to a known G protein alpha subunit, the alpha subunit of Go (Go alpha), was isolated and sequenced. The predicted amino acid sequence of C. elegans Go alpha is 80-87% identical to other Go alpha sequences. An mRNA that hybridizes to the C. elegans Go alpha cDNA can be detected on Northern blots. A C. elegans protein that crossreacts with antibovine Go alpha antibody can be detected on immunoblots. A cosmid clone containing the C. elegans Go alpha gene (goa-1) was isolated and mapped to chromosome I. The genomic fragments of three other C. elegans G protein alpha subunit genes (gpa-1, gpa-2, and gpa-3) have been isolated using the polymerase chain reaction. The corresponding cosmid clones were isolated and mapped to disperse locations on chromosome V. The sequences of two of the genes, gpa-1 and gpa-3, were determined. The predicted amino acid sequences of gpa-1 and gpa-3 are only 48% identical to each other. Therefore, they are likely to have distinct functions. In addition they are not homologous enough to G protein alpha subunits in other organisms to be classified. Thus C. elegans has G proteins that are identifiable homologues of mammalian G proteins as well as G proteins that appear to be unique to C. elegans. Study of identifiable G proteins in C. elegans may result in a further understanding of their function in other organisms, whereas study of the novel G proteins may provide an understanding of unique aspects of nematode physiology. Images PMID:1907494

  12. Glycine receptor subunits expression in the developing rat retina.

    PubMed

    Sánchez-Chávez, Gustavo; Velázquez-Flores, Miguel Ángel; Ruiz Esparza-Garrido, Ruth; Salceda, Rocío

    2017-09-01

    Glycine receptor (GlyR) consists of two α (1-4) and three β subunits. Considerable evidence indicates that the adult retina expresses the four types of α subunits; however, the proportion of these subunits in adult and immature retina is almost unknown. In this report we have studied mRNA and the protein expression of GlyR subunits in the retina during postnatal rat development by Real-Time qRT-PCR and western blot. mRNA and protein expression indicated a gradual increase of the α1, α3, α4 and β GlyR subunits during postnatal ages tested. The mRNA β subunit showed higher expression levels (∼3 fold) than those observed for the α1 and α3 subunits. Very interestingly, the α2 GlyR subunit had the highest expression in the retina, even in the adult. These results revealed the expression of GlyR at early postnatal ages, supporting its role in retina development. In addition, our results indicated that the adult retina expressed a high proportion of the α2 subunit, suggesting the expression of monomeric and/or heteromeric receptors. A variety of studies are needed to further characterize the role of the specific subunits in both adult and immature retina. Copyright © 2017 Elsevier Ltd. All rights reserved.

  13. Stoichiometry of δ subunit containing GABA(A) receptors.

    PubMed

    Patel, B; Mortensen, M; Smart, T G

    2014-02-01

    Although the stoichiometry of the major synaptic αβγ subunit-containing GABAA receptors has consensus support for 2α:2β:1γ, a clear view of the stoichiometry of extrasynaptic receptors containing δ subunits has remained elusive. Here we examine the subunit stoichiometry of recombinant α4β3δ receptors using a reporter mutation and a functional electrophysiological approach. Using site-directed mutagenesis, we inserted a highly characterized 9' serine to leucine mutation into the second transmembrane (M2) region of α4, β3 and δ subunits that increases receptor sensitivity to GABA. Whole-cell, GABA-activated currents were recorded from HEK-293 cells co-expressing different combinations of wild-type (WT) and/or mutant α4(L297S), β3(L284S) and δ(L288S) subunits. Recombinant receptors containing one or more mutant subunits showed increased GABA sensitivity relative to WT receptors by approximately fourfold, independent of the subunit class (α, β or δ) carrying the mutation. GABA dose-response curves of cells co-expressing WT subunits with their respective L9'S mutants exhibited multiple components, with the number of discernible components enabling a subunit stoichiometry of 2α, 2β and 1δ to be deduced for α4β3δ receptors. Varying the cDNA transfection ratio by 10-fold had no significant effect on the number of incorporated δ subunits. Subunit stoichiometry is an important determinant of GABAA receptor function and pharmacology, and δ subunit-containing receptors are important mediators of tonic inhibition in several brain regions. Here we demonstrate a preferred subunit stoichiometry for α4β3δ receptors of 2α, 2β and 1δ. © 2013 The British Pharmacological Society.

  14. Topographic mapping

    USGS Publications Warehouse

    ,

    2008-01-01

    The U.S. Geological Survey (USGS) produced its first topographic map in 1879, the same year it was established. Today, more than 100 years and millions of map copies later, topographic mapping is still a central activity for the USGS. The topographic map remains an indispensable tool for government, science, industry, and leisure. Much has changed since early topographers traveled the unsettled West and carefully plotted the first USGS maps by hand. Advances in survey techniques, instrumentation, and design and printing technologies, as well as the use of aerial photography and satellite data, have dramatically improved mapping coverage, accuracy, and efficiency. Yet cartography, the art and science of mapping, may never before have undergone change more profound than today.

  15. Calmodulin is a subunit of nitric oxide synthase from macrophages

    PubMed Central

    1992-01-01

    A central issue in nitric oxide (NO) research is to understand how NO can act in some settings as a servoregulator and in others as a cytotoxin. To answer this, we have sought a molecular basis for the differential regulation of the two known types of NO synthase (NOS). Constitutive NOS's in endothelium and neurons are activated by agonist- induced elevation of Ca2+ and resultant binding of calmodulin (CaM). In contrast, NOS in macrophages does not require added Ca2+ or CaM, but is regulated instead by transcription. We show here that macrophage NOS contains, as a tightly bound subunit, a molecule with the immunologic reactivity, high performance liquid chromatography retention time, tryptic map, partial amino acid sequence, and exact molecular mass of CaM. In contrast to most CaM-dependent enzymes, macrophage NOS binds CaM tightly without a requirement for elevated Ca2+. This may explain why NOS that is independent of Ca2+ and elevated CaM appears to be activated simply by being synthesized. PMID:1380065

  16. The {gamma}-aminobutyric acid receptor {gamma}3 subunit gene (GABRG3) is tightly linked to the {alpha}5 subunit gene (GABRA5) on human chromosome 15q11-q13 and is transcribed in the same orientation

    SciTech Connect

    Greger, V. |; Knoll, J.H.M.; Woolf, E. |

    1995-03-20

    GABA{sub A} receptors are heterooligomeric ligand-gated ion channels that mediate the effect of the inhibitory neurotransmitter {gamma}-aminobutyric acid. The GABA{sub A} receptors consist of at least 15 different receptor subunits that can be classified into 5 subfamilies ({alpha},{beta},{gamma},{delta},{rho}) on the basis of sequence similarity. Chromosomal mapping studies have revealed that several of the GABA{sub A} receptor subunit genes appear to be organized as clusters. One such cluster, which consists of the GABA{sub A} receptor {beta}3 (GABRB3) and {alpha}5 (GABRA5) sub-unit genes, is located in chromosome 15q11-q13. It is shown here that the GABA{sub A} receptor {gamma}3 subunit gene (GABRG3) also maps to this region. Lambda and P1 phage clones surrounding both ends of GABRG3 were isolated; the clones derived from the 5{prime} end of GABRG3 were linked to an existing phage contig spanning the 3{prime} end of GABRA5. The two genes are located within 35 kb of each other and are transcribed in the same orientation. 39 refs., 4 figs.

  17. Complementation of subunits from different bacterial luciferases. Evidence for the role of the. beta. subunit in the bioluminescent mechanism

    SciTech Connect

    Meighen, E.A.; Bartlet, I.

    1980-12-10

    Complementation of the nonidentical subunits (..cap alpha.. and ..beta..) of luciferases isolated from two different bioluminescent strains, Beneckea harveyi and Photobacterium phosphoreum, has resulted in the formation of a functional hybrid luciferase (..cap alpha../sub h/..beta../sub p/) containing the ..cap alpha.. subunit from B. harveyi luciferase (..cap alpha../sub h/) and the ..beta.. subunit from P. phosphoreum luciferase (..beta../sub p/). The complementation was unidirectional; activity could not be restored by complementing the ..cap alpha.. subunit of P. phosphoreum luciferase with the ..beta.. subunit of B. harveyi luciferase, showing that the subunits from these luciferases were not identical. Kinetic parameters of the hybrid luciferase reflecting the intermediate and later steps of the bioluminescent reaction as well as the overall activity and specificity were essentially identical to the same kinetic parameters for B. harveyi luciferase, the source of the ..cap alpha.. subunit, and quite distinct from those of P. phosphoreum luciferase. However, kinetic parameters that reflected the initial step in the reaction involving interaction of FMNH/sub 2/ and luciferase were altered in the hybrid luciferase compared to both the parental luciferases, the K/sub d/ for FMNH/sub 2/ actually being closer to that observed for the P. phosphoreum luciferase (the source of the ..beta.. subunit). These results provide direct evidence that modification or alteration of the ..beta.. subunit in a dimeric luciferase molecule can affect the kinetic properties and indicates that the ..beta.. subunit plays a functional role in the bioluminescent mechanism. It is proposed that both the ..cap alpha.. and ..beta.. subunits are involved with the initial interaction with FMNH/sub 2/, whereas subsequent steps in the mechanism are dictated exclusively by the ..cap alpha.. subunit and are unaffected by alterations in the ..beta.. subunit.

  18. Formation of active bacterial luciferase between interspecific subunits in vivo.

    PubMed

    Almashanu, S; Tuby, A; Hadar, R; Einy, R; Kuhn, J

    1995-01-01

    Interspecific complementation between luxAs and luxBs from Vibrio harveyi, Vibrio fischeri, Photobacterium leiognathi and Xenorhabdus luminescens was examined in vivo. The individual genes from these species were cloned on different compatible plasmids or amplified by PCR and brought together to yield cis combinations without extraneous DNA. The beta subunits from V. harveyi and X. luminescens form active enzyme only with alpha subunits from one of these species. All other combinations yield active enzymes. The lack of activity of the V. harveyi and X. luminescens beta subunits with the alpha subunits from V. fischeri and P. leiognathi results from a lack of association. This was shown by in vivo competition in which these beta subunits were overproduced in comparison with the beta and alpha of V. fischeri. No reduction in light was found. Overall, the in vivo results parallel those found in vitro using isolated denatured subunits and renaturation by removal of the denaturant.

  19. Sodium channel β subunits: emerging targets in channelopathies

    PubMed Central

    O’Malley, Heather A.; Isom, Lori L.

    2016-01-01

    Voltage-gated sodium channels (VGSCs) are responsible for initiation and propagation of action potentials in excitable cells. VGSCs in mammalian brain are heterotrimeric complexes of α and β subunits. Originally called “auxiliary,” we now know that β subunit proteins are multifunctional signaling molecules that play roles in both excitable and non-excitable cell types, and with or without the pore-forming α subunit present. β subunits function in VGSC and potassium channel modulation, cell adhesion, and gene regulation, with particularly important roles in brain development. Mutations in the genes encoding β subunits are linked to a number of diseases, including epilepsy, sudden death syndromes like SUDEP and SIDS, and cardiac arrhythmia. While VGSC β subunit-specific drugs have not yet been developed, this protein family is an emerging therapeutic target. PMID:25668026

  20. [Molecular cloning of activin betaA subunit mature peptide from peafowl and its application in taxonomy and phylogeny].

    PubMed

    Zou, Fang-Dong; Tong, Xin-Xin; Yue, Bi-Song

    2005-03-01

    The sequences of activin gene betaA subunit mature peptide have been amplified from white peafowl, blue peafowl (pavo cristatus) and green peafowl (pavo muticus) genomic DNA by polymerase chain reaction (PCR) with a pair of degenerate primers. The target fragments were cloned into the vector pMD18-T and sequenced. The length of activin gene betaA subunit mature peptide is 345bp, which encoded a peptide of 115 amino acid residues. Sequence analysis of activin gene betaA subunit mature peptide demonstrated that the identity of nucleotide is 98.0% between blue peaflowl and green peafowl, and the identity of that is 98.8% between blue peaflowl and white peafow. Sequences comparison in NCBI revealed that the sequences of activin gene betaA subunit mature peptides of different species are highly conserved during evolution process. In addition, the restriction enzyme map of activins is high similar between white peafowl and blue peafowl. Phylogenetic tree was constructed with Mega 2 and Clustalxldx software. The result showed that white peafowl has a closer relationship to blue peafowl than to green peafowl. Considered the nucleotide differences of peafowls' activin gene betaA subunit mature peptides, a highly conserved region, we supported that white peafowl was derived from blue peafowl, and it is more possible the hybrid but just the product of color mutation, or maybe as a subspecies of Pavo genus.

  1. Conformational changes in the C terminus of Shaker K+ channel bound to the rat Kvβ2-subunit

    PubMed Central

    Sokolova, Olga; Accardi, Alessio; Gutierrez, David; Lau, Adrian; Rigney, Mike; Grigorieff, Nikolaus

    2003-01-01

    We studied the structure of the C terminus of the Shaker potassium channel. The 3D structures of the full-length and a C-terminal deletion (ΔC) mutant of Shaker were determined by electron microscopy and single-particle analysis. The difference map between the full-length and the truncated channels clearly shows a compact density, located on the sides of the T1 domain, that corresponds to a large part of the C terminus. We also expressed and purified both WT and ΔC Shaker, assembled with the rat Kvβ2-subunit. By using a difference map between the full-length and truncated Shaker α–β complexes, a conformational change was identified that shifts a large part of the C terminus away from the membrane domain and into close contact with the β-subunit. This conformational change, induced by the binding of the Kvβ2-subunit, suggests a possible mechanism for the modulation of the K+ voltage-gated channel function by its β-subunit. PMID:14569011

  2. Diversity of heterotrimeric G-protein γ subunits in plants.

    PubMed

    Trusov, Yuri; Chakravorty, David; Botella, José Ramón

    2012-10-31

    Heterotrimeric G-proteins, consisting of three subunits Gα, Gβ and Gγ are present in most eukaryotes and mediate signaling in numerous biological processes. In plants, Gγ subunits were shown to provide functional selectivity to G-proteins. Three unconventional Gγ subunits were recently reported in Arabidopsis, rice and soybean but no structural analysis has been reported so far. Their relationship with conventional Gγ subunits and taxonomical distribution has not been yet demonstrated. After an extensive similarity search through plant genomes, transcriptomes and proteomes we assembled over 200 non-redundant proteins related to the known Gγ subunits. Structural analysis of these sequences revealed that most of them lack the obligatory C-terminal prenylation motif (CaaX). According to their C-terminal structures we classified the plant Gγ subunits into three distinct types. Type A consists of Gγ subunits with a putative prenylation motif. Type B subunits lack a prenylation motif and do not have any cysteine residues in the C-terminal region, while type C subunits contain an extended C-terminal domain highly enriched with cysteines. Comparative analysis of C-terminal domains of the proteins, intron-exon arrangement of the corresponding genes and phylogenetic studies suggested a common origin of all plant Gγ subunits. Phylogenetic analyses suggest that types C and B most probably originated independently from type A ancestors. We speculate on a potential mechanism used by those Gγ subunits lacking isoprenylation motifs to anchor the Gβγ dimer to the plasma membrane and propose a new flexible nomenclature for plant Gγ subunits. Finally, in the light of our new classification, we give a word of caution about the interpretation of Gγ research in Arabidopsis and its generalization to other plant species.

  3. Quantifying the cooperative subunit action in a multimeric membrane receptor

    PubMed Central

    Wongsamitkul, Nisa; Nache, Vasilica; Eick, Thomas; Hummert, Sabine; Schulz, Eckhard; Schmauder, Ralf; Schirmeyer, Jana; Zimmer, Thomas; Benndorf, Klaus

    2016-01-01

    In multimeric membrane receptors the cooperative action of the subunits prevents exact knowledge about the operation and the interaction of the individual subunits. We propose a method that permits quantification of ligand binding to and activation effects of the individual binding sites in a multimeric membrane receptor. The power of this method is demonstrated by gaining detailed insight into the subunit action in olfactory cyclic nucleotide-gated CNGA2 ion channels. PMID:26858151

  4. Respiratory syncytial virus fusion glycoprotein: nucleotide sequence of mRNA, identification of cleavage activation site and amino acid sequence of N-terminus of F1 subunit.

    PubMed Central

    Elango, N; Satake, M; Coligan, J E; Norrby, E; Camargo, E; Venkatesan, S

    1985-01-01

    The amino acid sequence of respiratory syncytial virus fusion protein (Fo) was deduced from the sequence of a partial cDNA clone of mRNA and from the 5' mRNA sequence obtained by primer extension and dideoxysequencing. The encoded protein of 574 amino acids is extremely hydrophobic and has a molecular weight of 63371 daltons. The site of proteolytic cleavage within this protein was accurately mapped by determining a partial amino acid sequence of the N-terminus of the larger subunit (F1) purified by radioimmunoprecipitation using monoclonal antibodies. Alignment of the N-terminus of the F1 subunit within the deduced amino acid sequence of Fo permitted us to identify a sequence of lys-lys-arg-lys-arg-arg at the C-terminus of the smaller N-terminal F2 subunit that appears to represent the cleavage/activation domain. Five potential sites of glycosylation, four within the F2 subunit, were also identified. Three extremely hydrophobic domains are present in the protein; a) the N-terminal signal sequence, b) the N-terminus of the F1 subunit that is analogous to the N-terminus of the paramyxovirus F1 subunit and the HA2 subunit of influenza virus hemagglutinin, and c) the putative membrane anchorage domain near the C-terminus of F1. Images PMID:2987829

  5. Genetic analysis of neuronal ionotropic glutamate receptor subunits.

    PubMed

    Granger, Adam J; Gray, John A; Lu, Wei; Nicoll, Roger A

    2011-09-01

    In the brain, fast, excitatory synaptic transmission occurs primarily through AMPA- and NMDA-type ionotropic glutamate receptors. These receptors are composed of subunit proteins that determine their biophysical properties and trafficking behaviour. Therefore, determining the function of these subunits and receptor subunit composition is essential for understanding the physiological properties of synaptic transmission. Here, we discuss and evaluate various genetic approaches that have been used to study AMPA and NMDA receptor subunits. These approaches have demonstrated that the GluA1 AMPA receptor subunit is required for activity-dependent trafficking and contributes to basal synaptic transmission, while the GluA2 subunit regulates Ca(2+) permeability, homeostasis and trafficking to the synapse under basal conditions. In contrast, the GluN2A and GluN2B NMDA receptor subunits regulate synaptic AMPA receptor content, both during synaptic development and plasticity. Ongoing research in this field is focusing on the molecular interactions and mechanisms that control these functions. To accomplish this, molecular replacement techniques are being used, where native subunits are replaced with receptors containing targeted mutations. In this review, we discuss a single-cell molecular replacement approach which should arguably advance our physiological understanding of ionotropic glutamate receptor subunits, but is generally applicable to study of any neuronal protein.

  6. Expression of GABA receptor rho subunits in rat brain.

    PubMed

    Boue-Grabot, E; Roudbaraki, M; Bascles, L; Tramu, G; Bloch, B; Garret, M

    1998-03-01

    The GABA receptor rho1, rho2, and rho3 subunits are expressed in the retina where they form bicuculline-insensitive GABA(C) receptors. We used northern blot, in situ hybridization, and RT-PCR analysis to study the expression of rho subunits in rat brains. In situ hybridization allowed us to detect rho-subunit expression in the superficial gray layer of the superior colliculus and in the cerebellar Purkinje cells. RT-PCR experiments indicated that (a) in retina and in domains that may contain functional GABA(C) receptors, rho2 and rho1 subunits are expressed at similar levels; and (b) in domains and in tissues that are unlikely to contain GABA(C) receptors, rho2 mRNA is enriched relative to rho1 mRNA. These results suggest that both rho1 and rho2 subunits are necessary to form a functional GABA(C) receptor. The use of RT-PCR also showed that, except in the superior colliculus, rho3 is expressed along with rho1 and rho2 subunits. We also raised an antibody against a peptide sequence unique to the rho1 subunit. The use of this antibody on cerebellum revealed the rat rho1 subunit in the soma and dendrites of Purkinje neurons. The allocation of GABA(C) receptor subunits to identified neurons paves the way for future electrophysiological studies.

  7. Exclusion of catalytic and regulatory subunits of cAMP-dependent protein kinase as candidate genes for the defect causing cystic fibrosis.

    PubMed

    Scambler, P; Oyen, O; Wainwright, B; Farrall, M; Law, H Y; Estivill, X; Sandberg, M; Williamson, R; Jahnsen, T

    1987-11-01

    Cystic fibrosis (CF) is a common autosomal recessive disease with significant morbidity and mortality. Defects in cAMP control mechanisms are implicated in the pathophysiology of the disease. The mutation causing CF has been localized to chromosome 7q22-7q31.1. We have used (1) somatic-cell hybrids containing this region of the human genome in a mouse background and (2) segregation analysis in families to exclude both the genes coding for a catalytic subunit and three distinct regulatory subunits of cAMP-dependent protein kinase as candidates for the gene defect in CF. Two of these genes--those for the human homologue of the mouse type I regulatory subunit and the human homologue of the rat type II regulatory subunit--map to human chromosome 7.

  8. Mapping Van

    NASA Technical Reports Server (NTRS)

    1994-01-01

    A NASA Center for the Commercial Development of Space (CCDS) - developed system for satellite mapping has been commercialized for the first time. Global Visions, Inc. maps an area while driving along a road in a sophisticated mapping van equipped with satellite signal receivers, video cameras and computer systems for collecting and storing mapping data. Data is fed into a computerized geographic information system (GIS). The resulting amps can be used for tax assessment purposes, emergency dispatch vehicles and fleet delivery companies as well as other applications.

  9. Rubisco small subunit gene family in cassava.

    PubMed

    Yeo, T W; Mak, Y M; Ho, K K

    1999-01-01

    Cassava leaves of two different cultivars, Brazil and Buloh, were used to isolate mRNA. The mRNA isolated was successfully used in the construction of cDNA libraries for each of the cultivars. The cDNA libraries were screened for members of ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit gene family and positive clones were sequenced. A total of seven different SSU genes, of which five were from cultivar Brazil and two were from cultivar Buloh, were isolated. Comparison results show that even though all the sequences are highly similar, they can be classified into three subfamilies. Homology between members of the same subfamily is higher than homology between members from the same cultivar.

  10. Alkaline-extracted influenza subunit vaccine.

    PubMed Central

    Eckert, E A

    1976-01-01

    Treatment of influenza virus concentrates with alkaline solvents releases a major fraction of the viral structural protein content. As determined by polyacrylamide gel electrophoresis, the surface glycoprotein substructures, hemagglutinin and neuraminidase, are the primary solubilized products. Two forms of hemagglutinin antigen are recovered, a 39S active hemagglutinin and a 23S blocking antigen. Dose-response assays in mice demonstrate that hemagglutination-inhibiting and neuraminidase antibodies are induced. Antibody responses are comparable to those resulting from immunization with inactivated whole virus. On the basis of demonstrated purity, high yields of protective antigens, immunogenic potency, and absence of deleterious reagents, alkaline-extracted influenza protein preparations merit consideration as subunit vaccines for human use. PMID:826484

  11. Compilation of small ribosomal subunit RNA structures.

    PubMed Central

    Neefs, J M; Van de Peer, Y; De Rijk, P; Chapelle, S; De Wachter, R

    1993-01-01

    The database on small ribosomal subunit RNA structure contained 1804 nucleotide sequences on April 23, 1993. This number comprises 365 eukaryotic, 65 archaeal, 1260 bacterial, 30 plastidial, and 84 mitochondrial sequences. These are stored in the form of an alignment in order to facilitate the use of the database as input for comparative studies on higher-order structure and for reconstruction of phylogenetic trees. The elements of the postulated secondary structure for each molecule are indicated by special symbols. The database is available on-line directly from the authors by ftp and can also be obtained from the EMBL nucleotide sequence library by electronic mail, ftp, and on CD ROM disk. PMID:8332525

  12. Geologic mapping of Europa

    USGS Publications Warehouse

    Greeley, R.; Figueredo, P.H.; Williams, D.A.; Chuang, F.C.; Klemaszewski, J.E.; Kadel, S.D.; Prockter, L.M.; Pappalardo, R.T.; Head, J. W.; Collins, G.C.; Spaun, N.A.; Sullivan, R.J.; Moore, Johnnie N.; Senske, D.A.; Tufts, B.R.; Johnson, T.V.; Belton, M.J.S.; Tanaka, K.L.

    2000-01-01

    Galileo data enable the major geological units, structures, and surface features to be identified on Europa. These include five primary units (plains, chaos, band, ridge, and crater materials) and their subunits, along with various tectonic structures such as faults. Plains units are the most widespread. Ridged plains material spans a wide range of geological ages, including the oldest recognizable features on Europa, and appears to represent a style of tectonic resurfacing, rather than cryovolcanism. Smooth plains material typically embays other terrains and units, possibly as a type of fluid emplacement, and is among the youngest material units observed. At global scales, plains are typically mapped as undifferentiated plains material, although in some areas differences can be discerned in the near infrared which might be related to differences in ice grain size. Chaos material is composed of plains and other preexisting materials that have been severely disrupted by inferred internal activity; chaos is characterized by blocks of icy material set in a hummocky matrix. Band material is arrayed in linear, curvilinear, wedge-shaped, or cuspate zones with contrasting albedo and surface textures with respect to the surrounding terrain. Bilateral symmetry observed in some bands and the relationships with the surrounding units suggest that band material forms by the lithosphere fracturing, spreading apart, and infilling with material derived from the subsurface. Ridge material is mapped as a unit on local and some regional maps but shown with symbols at global scales. Ridge material includes single ridges, doublet ridges, and ridge complexes. Ridge materials are considered to represent tectonic processes, possibly accompanied by the extrusion or intrusion of subsurface materials, such as diapirs. The tectonic processes might be related to tidal flexing of the icy lithosphere on diurnal or longer timescales. Crater materials include various interior (smooth central

  13. Subunit vaccine efficacy against Botulinum neurotoxin subtypes.

    PubMed

    Henkel, James S; Tepp, William H; Przedpelski, Amanda; Fritz, Robert B; Johnson, Eric A; Barbieri, Joseph T

    2011-10-13

    Botulinum neurotoxins (BoNT) are classified into 7 serotypes (A-G) based upon neutralization by serotype-specific anti-sera. Several recombinant serotype-specific subunit BoNT vaccines have been developed, including a subunit vaccine comprising the receptor binding domain (HCR) of the BoNTs. Sequencing of the genes encoding BoNTs has identified variants (subtypes) that possess up to 32% primary amino acid variation among different BoNT serotypes. Studies were conducted to characterize the ability of the HCR of BoNT/A to protect against challenge by heterologous BoNT/A subtypes (A1-A3). High dose vaccination with HCR/A subtypes A1-A4 protected mice from challenge by heterologous BoNT/A subtype A1-A3, while low dose HCR vaccination yielded partial protection to heterologous BoNT/A subtype challenge. Absolute IgG titers to HCRs correlated to the dose of HCR used for vaccination, where HCR/A1 elicited an A1 subtype-specific IgG response, which was not observed with HCR/A2 vaccination. Survival of mice challenged to heterologous BoNT/A2 following low dose HCR/A1 vaccination correlated with elevated IgG titers directed to the denatured C-terminal sub-domain of HCR/A2, while survival of mice to heterologous BoNT/A1 following low dose HCR/A2 vaccination correlated to elevated IgG titers directed to native HCRc/A1. This implies that low dose vaccinations with HCR/A subtypes elicit unique IgG responses, and provides a basis to define how the host develops a neutralizing immune response to BoNT intoxication. These results may provide a reference for the development of pan-BoNT vaccines.

  14. Dynamic regulation of β1 subunit trafficking controls vascular contractility.

    PubMed

    Leo, M Dennis; Bannister, John P; Narayanan, Damodaran; Nair, Anitha; Grubbs, Jordan E; Gabrick, Kyle S; Boop, Frederick A; Jaggar, Jonathan H

    2014-02-11

    Ion channels composed of pore-forming and auxiliary subunits control physiological functions in virtually all cell types. A conventional view is that channels assemble with their auxiliary subunits before anterograde plasma membrane trafficking of the protein complex. Whether the multisubunit composition of surface channels is fixed following protein synthesis or flexible and open to acute and, potentially, rapid modulation to control activity and cellular excitability is unclear. Arterial smooth muscle cells (myocytes) express large-conductance Ca(2+)-activated potassium (BK) channel α and auxiliary β1 subunits that are functionally significant modulators of arterial contractility. Here, we show that native BKα subunits are primarily (∼95%) plasma membrane-localized in human and rat arterial myocytes. In contrast, only a small fraction (∼10%) of total β1 subunits are located at the cell surface. Immunofluorescence resonance energy transfer microscopy demonstrated that intracellular β1 subunits are stored within Rab11A-postive recycling endosomes. Nitric oxide (NO), acting via cGMP-dependent protein kinase, and cAMP-dependent pathways stimulated rapid (≤1 min) anterograde trafficking of β1 subunit-containing recycling endosomes, which increased surface β1 almost threefold. These β1 subunits associated with surface-resident BKα proteins, elevating channel Ca(2+) sensitivity and activity. Our data also show that rapid β1 subunit anterograde trafficking is the primary mechanism by which NO activates myocyte BK channels and induces vasodilation. In summary, we show that rapid β1 subunit surface trafficking controls functional BK channel activity in arterial myocytes and vascular contractility. Conceivably, regulated auxiliary subunit trafficking may control ion channel activity in a wide variety of cell types.

  15. Bacteriophage HK97 structure: wholesale covalent cross-linking between the major head shell subunits.

    PubMed Central

    Popa, M P; McKelvey, T A; Hempel, J; Hendrix, R W

    1991-01-01

    We describe initial genetic and structural characterizations of HK97, a temperate bacteriophage of Escherichia coli. We isolated 28 amber mutants, characterized them with respect to what phage-related structures they make, and mapped many of them to restriction fragments of genomic DNA. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of HK97 virions revealed nine different protein species plus a substantial amount of material that failed to enter the gel, apparently because it is too large. Five proteins are tail components and are assigned functions as tail fiber subunit, tail length template, and major shaft subunit (two and possibly three species). The four remaining proteins and the material that did not enter the gel are head components. One of these proteins is assigned as the portal subunit, and the remaining three head proteins in the gel and the material that did not enter the gel are components of the head shell. All of the head shell protein species have apparent molecular masses well in excess of 100 kDa; they share amino acid sequence with each other and also with a 42-kDa protein that is found in infected lysates and as the major component of prohead structures that accumulate in infections by one of the amber mutants. We propose that all of the head shell species found in mature heads are covalently cross-linked oligomers derived from the 42-kDa precursor during head shell maturation. Images PMID:1709700

  16. Yeast ribosomal protein L10 helps coordinate tRNA movement through the large subunit

    PubMed Central

    Petrov, Alexey N.; Meskauskas, Arturas; Roshwalb, Sara C.; Dinman, Jonathan D.

    2008-01-01

    Yeast ribosomal protein L10 (E. coli L16) is located at the center of a topological nexus that connects many functional regions of the large subunit. This essential protein has previously been implicated in processes as diverse as ribosome biogenesis, translational fidelity and mRNA stability. Here, the inability to maintain the yeast Killer virus was used as a proxy for large subunit defects to identify a series of L10 mutants. These mapped to roughly four discrete regions of the protein. A detailed analysis of mutants located in the N-terminal ‘hook’ of L10, which inserts into the bulge of 25S rRNA helix 89, revealed strong effects on rRNA structure corresponding to the entire path taken by the tRNA 3′ end as it moves through the large subunit during the elongation cycle. The mutant-induced structural changes are wide-ranging, affecting ribosome biogenesis, elongation factor binding, drug resistance/hypersensitivity, translational fidelity and virus maintenance. The importance of L10 as a potential transducer of information through the ribosome, and of a possible role of its N-terminal domain in switching between the pre- and post-translocational states are discussed. PMID:18824477

  17. Cryo-EM structure of the large subunit of the spinach chloroplast ribosome

    PubMed Central

    Ahmed, Tofayel; Yin, Zhan; Bhushan, Shashi

    2016-01-01

    Protein synthesis in the chloroplast is mediated by the chloroplast ribosome (chloro-ribosome). Overall architecture of the chloro-ribosome is considerably similar to the Escherichia coli (E. coli) ribosome but certain differences are evident. The chloro-ribosome proteins are generally larger because of the presence of chloroplast-specific extensions in their N- and C-termini. The chloro-ribosome harbours six plastid-specific ribosomal proteins (PSRPs); four in the small subunit and two in the large subunit. Deletions and insertions occur throughout the rRNA sequence of the chloro-ribosome (except for the conserved peptidyl transferase center region) but the overall length of the rRNAs do not change significantly, compared to the E. coli. Although, recent advancements in cryo-electron microscopy (cryo-EM) have provided detailed high-resolution structures of ribosomes from many different sources, a high-resolution structure of the chloro-ribosome is still lacking. Here, we present a cryo-EM structure of the large subunit of the chloro-ribosome from spinach (Spinacia oleracea) at an average resolution of 3.5 Å. High-resolution map enabled us to localize and model chloro-ribosome proteins, chloroplast-specific protein extensions, two PSRPs (PSRP5 and 6) and three rRNA molecules present in the chloro-ribosome. Although comparable to E. coli, the polypeptide tunnel and the tunnel exit site show chloroplast-specific features. PMID:27762343

  18. Collection Mapping.

    ERIC Educational Resources Information Center

    Harbour, Denise

    2002-01-01

    Explains collection mapping for library media collections. Discusses purposes for creating collection maps, including helping with selection and weeding decisions, showing how the collection supports the curriculum, and making budget decisions; and methods of data collection, including evaluating a collaboratively taught unit with the classroom…

  19. Concept Mapping

    ERIC Educational Resources Information Center

    Technology & Learning, 2005

    2005-01-01

    Concept maps are graphical ways of working with ideas and presenting information. They reveal patterns and relationships and help students to clarify their thinking, and to process, organize and prioritize. Displaying information visually--in concept maps, word webs, or diagrams--stimulates creativity. Being able to think logically teaches…

  20. Undersea Mapping.

    ERIC Educational Resources Information Center

    DiSpezio, Michael A.

    1991-01-01

    Presented is a cooperative learning activity in which students assume different roles in an effort to produce a relief map of the ocean floor. Materials, procedures, definitions, student roles, and questions are discussed. A reproducible map for the activity is provided. (CW)

  1. Map Adventures.

    ERIC Educational Resources Information Center

    Geological Survey (Dept. of Interior), Reston, VA.

    This curriculum packet about maps, with seven accompanying lessons, is appropriate for students in grades K-3. Students learn basic concepts for visualizing objects from different perspectives and how to understand and use maps. Lessons in the packet center on a story about a little girl, Nikki, who rides in a hot-air balloon that gives her, and…

  2. Concept Mapping

    ERIC Educational Resources Information Center

    Technology & Learning, 2005

    2005-01-01

    Concept maps are graphical ways of working with ideas and presenting information. They reveal patterns and relationships and help students to clarify their thinking, and to process, organize and prioritize. Displaying information visually--in concept maps, word webs, or diagrams--stimulates creativity. Being able to think logically teaches…

  3. Question Mapping

    ERIC Educational Resources Information Center

    Martin, Josh

    2012-01-01

    After accepting the principal position at Farmersville (TX) Junior High, the author decided to increase instructional rigor through question mapping because of the success he saw using this instructional practice at his prior campus. Teachers are the number one influence on student achievement (Marzano, 2003), so question mapping provides a…

  4. Question Mapping

    ERIC Educational Resources Information Center

    Martin, Josh

    2012-01-01

    After accepting the principal position at Farmersville (TX) Junior High, the author decided to increase instructional rigor through question mapping because of the success he saw using this instructional practice at his prior campus. Teachers are the number one influence on student achievement (Marzano, 2003), so question mapping provides a…

  5. Collection Mapping.

    ERIC Educational Resources Information Center

    Harbour, Denise

    2002-01-01

    Explains collection mapping for library media collections. Discusses purposes for creating collection maps, including helping with selection and weeding decisions, showing how the collection supports the curriculum, and making budget decisions; and methods of data collection, including evaluating a collaboratively taught unit with the classroom…

  6. Undersea Mapping.

    ERIC Educational Resources Information Center

    DiSpezio, Michael A.

    1991-01-01

    Presented is a cooperative learning activity in which students assume different roles in an effort to produce a relief map of the ocean floor. Materials, procedures, definitions, student roles, and questions are discussed. A reproducible map for the activity is provided. (CW)

  7. Genome mapping

    USDA-ARS?s Scientific Manuscript database

    Genome maps can be thought of much like road maps except that, instead of traversing across land, they traverse across the chromosomes of an organism. Genetic markers serve as landmarks along the chromosome and provide researchers information as to how close they may be to a gene or region of inter...

  8. Structure of the archaeal Cascade subunit Csa5: relating the small subunits of CRISPR effector complexes.

    PubMed

    Reeks, Judith; Graham, Shirley; Anderson, Linzi; Liu, Huanting; White, Malcolm F; Naismith, James H

    2013-05-01

    The Cascade complex for CRISPR-mediated antiviral immunity uses CRISPR RNA (crRNA) to target invading DNA species from mobile elements such as viruses, leading to their destruction. The core of the Cascade effector complex consists of the Cas5 and Cas7 subunits, which are widely conserved in prokaryotes. Cas7 binds crRNA and forms the helical backbone of Cascade. Many archaea encode a version of the Cascade complex (denoted Type I-A) that includes a Csa5 (or small) subunit, which interacts weakly with the core proteins. Here, we report the crystal structure of the Csa5 protein from Sulfolobus solfataricus. Csa5 comprises a conserved α-helical domain with a small insertion consisting of a weakly conserved β-strand domain. In the crystal, the Csa5 monomers have multimerized into infinite helical threads. At each interface is a strictly conserved intersubunit salt bridge, deletion of which disrupts multimerization. Structural analysis indicates a shared evolutionary history among the small subunits of the CRISPR effector complexes. The same α-helical domain is found in the C-terminal domain of Cse2 (from Type I-E Cascade), while the N-terminal domain of Cse2 is found in Cmr5 of the CMR (Type III-B) effector complex. As Cmr5 shares no match with Csa5, two possibilities present themselves: selective domain loss from an ancestral Cse2 to create two new subfamilies or domain fusion of two separate families to create a new Cse2 family. A definitive answer awaits structural studies of further small subunits from other CRISPR effector complexes.

  9. Liposome-Based Adjuvants for Subunit Vaccines: Formulation Strategies for Subunit Antigens and Immunostimulators

    PubMed Central

    Tandrup Schmidt, Signe; Foged, Camilla; Smith Korsholm, Karen; Rades, Thomas; Christensen, Dennis

    2016-01-01

    The development of subunit vaccines has become very attractive in recent years due to their superior safety profiles as compared to traditional vaccines based on live attenuated or whole inactivated pathogens, and there is an unmet medical need for improved vaccines and vaccines against pathogens for which no effective vaccines exist. The subunit vaccine technology exploits pathogen subunits as antigens, e.g., recombinant proteins or synthetic peptides, allowing for highly specific immune responses against the pathogens. However, such antigens are usually not sufficiently immunogenic to induce protective immunity, and they are often combined with adjuvants to ensure robust immune responses. Adjuvants are capable of enhancing and/or modulating immune responses by exposing antigens to antigen-presenting cells (APCs) concomitantly with conferring immune activation signals. Few adjuvant systems have been licensed for use in human vaccines, and they mainly stimulate humoral immunity. Thus, there is an unmet demand for the development of safe and efficient adjuvant systems that can also stimulate cell-mediated immunity (CMI). Adjuvants constitute a heterogeneous group of compounds, which can broadly be classified into delivery systems or immunostimulators. Liposomes are versatile delivery systems for antigens, and they can carefully be customized towards desired immune profiles by combining them with immunostimulators and optimizing their composition, physicochemical properties and antigen-loading mode. Immunostimulators represent highly diverse classes of molecules, e.g., lipids, nucleic acids, proteins and peptides, and they are ligands for pattern-recognition receptors (PRRs), which are differentially expressed on APC subsets. Different formulation strategies might thus be required for incorporation of immunostimulators and antigens, respectively, into liposomes, and the choice of immunostimulator should ideally be based on knowledge regarding the specific PRR

  10. Liposome-Based Adjuvants for Subunit Vaccines: Formulation Strategies for Subunit Antigens and Immunostimulators.

    PubMed

    Tandrup Schmidt, Signe; Foged, Camilla; Korsholm, Karen Smith; Rades, Thomas; Christensen, Dennis

    2016-03-10

    The development of subunit vaccines has become very attractive in recent years due to their superior safety profiles as compared to traditional vaccines based on live attenuated or whole inactivated pathogens, and there is an unmet medical need for improved vaccines and vaccines against pathogens for which no effective vaccines exist. The subunit vaccine technology exploits pathogen subunits as antigens, e.g., recombinant proteins or synthetic peptides, allowing for highly specific immune responses against the pathogens. However, such antigens are usually not sufficiently immunogenic to induce protective immunity, and they are often combined with adjuvants to ensure robust immune responses. Adjuvants are capable of enhancing and/or modulating immune responses by exposing antigens to antigen-presenting cells (APCs) concomitantly with conferring immune activation signals. Few adjuvant systems have been licensed for use in human vaccines, and they mainly stimulate humoral immunity. Thus, there is an unmet demand for the development of safe and efficient adjuvant systems that can also stimulate cell-mediated immunity (CMI). Adjuvants constitute a heterogeneous group of compounds, which can broadly be classified into delivery systems or immunostimulators. Liposomes are versatile delivery systems for antigens, and they can carefully be customized towards desired immune profiles by combining them with immunostimulators and optimizing their composition, physicochemical properties and antigen-loading mode. Immunostimulators represent highly diverse classes of molecules, e.g., lipids, nucleic acids, proteins and peptides, and they are ligands for pattern-recognition receptors (PRRs), which are differentially expressed on APC subsets. Different formulation strategies might thus be required for incorporation of immunostimulators and antigens, respectively, into liposomes, and the choice of immunostimulator should ideally be based on knowledge regarding the specific PRR

  11. Amino Acid Substitutions in the Caenorhabditis elegans RNA Polymerase II Large Subunit AMA-1/RPB-1 that Result in α-Amanitin Resistance and/or Reduced Function.

    PubMed

    Bowman, Elizabeth Anne; Riddle, Donald L; Kelly, William

    2011-11-01

    Mutations in the Caenorhabditis elegans RNA polymerase II AMA-1/RPB-1 subunit that cause α-amanitin resistance and/or developmental defects were isolated previously. We identified 12 of these mutations and mapped them onto the Saccharomyces cerevisiae RPB1 structure to provide insight into AMA-1 regions that are essential for development in a multicellular organism.

  12. Amino Acid Substitutions in the Caenorhabditis elegans RNA Polymerase II Large Subunit AMA-1/RPB-1 that Result in α-Amanitin Resistance and/or Reduced Function

    PubMed Central

    Bowman, Elizabeth Anne; Riddle, Donald L.; Kelly, William

    2011-01-01

    Mutations in the Caenorhabditis elegans RNA polymerase II AMA-1/RPB-1 subunit that cause α-amanitin resistance and/or developmental defects were isolated previously. We identified 12 of these mutations and mapped them onto the Saccharomyces cerevisiae RPB1 structure to provide insight into AMA-1 regions that are essential for development in a multicellular organism. PMID:22384351

  13. Epitopes from two soybean glycinin subunits antigenic in pigs

    USDA-ARS?s Scientific Manuscript database

    Background: Glycinin is a seed storage protein in soybean (Glycine max) that is allergenic in pigs. Glycinin is a hexamer composed of subunits consisting of a basic and acidic portion joined by disulfide bridges. There are 5 glycinin subunits designated Gy1-Gy5. Results: Twenty seven out of 30 pi...

  14. Proteopedia Entry: The Large Ribosomal Subunit of "Haloarcula Marismortui"

    ERIC Educational Resources Information Center

    Decatur, Wayne A.

    2010-01-01

    This article presents a "Proteopedia" page that shows the refined version of the structure of the "Haloarcula" large ribosomal subunit as solved by the laboratories of Thomas Steitz and Peter Moore. The landmark structure is of great impact as it is the first atomic-resolution structure of the highly conserved ribosomal subunit which harbors…

  15. Proteopedia Entry: The Large Ribosomal Subunit of "Haloarcula Marismortui"

    ERIC Educational Resources Information Center

    Decatur, Wayne A.

    2010-01-01

    This article presents a "Proteopedia" page that shows the refined version of the structure of the "Haloarcula" large ribosomal subunit as solved by the laboratories of Thomas Steitz and Peter Moore. The landmark structure is of great impact as it is the first atomic-resolution structure of the highly conserved ribosomal subunit which harbors…

  16. Specific Roles of NMDA Receptor Subunits in Mental Disorders

    PubMed Central

    Yamamoto, H.; Hagino, Y.; Kasai, S.; Ikeda, K.

    2015-01-01

    N-methyl-D-aspartate (NMDA) receptor plays important roles in learning and memory. NMDA receptors are a tetramer that consists of two glycine-binding subunits GluN1, two glutamate-binding subunits (i.e., GluN2A, GluN2B, GluN2C, and GluN2D), a combination of a GluN2 subunit and glycine-binding GluN3 subunit (i.e., GluN3A or GluN3B), or two GluN3 subunits. Recent studies revealed that the specific expression and distribution of each subunit are deeply involved in neural excitability, plasticity, and synaptic deficits. The present article summarizes reports on the dysfunction of NMDA receptors and responsible subunits in various neurological and psychiatric disorders, including schizophrenia, autoimmune-induced glutamatergic receptor dysfunction, mood disorders, and autism. A key role for the GluN2D subunit in NMDA receptor antagonist-induced psychosis has been recently revealed. PMID:25817860

  17. The Development and Institutionalization of Subunit Power in Organizations.

    ERIC Educational Resources Information Center

    Boeker, Warren

    1989-01-01

    Examines the effects of founding events on the evolution of subunit importance in the semiconductor industry from 1958 to 1985. Distributions of power and subunit importance represent not only influences of current conditions, but also vestiges of earlier events, including the institution's founding. Includes 55 references. (MLH)

  18. Mutational analysis of muscle nicotinic acetylcholine receptor subunit assembly

    PubMed Central

    1990-01-01

    The structural elements required for normal maturation and assembly of the nicotinic acetylcholine receptor alpha subunit were investigated by expression of mutated subunits in transfected fibroblasts. Normally, the wild-type alpha subunit acquires high affinity alpha bungarotoxin binding in a time-dependent manner; however, mutation of the 128 and/or 142 cysteines to either serine or alanine, as well as deletion of the entire 14 amino acids in this region abolished all detectable high affinity binding. Nonglycosylated subunits that had a serine to glycine mutation in the consensus sequence also did not efficiently attain high affinity binding to toxin. In contrast, mutation of the proline at position 136 to glycine or alanine, or a double mutation of the cysteines at position 192 and 193 to serines had no effect on the acquisition of high affinity toxin binding. These data suggest that a disulfide bridge between cysteines 128 and 142 and oligosaccharide addition at asparagine 141 are required for the normal maturation of alpha subunit as assayed by high affinity toxin binding. The unassembled wild-type alpha subunit expressed in fibroblasts is normally degraded with a t1/2 of 2 h; upon assembly with the delta subunit, the degradation rate slows significantly (t1/2 greater than 13 h). All mutated alpha subunits retained the capacity to assemble with a delta subunit coexpressed in fibroblasts; however, mutated alpha subunits that were not glycosylated or did not acquire high affinity toxin binding were rapidly degraded (t1/2 = 20 min to 2 h) regardless of whether or not they assembled with the delta subunit. Assembly and rapid degradation of nonglycosylated acetylcholine receptor (AChR) subunits and subunit complexes were also observed in tunicamycin- treated BC3H-1 cells, a mouse musclelike cell line that normally expresses functional AChR. Hence, rapid degradation may be one form of regulation assuring that only correctly processed and assembled subunits

  19. INTRINSIC REGULATION OF HEMOGLOBIN EXPRESSION BY VARIABLE SUBUNIT INTERFACE STRENGTHS

    PubMed Central

    Manning, James M.; Popowicz, Anthony M.; Padovan, Julio C.; Chait, Brian T.; Manning, Lois R.

    2012-01-01

    SUMMARY The expression of the six types of human hemoglobin subunits over time is currently considered to be regulated mainly by transcription factors that bind to upstream control regions of the gene (the “extrinsic” component of regulation). Here we describe how subunit pairing and further assembly to tetramers in the liganded state is influenced by the affinity of subunits for one another (the “intrinsic” component of regulation). The adult hemoglobin dimers have the strongest subunit interfaces and the embryonic hemoglobins are the weakest with fetal hemoglobins of intermediate strength, corresponding to the temporal order of their expression. These variable subunit binding strengths and the attenuating effects of acetylation contribute to the differences with which these hemoglobin types form functional O2-binding tetramers consistent with gene switching. PMID:22129306

  20. [Penicillin acylase from Escherichia coli: catalytically active subunits].

    PubMed

    Kabakov, V E; Kliachko, N L; Levashov, A V

    1995-05-01

    Gel filtration under denaturing conditions was used to isolate the alpha- and beta-subunits of penicillin acylase (PA). Refolded subunits were obtained through removing urea by dialysis. Both renatured subunits were catalytically active during hydrolysis of phenylacetic acid p-nitroanilide; this activity decreased after addition of a serine-specific inhibitor--phenylmethanesulfonyl fluoride. The subunits were also active in reversed micelles of Aerosol OT (AOT) in octane, the optimum hydration degree being 11.9 and 17.5 for the light (alpha) and heavy (beta) subunits, respectively. The positions of the maxima were consistent with both theoretically calculated optimum hydration degrees and the earlier reported profile of enzymatic activity for native PA in reversed micelles.

  1. Geranyl diphosphate synthase large subunit, and methods of use

    DOEpatents

    Croteau, Rodney B.; Burke, Charles C.; Wildung, Mark R.

    2001-10-16

    A cDNA encoding geranyl diphosphate synthase large subunit from peppermint has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Replicable recombinant cloning vehicles are provided which code for geranyl diphosphate synthase large subunit). In another aspect, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding geranyl diphosphate synthase large subunit. In yet another aspect, the present invention provides isolated, recombinant geranyl diphosphate synthase protein comprising an isolated, recombinant geranyl diphosphate synthase large subunit protein and an isolated, recombinant geranyl diphosphate synthase small subunit protein. Thus, systems and methods are provided for the recombinant expression of geranyl diphosphate synthase.

  2. Subunit Structure Differences in RNA Polymerase II Purified from Ungerminated versus Germinated Wheat Embryos 1

    PubMed Central

    Jendrisak, Jerry; Skuzeski, Jim

    1983-01-01

    DNA-dependent RNA polymerase II (RNAP II) was purified from wheat embryos germinated for 0, 12, 24, and 36 hours and examined with several polyacrylamide gel electrophoretic systems. A changing electrophoretic pattern of RNAP II was observed on nondenaturing polyacrylamide gels. Subunit structure analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) indicated that from ungerminated embryos, RNAP IIA was almost exclusively obtained which has a subunit structure identical to that established for wheat germ RNAP II previously (Jendrisak, Burgess 1977 Biochemistry 16: 1959-1964). Twelve polypeptides with molecular weights × 10−3 of 220, 140, 42, 40, 27, 25, 21, 20, 17.8, 17.0, 16.3, and 16.0 were routinely found to be associated with the purified enzyme. From embryos germinated for 36 hours, RNAP IIB was almost exclusively obtained which has a largest subunit of 180,000 mol wt instead of 220,000. From embryos germinated for 24 hours, an approximately equimolar mixture of RNAP IIA and IIB was obtained. Peptide maps of the 220,000 and 180,000 mol wt polypeptides of RNAP IIA and IIB were virtually identical, indicative of a precursor-product relationship for the two polypeptides. In addition to these results, SDS-PAGE indicated that the stoichiometry of the 27,000 mol wt polypeptide increased at the expense of the 25,000 mol wt polypeptide during germination and concomitantly with the appearance of the 180,000 molecular weight polypeptide. No modifications (e.g. gain, loss, or altered mobilities on analytical gels) in any of the other RNAP II subunits were observed in enzyme purified from embryos after various times of germination as determined by a variety of electrophoretic analyses under denaturing conditions. Images Fig. 1 Fig. 3 Fig. 4 Fig. 5 PMID:16663122

  3. Mapping Children--Mapping Space.

    ERIC Educational Resources Information Center

    Pick, Herbert L., Jr.

    Research is underway concerning the way the perception, conception, and representation of spatial layout develops. Three concepts are important here--space itself, frame of reference, and cognitive map. Cognitive map refers to a form of representation of the behavioral space, not paired associate or serial response learning. Other criteria…

  4. A revised model for AMP-activated protein kinase structure: The alpha-subunit binds to both the beta- and gamma-subunits although there is no direct binding between the beta- and gamma-subunits.

    PubMed

    Wong, Kelly A; Lodish, Harvey F

    2006-11-24

    The 5'-AMP-activated protein kinase (AMPK) is a master sensor for cellular metabolic energy state. It is activated by a high AMP/ATP ratio and leads to metabolic changes that conserve energy and utilize alternative cellular fuel sources. The kinase is composed of a heterotrimeric protein complex containing a catalytic alpha-subunit, an AMP-binding gamma-subunit, and a scaffolding beta-subunit thought to bind directly both the alpha- and gamma-subunits. Here, we use coimmunoprecipitation of proteins in transiently transfected cells to show that the alpha2-subunit binds directly not only to the beta-subunit, confirming previous work, but also to the gamma1-subunit. Deletion analysis of the alpha2-subunit reveals that the C-terminal 386-552 residues are sufficient to bind to the beta-subunit. The gamma1-subunit binds directly to the alpha2-subunit at two interaction sites, one within the catalytic domain consisting of alpha2 amino acids 1-312 and a second within residues 386-552. Binding of the alpha2 and the gamma1-subunits was not affected by 400 mum AMP or ATP. Furthermore, we show that the beta-subunit C terminus is essential for binding to the alpha2-subunit but, in contrast to previous work, the beta-subunit does not bind directly to the gamma1-subunit. Taken together, this study presents a new model for AMPK heterotrimer structure where through its C terminus the beta-subunit binds to the alpha-subunit that, in turn, binds to the gamma-subunit. There is no direct interaction between the beta- and gamma-subunits.

  5. The subunit composition and function of mammalian cytochrome c oxidase.

    PubMed

    Kadenbach, Bernhard; Hüttemann, Maik

    2015-09-01

    Cytochrome c oxidase (COX) from mammals and birds is composed of 13 subunits. The three catalytic subunits I-III are encoded by mitochondrial DNA, the ten nuclear-coded subunits (IV, Va, Vb, VIa, VIb, VIc, VIIa, VIIb, VIIc, VIII) by nuclear DNA. The nuclear-coded subunits are essentially involved in the regulation of oxygen consumption and proton translocation by COX, since their removal or modification changes the activity and their mutation causes mitochondrial diseases. Respiration, the basis for ATP synthesis in mitochondria, is differently regulated in organs and species by expression of tissue-, developmental-, and species-specific isoforms for COX subunits IV, VIa, VIb, VIIa, VIIb, and VIII, but the holoenzyme in mammals is always composed of 13 subunits. Various proteins and enzymes were shown, e.g., by co-immunoprecipitation, to bind to specific COX subunits and modify its activity, but these interactions are reversible, in contrast to the tightly bound 13 subunits. In addition, the formation of supercomplexes with other oxidative phosphorylation complexes has been shown to be largely variable. The regulatory complexity of COX is increased by protein phosphorylation. Up to now 18 phosphorylation sites have been identified under in vivo conditions in mammals. However, only for a few phosphorylation sites and four nuclear-coded subunits could a specific function be identified. Research on the signaling pathways leading to specific COX phosphorylations remains a great challenge for understanding the regulation of respiration and ATP synthesis in mammalian organisms. This article reviews the function of the individual COX subunits and their isoforms, as well as proteins and small molecules interacting and regulating the enzyme.

  6. Subunit structure of the follitropin receptor

    SciTech Connect

    Shin, J.

    1985-01-01

    Both of the ..cap alpha.. and ..beta.. subunits of intact human follitropin (FSH) were radioiodinated with /sup 125/I-FSH-sodium iodide and chloramine-T, and could be resolved on polyacrylamide gels (SDS-PAGE). The electrophoretic mobility of radioiodinated FSH ..cap alpha.. and ..beta.. subunits as well as the ..cap alpha beta.. dimer changed markedly depending on the concentration of reducing agents. /sup 125/I-FSH (Ka = 1.4 x 10/sup 10/ M/sup -1/), complexes to the receptor on procine granulosa cells or in Triton X-100 extracts, was affinity-crosslinked with a cleavable (nondisulfide) homobifunctional reagent, bis(2-(succinimidooxycarbonyloxy)ethyl)sulfone, solubilized in sodium dodecyl sulfate with or without reducing agents, and electrophoresed. Crosslinked samples revealed three additional bands of slower electrophoretic mobility, corresponding to 65 (unreduced 62), 83 (unreduced 76) and 117 (unreduced 110)kDa, in addition to hormone bands. Formation of the three bands requires the /sup 125/I-FSH hormone to bind specifically to the receptor with subsequent cross-linking. The rate of formation and cleavage of the cross-linked complexes indicated a sequential and incremental addition of 22, 18, and 34 kDa components to the FSH ..cap alpha beta.. dimer. The results of reduction of cross-linked complexes demonstrated the existence of disulfide linkage between the three components. FSH was photoactively derivatized with N-hydroxysuccinimide ester of 4-azidobenzolyl-glycine and radioiodinated for photoaffinity labeling. When derivatized /sup 125/I-FSH (Ka = 1.12 10/sup 10/ M/sup -1/) bound to the cell was photolyzed for cross-linking and resolved on the SDS-PAGE, two new bands (106 and 61 kDa) under reducing condition appeared in addition to the hormone bands. Upon reduction with dithiotheitol and second-dimensional electrophoresis, the unreduced 104 kDa (reduced 106 kDa) band released two small components 31 and 14 kDa.

  7. Na, K ATPase beta3 subunit (CD298): association with alpha subunit and expression on peripheral blood cells.

    PubMed

    Chiampanichayakul, S; Khunkaewla, P; Pata, S; Kasinrerk, W

    2006-12-01

    Beta3 subunit is described as one of the Na, K ATPase subunits. Recently, we generated a monoclonal antibody (mAb), termed P-3E10. This mAb was shown to react with the Na, K ATPase beta3 subunit or CD298. By immunofluorescence analysis using mAb P-3E10, it was found that all peripheral blood leukocytes express Na, K ATPase beta3. The presence of beta3 subunit on leukocytes is not in a quantitative polymorphic manner. Upon phytohemagglutinin or phorbol myristate acetate activation, the expression level of the Na, K ATPase beta3 subunit on activated peripheral blood mononuclear cells was not altered in comparison with those of unstimulated cells. Red blood cells (RBCs) of healthy donors showed negative reactivity with mAb P-3E10. However, more than 80% of thalassemic RBCs showed positive reactivity. By immunoprecipitation, moreover, a protein band of 55-65 kDa was precipitated from normal RBC membrane using mAb P-3E10. These results evidenced that the beta3 subunit of Na, K ATPase is expressed on RBC membrane but the epitope recognized by mAb P-3E10 is hidden in normal RBCs. Furthermore, we showed the association of beta3 subunit and alpha subunit of Na, K ATPase. This information is important for further understanding of the functional roles of this molecule.

  8. Electronic spectra of PS I mutants: the peripheral subunits do not bind red chlorophylls in Synechocystis sp. PCC 6803.

    PubMed

    Soukoulis, V; Savikhin, S; Xu, W; Chitnis, P R; Struve, W S

    1999-05-01

    Steady-state fluorescence and absorption spectra have been obtained in the Qy spectral region (690-780 nm and 600-750 nm, respectively) for several subunit-deficient photosystem I mutants from the cyanobacterium Synechocystis sp. PCC 6803. The 77 K fluorescence spectra of the wild-type and subunit-deficient mutant photosystem I particles are all very similar, peaking at approximately 720 nm with essentially the same excitation spectrum. Because emission from far-red chlorophylls absorbing near 708 nm dominates low-temperature fluorescence in Synechocystis sp., these pigments are not coordinated to any the subunits PsaF, Psa I, PsaJ, PsaK, PsaL, or psaM. The room temperature (wild-type-mutant) absorption difference spectra for trimeric mutants lacking the PsaF/J, PsaK, and PsaM subunits suggest that these mutants are deficient in core antenna chlorophylls (Chls) absorbing near 685, 670, 675, and 700 nm, respectively. The absorption difference spectrum for the PsaF/J/I/L-deficient photosystem I complexes at 5 K reveals considerably more structure than the room-temperature spectrum. The integrated absorbance difference spectra (when normalized to the total PS I Qy spectral area) are comparable to the fractions of Chls bound by the respective (groups of) subunits, according to the 4-A density map of PS I from Synechococcus elongatus. The spectrum of the monomeric PsaL-deficient mutant suggests that this subunit may bind pigments absorbing near 700 nm.

  9. Stepwise and dynamic assembly of the earliest precursors of small ribosomal subunits in yeast.

    PubMed

    Zhang, Liman; Wu, Chen; Cai, Gaihong; Chen, She; Ye, Keqiong

    2016-03-15

    The eukaryotic ribosomal RNA (rRNA) is associated cotranscriptionally with numerous factors into an enormous 90S preribosomal particle that conducts early processing of small ribosomal subunits. The assembly pathway and structure of the 90S particle is poorly understood. Here, we affinity-purified and analyzed the constituents of yeast 90S particles that were assembled on a series of plasmid-encoded 3'-truncated pre-18S RNAs. We determined the assembly point of 65 proteins and the U3, U14, and snR30 small nucleolar RNAs (snoRNAs), revealing a stepwise and dynamic assembly map. The 5' external transcribed spacer (ETS) alone can nucleate a large complex. When the 18S rRNA is nearly complete, the 90S structure undergoes a dramatic reorganization, releasing U14, snR30, and 14 protein factors that bind earlier. We also identified a reference state of 90S that is fully assembled yet has not undergone 5'ETS processing. The assembly map present here provides a new framework to understand small subunit biogenesis. © 2016 Zhang et al.; Published by Cold Spring Harbor Laboratory Press.

  10. Stepwise and dynamic assembly of the earliest precursors of small ribosomal subunits in yeast

    PubMed Central

    Zhang, Liman; Wu, Chen; Cai, Gaihong; Chen, She

    2016-01-01

    The eukaryotic ribosomal RNA (rRNA) is associated cotranscriptionally with numerous factors into an enormous 90S preribosomal particle that conducts early processing of small ribosomal subunits. The assembly pathway and structure of the 90S particle is poorly understood. Here, we affinity-purified and analyzed the constituents of yeast 90S particles that were assembled on a series of plasmid-encoded 3′-truncated pre-18S RNAs. We determined the assembly point of 65 proteins and the U3, U14, and snR30 small nucleolar RNAs (snoRNAs), revealing a stepwise and dynamic assembly map. The 5′ external transcribed spacer (ETS) alone can nucleate a large complex. When the 18S rRNA is nearly complete, the 90S structure undergoes a dramatic reorganization, releasing U14, snR30, and 14 protein factors that bind earlier. We also identified a reference state of 90S that is fully assembled yet has not undergone 5′ETS processing. The assembly map present here provides a new framework to understand small subunit biogenesis. PMID:26980190

  11. [A promoter responsible for over-expression of cholera toxin B subunit in cholera toxin A subunit structure gene].

    PubMed

    Cao, C; Shi, C; Li, P; Ma, Q

    1997-01-01

    A promoter sequence, which promotes the transcription of cholera toxin B subunit gene, was found in cholera toxin A subunit structure gene. The transcription starts at the adenine Located at +833, that is 456bp upstream to the A of the initiation codon ATG of cholera toxin B gene. Under the control of the promoter, cholera toxin B subunit was over-expressed as high as 200 mg/L at an optimized culture condition. The chloramphenicol acetyl transferase gene and beta-galactosidase could also be efficiently expressed under the direction of the promoter. This promoter may be responsible for the 6 fold and 7 fold higher expression level of cholera toxin B subunit than cholera toxin A subunit in V. cholerae and Escheria coli respectively. The over-expression of CTB may be useful in preparing vaccine against cholera and facilitating the construction of peptide-bearing immunogenic hybrid proteins.

  12. Subunit recombinant vaccine protects against monkeypox.

    PubMed

    Heraud, Jean-Michel; Edghill-Smith, Yvette; Ayala, Victor; Kalisz, Irene; Parrino, Janie; Kalyanaraman, Vaniambadi S; Manischewitz, Jody; King, Lisa R; Hryniewicz, Anna; Trindade, Christopher J; Hassett, Meredith; Tsai, Wen-Po; Venzon, David; Nalca, Aysegul; Vaccari, Monica; Silvera, Peter; Bray, Mike; Graham, Barney S; Golding, Hana; Hooper, Jay W; Franchini, Genoveffa

    2006-08-15

    The smallpox vaccine Dryvax, a live vaccinia virus (VACV), protects against smallpox and monkeypox, but is contraindicated in immunocompromised individuals. Because Abs to VACV mediate protection, a live virus vaccine could be substituted by a safe subunit protein-based vaccine able to induce a protective Ab response. We immunized rhesus macaques with plasmid DNA encoding the monkeypox orthologs of the VACV L1R, A27L, A33R, and B5R proteins by the intradermal and i.m. routes, either alone or in combination with the equivalent recombinant proteins produced in Escherichia coli. Animals that received only DNA failed to produce high titer Abs, developed innumerable skin lesions after challenge, and died in a manner similar to placebo controls. By contrast, the animals vaccinated with proteins developed moderate to severe disease (20-155 skin lesions) but survived. Importantly, those immunized with DNA and boosted with proteins had mild disease with 15 or fewer lesions that resolved within days. DNA/protein immunization elicited Th responses and binding Ab titers to all four proteins that correlated negatively with the total lesion number. The sera of the immunized macaques recognized a limited number of linear B cell epitopes that are highly conserved among orthopoxviruses. Their identification may guide future efforts to develop simpler, safer, and more effective vaccines for monkeypox and smallpox.

  13. Mapping Biodiversity.

    ERIC Educational Resources Information Center

    World Wildlife Fund, Washington, DC.

    This document features a lesson plan that examines how maps help scientists protect biodiversity and how plants and animals are adapted to specific ecoregions by comparing biome, ecoregion, and habitat. Samples of instruction and assessment are included. (KHR)

  14. Planetary Mapping

    NASA Astrophysics Data System (ADS)

    Greeley, Ronald; Batson, Raymond M.

    2007-02-01

    Preface; List of contributors; 1. Introduction R. Greeley and R. M. Batson; 2. History of planetary cartography R. M. Batson, E. A. Whitaker and D. E. Wilhelms; 3. Cartography R. M. Batson; 4. Planetary nomenclature M. E. Strobell and H. Masursky; 5. Geodetic control M. E. Davies; 6. Topographic mapping S. S. C. Wu and F. J. Doyle; 7. Geologic mapping D. E. Wilhelms; Appendices R. M. Batson and J. L. Inge; Index.

  15. Map Separates

    USGS Publications Warehouse

    ,

    2001-01-01

    U.S. Geological Survey (USGS) topographic maps are printed using up to six colors (black, blue, green, red, brown, and purple). To prepare your own maps or artwork based on maps, you can order separate black-and-white film positives or negatives for any color printed on a USGS topographic map, or for one or more of the groups of related features printed in the same color on the map (such as drainage and drainage names from the blue plate.) In this document, examples are shown with appropriate ink color to illustrate the various separates. When purchased, separates are black-and-white film negatives or positives. After you receive a film separate or composite from the USGS, you can crop, enlarge or reduce, and edit to add or remove details to suit your special needs. For example, you can adapt the separates for making regional and local planning maps or for doing many kinds of studies or promotions by using the features you select and then printing them in colors of your choice.

  16. Venus mapping

    NASA Technical Reports Server (NTRS)

    Batson, R. M.; Morgan, H. F.; Sucharski, Robert

    1991-01-01

    Semicontrolled image mosaics of Venus, based on Magellan data, are being compiled at 1:50,000,000, 1:10,000,000, 1:5,000,000, and 1:1,000,000 scales to support the Magellan Radar Investigator (RADIG) team. The mosaics are semicontrolled in the sense that data gaps were not filled and significant cosmetic inconsistencies exist. Contours are based on preliminary radar altimetry data that is subjected to revision and improvement. Final maps to support geologic mapping and other scientific investigations, to be compiled as the dataset becomes complete, will be sponsored by the Planetary Geology and Geophysics Program and/or the Venus Data Analysis Program. All maps, both semicontrolled and final, will be published as I-maps by the United States Geological Survey. All of the mapping is based on existing knowledge of the spacecraft orbit; photogrammetric triangulation, a traditional basis for geodetic control on planets where framing cameras were used, is not feasible with the radar images of Venus, although an eventual shift of coordinate system to a revised spin-axis location is anticipated. This is expected to be small enough that it will affect only large-scale maps.

  17. Interactions between the human RNA polymerase II subunits.

    PubMed

    Acker, J; de Graaff, M; Cheynel, I; Khazak, V; Kedinger, C; Vigneron, M

    1997-07-04

    As an initial approach to characterizing the molecular structure of the human RNA polymerase II (hRPB), we systematically investigated the protein-protein contacts that the subunits of this enzyme may establish with each other. To this end, we applied a glutathione S-transferase-pulldown assay to extracts from Sf9 insect cells, which were coinfected with all possible combinations of recombinant baculoviruses expressing hRPB subunits, either as untagged polypeptides or as glutathione S-transferase fusion proteins. This is the first comprehensive study of interactions between eukaryotic RNA polymerase subunits; among the 116 combinations of hRPB subunits tested, 56 showed significant to strong interactions, whereas 60 were negative. Within the intricate network of interactions, subunits hRPB3 and hRPB5 play a central role in polymerase organization. These subunits, which are able to homodimerize and to interact, may constitute the nucleation center for polymerase assembly, by providing a large interface to most of the other subunits.

  18. Further evidence for clustering of human GABA[sub A] receptor subunit genes: Localization of the [alpha][sub 6]-subunit gene (GABRA6) to distal chromosome 5q by linkage analysis

    SciTech Connect

    Hicks, A.A.; Kamphuis, W.; Darlison, M.G. ); Bailey, M.E.S.; Johnson, K.J. ); Riley, B.P. ); Siciliano, M.J. )

    1994-03-15

    GABA[sub A] receptors are hetero-oligomeric ion-channel complexes that are composed of combinations of [alpha], [beta], [gamma], and [delta] subunits and play a major role in inhibitory neurotransmission in the mammalian brain. The authors report here a microsatellite polymorphism within the human [alpha][sub 6]-subunit gene (GABRA6). Mapping of this marker in a human-hamster hybrid cell-line panel and typing of the repeat in the Centre d'Etude du Polymorphisme Humain (CEPH) reference families enabled the localization of this gene to chromosome 5q and established its linkage to the GABA[sub A] receptor [alpha][sub 1]-subunit gene (GA-BRA1) with a maximum lod score (Z[sub max]) of 39.87 at a [theta] of 0.069 (males) and 0.100 (females). These results reveal the clustering of GABRA6, GABRA1, and the GABA[sub A] receptor [gamma][sub 2]-subunit gene (GABRG2) on distal chromosome 5q. 17 refs., 1 fig., 1 tab.

  19. Confirmation that DNA encoding the major fimbrial subunit of Av24 fimbriae is homologous to DNA encoding the major fimbrial subunit of F107 fimbriae.

    PubMed

    Kennan, R M; Moncktor, R P; McDougall, B M; Conway, P L

    1995-01-01

    Plasmid DNA from porcine enterotoxigenic Escherichia coli strain Av24 (O141:K85ab) was cloned into recipient E. coli strain JM105 using the plasmid vector pUC18. Clones were obtained that produced fimbriae which reacted with antisera specific to the fimbriae produced by strain Av24. Restriction mapping of cloned DNA, PCR with fedA primers and DNA sequencing showed a portion of the cloned DNA to be homologous to that encoding the major fimbrial subunit of F107 fimbriae. This confirms that the fimbriae possessed by strains of E. coli causing both edema disease and post-weaning diarrhoea in piglets are variants of the same fimbriae.

  20. Structural analysis and chromosomal localization of the mouse Psmb5 gene coding for the constitutively expressed beta-type proteasome subunit.

    PubMed

    Kohda, K; Matsuda, Y; Ishibashi, T; Tanaka, K; Kasahara, M

    1997-01-01

    The proteasome is a multi-subunit protease responsible for the production of peptides presented by major histocompatibility complex class I molecules. Accumulated evidence indicates that, upon stimulation with interferon-gamma (IFN-gamma), three beta-type subunits, designated LMP2, LMP7, and PSMB10, are incorporated into the 20S proteasome by displacing the housekeeping beta-type subunits designated PSMB6, PSMB5, and PSMB7, respectively. These changes in the subunit composition appear to facilitate class I-mediated antigen presentation, presumably by altering the cleavage specificities of the proteasome. In the present study, we determined the organization of the mouse gene Psmb5, coding for the PSMB5 subunit. Psmb5 is made up of three exons, spanning approximately 5 kilobases. Its exon-intron organization differs radically from those of the other IFN-gamma-regulated, beta-type subunit genes including Lmp7 with which Psmb5 is believed to share an immediate common ancestor. The structure of the mouse Psmb5 gene is identical to that of its recently characterized human counterpart. Thus, the unique organization of the gene coding for the PSMB5 subunit appears to have been established before mammalian radiation. As well as the Psmb5 gene, the mouse genome contains a processed pseudogene designated Psmb5-ps. Interspecific backcross mapping showed that Psmb5 maps close to the Gtrgal2 locus on chromosome 14 and that Psmb5-ps is located in the vicinity of the Psme3 locus on chromosome 11. These results were confirmed by fluorescent in situ hybridization analysis that localized Psmb5 to band C2 to proximal D1 of chromosome 14 and Psmb5-ps to band D of chromosome 11.

  1. Deficiency of subunits of Complex I and mitochondrial encephalomyopathy.

    PubMed

    Ichiki, T; Tanaka, M; Nishikimi, M; Suzuki, H; Ozawa, T; Kobayashi, M; Wada, Y

    1988-03-01

    Enzymic activities of the respiratory chain and content of immunochemically detectable subunits in NADH-ubiquinone oxidoreductase (Complex I) were measured in mitochondria from the skeletal muscles of 4 patients with mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes (MELAS). The rotenone-sensitive NADH-cytochrome c reductase activity was extremely decreased, ranging from 0% to 27% of the control value. In all patients, the content of subunits of Complex I was also reduced in parallel with the rotenone-sensitive NADH-cytochrome c reductase activity. It is suggested that the variation in the degree of deficiency of Complex I subunits could explain the clinical heterogeneity of patients with MELAS.

  2. Echinococcus granulosus Antigen B Structure: Subunit Composition and Oligomeric States

    PubMed Central

    Monteiro, Karina M.; Cardoso, Mateus B.; Follmer, Cristian; da Silveira, Nádya P.; Vargas, Daiani M.; Kitajima, Elliot W.; Zaha, Arnaldo; Ferreira, Henrique B.

    2012-01-01

    Background Antigen B (AgB) is the major protein secreted by the Echinococcus granulosus metacestode and is involved in key host-parasite interactions during infection. The full comprehension of AgB functions depends on the elucidation of several structural aspects that remain unknown, such as its subunit composition and oligomeric states. Methodology/Principal Findings The subunit composition of E. granulosus AgB oligomers from individual bovine and human cysts was assessed by mass spectrometry associated with electrophoretic analysis. AgB8/1, AgB8/2, AgB8/3 and AgB8/4 subunits were identified in all samples analyzed, and an AgB8/2 variant (AgB8/2v8) was found in one bovine sample. The exponentially modified protein abundance index (emPAI) was used to estimate the relative abundance of the AgB subunits, revealing that AgB8/1 subunit was relatively overrepresented in all samples. The abundance of AgB8/3 subunit varied between bovine and human cysts. The oligomeric states formed by E. granulosus AgB and recombinant subunits available, rAgB8/1, rAgB8/2 and rAgB8/3, were characterized by native PAGE, light scattering and microscopy. Recombinant subunits showed markedly distinct oligomerization behaviors, forming oligomers with a maximum size relation of rAgB8/3>rAgB8/2>rAgB8/1. Moreover, the oligomeric states formed by rAgB8/3 subunit were more similar to those observed for AgB purified from hydatid fluid. Pressure-induced dissociation experiments demonstrated that the molecular assemblies formed by the more aggregative subunits, rAgB8/2 and rAgB8/3, also display higher structural stability. Conclusions/Significance For the first time, AgB subunit composition was analyzed in samples from single hydatid cysts, revealing qualitative and quantitative differences between samples. We showed that AgB oligomers are formed by different subunits, which have distinct abundances and oligomerization properties. Overall, our findings have significantly contributed to increase the

  3. Conformational analysis of putative regulatory subunit D of the toluene/o-xylene-monooxygenase complex from Pseudomonas stutzeri OX1

    PubMed Central

    Scognamiglio, Roberta; Notomista, Eugenio; Barbieri, Paola; Pucci, Piero; Piaz, Fabrizio Dal; Tramontano, Anna; Di Donato, Alberto

    2001-01-01

    A gene cluster isolated from Pseudomonas stutzeri OX1 genomic DNA and containing six ORFs codes for toluene/o-xylene-monooxygenase. The putative regulatory D subunit was expressed in Escherichia coli and purified. Its protein sequence was verified by mass spectrometry mapping and found to be identical to the sequence predicted on the basis of the DNA sequence. The surface topology of subunit D in solution was probed by limited proteolysis carried out under strictly controlled conditions using several proteases as proteolytic probes. The same experiments were carried out on the homologous P2 component of the multicomponent phenol hydroxylase from Pseudomonas putida CF600. The proteolytic fragments released from both proteins in their native state were analyzed by electrospray mass spectrometry, and the preferential cleavage sites were assessed. The results indicated that despite the relatively high similarity between the sequences of the two proteins, some differences in the distribution of preferential proteolytic cleavages were detected, and a much higher conformational flexibility of subunit D was inferred. Moreover, automatic modeling of subunit D was attempted, based on the known three-dimensional structure of P2. Our results indicate that, at least in this case, standard modeling procedures based on automatic alignment on the structure of P2 fail to produce a model consistent with limited proteolysis experimental data. Thus, it is our opinion that reliable techniques such as limited proteolysis can be employed to test three-dimensional models and highlight problems in automatic model building. PMID:11344317

  4. NADH dehydrogenase subunit 1 and cytochrome c oxidase subunit I sequences compared for members of the genus Taenia (Cestoda).

    PubMed

    Gasser, R B; Zhu, X; McManus, D P

    1999-12-01

    Nine members of the genus Taenia (Taenia taeniaeformis, Taenia hydatigena, Taenia pisiformis, Taenia ovis, Taenia multiceps, Taenia serialis, Taenia saginata, Taenia solium and the Asian Taenia) were characterised by their mitochondrial NADH dehydrogenase subunit 1 gene sequences and their genetic relationships were compared with those derived from the cytochrome c oxidase subunit 1 sequence data. The extent of inter-taxon sequence difference in NADH dehydrogenase subunit 1 (approximately 5.9-30.8%) was usually greater than in cytochrome c oxidase subunit 1 (approximately 2.5-18%). Although topology of the phenograms derived from NADH dehydrogenase subunit 1 and cytochrome c oxidase subunit 1 sequence data differed, there was concordance in that T. multiceps, T. serialis (of canids), T. saginata and the Asian Taenia (of humans) were genetically most similar, and those four members were genetically more similar to T. ovis and T. solium than they were to T. hydatigena and T. pisiformis (of canids) or T. taeniaeformis (of cats). The NADH dehydrogenase subunit 1 sequence data may prove useful in studies of the systematics and population genetic structure of the Taeniidae.

  5. Mapping Potassium

    NASA Image and Video Library

    2015-04-16

    During the first year of NASA MESSENGER orbital mission, the spacecraft GRS instrument measured the elemental composition of Mercury surface materials. mong the most important discoveries from the GRS was the observation of higher abundances of the moderately volatile elements potassium, sodium, and chlorine than expected from previous scientific models and theories. Particularly high concentrations of these elements were observed at high northern latitudes, as illustrated in this potassium abundance map, which provides a view of the surface centered at 60° N latitude and 120° E longitude. This map was the first elemental map ever made of Mercury's surface and is to-date the only map to report absolute elemental concentrations, in comparison to element ratios. Prior to MESSENGER's arrival at Mercury, scientists expected that the planet would be depleted in moderately volatile elements, as is the case for our Moon. The unexpectedly high abundances observed with the GRS have forced a reevaluation of our understanding of the formation and evolution of Mercury. In addition, the K map provided the first evidence for distinct geochemical terranes on Mercury, as the high-potassium region was later found to also be distinct in its low Mg/Si, Ca/Si, S/Si, and high Na/Si and Cl/Si abundances. Instrument: Gamma-Ray Spectrometer (GRS) http://photojournal.jpl.nasa.gov/catalog/PIA19414

  6. Quality control of a cytoplasmic protein complex: chaperone motors and the ubiquitin-proteasome system govern the fate of orphan fatty acid synthase subunit Fas2 of yeast.

    PubMed

    Scazzari, Mario; Amm, Ingo; Wolf, Dieter H

    2015-02-20

    For the assembly of protein complexes in the cell, the presence of stoichiometric amounts of the respective protein subunits is of utmost importance. A surplus of any of the subunits may trigger unspecific and harmful protein interactions and has to be avoided. A stoichiometric amount of subunits must finally be reached via transcriptional, translational, and/or post-translational regulation. Synthesis of saturated 16 and 18 carbon fatty acids is carried out by fatty acid synthase: in yeast Saccharomyces cerevisiae, a 2.6-MDa molecular mass assembly containing six protomers each of two different subunits, Fas1 (β) and Fas2 (α). The (α)6(β)6 complex carries six copies of all eight enzymatic activities required for fatty acid synthesis. The FAS1 and FAS2 genes in yeast are unlinked and map on two different chromosomes. Here we study the fate of the α-subunit of the complex, Fas2, when its partner, the β-subunit Fas1, is absent. Individual subunits of fatty acid synthase are proteolytically degraded when the respective partner is missing. Elimination of Fas2 is achieved by the proteasome. Here we show that a ubiquitin transfer machinery is required for Fas2 elimination. The major ubiquitin ligase targeting the superfluous Fas2 subunit to the proteasome is Ubr1. The ubiquitin-conjugating enzymes Ubc2 and Ubc4 assist the degradation process. The AAA-ATPase Cdc48 and the Hsp70 chaperone Ssa1 are crucially involved in the elimination of Fas2. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. 2 n -rational maps

    NASA Astrophysics Data System (ADS)

    Kassotakis, Pavlos; Nieszporski, Maciej

    2017-05-01

    We present a natural extension of the notion of nondegenerate rational maps (quadrirational maps) to arbitrary dimensions. We refer to these maps as 2 n -rational maps. In this note we construct a rich family of 2 n -rational maps. These maps by construction are involutions and highly symmetric in the sense that the maps and their companion maps have the same functional form.

  8. Database on the structure of large ribosomal subunit RNA.

    PubMed Central

    De Rijk, P; Van de Peer, Y; Chapelle, S; De Wachter, R

    1994-01-01

    A database on large ribosomal subunit RNA is made available. It contains 258 sequences. It provides sequence, alignment and secondary structure information in computer-readable formats. Files can be obtained using ftp. PMID:7524023

  9. Genetic Analysis of the Cytoplasmic Dynein Subunit Families

    PubMed Central

    Pfister, K. Kevin; Shah, Paresh R; Hummerich, Holger; Russ, Andreas; Cotton, James; Annuar, Azlina Ahmad; King, Stephen M; Fisher, Elizabeth M. C

    2006-01-01

    Cytoplasmic dyneins, the principal microtubule minus-end-directed motor proteins of the cell, are involved in many essential cellular processes. The major form of this enzyme is a complex of at least six protein subunits, and in mammals all but one of the subunits are encoded by at least two genes. Here we review current knowledge concerning the subunits, their interactions, and their functional roles as derived from biochemical and genetic analyses. We also carried out extensive database searches to look for new genes and to clarify anomalies in the databases. Our analysis documents evolutionary relationships among the dynein subunits of mammals and other model organisms, and sheds new light on the role of this diverse group of proteins, highlighting the existence of two cytoplasmic dynein complexes with distinct cellular roles. PMID:16440056

  10. The eukaryotic RNA exosome: same scaffold but variable catalytic subunits.

    PubMed

    Lykke-Andersen, Søren; Tomecki, Rafal; Jensen, Torben Heick; Dziembowski, Andrzej

    2011-01-01

    The RNA exosome is a versatile ribonucleolytic protein complex that participates in a multitude of cellular RNA processing and degradation events. It consists of an invariable nine-subunit core that associates with a variety of enzymatically active subunits and co-factors. These contribute to or even provide the catalytic activity and substrate specificity of the complex. The S. cerevisiae exosome has been intensively studied since its discovery in 1997 and thus serves as the archetype of eukaryotic exosomes. Notably, its catalytic potential, derived exclusively from associated subunits, differs between the nuclear and cytoplasmic versions of the complex. The same holds true for other eukaryotes, however, recent discoveries from various laboratories including our own have revealed that there are variations on this theme. Here, we review the latest findings concerning catalytic subunits of eukaryotic exosomes, and we discuss the apparent need for differential composition and subcellular distribution of exosome variants.

  11. A process yields large quantities of pure ribosome subunits

    NASA Technical Reports Server (NTRS)

    Friedman, M.; Lu, P.; Rich, A.

    1972-01-01

    Development of process for in-vitro protein synthesis from living cells followed by dissociation of ribosomes into subunits is discussed. Process depends on dialysis or use of chelating agents. Operation of process and advantages over previous methods are outlined.

  12. Subunit-Specific Trafficking of GABAA Receptors during Status Epilepticus

    PubMed Central

    Goodkin, Howard P.; Joshi, Suchitra; Mtchedlishvili, Zakaria; Brar, Jasmit; Kapur, Jaideep

    2010-01-01

    It is proposed that a reduced surface expression of GABAA receptors (GABARs) contributes to the pathogenesis of status epilepticus (SE), a condition characterized by prolonged seizures. This hypothesis was based on the finding that prolonged epileptiform bursting (repetitive bursts of prolonged depolarizations with superimposed action potentials) in cultures of dissociated hippocampal pyramidal neurons (dissociated cultures) results in the increased intracellular accumulation of GABARs. However, it is not known whether this rapid modification in the surface-expressed GABAR pool results from selective, subunit-dependent or nonselective, subunit-independent internalization of GABARs. In hippocampal slices obtained from animals undergoing prolonged SE (SE-treated slices), we found that the surface expression of the GABARβ2/3 and γ2 subunits was reduced, whereas that of the δ subunit was not. Complementary electrophysiological recordings from dentate granule cells in SE-treated slices demonstrated a reduction in GABAR-mediated synaptic inhibition, but not tonic inhibition. A reduction in the surface expression of the γ2 subunit, but not the δ subunit was also observed in dissociated cultures and organotypic hippocampal slice cultures when incubated in an elevated KCl external medium or an elevated KCl external medium supplemented with NMDA, respectively. Additional studies demonstrated that the reduction in the surface expression of the γ2 subunit was independent of direct ligand binding of the GABAR. These findings demonstrate that the regulation of surface-expressed GABAR pool during SE is subunit-specific and occurs independent of ligand binding. The differential modulation of the surface expression of GABARs during SE has potential implications for the treatment of this neurological emergency. PMID:18322097

  13. F-subunit reinforces torque generation in V-ATPase.

    PubMed

    Kishikawa, Jun-ichi; Seino, Akihiko; Nakanishi, Atsuko; Tirtom, Naciye Esma; Noji, Hiroyuki; Yokoyama, Ken; Hayashi, Kumiko

    2014-09-01

    Vacuolar-type H(+)-pumping ATPases (V-ATPases) perform remarkably diverse functions in eukaryotic organisms. They are present in the membranes of many organelles and regulate the pH of several intracellular compartments. A family of V-ATPases is also present in the plasma membranes of some bacteria. Such V-ATPases function as ATP-synthases. Each V-ATPase is composed of a water-soluble domain (V1) and a membrane-embedded domain (Vo). The ATP-driven rotary unit, V[Formula: see text], is composed of A, B, D, and F subunits. The rotary shaft (the DF subcomplex) rotates in the central cavity of the A3B3-ring (the catalytic hexamer ring). The D-subunit, which has a coiled-coil domain, penetrates into the ring, while the F-subunit is a globular-shaped domain protruding from the ring. The minimal ATP-driven rotary unit of V[Formula: see text] is comprised of the A3B3D subunits, and we therefore investigated how the absence of the globular-shaped F-subunit affects the rotary torque generation of V[Formula: see text]. Using a single-molecule technique, we observed the motion of the rotary motors. To obtain the torque values, we then analyzed the measured motion trajectories based on the fluctuation theorem, which states that the law of entropy production in non-equilibrium conditions and has been suggested as a novel and effective method for measuring torque. The measured torque of A3B3D was half that of the wild-type V1, and full torque was recovered in the mutant V1, in which the F-subunit was genetically fused with the D-subunit, indicating that the globular-shaped F-subunit reinforces torque generation in V1.

  14. Transcription Activator Interactions with Multiple SWI/SNF Subunits

    PubMed Central

    Neely, Kristen E.; Hassan, Ahmed H.; Brown, Christine E.; Howe, LeAnn; Workman, Jerry L.

    2002-01-01

    We have previously shown that the yeast SWI/SNF complex stimulates in vitro transcription from chromatin templates in an ATP-dependent manner. SWI/SNF function in this regard requires the presence of an activator with which it can interact directly, linking activator recruitment of SWI/SNF to transcriptional stimulation. In this study, we determine the SWI/SNF subunits that mediate its interaction with activators. Using a photo-cross-linking label transfer strategy, we show that the Snf5, Swi1, and Swi2/Snf2 subunits are contacted by the yeast acidic activators, Gcn4 and Hap4, in the context of the intact native SWI/SNF complex. In addition, we show that the same three subunits can interact individually with acidic activation domains, indicating that each subunit contributes to binding activators. Furthermore, mutations that reduce the activation potential of these activators also diminish its interaction with each of these SWI/SNF subunits. Thus, three distinct subunits of the SWI/SNF complex contribute to its interactions with activation domains. PMID:11865042

  15. Subunit structure of the acetylcholine receptor from Electrophorus electricus.

    PubMed

    Conti-Tronconi, B M; Hunkapiller, M W; Lindstrom, J M; Raftery, M A

    1982-11-01

    The amino-terminal amino acid sequences of the four major peptides (Mr 41,000, 50,000, 55,000, and 62,000) present in purified preparations of Electrophorus electricus nicotinic acetylcholine receptor (AcChoR) have been determined for 24 cycles by automated sequence analysis procedures yielding four unique polypeptide sequences. The sequences showed a high degree of similarity, having identical residues in a number of positions ranging between 37% and 50% for specific pairs of subunits. Comparison of the sequences obtained with those of the subunits of similar molecular weight from Torpedo californica AcChoR revealed an even higher degree of homology (from 46% to 71%) for these two highly diverged species. Simultaneous sequence analysis of the amino termini present in native, purified Electrophorus AcChoR showed that these four related sequences were the only ones present and that they occur in a ratio of 2:1:1:1, with the smallest subunit ("alpha 1") being present in two copies. Genealogical analysis suggests that the subunits of both Torpedo and Electrophorus AcChoRs derive from a common ancestral gene, the divergence having occurred early in the evolution of the receptor. This shared ancestry and the very early divergence of the four subunits, as well as the highly conserved structure of the AcChoR complex along animal evolution, suggest that each of the subunits evolved to perform discrete crucial roles in the physiological function of the AcChoR.

  16. Effects of detergents on ribosomal precursor subunits of Bacillus megaterium.

    PubMed

    Body, A; Brownstein, B H

    1978-01-01

    Cell extracts prepared by osmotic lysis of protoplasts were analyzed by sucrose gradient sedimentation. In the absence of detergents, ribosomal precursor particles were found in a gradient fraction which sedimented faster than mature 50S subunits and in two other fractions coincident with mature 50S and 30S ribosomal subunits. Phospholipid, an indicator of membrane, was shown to be associated with only the fastest-sedimenting ribosomal precursor particle fraction. After the extracts were treated with detergents, all phospholipid was found at the top of the gradients. Brij 58, Triton X-100, and Nonidet P-40 did not cause a change in the sedimentation values of precursors; however, the detergents deoxycholate or LOC (Amway Corp.) disrupted the fastest-sedimenting precursor and converted the ribosomal precursor subunits which sedimented at the 50S and 30S positions to five different classes of more slowly sedimenting particles. Earlier reports on the in vivo assembly of ribosomal subunits have shown that several stages of ribosomal precursor subunits exist, and, in the presence of the detergents deoxycholate and LOC, which had been used to prepare cell extracts, the precursors sedimented more slowly. Our data are consistent with the hypothesis that those detergents selectively modify the structure of ribosomal precursors and lend further support to the hypothesis that the in vivo ribosomal precursor subunits have 50S and 30S sedimentation values. In addition, these data support the idea that the ribosomal precursor particles found in the fast-sedimenting fraction may constitute a unique precursor fraction.

  17. Effects of Detergents on Ribosomal Precursor Subunits of Bacillus megaterium

    PubMed Central

    Body, Barbara A.; Brownstein, Bernard H.

    1978-01-01

    Cell extracts prepared by osmotic lysis of protoplasts were analyzed by sucrose gradient sedimentation. In the absence of detergents, ribosomal precursor particles were found in a gradient fraction which sedimented faster than mature 50S subunits and in two other fractions coincident with mature 50S and 30S ribosomal subunits. Phospholipid, an indicator of membrane, was shown to be associated with only the fastest-sedimenting ribosomal precursor particle fraction. After the extracts were treated with detergents, all phospholipid was found at the top of the gradients. Brij 58, Triton X-100, and Nonidet P-40 did not cause a change in the sedimentation values of precursors; however, the detergents deoxycholate or LOC (Amway Corp.) disrupted the fastest-sedimenting precursor and converted the ribosomal precursor subunits which sedimented at the 50S and 30S positions to five different classes of more slowly sedimenting particles. Earlier reports on the in vivo assembly of ribosomal subunits have shown that several stages of ribosomal precursor subunits exist, and, in the presence of the detergents deoxycholate and LOC, which had been used to prepare cell extracts, the precursors sedimented more slowly. Our data are consistent with the hypothesis that those detergents selectively modify the structure of ribosomal precursors and lend further support to the hypothesis that the in vivo ribosomal precursor subunits have 50S and 30S sedimentation values. In addition, these data support the idea that the ribosomal precursor particles found in the fast-sedimenting fraction may constitute a unique precursor fraction. PMID:412833

  18. The Subunit Structure of Potato Tuber ADPglucose Pyrophosphorylase 1

    PubMed Central

    Okita, Thomas W.; Nakata, Paul A.; Anderson, Joseph M.; Sowokinos, Joseph; Morell, Matthew; Preiss, Jack

    1990-01-01

    ADPglucose pyrophosphorylase has been extensively purified from potato (Solanum tuberosum L.) tuber tissue to study its structure. By employing a modified published procedure (JR Sowokinos, J Preiss [1982] Plant Physiol 69: 1459-1466) together with Mono Q chromatography, a near homogeneous enzyme preparation was obtained with substantial improvement in enzyme yield and specific activity. In single dimensional sodium dodecyl sulfate polyacrylamide gels, the enzyme migrated as a single polypeptide band with a mobility of about 50,000 daltons. Analysis by two-dimensional polyacrylamide gel electrophoresis, however, revealed the presence of two types of subunits which could be distinguished by their slight differences in net charge and molecular weight. The smaller potato tuber subunit was recognized by antiserum prepared against the smaller spinach leaf 51 kilodalton ADPglucose pyrophosphorylase subunit. In contrast, the anti-54 kilodalton raised against the spinach leaf subunit did not significantly react to the tuber enzyme subunits. The results are consistent with the hypothesis that the potato tuber ADPglucose pyrophosphorylase is not composed of a simple homotetramer as previously suggested, but is a product of two separate and distinct subunits as observed for the spinach leaf and maize enzymes. Images Figure 1 Figure 2 Figure 3 PMID:16667537

  19. Transcriptional regulators of Na,K-ATPase subunits

    PubMed Central

    Li, Zhiqin; Langhans, Sigrid A.

    2015-01-01

    The Na,K-ATPase classically serves as an ion pump creating an electrochemical gradient across the plasma membrane that is essential for transepithelial transport, nutrient uptake and membrane potential. In addition, Na,K-ATPase also functions as a receptor, a signal transducer and a cell adhesion molecule. With such diverse roles, it is understandable that the Na,K-ATPase subunits, the catalytic α-subunit, the β-subunit and the FXYD proteins, are controlled extensively during development and to accommodate physiological needs. The spatial and temporal expression of Na,K-ATPase is partially regulated at the transcriptional level. Numerous transcription factors, hormones, growth factors, lipids, and extracellular stimuli modulate the transcription of the Na,K-ATPase subunits. Moreover, epigenetic mechanisms also contribute to the regulation of Na,K-ATPase expression. With the ever growing knowledge about diseases associated with the malfunction of Na,K-ATPase, this review aims at summarizing the best-characterized transcription regulators that modulate Na,K-ATPase subunit levels. As abnormal expression of Na,K-ATPase subunits has been observed in many carcinoma, we will also discuss transcription factors that are associated with epithelial-mesenchymal transition, a crucial step in the progression of many tumors to malignant disease. PMID:26579519

  20. Calcium channel beta subunits differentially modulate recovery of the channel from inactivation.

    PubMed

    Jeziorski, M C; Greenberg, R M; Anderson, P A

    2000-10-20

    We examined the effects of calcium channel beta subunits upon the recovery from inactivation of alpha(1) subunits expressed in Xenopus oocytes. Recovery of the current carried by the L-type alpha(1) subunit (cyCa(v)1) from the jellyfish Cyanea capillata was accelerated by coexpression of any beta subunit, but the degree of potentiation differed according to which beta isoform was coexpressed. The Cyanea beta subunit was most effective, followed by the mammalian b(3), b(4), and beta(2a) subtypes. Recovery of the human Ca(v)2.3 subunit was also modulated by beta subunits, but was slowed instead. beta(3) was the most potent subunit tested, followed by beta(4), then beta(2a), which had virtually no effect. These results demonstrate that different beta subunit isoforms can affect recovery of the channel to varying degrees, and provide an additional mechanism by which beta subunits can differentially regulate alpha(1) subunits.

  1. Hybrid Rubisco of tomato large subunits and tobacco small subunits is functional in tobacco plants.

    PubMed

    Zhang, Xing-Hai; Webb, James; Huang, Yi-Hong; Lin, Li; Tang, Ri-Sheng; Liu, Aimin

    2011-03-01

    Biogenesis of functional ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in plants requires specific assembly in the chloroplast of the imported, cytosol-synthesized small subunits (SS) with the chloroplast-made large subunits (LS). Accumulating evidence indicates that chloroplasts (plastids) generally have a low tolerance for assembling foreign or modified Rubisco. To explore Rubisco engineering, we created two lines of transplastomic tobacco plants whose rbcL gene was replaced by tomato-derived rbcL: plant LLS2 with Rubisco composed of tobacco SS and Q437R LS and plant LLS4 with a hybrid Rubisco of tobacco SS and tomato LS (representing four substitutions of Y226F, A230T, S279T and Q437R from tobacco LS). Plant LLS2 exhibited similar phenotypes as the wild type. Plant LLS4 showed lower chlorophyll and Rubisco levels particularly in young emerging leaves, lower photosynthesis rates and biomass during early stages of development, but was able to reach reproductive maturity and somewhat wild type-like phenotype under ambient CO₂ condition. In vitro assays detected similar carboxylase activity and RuBP affinity in LLS2 and LLS4 plants as in wild type. Our studies demonstrated that tomato LS was sufficiently assembled with tobacco SS into functional Rubisco. The hybrid Rubisco of tomato LS and tobacco SS can drive photosynthesis that supports photoautotrophic growth and reproduction of tobacco plants under ambient CO₂ and light conditions. We discuss the effect of these residue substitutions on Rubisco activity and the possible attribution of chlorophyll deficiency to the in planta photosynthesis performance in the hybrid Rubisco plants.

  2. Horizontal membrane-intrinsic α-helices in the stator a-subunit of an F-type ATP synthase.

    PubMed

    Allegretti, Matteo; Klusch, Niklas; Mills, Deryck J; Vonck, Janet; Kühlbrandt, Werner; Davies, Karen M

    2015-05-14

    ATP, the universal energy currency of cells, is produced by F-type ATP synthases, which are ancient, membrane-bound nanomachines. F-type ATP synthases use the energy of a transmembrane electrochemical gradient to generate ATP by rotary catalysis. Protons moving across the membrane drive a rotor ring composed of 8-15 c-subunits. A central stalk transmits the rotation of the c-ring to the catalytic F1 head, where a series of conformational changes results in ATP synthesis. A key unresolved question in this fundamental process is how protons pass through the membrane to drive ATP production. Mitochondrial ATP synthases form V-shaped homodimers in cristae membranes. Here we report the structure of a native and active mitochondrial ATP synthase dimer, determined by single-particle electron cryomicroscopy at 6.2 Å resolution. Our structure shows four long, horizontal membrane-intrinsic α-helices in the a-subunit, arranged in two hairpins at an angle of approximately 70° relative to the c-ring helices. It has been proposed that a strictly conserved membrane-embedded arginine in the a-subunit couples proton translocation to c-ring rotation. A fit of the conserved carboxy-terminal a-subunit sequence places the conserved arginine next to a proton-binding c-subunit glutamate. The map shows a slanting solvent-accessible channel that extends from the mitochondrial matrix to the conserved arginine. Another hydrophilic cavity on the lumenal membrane surface defines a direct route for the protons to an essential histidine-glutamate pair. Our results provide unique new insights into the structure and function of rotary ATP synthases and explain how ATP production is coupled to proton translocation.

  3. Molecular cloning and nucleotide sequence of the alpha and beta subunits of allophycocyanin from the cyanelle genome of Cyanophora paradoxa.

    PubMed Central

    Bryant, D A; de Lorimier, R; Lambert, D H; Dubbs, J M; Stirewalt, V L; Stevens, S E; Porter, R D; Tam, J; Jay, E

    1985-01-01

    The genes for the alpha- and beta-subunit apoproteins of allophycocyanin (AP) were isolated from the cyanelle genome of Cyanophora paradoxa and subjected to nucleotide sequence analysis. The AP beta-subunit apoprotein gene was localized to a 7.8-kilobase-pair Pst I restriction fragment from cyanelle DNA by hybridization with a tetradecameric oligonucleotide probe. Sequence analysis using that oligonucleotide and its complement as primers for the dideoxy chain-termination sequencing method confirmed the presence of both AP alpha- and beta-subunit genes on this restriction fragment. Additional oligonucleotide primers were synthesized as sequencing progressed and were used to determine rapidly the nucleotide sequence of a 1336-base-pair region of this cloned fragment. This strategy allowed the sequencing to be completed without a detailed restriction map and without extensive and time-consuming subcloning. The sequenced region contains two open reading frames whose deduced amino acid sequences are 81-85% homologous to cyanobacterial and red algal AP subunits whose amino acid sequences have been determined. The two open reading frames are in the same orientation and are separated by 39 base pairs. AP alpha is 5' to AP beta and both coding sequences are preceded by a polypurine, Shine-Dalgarno-type sequence. Sequences upstream from AP alpha closely resemble the Escherichia coli consensus promoter sequences and also show considerable homology to promoter sequences for several chloroplast-encoded psbA genes. A 56-base-pair palindromic sequence downstream from the AP beta gene could play a role in the termination of transcription or translation. The allophycocyanin apoprotein subunit genes are located on the large single-copy region of the cyanelle genome. PMID:2987916

  4. Random mutagenesis of yeast 25S rRNA identify bases critical for 60S subunit structural integrity and function

    PubMed Central

    Nemoto, Naoki; Udagawa, Tsuyoshi; Chowdhury, Wasimul; Kitabatake, Makoto; Shin, Byung-shik; Hiraishi, Hiroyuki; Wang, Suzhi; Singh, Chingakham Ranjit; Brown, Susan J.; Ohno, Mutsuhito; Asano, Katsura

    2013-01-01

    In yeast Saccharomyces cerevisiae, 25S rRNA makes up the major mass and shape of the 60S ribosomal subunit. During translation initiation, the 60S subunit joins the 40S initiation complex, producing the 80S initiation complex. During elongation, the 60S subunit binds the CCA-ends of aminoacyl- and peptidyl-tRNAs at the A-loop and P-loop, respectively, transferring the peptide onto the α-amino group of the aminoacyl-tRNA. To study the role of 25S rRNA in translation in vivo, we randomly mutated 25S rRNA and isolated and characterized seven point mutations that affected yeast cell growth and polysome profiles. Four of these mutations, G651A, A1435U, A1446G and A1587G, change a base involved in base triples crucial for structural integrity. Three other mutations change bases near the ribosomal surface: C2879U and U2408C alter the A-loop and P-loop, respectively, and G1735A maps near a Eukarya-specific bridge to the 40S subunit. By polysome profiling in mmslΔ mutants defective in nonfunctional 25S rRNA decay, we show that some of these mutations are defective in both the initiation and elongation phases of translation. Of the mutants characterized, C2879U displays the strongest defect in translation initiation. The ribosome transit-time assay directly shows that this mutation is also defective in peptide elongation/termination. Thus, our genetic analysis not only identifies bases critical for structural integrity of the 60S subunit, but also suggests a role for bases near the peptidyl transferase center in translation initiation. PMID:26824023

  5. Functional Properties of a New Voltage-dependent Calcium Channel α2δ Auxiliary Subunit Gene (CACNA2D2)*

    PubMed Central

    Gao, Boning; Sekido, Yoshitaka; Maximov, Anton; Saad, Mohamad; Forgacs, Eva; Latif, Farida; Wei, Ming H.; Lerman, Michael; Lee, Jung-Ha; Perez-Reyes, Edward; Bezprozvanny, Ilya; Minna, John D.

    2012-01-01

    We have positionally cloned and characterized a new calcium channel auxiliary subunit, α2δ-2 (CACNA2D2), which shares 56% amino acid identity with the known α2δ-1 subunit. The gene maps to the critical human tumor suppressor gene region in chromosome 3p21.3, showing very frequent allele loss and occasional homozygous deletions in lung, breast, and other cancers. The tissue distribution of α2δ-2 expression is different from α2δ-1, and α2δ-2 mRNA is most abundantly expressed in lung and testis and well expressed in brain, heart, and pancreas. In contrast, α2δ-1 is expressed predominantly in brain, heart, and skeletal muscle. When co-expressed (via cRNA injections) with α1B and β3 subunits in Xenopus oocytes, α2δ-2 increased peak size of the N-type Ca2+ currents 9-fold, and when co-expressed with α1C or α1G subunits in Xenopus oocytes increased peak size of L-type channels 2-fold and T-type channels 1.8-fold, respectively. Anti-peptide antibodies detect the expression of a 129-kDa α2δ-2 polypeptide in some but not all lung tumor cells. We conclude that the α2δ-2 gene encodes a functional auxiliary subunit of voltage-gated Ca2+ channels. Because of its chromosomal location and expression patterns, CACNA2D2 needs to be explored as a potential tumor suppressor gene linking Ca2+ signaling and lung, breast, and other cancer pathogenesis. The homologous location on mouse chromosome 9 is also the site of the mouse neurologic mutant ducky (du), and thus, CACNA2D2 is also a candidate gene for this inherited idiopathic generalized epilepsy syndrome. PMID:10766861

  6. Topology of yeast RNA polymerase II subunits in transcription elongation complexes studied by photoaffinity cross-linking.

    PubMed

    Wooddell, C I; Burgess, R R

    2000-11-07

    The subunits of Saccharomyces cerevisiae RNA polymerase II (RNAP II) in proximity to the DNA during transcription elongation have been identified by photoaffinity cross-linking. In the absence of transcription factors, RNAP II will transcribe a double-stranded DNA fragment containing a 3'-extension of deoxycytidines, a "tailed template". We designed a DNA template allowing the RNAP to transcribe 76 bases before it was stalled by omission of CTP in the transcription reaction. This stall site oriented the RNAP on the DNA template and allowed us to map the RNAP subunits along the DNA. The DNA analogue 5-[N-(p-azidobenzoyl)-3-aminoallyl]-dUTP (N(3)RdUTP) [Bartholomew, B., Kassavetis, G. A., Braun, B. R., and Geiduschek, E. P. (1990) EMBO J. 9, 2197-205] was synthesized and enzymatically incorporated into the DNA at specified positions upstream or downstream of the stall site, in either the template or nontemplate strand of the DNA. Radioactive nucleotides were positioned beside the photoactivatable nucleotides, and cross-linking by brief ultraviolet irradiation transferred the radioactive tag from the DNA onto the RNAP subunits. In addition to N(3)RdUTP, which has a photoreactive azido group 9 A from the uridine base, we used the photoaffinity cross-linker 5N(3)dUTP with an azido group directly on the uridine ring to identify the RNAP II subunits closest to the DNA at positions where multiple subunits cross-linked. In cross-linking reactions dependent on transcription, RPB1, RPB2, and RPB5 were cross-linked with N(3)RdUTP. With 5N(3)dUTP, only RPB1 and RPB2 were cross-linked. Under certain circumstances, RPB3, RPB4, and RPB7 were cross-linked. From the information obtained in this topological study, we developed a model of yeast RNAP II in a transcription elongation complex.

  7. Streptococcus suis Bacterin and Subunit Vaccine Immunogenicities and Protective Efficacies against Serotypes 2 and 9▿†

    PubMed Central

    Baums, Christoph Georg; Kock, Christoph; Beineke, Andreas; Bennecke, Katharina; Goethe, Ralph; Schröder, Charlotte; Waldmann, Karl-Heinz; Valentin-Weigand, Peter

    2009-01-01

    Streptococcus suis causes numerous diseases in pigs, most importantly, meningitis, arthritis, septicemia, and bronchopneumonia. One of the major problems in modern swine production is the lack of a vaccine protecting against more than one S. suis serotype. The objective of this study was to determine the protective efficacy of a serotype 2 murein-associated protein (MAP) fraction subunit vaccine in comparison to that of a bacterin against experimental challenge with serotype 2 (containing muramidase-released protein [MRP], extracellular factor, and suilysin [SLY]) and serotype 9 (containing MRP variant MRP* and SLY) strains. MAP was shown to include different surface-associated proteins, such as the MRP and surface antigen one (SAO) expressed by both pathotypes used for challenge. The results of this study demonstrated that the serotype 2 bacterin induced protective immunity against homologous challenge. In contrast, the protective efficacy of the MAP subunit vaccine was low, though MAP immunization resulted in high serum immunoglobulin G2 titers against MRP and SAO. Importantly, immunization with bacterin but not with MAP induced opsonizing antibody titers against the serotype 2 strain, and these antibody titers were found to correlate with protection. However, after absorption with a nonencapsulated isogenic mutant, the sera from bacterin-immunized piglets failed to facilitate neutrophil killing, indicating that antibodies directed against capsule may not have been essential for opsonophagocytosis. Furthermore, induction of opsonizing antibodies against serotype 9 was not detectable in the group receiving bacterin or in the group receiving the MAP vaccine. In agreement, protection against the heterologous serotype 9 strain was low in both groups. Thus, identification of an antigen protecting against these two important S. suis pathotypes remains an important goal of future studies. PMID:19109449

  8. NOX Activation by Subunit Interaction and Underlying Mechanisms in Disease

    PubMed Central

    Rastogi, Radhika; Geng, Xiaokun; Li, Fengwu; Ding, Yuchuan

    2017-01-01

    Nicotinamide adenine dinucleotide phosphate (NAPDH) oxidase (NOX) is an enzyme complex with the sole function of producing superoxide anion and reactive oxygen species (ROS) at the expense of NADPH. Vital to the immune system as well as cellular signaling, NOX is also involved in the pathologies of a wide variety of disease states. Particularly, it is an integral player in many neurological diseases, including stroke, TBI, and neurodegenerative diseases. Pathologically, NOX produces an excessive amount of ROS that exceed the body’s antioxidant ability to neutralize them, leading to oxidative stress and aberrant signaling. This prevalence makes it an attractive therapeutic target and as such, NOX inhibitors have been studied and developed to counter NOX’s deleterious effects. However, recent studies of NOX have created a better understanding of the NOX complex. Comprised of independent cytosolic subunits, p47-phox, p67-phox, p40-phox and Rac, and membrane subunits, gp91-phox and p22-phox, the NOX complex requires a unique activation process through subunit interaction. Of these subunits, p47-phox plays the most important role in activation, binding and translocating the cytosolic subunits to the membrane and anchoring to p22-phox to organize the complex for NOX activation and function. Moreover, these interactions, particularly that between p47-phox and p22-phox, are dependent on phosphorylation initiated by upstream processes involving protein kinase C (PKC). This review will look at these interactions between subunits and with PKC. It will focus on the interaction involving p47-phox with p22-phox, key in bringing the cytosolic subunits to the membrane. Furthermore, the implication of these interactions as a target for NOX inhibitors such as apocynin will be discussed as a potential avenue for further investigation, in order to develop more specific NOX inhibitors based on the inhibition of NOX assembly and activation. PMID:28119569

  9. Protein Expression of Proteasome Subunits in Elderly Patients with Schizophrenia

    PubMed Central

    Scott, Madeline R; Rubio, Maria D; Haroutunian, Vahram; Meador-Woodruff, James H

    2016-01-01

    The ubiquitin proteasome system (UPS) is a major regulator of protein processing, trafficking, and degradation. While protein ubiquitination is utilized for many cellular processes, one major function of this system is to target proteins to the proteasome for degradation. In schizophrenia, studies have found UPS transcript abnormalities in both blood and brain, and we have previously reported decreased protein expression of ubiquitin-associated proteins in brain. To test whether the proteasome is similarly dysregulated, we measured the protein expression of proteasome catalytic subunits as well as essential subunits from proteasome regulatory complexes in 14 pair-matched schizophrenia and comparison subjects in superior temporal cortex. We found decreased expression of Rpt1, Rpt3, and Rpt6, subunits of the 19S regulatory particle essential for ubiquitin-dependent degradation by the proteasome. Additionally, the α subunit of the 11S αβ regulatory particle, which enhances proteasomal degradation of small peptides and unfolded proteins, was also decreased. Haloperidol-treated rats did not have altered expression of these subunits, suggesting the changes we observed in schizophrenia are likely not due to chronic antipsychotic treatment. Interestingly, expression of the catalytic subunits of both the standard and immunoproteasome were unchanged, suggesting the abnormalities we observed may be specific to the complexed state of the proteasome. Aging has significant effects on the proteasome, and several subunits (20S β2, Rpn10, Rpn13, 11Sβ, and 11Sγ) were significantly correlated with subject age. These data provide further evidence of dysfunction of the ubiquitin-proteasome system in schizophrenia, and suggest that altered proteasome activity may be associated with the pathophysiology of this illness. PMID:26202105

  10. Cytochrome c oxidase: Evolution of control via nuclear subunit addition☆

    PubMed Central

    Pierron, Denis; Wildman, Derek E.; Hüttemann, Maik; Markondapatnaikuni, Gopi Chand; Aras, Siddhesh; Grossman, Lawrence I.

    2014-01-01

    According to theory, present eukaryotic cells originated from a beneficial association between two free-living cells. Due to this endosymbiotic event the pre-eukaryotic cell gained access to oxidative phosphorylation (OXPHOS), which produces more than 15 times as much ATP as glycolysis. Because cellular ATP needs fluctuate and OXPHOS both requires and produces entities that can be toxic for eukaryotic cells such as ROS or NADH, we propose that the success of endosymbiosis has largely depended on the regulation of endosymbiont OXPHOS. Several studies have presented cytochrome c oxidase as a key regulator of OXPHOS; for example, COX is the only complex of mammalian OXPHOS with known tissue-specific isoforms of nuclear encoded subunits. We here discuss current knowledge about the origin of nuclear encoded subunits and the appearance of different isozymes promoted by tissue and cellular environments such as hypoxia. We also review evidence for recent selective pressure acting on COX among vertebrates, particularly in primate lineages, and discuss the unique pattern of co-evolution between the nuclear and mitochondrial genomes. Finally, even though the addition of nuclear encoded subunits was a major event in eukaryotic COX evolution, this does not lead to emergence of a more efficient COX, as might be expected from an anthropocentric point of view, for the “higher” organism possessing large brains and muscles. The main function of these subunits appears to be “only” to control the activity of the mitochondrial subunits. We propose that this control function is an as yet underappreciated key point of evolution. Moreover, the importance of regulating energy supply may have caused the addition of subunits encoded by the nucleus in a process comparable to a “domestication scenario” such that the host tends to control more and more tightly the ancestral activity of COX performed by the mtDNA encoded subunits. This article is part of a Special Issue entitled

  11. Cytochrome c oxidase: evolution of control via nuclear subunit addition.

    PubMed

    Pierron, Denis; Wildman, Derek E; Hüttemann, Maik; Markondapatnaikuni, Gopi Chand; Aras, Siddhesh; Grossman, Lawrence I

    2012-04-01

    According to theory, present eukaryotic cells originated from a beneficial association between two free-living cells. Due to this endosymbiotic event the pre-eukaryotic cell gained access to oxidative phosphorylation (OXPHOS), which produces more than 15 times as much ATP as glycolysis. Because cellular ATP needs fluctuate and OXPHOS both requires and produces entities that can be toxic for eukaryotic cells such as ROS or NADH, we propose that the success of endosymbiosis has largely depended on the regulation of endosymbiont OXPHOS. Several studies have presented cytochrome c oxidase as a key regulator of OXPHOS; for example, COX is the only complex of mammalian OXPHOS with known tissue-specific isoforms of nuclear encoded subunits. We here discuss current knowledge about the origin of nuclear encoded subunits and the appearance of different isozymes promoted by tissue and cellular environments such as hypoxia. We also review evidence for recent selective pressure acting on COX among vertebrates, particularly in primate lineages, and discuss the unique pattern of co-evolution between the nuclear and mitochondrial genomes. Finally, even though the addition of nuclear encoded subunits was a major event in eukaryotic COX evolution, this does not lead to emergence of a more efficient COX, as might be expected from an anthropocentric point of view, for the "higher" organism possessing large brains and muscles. The main function of these subunits appears to be "only" to control the activity of the mitochondrial subunits. We propose that this control function is an as yet under appreciated key point of evolution. Moreover, the importance of regulating energy supply may have caused the addition of subunits encoded by the nucleus in a process comparable to a "domestication scenario" such that the host tends to control more and more tightly the ancestral activity of COX performed by the mtDNA encoded subunits. Copyright © 2011 Elsevier B.V. All rights reserved.

  12. Inhibition of the catalytic subunit of cAMP-dependent protein kinase by dicyclohexylcarbodiimide

    SciTech Connect

    Toner-Webb, J.; Taylor, S.S.

    1987-11-17

    The hydrophobic carbodiimide dicyclohexylcarbodiimide (DCCD) has been shown to inhibit the catalytic (C) subunit of adenosine cyclic 3',5'-phosphate dependent protein kinase in a time-dependent, irreversible manner. The rate of inactivation was first order and showed saturation kinetics with an apparent K/sub i/ of 60 ..mu..M. Magnesium adenosine 5'-triphosphate (MgATP) was capable of protecting against this inhibition, whereas neither a synthetic peptide substrate nor histone afforded protection. Mg alone afforded some protection. When the catalytic subunit was aggregated with the regulatory subunit in the holoenzyme complex, no inhibition was observed. The inhibition was enhanced at low pH, suggesting that a carboxylic acid group was the target for interaction with DCCD. On the basis of the protection studies, it is most likely that this carboxylic acid group is associated with the MgATP binding site, perhaps serving as a ligand for the metal. Efforts to identify the site that was modified by DCCD were made. In no case was radioactivity incorporated into the protein, suggesting that the irreversible inhibition was due to an intramolecular cross-link between a reactive carboxylic acid group and a nearby amino group. Differential peptide mapping identified a single peptide that was consistently lost as a consequence of DCCD inhibition. This peptide (residues 166-189) contained four carboxylic acid residues as well as an internal Lys. Two of these carboxyl groups, Asp-166 and Asp-184, are conserved in all protein kinases, including oncogene transforming proteins and growth factor receptors, and thus are likely to play an essential role.

  13. Identification of structural domains within the large subunit of herpes simplex virus ribonucleotide reductase.

    PubMed

    Conner, J; Cross, A; Murray, J; Marsden, H

    1994-12-01

    The large subunit (R1) of herpes simplex virus (HSV) ribonucleotide reductase is a bifunctional protein consisting of a unique N-terminal protein kinase domain and a ribonucleotide reductase domain. Previous studies showed that the two functional domains are linked by a protease sensitive site. Here we provide evidence for two subdomains, of 30K and 53K, within the reductase domain. The two fragments, which were produced by limited proteolysis and were resistant to further degradation, remained tightly associated in a complex containing two molecules of each. They were capable of binding the R2 subunit of HSV ribonucleotide reductase with approximately the same affinity as the intact protein but the complex did not complement the small subunit (R2) to give an active enzyme. At low concentrations (0.4 micrograms/ml) of trypsin or V8 protease, cleavage between the subdomains was prevented by the presence of the N-terminal protein kinase domain. At higher protease concentrations (1 micrograms/ml) the N-terminal domain is extensively proteolysed and the 30K and 53K domains were generated. Identical results were obtained using purified R1 isolated from infected cell extracts or following expression in Escherichia coli. The origin of the two domains was investigated by N-terminal sequencing of the 53K fragment and by examining their reactivity with a panel of R1-specific monoclonal antibodies which we isolated and epitope mapped for that purpose. The trypsin cleavage site was found to lie between arginine 575 and asparagine 576, and proteolysis in this region was not prevented by the presence of R2 or the nonapeptide YAGAVVNDL. We propose that the ribonucleotide reductase region of HSV R1 exists in a two domain structure, and that the interdomain linking region is protected by the unique N terminus.

  14. Crystal Structure of the 30S Ribosomal Subunit from Thermus Thermophilus. Purification, Crystallization and Structure Determination

    SciTech Connect

    Clemons, William M.; Brodersen, Ditlev E.; McCutcheonn, John P.; May, Joanna L.C.; Carter, Andrew P.; Morgan-Warren, Robert J.; Wimberly, Brian T.; Ramakrishnan, Venki

    2009-10-07

    We describe the crystallization and structure determination of the 30 S ribosomal subunit from Thermus thermophilus. Previous reports of crystals that diffracted to 10 {angstrom} resolution were used as a starting point to improve the quality of the diffraction. Eventually, ideas such as the addition of substrates or factors to eliminate conformational heterogeneity proved less important than attention to detail in yielding crystals that diffracted beyond 3 {angstrom} resolution. Despite improvements in technology and methodology in the last decade, the structure determination of the 30 S subunit presented some very challenging technical problems because of the size of the asymmetric unit, crystal variability and sensitivity to radiation damage. Some steps that were useful for determination of the atomic structure were: the use of anomalous scattering from the LIII edges of osmium and lutetium to obtain the necessary phasing signal; the use of tunable, third-generation synchrotron sources to obtain data of reasonable quality at high resolution; collection of derivative data precisely about a mirror plane to preserve small anomalous differences between Bijvoet mates despite extensive radiation damage and multi-crystal scaling; the pre-screening of crystals to ensure quality, isomorphism and the efficient use of scarce third-generation synchrotron time; pre-incubation of crystals in cobalt hexaammine to ensure isomorphism with other derivatives; and finally, the placement of proteins whose structures had been previously solved in isolation, in conjunction with biochemical data on protein-RNA interactions, to map out the architecture of the 30 S subunit prior to the construction of a detailed atomic-resolution model.

  15. Mapping of Rhodopseudomonas capsulata nif genes.

    PubMed Central

    Wall, J D; Braddock, K

    1984-01-01

    The endogenous gene transfer system of Rhodopseudomonas capsulata was used to analyze mutations which block the ability to use molecular nitrogen as the sole nitrogen source (nif). With this fine-structure mapping tool, linkage of nif mutations could be reliably established if separated by 2,700 base pairs or less. Eleven independent mutations were analyzed, and five linkage groups were found. The overall chromosomal arrangement of these groups awaits conjugational or physical analysis. A candidate for the inactive subunit of R. capsulata Fe protein was located in gels at a position of about 38,000 molecular weight, 5,000 more than that of the presumed active subunit. Images PMID:6586713

  16. Map projections

    USGS Publications Warehouse

    ,

    1993-01-01

    A map projection is used to portray all or part of the round Earth on a flat surface. This cannot be done without some distortion. Every projection has its own set of advantages and disadvantages. There is no "best" projection. The mapmaker must select the one best suited to the needs, reducing distortion of the most important features. Mapmakers and mathematicians have devised almost limitless ways to project the image of the globe onto paper. Scientists at the U. S. Geological Survey have designed projections for their specific needs—such as the Space Oblique Mercator, which allows mapping from satellites with little or no distortion. This document gives the key properties, characteristics, and preferred uses of many historically important projections and of those frequently used by mapmakers today.

  17. Phenotypic consequences of deletion of the gamma 3, alpha 5, or beta 3 subunit of the type A gamma-aminobutyric acid receptor in mice.

    PubMed

    Culiat, C T; Stubbs, L J; Montgomery, C S; Russell, L B; Rinchik, E M

    1994-03-29

    Three genes (Gabrg3, Gabra5, and Gabrb3) encoding the gamma 3, alpha 5, and beta 3 subunits of the type A gamma-aminobutyric acid receptor, respectively, are known to map near the pink-eyed dilution (p) locus in mouse chromosome 7. This region shares homology with a segment of human chromosome 15 that is implicated in Angelman syndrome, an inherited neurobehavioral disorder. By mapping Gabrg3 on a panel of p-locus deletions, we have determined that the order of genes within this cluster is centromere-p(D15S12h)-Gabrg3-Gabra5-Gabrb3-telom ere. Like Gabrb3, neither the Gabra5 nor Gabrg3 gene is functionally imprinted in adult mouse brain. Mice deleted for all three subunits die at birth with a cleft palate, although there are rare survivors (approximately 5%) that do not have a cleft palate but do exhibit a neurological abnormality characterized by tremor, jerky gait, and runtiness. We have previously suggested that deficiency of the beta 3 subunit may be responsible for the clefting defect. Most notably, however, in this report we describe mice carrying two overlapping, complementing p deletions that fail to express the gamma 3 transcript, as well as mice from another line that express neither the gamma 3 nor alpha 5 transcripts. Surprisingly, mice from both of these lines are phenotypically normal and do not exhibit any of the neurological symptoms characteristic of the rare survivors that are deleted for all three (gamma 3, alpha 5, and beta 3) subunits. These mice therefore provide a whole-organism type A gamma-aminobutyric-acid receptor background that is devoid of any receptor subtypes that normally contain the gamma 3 and/or alpha 5 subunits. The absence of an overt neurological phenotype in mice lacking the gamma 3 and/or alpha 5 subunits also suggests that mutations in these genes are unlikely to provide useful animal models for Angelman syndrome in humans.

  18. Molecular architecture of the 90S small subunit pre-ribosome

    PubMed Central

    Sun, Qi; Zhu, Xing; Qi, Jia; An, Weidong; Lan, Pengfei; Tan, Dan; Chen, Rongchang; Wang, Bing; Zheng, Sanduo; Zhang, Cheng; Chen, Xining; Zhang, Wei; Chen, Jing; Dong, Meng-Qiu; Ye, Keqiong

    2017-01-01

    Eukaryotic small ribosomal subunits are first assembled into 90S pre-ribosomes. The complete 90S is a gigantic complex with a molecular mass of approximately five megadaltons. Here, we report the nearly complete architecture of Saccharomyces cerevisiae 90S determined from three cryo-electron microscopy single particle reconstructions at 4.5 to 8.7 angstrom resolution. The majority of the density maps were modeled and assigned to specific RNA and protein components. The nascent ribosome is assembled into isolated native-like substructures that are stabilized by abundant assembly factors. The 5' external transcribed spacer and U3 snoRNA nucleate a large subcomplex that scaffolds the nascent ribosome. U3 binds four sites of pre-rRNA, including a novel site on helix 27 but not the 3' side of the central pseudoknot, and crucially organizes the 90S structure. The 90S model provides significant insight into the principle of small subunit assembly and the function of assembly factors. DOI: http://dx.doi.org/10.7554/eLife.22086.001 PMID:28244370

  19. Molecular architecture of the 90S small subunit pre-ribosome.

    PubMed

    Sun, Qi; Zhu, Xing; Qi, Jia; An, Weidong; Lan, Pengfei; Tan, Dan; Chen, Rongchang; Wang, Bing; Zheng, Sanduo; Zhang, Cheng; Chen, Xining; Zhang, Wei; Chen, Jing; Dong, Meng-Qiu; Ye, Keqiong

    2017-02-28

    Eukaryotic small ribosomal subunits are first assembled into 90S pre-ribosomes. The complete 90S is a gigantic complex with a molecular mass of approximately five megadaltons. Here, we report the nearly complete architecture of Saccharomyces cerevisiae 90S determined from three cryo-electron microscopy single particle reconstructions at 4.5 to 8.7 angstrom resolution. The majority of the density maps were modeled and assigned to specific RNA and protein components. The nascent ribosome is assembled into isolated native-like substructures that are stabilized by abundant assembly factors. The 5' external transcribed spacer and U3 snoRNA nucleate a large subcomplex that scaffolds the nascent ribosome. U3 binds four sites of pre-rRNA, including a novel site on helix 27 but not the 3' side of the central pseudoknot, and crucially organizes the 90S structure. The 90S model provides significant insight into the principle of small subunit assembly and the function of assembly factors.

  20. Integrative structural analysis of the UTPB complex, an early assembly factor for eukaryotic small ribosomal subunits

    PubMed Central

    Zhang, Cheng; Sun, Qi; Chen, Rongchang; Chen, Xining; Lin, Jinzhong; Ye, Keqiong

    2016-01-01

    Ribosome assembly is an essential and conserved cellular process in eukaryotes that requires numerous assembly factors. The six-subunit UTPB complex is an essential component of the 90S precursor of the small ribosomal subunit. Here, we analyzed the molecular architecture of UTPB using an integrative structural biology approach. We mapped the major interactions that associate each of six UTPB proteins. Crystallographic studies showed that Utp1, Utp21, Utp12 and Utp13 are evolutionarily related and form a dimer of dimers (Utp1–Utp21, Utp12–Utp13) through their homologous helical C-terminal domains. Molecular docking with crosslinking restraints showed that the WD domains of Utp12 and Utp13 are associated, as are the WD domains of Utp1, Utp21 and Utp18. Electron microscopy images of the entire UTPB complex revealed that it predominantly adopts elongated conformations and possesses internal flexibility. We also determined crystal structures of the WD domain of Utp18 and the HAT and deviant HAT domains of Utp6. A structural model of UTPB was derived based on these data. PMID:27330138

  1. Integrative structural analysis of the UTPB complex, an early assembly factor for eukaryotic small ribosomal subunits.

    PubMed

    Zhang, Cheng; Sun, Qi; Chen, Rongchang; Chen, Xining; Lin, Jinzhong; Ye, Keqiong

    2016-09-06

    Ribosome assembly is an essential and conserved cellular process in eukaryotes that requires numerous assembly factors. The six-subunit UTPB complex is an essential component of the 90S precursor of the small ribosomal subunit. Here, we analyzed the molecular architecture of UTPB using an integrative structural biology approach. We mapped the major interactions that associate each of six UTPB proteins. Crystallographic studies showed that Utp1, Utp21, Utp12 and Utp13 are evolutionarily related and form a dimer of dimers (Utp1-Utp21, Utp12-Utp13) through their homologous helical C-terminal domains. Molecular docking with crosslinking restraints showed that the WD domains of Utp12 and Utp13 are associated, as are the WD domains of Utp1, Utp21 and Utp18. Electron microscopy images of the entire UTPB complex revealed that it predominantly adopts elongated conformations and possesses internal flexibility. We also determined crystal structures of the WD domain of Utp18 and the HAT and deviant HAT domains of Utp6. A structural model of UTPB was derived based on these data.

  2. RILP interacts with HOPS complex via VPS41 subunit to regulate endocytic trafficking.

    PubMed

    Lin, Xiaosi; Yang, Ting; Wang, Shicong; Wang, Zhen; Yun, Ye; Sun, Lixiang; Zhou, Yunhe; Xu, Xiaohui; Akazawa, Chihiro; Hong, Wanjin; Wang, Tuanlao

    2014-12-02

    The HOPS complex serves as a tethering complex with GEF activity for Ypt7p in yeast to regulate late endosomal membrane maturation. While the role of HOPS complex is well established in yeast cells, its functional and mechanistic aspects in mammalian cells are less well defined. In this study, we report that RILP, a downstream effector of Rab7, interacts with HOPS complex and recruits HOPS subunits to the late endosomal compartment. Structurally, the amino-terminal portion of RILP interacts with HOPS complex. Unexpectedly, this interaction is independent of Rab7. VPS41 subunit of HOPS complex was defined to be the major partner for interacting with RILP. The carboxyl-terminal region of VPS41 was mapped to be responsible for the interaction. Functionally, either depletion of VPS41 by shRNA or overexpression of VPS41 C-terminal half retarded EGF-induced degradation of EGFR. These results suggest that interaction of RILP with HOPS complex via VPS41 plays a role in endocytic trafficking of EGFR.

  3. The bacteriophage T4 gene for the small subunit of ribonucleotide reductase contains an intron.

    PubMed Central

    Sjöberg, B M; Hahne, S; Mathews, C Z; Mathews, C K; Rand, K N; Gait, M J

    1986-01-01

    The bacteriophage T4 gene nrdB codes for the small subunit of the enzyme ribonucleotide reductase. The T4 nrdB gene was localized between 136.1 kb and 137.8 kb in the T4 genetic map according to the deduced structural homology of the protein to the amino acid sequence of its bacterial counterpart, the B2 subunit of Escherichia coli. This positions the C-terminal end of the T4 nrdB gene approximately 2 kb closer to the T4 gene 63 than earlier anticipated from genetic recombinational analyses. The most surprising feature of the T4 nrdB gene is the presence of an approximately 625 bp intron which divides the structural gene into two parts. This is the second example of a prokaryotic structural gene with an intron. The first prokaryotic intron was reported in the nearby td gene, coding for the bacteriophage T4-specific thymidylate synthase enzyme. The nucleotide sequence at the exon-intron junctions of the T4 nrdB gene is similar to that of the junctions of the T4 td gene: the anticipated exon-intron boundary at the donor site ends with a TAA stop codon and there is an ATG start codon at the putative downstream intron-exon boundary of the acceptor site. In the course of this work the denA gene of T4 (endonuclease II) was also located. PMID:3530746

  4. The first transmembrane domain (TM1) of β2-subunit binds to the transmembrane domain S1 of α-subunit in BK potassium channels

    PubMed Central

    Morera, Francisco J.; Alioua, Abderrahmane; Kundu, Pallob; Salazar, Marcelo; Gonzalez, Carlos; Martinez, Agustin D.; Stefani, Enrico; Toro, Ligia; Latorre, Ramon

    2012-01-01

    The BK channel is one of the most broadly expressed ion channels in mammals. In many tissues, the BK channel pore-forming α-subunit is associated to an auxiliary β-subunit that modulates the voltage- and Ca2+-dependent activation of the channel. Structural components present in β-subunits that are important for the physical association with the α-subunit are yet unknown. Here, we show through co-immunoprecipitation that the intracellular C-terminus, the second transmembrane domain (TM2) and the extracellular loop of the β2-subunit are dispensable for association with the α-subunit pointing transmembrane domain 1 (TM1) as responsible for the interaction. Indeed, the TOXCAT assay for transmembrane protein–protein interactions demonstrated for the first time that TM1 of the β2-subunit physically binds to the transmembrane S1 domain of the α-subunit. PMID:22710124

  5. A distinct holoenzyme organization for two-subunit pyruvate carboxylase

    PubMed Central

    Choi, Philip H.; Jo, Jeanyoung; Lin, Yu-Cheng; Lin, Min-Han; Chou, Chi-Yuan; Dietrich, Lars E. P.; Tong, Liang

    2016-01-01

    Pyruvate carboxylase (PC) has important roles in metabolism and is crucial for virulence for some pathogenic bacteria. PC contains biotin carboxylase (BC), carboxyltransferase (CT) and biotin carboxyl carrier protein (BCCP) components. It is a single-chain enzyme in eukaryotes and most bacteria, and functions as a 500 kD homo-tetramer. In contrast, PC is a two-subunit enzyme in a collection of Gram-negative bacteria, with the α subunit containing the BC and the β subunit the CT and BCCP domains, and it is believed that the holoenzyme has α4β4 stoichiometry. We report here the crystal structures of a two-subunit PC from Methylobacillus flagellatus. Surprisingly, our structures reveal an α2β4 stoichiometry, and the overall architecture of the holoenzyme is strikingly different from that of the homo-tetrameric PCs. Biochemical and mutagenesis studies confirm the stoichiometry and other structural observations. Our functional studies in Pseudomonas aeruginosa show that its two-subunit PC is important for colony morphogenesis. PMID:27708276

  6. Expression and function of two nicotinic subunits in insect neurons.

    PubMed

    Vermehren, A; Trimmer, B A

    2005-02-15

    Nicotinic acetylcholine receptors (nAChRs) in insects are neuron-specific oligomeric proteins essential for the central transmission of sensory information. Little is known about their subunit composition because it is difficult to express functional insect nAChRs in heterologous systems. As an alternative approach we have examined the native expression of two subunits in neurons of the nicotinic-resistant, tobacco-feeding insect Manduca sexta. Both the alpha-subunit MARA1 and the beta-subunit MARB can be detected by in situ hybridization in the majority of cultured neurons with an overlapping, but not identical, distribution. Changes in intracellular Ca(2+) evoked by nicotinic stimulation are more strongly correlated to the expression of MARA1 than MARB and are independent of cell size. Unlike the previously reported critical role of MARA1 in mediating nicotinic Ca(2+) responses, down-regulation of MARB by RNA interference (RNAi) did not reduce the number of responding neurons or the size of evoked responses, suggesting that additional subunits remain to be identified in Manduca.

  7. Interacting cytoplasmic loops of subunits a and c of Escherichia coli F1F0 ATP synthase gate H+ transport to the cytoplasm.

    PubMed

    Steed, P Ryan; Kraft, Kaitlin A; Fillingame, Robert H

    2014-11-25

    H(+)-transporting F1F0 ATP synthase catalyzes the synthesis of ATP via coupled rotary motors within F0 and F1. H(+) transport at the subunit a-c interface in transmembranous F0 drives rotation of a cylindrical c10 oligomer within the membrane, which is coupled to rotation of subunit γ within the α3β3 sector of F1 to mechanically drive ATP synthesis. F1F0 functions in a reversible manner, with ATP hydrolysis driving H(+) transport. ATP-driven H(+) transport in a select group of cysteine mutants in subunits a and c is inhibited after chelation of Ag(+) and/or Cd(+2) with the substituted sulfhydryl groups. The H(+) transport pathway mapped via these Ag(+)(Cd(+2))-sensitive Cys extends from the transmembrane helices (TMHs) of subunits a and c into cytoplasmic loops connecting the TMHs, suggesting these loop regions could be involved in gating H(+) release to the cytoplasm. Here, using select loop-region Cys from the single cytoplasmic loop of subunit c and multiple cytoplasmic loops of subunit a, we show that Cd(+2) directly inhibits passive H(+) transport mediated by F0 reconstituted in liposomes. Further, in extensions of previous studies, we show that the regions mediating passive H(+) transport can be cross-linked to each other. We conclude that the loop-regions in subunits a and c that are implicated in H(+) transport likely interact in a single structural domain, which then functions in gating H(+) release to the cytoplasm.

  8. Retention of duplicated ITAM-containing transmembrane signaling subunits in the tetraploid amphibian species Xenopus laevis.

    PubMed

    Guselnikov, S V; Grayfer, L; De Jesús Andino, F; Rogozin, I B; Robert, J; Taranin, A V

    2015-11-01

    The ITAM-bearing transmembrane signaling subunits (TSS) are indispensable components of activating leukocyte receptor complexes. The TSS-encoding genes map to paralogous chromosomal regions, which are thought to arise from ancient genome tetraploidization(s). To assess a possible role of tetraploidization in the TSS evolution, we studied TSS and other functionally linked genes in the amphibian species Xenopus laevis whose genome was duplicated about 40 MYR ago. We found that X. laevis has retained a duplicated set of sixteen TSS genes, all except one being transcribed. Furthermore, duplicated TCRα loci and genes encoding TSS-coupling protein kinases have also been retained. No clear evidence for functional divergence of the TSS paralogs was obtained from gene expression and sequence analyses. We suggest that the main factor of maintenance of duplicated TSS genes in X. laevis was a protein dosage effect and that this effect might have facilitated the TSS set expansion in early vertebrates.

  9. YphC and YsxC GTPases assist the maturation of the central protuberance, GTPase associated region and functional core of the 50S ribosomal subunit

    PubMed Central

    Ni, Xiaodan; Davis, Joseph H.; Jain, Nikhil; Razi, Aida; Benlekbir, Samir; McArthur, Andrew G.; Rubinstein, John L.; Britton, Robert A.; Williamson, James R.; Ortega, Joaquin

    2016-01-01

    YphC and YsxC are GTPases in Bacillus subtilis that facilitate the assembly of the 50S ribosomal subunit, however their roles in this process are still uncharacterized. To explore their function, we used strains in which the only copy of the yphC or ysxC genes were under the control of an inducible promoter. Under depletion conditions, they accumulated incomplete ribosomal subunits that we named 45SYphC and 44.5SYsxC particles. Quantitative mass spectrometry analysis and the 5–6 Å resolution cryo-EM maps of the 45SYphC and 44.5SYsxC particles revealed that the two GTPases participate in the maturation of the central protuberance, GTPase associated region and key RNA helices in the A, P and E functional sites of the 50S subunit. We observed that YphC and YsxC bind specifically to the two immature particles, suggesting that they represent either on-pathway intermediates or that their structure has not significantly diverged from that of the actual substrate. These results describe the nature of these immature particles, a widely used tool to study the assembly process of the ribosome. They also provide the first insights into the function of YphC and YsxC in 50S subunit assembly and are consistent with this process occurring through multiple parallel pathways, as it has been described for the 30S subunit. PMID:27484475

  10. A protein-protein interaction map of yeast RNA polymerase III.

    PubMed

    Flores, A; Briand, J F; Gadal, O; Andrau, J C; Rubbi, L; Van Mullem, V; Boschiero, C; Goussot, M; Marck, C; Carles, C; Thuriaux, P; Sentenac, A; Werner, M

    1999-07-06

    The structure of the yeast RNA polymerase (pol) III was investigated by exhaustive two-hybrid screening using a library of random genomic fragments fused to the Gal4 activation domain. This procedure allowed us to identify contacts between individual polypeptides, localize the contact domains, and deduce a protein-protein interaction map of the multisubunit enzyme. In all but one case, pol III subunits were able to interact in vivo with one or sometimes two partner subunits of the enzyme or with subunits of TFIIIC. Four subunits that are common to pol I, II, and III (ABC27, ABC14.5, ABC10alpha, and ABC10beta), two that are common to pol I and III (AC40 and AC19), and one pol III-specific subunit (C11) can associate with defined regions of the two large subunits. These regions overlapped with highly conserved domains. C53, a pol III-specific subunit, interacted with a 37-kDa polypeptide that copurifies with the enzyme and therefore appears to be a unique pol III subunit (C37). Together with parallel interaction studies based on dosage-dependent suppression of conditional mutants, our data suggest a model of the pol III preinitiation complex.

  11. Assignment of human genes for phosphorylase kinase subunits alpha (PHKA) to Xq12-q13 and beta (PHKB) to 16q12-q13.

    PubMed Central

    Francke, U; Darras, B T; Zander, N F; Kilimann, M W

    1989-01-01

    Phosphorylase kinase (PHK), the enzyme that activates glycogen phosphorylases in muscle, liver, and other tissues, is composed of four different subunits. Recently isolated rabbit muscle cDNAs for the larger two subunits, alpha and beta, have been used to map the location of their cognate sequences on human chromosomes. Southern blot analysis of rodent x human somatic cell hybrid panels, as well as in situ chromosomal hybridization, have provided evidence of single sites for both genes. The alpha subunit gene (PHKA) is located on the proximal long arm of the X chromosome in region Xq12-q13 near the locus for phosphoglycerate kinase (PGK1). X-linked mutations leading to PHK deficiency, known to exist in humans and mice, are likely to involve this locus. This hypothesis is consistent with the proximity of the Phk and Pgk-1 loci on the mouse X chromosome. In contrast, the beta subunit gene (PHKB) was found to be autosomal and was mapped to chromosome 16, region q12-q13 on the proximal long arm. Several different autosomally inherited forms of PHK deficiency for which the PHKB could be a candidate gene have been described in humans and rats. PMID:2757032

  12. Separation and characterization of alpha-chain subunits from tilapia (Tilapia zillii) skin gelatin using ultrafiltration.

    PubMed

    Chen, Shulin; Tang, Lanlan; Su, Wenjin; Weng, Wuyin; Osako, Kazufumi; Tanaka, Munehiko

    2015-12-01

    Alpha-chain subunits were separated from tilapia skin gelatin using ultrafiltration, and the physicochemical properties of obtained subunits were investigated. As a result, α1-subunit and α2-subunit could be successfully separated by 100 kDa MWCO regenerated cellulose membranes and 150 kDa MWCO polyethersulfone membranes, respectively. Glycine was the most dominant amino acid in both α1-subunit and α2-subunit. However, the tyrosine content was higher in α2-subunit than in α1-subunit, resulting in strong absorption near 280 nm observed in the UV absorption spectrum. Based on the DSC analysis, it was found that the glass transition temperatures of gelatin, α1-subunit and α2-subunit were 136.48 °C, 126.77 °C and 119.43 °C, respectively. Moreover, the reduced viscosity and denaturation temperature of α1-subunit were higher than those of α2-subunit, and the reduced viscosity reached the highest when α-subunits were mixed with α1/α2 ratio of approximately 2, suggesting that α1-subunit plays a more important role in the thermostability of gelatin than α2-subunit.

  13. Antigenic breadth: a missing ingredient in HSV-2 subunit vaccines?

    PubMed

    Halford, William P

    2014-06-01

    The successful human papillomavirus and hepatitis B virus subunit vaccines contain single viral proteins that represent 22 and 12%, respectively, of the antigens encoded by these tiny viruses. The herpes simplex virus 2 (HSV-2) genome is >20 times larger. Thus, a single protein subunit represents 1% of HSV-2's total antigenic breadth. Antigenic breadth may explain why HSV-2 glycoprotein subunit vaccines have failed in clinical trials, and why live HSV-2 vaccines that express 99% of HSV-2's proteome may be more effective. I review the mounting evidence that live HSV-2 vaccines offer a greater opportunity to stop the spread of genital herpes, and I consider the unfounded 'safety concerns' that have kept live HSV-2 vaccines out of U.S. clinical trials for 25 years.

  14. Dengue vaccine: an update on recombinant subunit strategies.

    PubMed

    Martin, J; Hermida, L

    2016-03-01

    Dengue is an increasing public health problem worldwide, with the four serotypes of the virus infecting over 390 million people annually. There is no specific treatment or antiviral drug for dengue, and prevention is largely limited to controlling the mosquito vectors or disrupting the human-vector contact. Despite the considerable progress made in recent years, an effective vaccine against the virus is not yet available. The development of a dengue vaccine has been hampered by many unique challenges, including the need to ensure the absence of vaccine-induced enhanced severity of disease. Recombinant protein subunit vaccines offer a safer alternative to other vaccine approaches. Several subunit vaccine candidates are presently under development, based on different structural and non-structural proteins of the virus. Novel adjuvants or immunopotentiating strategies are also being tested to improve their immunogenicity. This review summarizes the current status and development trends of subunit dengue vaccines.

  15. Cholera toxin B: one subunit with many pharmaceutical applications.

    PubMed

    Baldauf, Keegan J; Royal, Joshua M; Hamorsky, Krystal Teasley; Matoba, Nobuyuki

    2015-03-20

    Cholera, a waterborne acute diarrheal disease caused by Vibrio cholerae, remains prevalent in underdeveloped countries and is a serious health threat to those living in unsanitary conditions. The major virulence factor is cholera toxin (CT), which consists of two subunits: the A subunit (CTA) and the B subunit (CTB). CTB is a 55 kD homopentameric, non-toxic protein binding to the GM1 ganglioside on mammalian cells with high affinity. Currently, recombinantly produced CTB is used as a component of an internationally licensed oral cholera vaccine, as the protein induces potent humoral immunity that can neutralize CT in the gut. Additionally, recent studies have revealed that CTB administration leads to the induction of anti-inflammatory mechanisms in vivo. This review will cover the potential of CTB as an immunomodulatory and anti-inflammatory agent. We will also summarize various recombinant expression systems available for recombinant CTB bioproduction.

  16. Cholera Toxin B: One Subunit with Many Pharmaceutical Applications

    PubMed Central

    Baldauf, Keegan J.; Royal, Joshua M.; Hamorsky, Krystal Teasley; Matoba, Nobuyuki

    2015-01-01

    Cholera, a waterborne acute diarrheal disease caused by Vibrio cholerae, remains prevalent in underdeveloped countries and is a serious health threat to those living in unsanitary conditions. The major virulence factor is cholera toxin (CT), which consists of two subunits: the A subunit (CTA) and the B subunit (CTB). CTB is a 55 kD homopentameric, non-toxic protein binding to the GM1 ganglioside on mammalian cells with high affinity. Currently, recombinantly produced CTB is used as a component of an internationally licensed oral cholera vaccine, as the protein induces potent humoral immunity that can neutralize CT in the gut. Additionally, recent studies have revealed that CTB administration leads to the induction of anti-inflammatory mechanisms in vivo. This review will cover the potential of CTB as an immunomodulatory and anti-inflammatory agent. We will also summarize various recombinant expression systems available for recombinant CTB bioproduction. PMID:25802972

  17. Subunits of the Schizosaccharomyces pombe RNA polymerase II: enzyme purification and structure of the subunit 3 gene.

    PubMed Central

    Azuma, Y; Yamagishi, M; Ishihama, A

    1993-01-01

    To improve our understanding of the structure and function of eukaryotic RNA polymerase II, we purified the enzyme from the fission yeast Schizosaccharomyces pombe. The highly purified RNA polymerase II contained more than eleven polypeptides. The sizes of the largest the second-, and the third-largest polypeptides as measured by SDS-polyacrylamide gel electrophoresis were about 210, 150, and 40 kilodaltons (kDa), respectively, and are similar to those of RPB1, 2, and 3 subunits of Saccharomyces cerevisiae RNA polymerase II. Using the degenerated primers designed after amino acid micro-sequencing of the 40 kDa third-largest polypeptide (subunit 3), we cloned the subunit 3 gene (rpb3) and determined its DNA sequence. Taken together with the sequence of parts of PCR-amplified cDNA, the predicted coding sequence of rpb3, interrupted by two introns, was found to encode a polypeptide of 297 amino acid residues in length with a molecular weight of 34 kDa. The S. pombe subunit 3 contains four structural domains conserved for the alpha-subunit family of RNA polymerase from both eukaryotes and prokaryotes. A putative leucine zipper motif was found to exist in the C-terminal proximal conserved region (domain D). Possible functions of the conserved domains are discussed. Images PMID:8367291

  18. Rod Phosphodiesterase-6 (PDE6) Catalytic Subunits Restore Cone Function in a Mouse Model Lacking Cone PDE6 Catalytic Subunit*

    PubMed Central

    Kolandaivelu, Saravanan; Chang, Bo; Ramamurthy, Visvanathan

    2011-01-01

    Rod and cone photoreceptor neurons utilize discrete PDE6 enzymes that are crucial for phototransduction. Rod PDE6 is composed of heterodimeric catalytic subunits (αβ), while the catalytic core of cone PDE6 (α′) is a homodimer. It is not known if variations between PDE6 subunits preclude rod PDE6 catalytic subunits from coupling to the cone phototransduction pathway. To study this issue, we generated a cone-dominated mouse model lacking cone PDE6 (Nrl−/− cpfl1). In this animal model, using several independent experimental approaches, we demonstrated the expression of rod PDE6 (αβ) and the absence of cone PDE6 (α′) catalytic subunits. The rod PDE6 enzyme expressed in cone cells is active and contributes to the hydrolysis of cGMP in response to light. In addition, rod PDE6 expressed in cone cells couples to the light signaling pathway to produce S-cone responses. However, S-cone responses and light-dependent cGMP hydrolysis were eliminated when the β-subunit of rod PDE6 was removed (Nrl−/− cpfl1 rd). We conclude that either rod or cone PDE6 can effectively couple to the cone phototransduction pathway to mediate visual signaling. Interestingly, we also found that functional PDE6 is required for trafficking of M-opsin to cone outer segments. PMID:21799013

  19. G alpha 12 and G alpha 13 subunits define a fourth class of G protein alpha subunits.

    PubMed Central

    Strathmann, M P; Simon, M I

    1991-01-01

    Heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins) are central to the signaling processes of multicellular organisms. We have explored the diversity of the G protein subunits in mammals and found evidence for a large family of genes that encode the alpha subunits. Amino acid sequence comparisons show that the different alpha subunits fall into at least three classes. These classes have been conserved in animals separated by considerable evolutionary distances; they are present in mammals, Drosophila, and nematodes. We have now obtained cDNA clones encoding two murine alpha subunits, G alpha 12 and G alpha 13, that define a fourth class. The translation products are predicted to have molecular masses of 44 kDa and to be insensitive to ADP-ribosylation by pertussis toxin. They share 67% amino acid sequence identity with each other and less than 45% identity with other alpha subunits. Their transcripts can be detected in every tissue examined, although the relative levels of the G alpha 13 message appear somewhat variable. Images PMID:1905812

  20. Stiffness of γ subunit of F(1)-ATPase.

    PubMed

    Okuno, Daichi; Iino, Ryota; Noji, Hiroyuki

    2010-11-01

    F(1)-ATPase is a molecular motor in which the γ subunit rotates inside the α(3)β(3) ring upon adenosine triphosphate (ATP) hydrolysis. Recent works on single-molecule manipulation of F(1)-ATPase have shown that kinetic parameters such as the on-rate of ATP and the off-rate of adenosine diphosphate (ADP) strongly depend on the rotary angle of the γ subunit (Hirono-Hara et al. 2005; Iko et al. 2009). These findings provide important insight into how individual reaction steps release energy to power F(1) and also have implications regarding ATP synthesis and how reaction steps are reversed upon reverse rotation. An important issue regarding the angular dependence of kinetic parameters is that the angular position of a magnetic bead rotation probe could be larger than the actual position of the γ subunit due to the torsional elasticity of the system. In the present study, we assessed the stiffness of two different portions of F(1) from thermophilic Bacillus PS3: the internal part of the γ subunit embedded in the α(3)β(3) ring, and the complex of the external part of the γ subunit and the α(3)β(3) ring (and streptavidin and magnetic bead), by comparing rotational fluctuations before and after crosslinkage between the rotor and stator. The torsional stiffnesses of the internal and remaining parts were determined to be around 223 and 73 pNnm/radian, respectively. Based on these values, it was estimated that the actual angular position of the internal part of the γ subunit is one-fourth of the magnetic bead position upon stalling using an external magnetic field. The estimated elasticity also partially explains the accommodation of the intrinsic step size mismatch between F(o) and F(1)-ATPase.

  1. Subunit structure of the acetylcholine receptor from Electrophorus electricus.

    PubMed Central

    Conti-Tronconi, B M; Hunkapiller, M W; Lindstrom, J M; Raftery, M A

    1982-01-01

    The amino-terminal amino acid sequences of the four major peptides (Mr 41,000, 50,000, 55,000, and 62,000) present in purified preparations of Electrophorus electricus nicotinic acetylcholine receptor (AcChoR) have been determined for 24 cycles by automated sequence analysis procedures yielding four unique polypeptide sequences. The sequences showed a high degree of similarity, having identical residues in a number of positions ranging between 37% and 50% for specific pairs of subunits. Comparison of the sequences obtained with those of the subunits of similar molecular weight from Torpedo californica AcChoR revealed an even higher degree of homology (from 46% to 71%) for these two highly diverged species. Simultaneous sequence analysis of the amino termini present in native, purified Electrophorus AcChoR showed that these four related sequences were the only ones present and that they occur in a ratio of 2:1:1:1, with the smallest subunit ("alpha 1") being present in two copies. Genealogical analysis suggests that the subunits of both Torpedo and Electrophorus AcChoRs derive from a common ancestral gene, the divergence having occurred early in the evolution of the receptor. This shared ancestry and the very early divergence of the four subunits, as well as the highly conserved structure of the AcChoR complex along animal evolution, suggest that each of the subunits evolved to perform discrete crucial roles in the physiological function of the AcChoR. Images PMID:6959131

  2. Subunit composition of Kv1 channels in human CNS.

    PubMed

    Coleman, S K; Newcombe, J; Pryke, J; Dolly, J O

    1999-08-01

    The alpha subunits of Shaker-related K+ channels (Kv1.X) show characteristic distributions in mammalian brain and restricted coassembly. Despite the functional importance of these voltage-sensitive K+ channels and involvement in a number of diseases, little progress has been achieved in deciphering the subunit composition of the (alpha)4(beta)4 oligomers occurring in human CNS. Thus, the association of alpha and beta subunits was investigated in cerebral grey and white matter and spinal cord from autopsy samples. Immunoblotting established the presence of Kv1.1, 1.2, and 1.4 in all the tissues, with varying abundance. Sequential immunoprecipitations identified the subunits coassembled. A putative tetramer of Kv1.3/1.4/1.1/1.2 was found in grey matter. Both cerebral white matter and spinal cord contained the heterooligomers Kv1.1/1.4 and Kv1.1/1.2, similar to grey matter, but both lacked Kv1.3 and the Kv1.4/1.2 combination. An apparent Kv1.4 homooligomer was detected in all the samples, whereas only the brain tissue possessed a putative Kv1.2 homomer. In grey matter, Kvbeta2.1 was coassociated with the Kv1.1/1.2 combination and Kv1.2 homooligomer. In white matter, Kvbeta2.1 was associated with Kv1.2 only, whereas Kvbeta1.1 coprecipitated with all the alpha subunits present. This represents the first description of Kv1 subunit complexes in the human CNS and demonstrates regional variations, indicative of functional specialisation.

  3. Localisation of AMPK γ subunits in cardiac and skeletal muscles.

    PubMed

    Pinter, Katalin; Grignani, Robert T; Watkins, Hugh; Redwood, Charles

    2013-12-01

    The trimeric protein AMP-activated protein kinase (AMPK) is an important sensor of energetic status and cellular stress, and mutations in genes encoding two of the regulatory γ subunits cause inherited disorders of either cardiac or skeletal muscle. AMPKγ2 mutations cause hypertrophic cardiomyopathy with glycogen deposition and conduction abnormalities; mutations in AMPKγ3 result in increased skeletal muscle glycogen. In order to gain further insight into the roles of the different γ subunits in muscle and into possible disease mechanisms, we localised the γ2 and γ3 subunits, along with the more abundant γ1 subunit, by immunofluorescence in cardiomyocytes and skeletal muscle fibres. The predominant cardiac γ2 variant, γ2-3B, gave a striated pattern in cardiomyocytes, aligning with the Z-disk but with punctate staining similar to T-tubule (L-type Ca(2+) channel) and sarcoplasmic reticulum (SERCA2) markers. In skeletal muscle fibres AMPKγ3 localises to the I band, presenting a uniform staining that flanks the Z-disk, also coinciding with the position of Ca(2+) influx in these muscles. The localisation of γ2-3B- and γ3-containing AMPK suggests that these trimers may have similar functions in the different muscles. AMPK containing γ2-3B was detected in oxidative skeletal muscles which had low expression of γ3, confirming that these two regulatory subunits may be co-ordinately regulated in response to metabolic requirements. Compartmentalisation of AMPK complexes is most likely dependent on the regulatory γ subunit and this differential localisation may direct substrate selection and specify particular functional roles.

  4. Micelle-Based Adjuvants for Subunit Vaccine Delivery

    PubMed Central

    Trimaille, Thomas; Verrier, Bernard

    2015-01-01

    In the development of subunit vaccines with purified or recombinant antigens for cancer and infectious diseases, the design of improved and safe adjuvants able to efficiently target the antigen presenting cells, such as dendritic cells, represents a crucial challenge. Nanoparticle-based antigen delivery systems have been identified as an innovative strategy to improve the efficacy of subunit vaccines. Among them, self-assembled micellar nanoparticles from amphiphilic (macro)molecules have recently emerged as promising candidates. In this short review, we report on the recent research findings highlighting the versatility and potential of such systems in vaccine delivery. PMID:26426060

  5. Dissociation of Mammalian Polyribosomes into Subunits by Puromycin

    PubMed Central

    Blobel, Günter; Sabatini, David

    1971-01-01

    Hepatic ribosomes have been dissociated into biologically active subunits as follows. Polysomes were treated at 0°C with puromycin at high ionic strength. This released most of the nascent polypeptide chains without dissociating the polysomes, which retained the mRNA and the tRNA moiety of peptidyl tRNA, but were unable to continue the translation of mRNA. The polysomes were then heated to 37°C, when they dissociated completely into subunits. Similar treatment without puromycin resulted in only partial dissociation. PMID:5277091

  6. Advancements in the development of subunit influenza vaccines

    PubMed Central

    Zhang, Naru; Zheng, Bo-Jian; Lu, Lu; Zhou, Yusen; Jiang, Shibo; Du, Lanying

    2014-01-01

    The ongoing threat of influenza epidemics and pandemics has emphasized the importance of developing safe and effective vaccines against infections from divergent influenza viruses. In this review, we first introduce the structure and life cycle of influenza A viruses, describing major influenza A virus-caused pandemics. We then compare different types of influenza vaccines and discuss current advancements in the development of subunit influenza vaccines, particularly those based on nucleoprotein (NP), extracellular domain of matrix protein 2 (M2e) and hemagglutinin (HA) proteins. We also illustrate potential strategies for improving the efficacy of subunit influenza vaccines. PMID:25529753

  7. Carrier Subunit of Plasma Membrane Transporter Is Required for Oxidative Folding of Its Helper Subunit*

    PubMed Central

    Rius, Mònica; Chillarón, Josep

    2012-01-01

    We study the amino acid transport system b0,+ as a model for folding, assembly, and early traffic of membrane protein complexes. System b0,+ is made of two disulfide-linked membrane subunits: the carrier, b0,+ amino acid transporter (b0,+AT), a polytopic protein, and the helper, related to b0,+ amino acid transporter (rBAT), a type II glycoprotein. rBAT ectodomain mutants display folding/trafficking defects that lead to type I cystinuria. Here we show that, in the presence of b0,+AT, three disulfides were formed in the rBAT ectodomain. Disulfides Cys-242-Cys-273 and Cys-571-Cys-666 were essential for biogenesis. Cys-673-Cys-685 was dispensable, but the single mutants C673S, and C685S showed compromised stability and trafficking. Cys-242-Cys-273 likely was the first disulfide to form, and unpaired Cys-242 or Cys-273 disrupted oxidative folding. Strikingly, unassembled rBAT was found as an ensemble of different redox species, mainly monomeric. The ensemble did not change upon inhibition of rBAT degradation. Overall, these results indicated a b0,+AT-dependent oxidative folding of the rBAT ectodomain, with the initial and probably cotranslational formation of Cys-242-Cys-273, followed by the oxidation of Cys-571-Cys-666 and Cys-673-Cys-685, that was completed posttranslationally. PMID:22493502

  8. Carrier subunit of plasma membrane transporter is required for oxidative folding of its helper subunit.

    PubMed

    Rius, Mònica; Chillarón, Josep

    2012-05-25

    We study the amino acid transport system b(0,+) as a model for folding, assembly, and early traffic of membrane protein complexes. System b(0,+) is made of two disulfide-linked membrane subunits: the carrier, b(0,+) amino acid transporter (b(0,+)AT), a polytopic protein, and the helper, related to b(0,+) amino acid transporter (rBAT), a type II glycoprotein. rBAT ectodomain mutants display folding/trafficking defects that lead to type I cystinuria. Here we show that, in the presence of b(0,+)AT, three disulfides were formed in the rBAT ectodomain. Disulfides Cys-242-Cys-273 and Cys-571-Cys-666 were essential for biogenesis. Cys-673-Cys-685 was dispensable, but the single mutants C673S, and C685S showed compromised stability and trafficking. Cys-242-Cys-273 likely was the first disulfide to form, and unpaired Cys-242 or Cys-273 disrupted oxidative folding. Strikingly, unassembled rBAT was found as an ensemble of different redox species, mainly monomeric. The ensemble did not change upon inhibition of rBAT degradation. Overall, these results indicated a b(0,+)AT-dependent oxidative folding of the rBAT ectodomain, with the initial and probably cotranslational formation of Cys-242-Cys-273, followed by the oxidation of Cys-571-Cys-666 and Cys-673-Cys-685, that was completed posttranslationally.

  9. Probing the proton channels in subunit N of Complex I from Escherichia coli through intra-subunit cross-linking.

    PubMed

    Tursun, Ablat; Zhu, Shaotong; Vik, Steven B

    2016-12-01

    Respiratory Complex I appears to have 4 sites for proton translocation, which are coupled to the oxidation of NADH and reduction of coenzyme Q. The proton pathways are thought to be made of offset half-channels that connect to the membrane surfaces, and are connected by a horizontal path through the center of the membrane. In this study of the enzyme from Escherichia coli, subunit N, containing one of the sites, was targeted. Pairs of cysteine residues were introduced into neighboring α-helices along the proposed proton pathways. In an effort to constrain conformational changes that might occur during proton translocation, we attempted to form disulfide bonds or methanethiosulfonate bridges between two engineered cysteine residues. Cysteine modification was inferred by the inability of PEG-maleimide to shift the electrophoretic mobility of subunit N, which will occur upon reaction with free sulfhydryl groups. After the cross-linking treatment, NADH oxidase and NADH-driven proton translocation were measured. Ten different pairs of cysteine residues showed evidence of cross-linking. The most significant loss of enzyme activity was seen for residues near the essential Lys 395. This residue is positioned between the proposed proton half-channel to the periplasm and the horizontal connection through subunit N, and is also near the essential Glu 144 of subunit M. The results suggest important conformational changes in this region for the delivery of protons to the periplasm, or for coupling the actions of subunit N to subunit M. Copyright © 2016 Elsevier B.V. All rights reserved.

  10. Pea chloroplast DNA encodes homologues of Escherichia coli ribosomal subunit S2 and the beta'-subunit of RNA polymerase.

    PubMed Central

    Cozens, A L; Walker, J E

    1986-01-01

    The nucleotide sequence has been determined of a segment of 4680 bases of the pea chloroplast genome. It adjoins a sequence described elsewhere that encodes subunits of the F0 membrane domain of the ATP-synthase complex. The sequence contains a potential gene encoding a protein which is strongly related to the S2 polypeptide of Escherichia coli ribosomes. It also encodes an incomplete protein which contains segments that are homologous to the beta'-subunit of E. coli RNA polymerase and to yeast RNA polymerases II and III. PMID:3530249

  11. Catalytic Subunit 1 of Protein Phosphatase 2A Is a Subunit of the STRIPAK Complex and Governs Fungal Sexual Development.

    PubMed

    Beier, Anna; Teichert, Ines; Krisp, Christoph; Wolters, Dirk A; Kück, Ulrich

    2016-06-21

    The generation of complex three-dimensional structures is a key developmental step for most eukaryotic organisms. The details of the molecular machinery controlling this step remain to be determined. An excellent model system to study this general process is the generation of three-dimensional fruiting bodies in filamentous fungi like Sordaria macrospora Fruiting body development is controlled by subunits of the highly conserved striatin-interacting phosphatase and kinase (STRIPAK) complex, which has been described in organisms ranging from yeasts to humans. The highly conserved heterotrimeric protein phosphatase PP2A is a subunit of STRIPAK. Here, catalytic subunit 1 of PP2A was functionally characterized. The Δpp2Ac1 strain is sterile, unable to undergo hyphal fusion, and devoid of ascogonial septation. Further, PP2Ac1, together with STRIPAK subunit PRO22, governs vegetative and stress-related growth. We revealed in vitro catalytic activity of wild-type PP2Ac1, and our in vivo analysis showed that inactive PP2Ac1 blocks the complementation of the sterile deletion strain. Tandem affinity purification, followed by mass spectrometry and yeast two-hybrid analysis, verified that PP2Ac1 is a subunit of STRIPAK. Further, these data indicate links between the STRIPAK complex and other developmental signaling pathways, implying the presence of a large interconnected signaling network that controls eukaryotic developmental processes. The insights gained in our study can be transferred to higher eukaryotes and will be important for understanding eukaryotic cellular development in general. The striatin-interacting phosphatase and kinase (STRIPAK) complex is highly conserved from yeasts to humans and is an important regulator of numerous eukaryotic developmental processes, such as cellular signaling and cell development. Although functional insights into the STRIPAK complex are accumulating, the detailed molecular mechanisms of single subunits are only partially understood

  12. Mg-chelatase I subunit 1 and Mg-protoporphyrin IX methyltransferase affect the stomatal aperture in Arabidopsis thaliana.

    PubMed

    Tomiyama, Masakazu; Inoue, Shin-Ichiro; Tsuzuki, Tomo; Soda, Midori; Morimoto, Sayuri; Okigaki, Yukiko; Ohishi, Takaya; Mochizuki, Nobuyoshi; Takahashi, Koji; Kinoshita, Toshinori

    2014-07-01

    To elucidate the molecular mechanisms of stomatal opening and closure, we performed a genetic screen using infrared thermography to isolate stomatal aperture mutants. We identified a mutant designated low temperature with open-stomata 1 (lost1), which exhibited reduced leaf temperature, wider stomatal aperture, and a pale green phenotype. Map-based analysis of the LOST1 locus revealed that the lost1 mutant resulted from a missense mutation in the Mg-chelatase I subunit 1 (CHLI1) gene, which encodes a subunit of the Mg-chelatase complex involved in chlorophyll synthesis. Transformation of the wild-type CHLI1 gene into lost1 complemented all lost1 phenotypes. Stomata in lost1 exhibited a partial ABA-insensitive phenotype similar to that of rtl1, a Mg-chelatase H subunit missense mutant. The Mg-protoporphyrin IX methyltransferase (CHLM) gene encodes a subsequent enzyme in the chlorophyll synthesis pathway. We examined stomatal movement in a CHLM knockdown mutant, chlm, and found that it also exhibited an ABA-insensitive phenotype. However, lost1 and chlm seedlings all showed normal expression of ABA-induced genes, such as RAB18 and RD29B, in response to ABA. These results suggest that the chlorophyll synthesis enzymes, Mg-chelatase complex and CHLM, specifically affect ABA signaling in the control of stomatal aperture and have no effect on ABA-induced gene expression.

  13. Mass spectrometry-based identification of native cardiac Nav1.5 channel α subunit phosphorylation sites.

    PubMed

    Marionneau, Céline; Lichti, Cheryl F; Lindenbaum, Pierre; Charpentier, Flavien; Nerbonne, Jeanne M; Townsend, R Reid; Mérot, Jean

    2012-12-07

    Cardiac voltage-gated Na+ (Nav) channels are key determinants of action potential waveforms, refractoriness and propagation, and Nav1.5 is the main Nav pore-forming (α) subunit in the mammalian heart. Although direct phosphorylation of the Nav1.5 protein has been suggested to modulate various aspects of Nav channel physiology and pathophysiology, native Nav1.5 phosphorylation sites have not been identified. In the experiments here, a mass spectrometry (MS)-based proteomic approach was developed to identify native Nav1.5 phosphorylation sites directly. Using an anti-NavPAN antibody, Nav channel complexes were immunoprecipitated from adult mouse cardiac ventricles. The MS analyses revealed that this antibody immunoprecipitates several Nav α subunits in addition to Nav1.5, as well as several previously identified Nav channel associated/regulatory proteins. Label-free comparative and data-driven phosphoproteomic analyses of purified cardiac Nav1.5 protein identified 11 phosphorylation sites, 8 of which are novel. All the phosphorylation sites identified except one in the N-terminus are in the first intracellular linker loop, suggesting critical roles for this region in phosphorylation-dependent cardiac Nav channel regulation. Interestingly, commonly used prediction algorithms did not reliably predict these newly identified in situ phosphorylation sites. Taken together, the results presented provide the first in situ map of basal phosphorylation sites on the mouse cardiac Nav1.5 α subunit.

  14. Cycloheximide resistance can be mediated through either ribosomal subunit.

    PubMed Central

    Sutton, C A; Ares, M; Hallberg, R L

    1978-01-01

    Two cycloheximide-resistant mutants of Tetrahymena thermophila were analyzed to determine the site of their cycloheximide resistance. The mutations in both strains had been previously shown to be genetically dominant and located at separate loci (denoted Chx-A and Chx-B). Strains carrying these mutations were readily distinguished by the extent to which they were resistant to the drug. The homozygous double mutant was more resistant than either single mutant. Cell-free extracts of wild type and of the three mutant strains, assayed for protein synthetic activity by both runoff of natural mRNA and poly(U)-dependent phenylalanine polymerization, demonstrated that in vitro the mutants were all more resistant than the wild type. Further fractionation of the cell-free systems into ribosomes and supernates localized cycloheximide resistance to the ribosome for both Chx-A and Chx-B homozygotes. Ribosome dissociation and pairwise subunit mixing in the in vitro system indicated that ribosome resistance was conferred by the 60S subunit from one strain whereas resistance in the other strain was mediated through the 40S subunit. This was further confirmed by reconstruction of all four cycloheximide-resistance "phenotypes" by mixing ribosomal subunits from appropriate strains. This finding suggests that the mechanisms by which these mutations confer resistance to cycloheximide are different. PMID:277918

  15. Emergence of ion channel modal gating from independent subunit kinetics.

    PubMed

    Bicknell, Brendan A; Goodhill, Geoffrey J

    2016-09-06

    Many ion channels exhibit a slow stochastic switching between distinct modes of gating activity. This feature of channel behavior has pronounced implications for the dynamics of ionic currents and the signaling pathways that they regulate. A canonical example is the inositol 1,4,5-trisphosphate receptor (IP3R) channel, whose regulation of intracellular Ca(2+) concentration is essential for numerous cellular processes. However, the underlying biophysical mechanisms that give rise to modal gating in this and most other channels remain unknown. Although ion channels are composed of protein subunits, previous mathematical models of modal gating are coarse grained at the level of whole-channel states, limiting further dialogue between theory and experiment. Here we propose an origin for modal gating, by modeling the kinetics of ligand binding and conformational change in the IP3R at the subunit level. We find good agreement with experimental data over a wide range of ligand concentrations, accounting for equilibrium channel properties, transient responses to changing ligand conditions, and modal gating statistics. We show how this can be understood within a simple analytical framework and confirm our results with stochastic simulations. The model assumes that channel subunits are independent, demonstrating that cooperative binding or concerted conformational changes are not required for modal gating. Moreover, the model embodies a generally applicable principle: If a timescale separation exists in the kinetics of individual subunits, then modal gating can arise as an emergent property of channel behavior.

  16. Calcium channel gamma subunits: a functionally diverse protein family.

    PubMed

    Chen, Ren-Shiang; Deng, Tzyy-Chyn; Garcia, Thomas; Sellers, Zachary M; Best, Philip M

    2007-01-01

    The calcium channel gamma subunits comprise an eight-member protein family that share a common topology consisting of four transmembrane domains and intracellular N- and C-termini. Although the first gamma subunit was identified as an auxiliary subunit of a voltage-dependent calcium channel, a review of phylogenetic, bioinformatic, and functional studies indicates that they are a functionally diverse protein family. A cluster containing gamma1 and gamma6 conforms to the original description of the protein family as they seem to act primarily as subunits of calcium channels expressed in muscle. Members of a second cluster (gamma2, gamma3, gamma4, gamma8) function as regulators of AMPA receptor localization and function in the brain and are collectively known as TARPs. The function of members of the third cluster (gamma5, gamma7) remains unclear. Our analysis shows that the members of each cluster contain conserved regulatory motifs that help to differentiate the groups. However, the physiological significance of these motifs in many cases remains to be demonstrated.

  17. Emergence of ion channel modal gating from independent subunit kinetics

    PubMed Central

    Bicknell, Brendan A.

    2016-01-01

    Many ion channels exhibit a slow stochastic switching between distinct modes of gating activity. This feature of channel behavior has pronounced implications for the dynamics of ionic currents and the signaling pathways that they regulate. A canonical example is the inositol 1,4,5-trisphosphate receptor (IP3R) channel, whose regulation of intracellular Ca2+ concentration is essential for numerous cellular processes. However, the underlying biophysical mechanisms that give rise to modal gating in this and most other channels remain unknown. Although ion channels are composed of protein subunits, previous mathematical models of modal gating are coarse grained at the level of whole-channel states, limiting further dialogue between theory and experiment. Here we propose an origin for modal gating, by modeling the kinetics of ligand binding and conformational change in the IP3R at the subunit level. We find good agreement with experimental data over a wide range of ligand concentrations, accounting for equilibrium channel properties, transient responses to changing ligand conditions, and modal gating statistics. We show how this can be understood within a simple analytical framework and confirm our results with stochastic simulations. The model assumes that channel subunits are independent, demonstrating that cooperative binding or concerted conformational changes are not required for modal gating. Moreover, the model embodies a generally applicable principle: If a timescale separation exists in the kinetics of individual subunits, then modal gating can arise as an emergent property of channel behavior. PMID:27551100

  18. Bacterial cellulose biosynthesis: diversity of operons, subunits, products and functions

    PubMed Central

    Römling, Ute; Galperin, Michael Y.

    2015-01-01

    Summary Recent studies of bacterial cellulose biosynthesis, including structural characterization of a functional cellulose synthase complex, provided the first mechanistic insight into this fascinating process. In most studied bacteria, just two subunits, BcsA and BcsB, are necessary and sufficient for the formation of the polysaccharide chain in vitro. Other subunits – which differ among various taxa – affect the enzymatic activity and product yield in vivo by modulating expression of biosynthesis apparatus, export of the nascent β-D-glucan polymer to the cell surface, and the organization of cellulose fibers into a higher-order structure. These auxiliary subunits play key roles in determining the quantity and structure of the resulting biofilm, which is particularly important for interactions of bacteria with higher organisms that lead to rhizosphere colonization and modulate virulence of cellulose-producing bacterial pathogens inside and outside of host cells. Here we review the organization of four principal types of cellulose synthase operons found in various bacterial genomes, identify additional bcs genes that encode likely components of the cellulose biosynthesis and secretion machinery, and propose a unified nomenclature for these genes and subunits. We also discuss the role of cellulose as a key component of biofilms formed by a variety of free-living and pathogenic bacteria and, for the latter, in the choice between acute infection and persistence in the host. PMID:26077867

  19. CMF70 is a subunit of the dynein regulatory complex

    PubMed Central

    Kabututu, Zakayi P.; Thayer, Michelle; Melehani, Jason H.; Hill, Kent L.

    2010-01-01

    Flagellar motility drives propulsion of several important pathogens and is essential for human development and physiology. Motility of the eukaryotic flagellum requires coordinate regulation of thousands of dynein motors arrayed along the axoneme, but the proteins underlying dynein regulation are largely unknown. The dynein regulatory complex, DRC, is recognized as a focal point of axonemal dynein regulation, but only a single DRC subunit, trypanin/PF2, is currently known. The component of motile flagella 70 protein, CMF70, is broadly and uniquely conserved among organisms with motile flagella, suggesting a role in axonemal motility. Here we demonstrate that CMF70 is part of the DRC from Trypanosoma brucei. CMF70 is located along the flagellum, co-sediments with trypanin in sucrose gradients and co-immunoprecipitates with trypanin. RNAi knockdown of CMF70 causes motility defects in a wild-type background and suppresses flagellar paralysis in cells with central pair defects, thus meeting the functional definition of a DRC subunit. Trypanin and CMF70 are mutually conserved in at least five of six extant eukaryotic clades, indicating that the DRC was probably present in the last common eukaryotic ancestor. We have identified only the second known subunit of this ubiquitous dynein regulatory system, highlighting the utility of combined genomic and functional analyses for identifying novel subunits of axonemal sub-complexes. PMID:20876659

  20. Effects of cations and cosolvents on eukaryotic ribosomal subunit conformation

    SciTech Connect

    Moore, M.N.; Spremulli, L.L.

    1985-01-01

    The effects of various cations and cosolvents on the conformation of wheat germ ribosomes and ribosomal subunits have been investigated by using the techniques of circular dichroism and differential hydrogen exchange. A class of hydrogens on both the 40S and 60S subunits exchange out more rapidly as the Mg/sup 2 +/ concentration is raised, indicating that Mg/sup 2 +/ causes a change in subunit conformation. Ca/sup 2 +/ and the polyamines produce a similar increase in the rate of hydrogen exchange. These results suggest that increases in cation concentrations permit a tightening of ribosome structure and a greater degree of internalization of the rRNA. The cosolvent glycerol causes an alteration in the CD spectrum of 80S ribosomes in both the nucleic acid and protein portions of the spectrum. Glycerol also causes a decrease in the rate of exchange of a number of hydrogens on both the 40S and 60S subunits. These results are interpreted to mean that glycerol favors a more native, less denatured structure in the ribosome.

  1. Spectroscopic properties of Carcinus aestuarii hemocyanin and its structural subunits

    NASA Astrophysics Data System (ADS)

    Dolashka-Angelova, Pavlina; Hristova, Rumiyana; Stoeva, Stanka; Voelter, Wolfgang

    1999-12-01

    Hemocyanin (Hc) of Carcinus aestuarii contains three major and one minor electrophoretically separable polypeptide chains which were purified by fast protein liquid chromatography (FPLC) ion exchange chromatography. N-terminal amino acid sequences of four structural subunits (SSs) from C. aestuarii were compared with known N-terminal sequences from other arthropodan hemocyanins. The conformational changes, induced by various treatments, were monitored by far UV, CD and fluorescence spectroscopy. The critical temperatures for the structural subunits, Tc, determined by fluorescence spectroscopy, are in the region of 52-59°C and coincide with the melting temperatures, Tm (49-55°C), determined by CD spectroscopy. The free energy of stabilization in water, Δ GDH 2O , toward guanidinium hydrochloride is about 1.3 times higher for the dodecameric Hc as compared to the isolated subunits and about one time higher for Ca1, comparing with other SSs. The studies reveal that the conformational stability of the native dodecamer towards various denaturants (temperature and guanidinium hydrochloride) indicate that the quaternary structure is stabilized by oligomerization between structural subunits, and the possibility of a structural role of the sugar mojeties cannot be excluded.

  2. GABAB(1) receptor subunit isoforms differentially regulate stress resilience

    PubMed Central

    O’Leary, Olivia F.; Felice, Daniela; Galimberti, Stefano; Savignac, Hélène M.; Bravo, Javier A.; Crowley, Tadhg; El Yacoubi, Malika; Vaugeois, Jean-Marie; Gassmann, Martin; Bettler, Bernhard; Dinan, Timothy G.; Cryan, John F.

    2014-01-01

    Stressful life events increase the susceptibility to developing psychiatric disorders such as depression; however, many individuals are resilient to such negative effects of stress. Determining the neurobiology underlying this resilience is instrumental to the development of novel and more effective treatments for stress-related psychiatric disorders. GABAB receptors are emerging therapeutic targets for the treatment of stress-related disorders such as depression. These receptors are predominantly expressed as heterodimers of a GABAB(2) subunit with either a GABAB(1a) or a GABAB(1b) subunit. Here we show that mice lacking the GABAB(1b) receptor isoform are more resilient to both early-life stress and chronic psychosocial stress in adulthood, whereas mice lacking GABAB(1a) receptors are more susceptible to stress-induced anhedonia and social avoidance compared with wild-type mice. In addition, increased hippocampal expression of the GABAB(1b) receptor subunit is associated with a depression-like phenotype in the helpless H/Rouen genetic mouse model of depression. Stress resilience in GABAB(1b)−/− mice is coupled with increased proliferation and survival of newly born cells in the adult ventral hippocampus and increased stress-induced c-Fos activation in the hippocampus following early-life stress. Taken together, the data suggest that GABAB(1) receptor subunit isoforms differentially regulate the deleterious effects of stress and, thus, may be important therapeutic targets for the treatment of depression. PMID:25288769

  3. The multifaceted subunit interfaces of ionotropic glutamate receptors.

    PubMed

    Green, Tim; Nayeem, Naushaba

    2015-01-01

    The past fifteen years has seen a revolution in our understanding of ionotropic glutamate receptor (iGluR) structure, starting with the first view of the ligand binding domain (LBD) published in 1998, and in many ways culminating in the publication of the full-length structure of GluA2 in 2009. These reports have revealed not only the central role played by subunit interfaces in iGluR function, but also myriad binding sites within interfaces for endogenous and exogenous factors. Changes in the conformation of inter-subunit interfaces are central to transmission of ligand gating into pore opening (itself a rearrangement of interfaces), and subsequent closure through desensitization. With the exception of the agonist binding site, which is located entirely within individual subunits, almost all modulatory factors affecting iGluRs appear to bind to sites in subunit interfaces. This review seeks to summarize what we currently understand about the diverse roles interfaces play in iGluR function, and to highlight questions for future research.

  4. The multifaceted subunit interfaces of ionotropic glutamate receptors.

    PubMed

    Green, Tim; Nayeem, Naushaba

    2014-06-06

    The past fifteen years has seen a revolution in our understanding of ionotropic glutamate receptor (iGluR) structure, starting with the first view of the ligand binding domain (LBD) published in 1998, and in many ways culminating in the publication of the full-length structure of GluA2 in 2009. These reports have revealed not only the central role played by subunit interfaces in iGluR function, but also myriad binding sites within interfaces for endogenous and exogenous factors. Changes in the conformation of inter-subunit interfaces are central to transmission of ligand gating into pore opening (itself a rearrangement of interfaces), and subsequent closure through desensitization. With the exception of the agonist binding site, which is located entirely within individual subunits, almost all modulatory factors affecting iGluRs appear to bind to sites in subunit interfaces. This review seeks to summarize what we currently understand about the diverse roles interfaces play in iGluR function, and to highlight questions for future research.

  5. ATP-induced helicase slippage reveals highly coordinated subunits

    NASA Astrophysics Data System (ADS)

    Wang, Michelle D.

    2012-02-01

    Helicases are vital enzymes that carry out strand separation of duplex nucleic acids during replication, repair and recombination. T7 helicase, a model hexameric motor, has been observed to use dTTP, but not ATP, to unwind dsDNA as it translocates along ssDNA. Whether and how different subunits of the helicase coordinate their chemo-mechanical activities and DNA binding during translocation is still under debate. Here we address this question using a single-molecule approach to monitor helicase unwinding. We found that T7 helicase does in fact unwind dsDNA in the presence of ATP and that the unwinding rate is even faster than that with dTTP. However, unwinding was repeatedly interrupted by sudden slippage events, ultimately preventing unwinding over a substantial distance. This behaviour was greatly reduced with the supplement of a small amount of dTTP. These findings presented an opportunity to use nucleotide mixtures to investigate helicase subunit coordination. Our results support a model where nearly all subunits coordinate their chemo-mechanical activities and DNA binding. Such subunit coordination may be general to many ring-shaped helicases and reveals a potential mechanism for regulation of DNA unwinding during replication.

  6. A World History Sub-Unit: Teaching about Turkey.

    ERIC Educational Resources Information Center

    Lynn, Karen

    This document is a sub-unit teaching plan for world history teachers who want to use multicultural concepts in the world history curriculum. The objective explored includes a student response to the Turkish question of "Who are we"? Teacher preparation involves defining social and cultural roots and outlining periods of Turkish history.…

  7. Succinate dehydrogenase subunit D and succinate dehydrogenase subunit B mutation analysis in canine phaeochromocytoma and paraganglioma.

    PubMed

    Holt, D E; Henthorn, P; Howell, V M; Robinson, B G; Benn, D E

    2014-07-01

    Phaeochromocytomas (PCs) are tumours of the adrenal medulla chromaffin cells. Paragangliomas (PGLs) arise in sympathetic ganglia (previously called extra-adrenal PCs) or in non-chromaffin parasympathetic ganglia cells that are usually non-secretory. Parenchymal cells from these tumours have a common embryological origin from neural crest ectoderm. Several case series of canine PCs and PGLs have been published and a link between the increased incidence of chemoreceptor neoplasia in brachycephalic dog breeds and chronic hypoxia has been postulated. A similar link to hypoxia in man led to the identification of germline heterozygous mutations in the gene encoding succinate dehydrogenase subunit D (SDHD) and subsequently SDHA, SDHB and SDHC in similar tumours. We investigated canine PCs (n = 6) and PGLs (n = 2) for SDHD and SDHB mutations and in one PGL found a somatic SDHD mutation c.365A>G (p.Lys122Arg) in exon 4, which was not present in normal tissue from this brachycephalic dog. Two PCs were heterozygous for both c.365A>G (p.Lys122Arg) mutation and an exon 3 silent variant c.291G>A. We also identified the heterozygous SDHB exon 2 mutation c.113G>A (p.Arg38Gln) in a PC. These results illustrate that genetic mutations may underlie tumourigenesis in canine PCs and PGLs. The spontaneous nature of these canine diseases and possible association of PGLs with hypoxia in brachycephalic breeds may make them an attractive model for studying the corresponding human tumours. Copyright © 2014 Elsevier Ltd. All rights reserved.

  8. ATP-Induced Helicase Slippage Reveals Highly Coordinated Subunits

    PubMed Central

    Sun, Bo; Johnson, Daniel S.; Patel, Gayatri; Smith, Benjamin Y.; Pandey, Manjula; Patel, Smita S.; Wang, Michelle D.

    2011-01-01

    Helicases are vital enzymes that carry out strand separation of duplex nucleic acids during replication, repair, and recombination1,2. Bacteriophage T7 gene product 4 is a model hexameric helicase which has been observed to utilize dTTP, but not ATP, to unwind dsDNA as it translocates from 5′ to 3′ along ssDNA2–6. Whether and how different subunits of the helicase coordinate their chemo-mechanical activities and DNA binding during translocation is still under debate1,7. Here we address this question using a single molecule approach to monitor helicase unwinding. We discovered that T7 helicase does in fact unwind dsDNA in the presence of ATP and the unwinding rate is even faster than that with dTTP. However unwinding traces showed a remarkable sawtooth pattern where processive unwinding was repeatedly interrupted by sudden slippage events, ultimately preventing unwinding over a substantial distance. This behavior was not observed with dTTP alone and was greatly reduced when ATP solution was supplemented with a small amount of dTTP. These findings presented an opportunity to use nucleotide mixtures to investigate helicase subunit coordination. We found T7 helicase binds and hydrolyzes ATP and dTTP by competitive kinetics such that the unwinding rate is dictated simply by their respective Vmax, KM, and concentrations. In contrast, processivity does not follow a simple competitive behavior and shows a cooperative dependence on nucleotide concentrations. This does not agree with an uncoordinated mechanism where each subunit functions independently, but supports a model where nearly all subunits coordinate their chemo-mechanical activities and DNA binding. Our data indicate that only one subunit at a time can accept a nucleotide while other subunits are nucleotide-ligated and thus interact with the DNA to ensure processivity. Such subunit coordination may be general to many ring-shaped helicases and reveals a potential mechanism for regulation of DNA unwinding

  9. Analysis of the Subunit Stoichiometries in Viral Entry

    PubMed Central

    Magnus, Carsten; Regoes, Roland R.

    2012-01-01

    Virions of the Human Immunodeficiency Virus (HIV) infect cells by first attaching with their surface spikes to the CD4 receptor on target cells. This leads to conformational changes in the viral spikes, enabling the virus to engage a coreceptor, commonly CCR5 or CXCR4, and consecutively to insert the fusion peptide into the cellular membrane. Finally, the viral and the cellular membranes fuse. The HIV spike is a trimer consisting of three identical heterodimers composed of the gp120 and gp41 envelope proteins. Each of the gp120 proteins in the trimer is capable of attaching to the CD4 receptor and the coreceptor, and each of the three gp41 units harbors a fusion domain. It is still under debate how many of the envelope subunits within a given trimer have to bind to the CD4 receptors and to the coreceptors, and how many gp41 protein fusion domains are required for fusion. These numbers are referred to as subunit stoichiometries. We present a mathematical framework for estimating these parameters individually by analyzing infectivity assays with pseudotyped viruses. We find that the number of spikes that are engaged in mediating cell entry and the distribution of the spike number play important roles for the estimation of the subunit stoichiometries. Our model framework also shows why it is important to subdivide the question of the number of functional subunits within one trimer into the three different subunit stoichiometries. In a second step, we extend our models to study whether the subunits within one trimer cooperate during receptor binding and fusion. As an example for how our models can be applied, we reanalyze a data set on subunit stoichiometries. We find that two envelope proteins have to engage with CD4-receptors and coreceptors and that two fusion proteins must be revealed within one trimer for viral entry. Our study is motivated by the mechanism of HIV entry but the experimental technique and the model framework can be extended to other viral systems

  10. Glycoprotein hormone assembly in the endoplasmic reticulum: I. The glycosylated end of human alpha-subunit loop 2 is threaded through a beta-subunit hole.

    PubMed

    Xing, Yongna; Myers, Rebecca V; Cao, Donghui; Lin, Win; Jiang, Mei; Bernard, Michael P; Moyle, William R

    2004-08-20

    Glycoprotein hormone heterodimers are stabilized by their unusual structures in which a glycosylated loop of the alpha-subunit straddles a hole in the beta-subunit. This hole is formed when a cysteine at the end of a beta-subunit strand known as the "seatbelt" becomes "latched" by a disulfide to a cysteine in the beta-subunit core. The heterodimer is stabilized in part by the difficulty of threading the glycosylated end of the alpha-subunit loop 2 through this hole, a phenomenon required for subunit dissociation. Subunit combination in vitro, which occurs by the reverse process, can be accelerated by removing the alpha-subunit oligosaccharide. In cells, heterodimer assembly was thought to occur primarily by a mechanism in which the seatbelt is wrapped around the alpha-subunit after the subunits dock. Here we show that this "wraparound" process can be used to assemble disulfide cross-linked human choriogonadotropin analogs that contain an additional alpha-subunit cysteine, but only if the normal beta-subunit latch site has been removed. Normally, the seatbelt is latched before the subunits dock and assembly is completed when the glycosylated end of alpha-subunit loop 2 is threaded beneath the seatbelt. The unexpected finding that most assembly of human choriogonadotropin, human follitropin, and human thyrotropin heterodimers occurs in this fashion, indicates that threading may be an important phenomenon during protein folding and macromolecule assembly in the endoplasmic reticulum. We suggest that the unusual structures of the glycoprotein hormones makes them useful for identifying factors that influence this process in living cells.

  11. High molecular weight glutenin subunits in some durum wheat cultivars investigated by means of mass spectrometric techniques.

    PubMed

    Muccilli, Vera; Lo Bianco, Marisol; Cunsolo, Vincenzo; Saletti, Rosaria; Gallo, Giulia; Foti, Salvatore

    2011-11-23

    The primary structures of high molecular weight glutenin subunits (HMW-GS) of 5 Triticum durum Desf. cultivars (Simeto, Svevo, Duilio, Bronte, and Sant'Agata), largely cultivated in the south of Italy, and of 13 populations of the old spring Sicilian durum wheat landrace Timilia (Triticum durum Desf.) (accession nos. 1, 2, 3, 4, 7, 8, 9, 13, 14, 15, SG1, SG2, and SG3) were investigated using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and reversed-phase high performance liquid chromatography/nanoelectrospray ionization mass spectrometry (RP-HPLC/nESI-MS/MS). M(r) of the intact proteins determined by MALDI mass spectrometry showed that all the 13 populations of Timilia contained the same two HMW-GS with 75.2 kDa and 86.4 kDa, whereas the other durum wheat cultivars showed the presence of the expected HMW-GS 1By8 and 1Bx7 at 75.1 kDa and 83.1 kDa, respectively. By MALDI mass spectrometry of the tryptic digestion peptides of the isolated HMW-GS of Timilia, the 1Bx and 1By subunits were identified as the NCBInr Acc. No AAQ93629, and AAQ93633, respectively. Sequence verification for HMW-GS 1Bx and 1By both in Simeto and Timilia was obtained by MALDI mass mapping and HPLC/nESI-MSMS of the tryptic peptides. The Bx subunit of Timila presents a sequence similarity of 96% with respect to Simeto, with differences in the insertion of 3 peptides of 5, 9, and 15 amino acids, for a total insertion of 29 amino acids and 25 amino acid substitutions. These differences in the amino acidic sequence account for the determined Δm of 3294 Da between the M(r) of the 1Bx subunits in Timilia and Simeto. Sequence alignment between the two By subunits shows 10 amino acid substitutions and is consistent with the Δm of 148 Da found in the MALDI mass spectra of the intact subunits.

  12. Heterotrimeric G protein subunit Gγ13 is critical to olfaction

    PubMed Central

    Li, Feng; Ponissery-Saidu, Samsudeen; Yee, Karen; Wang, Hong; Chen, Meng-Ling; Iguchi, Naoko; Zhang, Genhua; Jiang, Ping; Reisert, Johannes; Huang, Liquan

    2013-01-01

    The activation of G-protein-coupled olfactory receptors on the olfactory sensory neurons (OSNs) triggers a signaling cascade, which is mediated by a heterotrimeric G protein consisting of α, β and γ subunits. Although its α subunit, Gαolf, has been identified and well characterized, the identities of its β and γ subunits and their function in olfactory signal transduction, however, have not been well established yet. We and others have found the expression of Gγ13 in the olfactory epithelium, particularly in the cilia of the OSNs. In this study, we generated a conditional gene knockout mouse line to specifically nullify Gγ13 expression in the olfactory marker protein-expressing OSNs. Immunohistochemical and Western blot results showed that Gγ13 subunit was indeed eliminated in the mutant mice’s olfactory epithelium. Intriguingly, Gαolf, β1 subunits, Ric-8B and CEP290 proteins were also absent in the epithelium whereas the presence of the effector enzyme adenylyl cyclase III remained largely unaltered. Electro-olfactogram studies showed that the mutant animals had greatly reduced responses to a battery of odorants including three presumable pheromones. Behavioral tests indicated that the mutant mice had a remarkably reduced ability to perform an odor-guided search task although their motivation and agility seemed normal. Our results indicate that Gαolf exclusively forms a functional heterotrimeric G protein with Gβ1 and Gγ13 in OSNs, mediating olfactory signal transduction. The identification of the olfactory G protein’s βγ moiety has provided a novel approach to understanding the feedback regulation of olfactory signal transduction pathways as well as the control of subcellular structures of OSNs. PMID:23637188

  13. Binding interactions with the complementary subunit of nicotinic receptors.

    PubMed

    Blum, Angela P; Van Arnam, Ethan B; German, Laurel A; Lester, Henry A; Dougherty, Dennis A

    2013-03-08

    The agonist-binding site of nicotinic acetylcholine receptors (nAChRs) spans an interface between two subunits of the pentameric receptor. The principal component of this binding site is contributed by an α subunit, and it binds the cationic moiety of the nicotinic pharmacophore. The other part of the pharmacophore, a hydrogen bond acceptor, has recently been shown to bind to the complementary non-α subunit via the backbone NH of a conserved Leu. This interaction was predicted by studies of ACh-binding proteins and confirmed by functional studies of the neuronal (CNS) nAChR, α4β2. The ACh-binding protein structures further suggested that the hydrogen bond to the backbone NH is mediated by a water molecule and that a second hydrogen bonding interaction occurs between the water molecule and the backbone CO of a conserved Asn, also on the non-α subunit. Here, we provide new insights into the nature of the interactions between the hydrogen bond acceptor of nicotinic agonists and the complementary subunit backbone. We studied both the nAChR of the neuromuscular junction (muscle-type) and a neuronal subtype, (α4)2(β4)3. In the muscle-type receptor, both ACh and nicotine showed a strong interaction with the Leu NH, but the potent nicotine analog epibatidine did not. This interaction was much attenuated in the α4β4 receptor. Surprisingly, we found no evidence for a functionally significant interaction with the backbone carbonyl of the relevant Asn in either receptor with an array of agonists.

  14. The Biochemistry, Ultrastructure, and Subunit Assembly Mechanism of AMPA Receptors

    PubMed Central

    2010-01-01

    The AMPA-type ionotropic glutamate receptors (AMPA-Rs) are tetrameric ligand-gated ion channels that play crucial roles in synaptic transmission and plasticity. Our knowledge about the ultrastructure and subunit assembly mechanisms of intact AMPA-Rs was very limited. However, the new studies using single particle EM and X-ray crystallography are revealing important insights. For example, the tetrameric crystal structure of the GluA2cryst construct provided the atomic view of the intact receptor. In addition, the single particle EM structures of the subunit assembly intermediates revealed the conformational requirement for the dimer-to-tetramer transition during the maturation of AMPA-Rs. These new data in the field provide new models and interpretations. In the brain, the native AMPA-R complexes contain auxiliary subunits that influence subunit assembly, gating, and trafficking of the AMPA-Rs. Understanding the mechanisms of the auxiliary subunits will become increasingly important to precisely describe the function of AMPA-Rs in the brain. The AMPA-R proteomics studies continuously reveal a previously unexpected degree of molecular heterogeneity of the complex. Because the AMPA-Rs are important drug targets for treating various neurological and psychiatric diseases, it is likely that these new native complexes will require detailed mechanistic analysis in the future. The current ultrastructural data on the receptors and the receptor-expressing stable cell lines that were developed during the course of these studies are useful resources for high throughput drug screening and further drug designing. Moreover, we are getting closer to understanding the precise mechanisms of AMPA-R-mediated synaptic plasticity. PMID:21080238

  15. Expression of glutamate receptor subunits in human cancers.

    PubMed

    Stepulak, Andrzej; Luksch, Hella; Gebhardt, Christine; Uckermann, Ortrud; Marzahn, Jenny; Sifringer, Marco; Rzeski, Wojciech; Staufner, Christian; Brocke, Katja S; Turski, Lechoslaw; Ikonomidou, Chrysanthy

    2009-10-01

    Emerging evidence suggests a role for glutamate and its receptors in the biology of cancer. This study was designed to systematically analyze the expression of ionotropic and metabotropic glutamate receptor subunits in various human cancer cell lines, compare expression levels to those in human brain tissue and, using electrophysiological techniques, explore whether cancer cells respond to glutamate receptor agonists and antagonists. Expression analysis of glutamate receptor subunits NR1-NR3B, GluR1-GluR7, KA1, KA2 and mGluR1-mGluR8 was performed by means of RT-PCR in human rhabdomyosarcoma/medulloblastoma (TE671), neuroblastoma (SK-NA-S), thyroid carcinoma (FTC 238), lung carcinoma (SK-LU-1), astrocytoma (MOGGCCM), multiple myeloma (RPMI 8226), glioma (U87-MG and U343), lung carcinoma (A549), colon adenocarcinoma (HT 29), T cell leukemia cells (Jurkat E6.1), breast carcinoma (T47D) and colon adenocarcinoma (LS180). Analysis revealed that all glutamate receptor subunits were differentially expressed in the tumor cell lines. For the majority of tumors, expression levels of NR2B, GluR4, GluR6 and KA2 were lower compared to human brain tissue. Confocal imaging revealed that selected glutamate receptor subunit proteins were expressed in tumor cells. By means of patch-clamp analysis, it was shown that A549 and TE671 cells depolarized in response to application of glutamate agonists and that this effect was reversed by glutamate receptor antagonists. This study reveals that glutamate receptor subunits are differentially expressed in human tumor cell lines at the mRNA and the protein level, and that their expression is associated with the formation of functional channels. The potential role of glutamate receptor antagonists in cancer therapy is a feasible goal to be explored in clinical trials.

  16. Targeting signals and subunit interactions in coated vesicle adaptor complexes

    PubMed Central

    1995-01-01

    There are two clathrin-coated vesicle adaptor complexes in the cell, one associated with the plasma membrane and one associated with the TGN. The subunit composition of the plasma membrane adaptor complex is alpha-adaptin, beta-adaptin, AP50, and AP17; while that of the TGN adaptor complex is gamma-adaptin, beta'-adaptin, AP47, and AP19. To search for adaptor targeting signals, we have constructed chimeras between alpha-adaptin and gamma-adaptin within their NH2-terminal domains. We have identified stretches of sequence in the two proteins between amino acids approximately 130 and 330-350 that are essential for targeting. Immunoprecipitation reveals that this region determines whether a construct coassemblies with AP50 and AP17, or with AP47 and AP19. These observations suggest that these other subunits may play an important role in targeting. In contrast, beta- and beta'-adaptins are clearly not involved in this event. Chimeras between the alpha- and gamma-adaptin COOH-terminal domains reveal the presence of a second targeting signal. We have further investigated the interactions between the adaptor subunits using the yeast two-hybrid system. Interactions can be detected between the beta/beta'-adaptins and the alpha/gamma- adaptins, between the beta/beta'-adaptins and the AP50/AP47 subunits, between alpha-adaptin and AP17, and between gamma-adaptin and AP19. These results indicate that the adaptor subunits act in concert to target the complex to the appropriate membrane. PMID:7593184

  17. The centromere-kinetochore complex: a repeat subunit model

    PubMed Central

    1991-01-01

    The three-dimensional structure of the kinetochore and the DNA/protein composition of the centromere-kinetochore region was investigated using two novel techniques, caffeine-induced detachment of unreplicated kinetochores and stretching of kinetochores by hypotonic and/or shear forces generated in a cytocentrifuge. Kinetochore detachment was confirmed by EM and immunostaining with CREST autoantibodies. Electron microscopic analyses of serial sections demonstrated that detached kinetochores represented fragments derived from whole kinetochores. This was especially evident for the seven large kinetochores in the male Indian muntjac that gave rise to 80-100 fragments upon detachment. The kinetochore fragments, all of which interacted with spindle microtubules and progressed through the entire repertoire of mitotic movements, provide evidence for a subunit organization within the kinetochore. Further support for a repeat subunit model was obtained by stretching or uncoiling the metaphase centromere-kinetochore complex by hypotonic treatments. When immunostained with CREST autoantibodies and subsequently processed for in situ hybridization using synthetic centromere probes, stretched kinetochores displayed a linear array of fluorescent subunits arranged in a repetitive pattern along a centromeric DNA fiber. In addition to CREST antigens, each repetitive subunit was found to bind tubulin and contain cytoplasmic dynein, a microtubule motor localized in the zone of the corona. Collectively, the data suggest that the kinetochore, a plate-like structure seen by EM on many eukaryotic chromosomes is formed by the folding of a linear DNA fiber consisting of tandemly repeated subunits interspersed by DNA linkers. This model, unlike any previously proposed, can account for the structural and evolutional diversity of the kinetochore and its relationship to the centromere of eukaryotic chromosomes of many species. PMID:1828250

  18. The catalytically active domain in the A subunit of calcineurin.

    PubMed

    Xiang, Benqiong; Liu, Ping; Jiang, Guohua; Zou, Ke; Yi, Fang; Yang, Shujie; Wei, Qun

    2003-01-01

    Calcineurin (CaN) is a heterodimer composed of a catalytic subunit A (CaNA) and a regulatory subunit B (CaNB). We report here an active truncated mutation of the rat CaNAdelta that contains only the catalytic domain (residues 1-347, also known as a/CaNA). The p-nitrophenyl phosphatase activity and protein phosphatase activity of a/CaNA were higher than that of CaNA. Both p-nitrophenyl phosphatase activity and protein phosphatase activity of a/CaNA were unaffected by CaM and the B-subunit; the B-subunit and CaM have relatively little effect on p-nitrophenyl phosphatase activity and a crucial effect on protein phosphatase activity of CaNA. Mn2+ and Ni2+ ions effeciently activated CaNA. The Km of a/CaNA was about 16 mM, and the k(cat) of a/CaNA was 10.03 s(-1) using pNPP as substrate. With RII peptide as a substrate, the Km of a/CaNA was about 21 microM and the k(cat) of a/CaNA was 0.51 s(-1). The optimum reaction temperature was about 45 degrees C, and the optimum reaction pH was about 7.2. Our results indicate that a/CaNA is the catalytic core of CaNA, and CaN and the B-subunit binding domain itself might play roles in the negative regulation of the phosphatase activity of CaN. The results provide the basis for future studies on the catalytic domain of CaN.

  19. A single gene encodes two different transcripts for the ADP-glucose pyrophosphorylase small subunit from barley (Hordeum vulgare).

    PubMed Central

    Thorbjørnsen, T; Villand, P; Kleczkowski, L A; Olsen, O A

    1996-01-01

    ADP-glucose pyrophosphorylase (AGPase), a heterotetrameric enzyme composed of two small and two large subunits, catalyses the first committed step of starch synthesis in plant tissues. In an attempt to learn more about the organization and expression of the small-subunit gene of AGPase, we have studied the small-subunit transcripts as well as the structure of the gene encoding these transcripts in barley (Hordeum vulgare L. cv. Bomi). Two different transcripts (bepsF1 and blps14) were identified: bepF1 was abundantly expressed in the starchy endosperm but not in leaves, whereas blps14 was isolated from leaves but was also found to be present at a moderate level in the starchy endosperm. The sequences for the two transcripts are identical over approx. 90% of the length, with differences being confined solely to their 5' ends. In blps14, the unique 5' end is 259 nt long and encodes a putative plastid transit peptide sequence. For the 178-nt 5' end of bepsF1, on the other hand, no transit peptide sequence could be recognized. A lambda clone that hybridized to the AGPase transcripts was isolated from a barley genomic library and characterized. The restriction map has suggested a complex organization of the gene, with alternative exons encoding the different 5' ends of the two transcripts followed by nine exons coding for the common part of the transcripts. The sequence of a portion of the genomic clone, covering the alternative 5'-end exons as well as upstream regions, has verified that both transcripts are encoded by the gene. The results suggest that the small-subunit gene of barley AGPase transcribes two different mRNAs by a mechanism classified as alternative splicing. PMID:8546676

  20. Specific interaction between EF-G and RRF and its implication for GTP-dependent ribosome splitting into subunits

    PubMed Central

    Gao, Ning; Zavialov, Andrey V.; Ehrenberg, Måns; Frank, Joachim

    2008-01-01

    Summary After termination of protein synthesis, the bacterial ribosome is split into its 30S and 50S subunits by the action of ribosome recycling factor (RRF) and elongation factor G (EF-G) in a GTP-hydrolysis dependent manner. Based on a previous cryo-electron microscopy (cryo-EM) study of ribosomal complexes, we have proposed that the binding of EF-G to an RRF containing post-termination ribosome triggers an inter-domain rotation of RRF, which destabilizes two strong intersubunit bridges (B2a and B3) and, ultimately, separates the two subunits. Here, we present a 9 Å (FSC at 0.5 cutoff) cryo-EM map of a 50S EFG GDPNP RRF complex and a quasi-atomic model derived from it, showing the interaction between EF-G and RRF on the 50S subunit in the presence of the non-cleavable GTP analogue GDPNP. The detailed information in this model and a comparative analysis of EF-G structures in various nucleotide- and ribosome-bound states show how rotation of the RRF head domain may be triggered by various domains of EF-G. For validation of our structural model, all known mutations in EF-G and RRF that relate to ribosome recycling have been taken into account. More importantly, our results indicate a substantial conformational change in the Switch I region of EF-G, suggesting that a conformational signal transduction mechanism, similar to that employed in tRNA translocation on the ribosome by EF-G, translates a large-scale movement of EF-G’s domain IV, induced by GTP hydrolysis, into the domain rotation of RRF that eventually splits the ribosome into subunits. PMID:17996252

  1. Caspase-dependent Proteolysis of Human Ribonucleotide Reductase Small Subunits R2 and p53R2 during Apoptosis*

    PubMed Central

    Tebbi, Ali; Guittet, Olivier; Tuphile, Karine; Cabrié, Aimeric; Lepoivre, Michel

    2015-01-01

    Ribonucleotide reductase (RnR) is a key enzyme synthesizing deoxyribonucleotides for DNA replication and repair. In mammals, the R1 catalytic subunit forms an active complex with either one of the two small subunits R2 and p53R2. Expression of R2 is S phase-specific and required for DNA replication. The p53R2 protein is expressed throughout the cell cycle and in quiescent cells where it provides dNTPs for mitochondrial DNA synthesis. Participation of R2 and p53R2 in DNA repair has also been suggested. In this study, we investigated the fate of the RnR subunits during apoptosis. The p53R2 protein was cleaved in a caspase-dependent manner in K-562 cells treated with inhibitors of the Bcr-Abl oncogenic kinase and in HeLa 229 cells incubated with TNF-α and cycloheximide. The cleavage site was mapped between Asp342 and Asn343. Caspase attack released a C-terminal p53R2 peptide of nine residues containing the conserved heptapeptide essential for R1 binding. As a consequence, the cleaved p53R2 protein was inactive. In vitro, purified caspase-3 and -8 could release the C-terminal tail of p53R2. Knocking down these caspases, but not caspase-2, -7, and -10, also inhibited p53R2 cleavage in cells committed to die via the extrinsic death receptor pathway. The R2 subunit was subjected to caspase- and proteasome-dependent proteolysis, which was prevented by siRNA targeting caspase-8. Knocking down caspase-3 was ineffective. Protein R1 was not subjected to degradation. Adding deoxyribonucleosides to restore dNTP pools transiently protected cells from apoptosis. These data identify RnR activity as a prosurvival function inactivated by proteolysis during apoptosis. PMID:25878246

  2. Localization of Saccharomyces cerevisiae Protein Phosphatase 2A Subunits throughout Mitotic Cell Cycle

    PubMed Central

    Gentry, Matthew S.; Hallberg, Richard L.

    2002-01-01

    Protein phosphatase 2A (PP2A) regulates a broad spectrum of cellular processes. This enzyme is a collection of varied heterotrimeric complexes, each composed of a catalytic (C) and regulatory (B) subunit bound together by a structural (A) subunit. To understand the cell cycle dynamics of this enzyme population, we carried out quantitative and qualitative analyses of the PP2A subunits of Saccharomyces cerevisiae. We found the following: the level of each subunit remained constant throughout the cell cycle; there is at least 10 times more of one of the regulatory subunits (Rts1p) than the other (Cdc55p); Tpd3p, the structural subunit, is limiting for both catalytic and regulatory subunit binding. Using green fluorescent protein-tagged forms of each subunit, we monitored the sites of significant accumulation of each protein throughout the cell cycle. The two regulatory subunits displayed distinctly different dynamic localization patterns that overlap with the A and C subunits at the bud tip, kinetochore, bud neck, and nucleus. Using strains null for single subunit genes, we confirmed the hypothesis that regulatory subunits determine sites of PP2A accumulation. Although Rts1p and Tpd3p required heterotrimer formation to achieve normal localization, Cdc55p achieved its normal localization in the absence of either an A or C subunit. PMID:12388751

  3. Localization of alpha integrin subunits in the neural retina of the tiger salamander.

    PubMed

    Sherry, D M; Proske, P A

    2001-04-01

    Integrin receptors mediate cell-extracellular matrix interactions and regulate many events, including cell growth, proliferation, and differentiation. Retinal integrins are incompletely understood, although these receptors are potentially important factors in normal retinal function and pathology. Immunocytochemistry was used to localize alpha integrin subunits 1-6 in the neural retina. Each alpha integrin subunit had a unique distribution in the retina, although there was considerable overlap among subunits. The alpha 1 subunit was broadly distributed throughout the retina, with some presumptive ganglion cells showing enriched labeling. The alpha 2 subunit was present on all retinal cell bodies, but was reduced in synaptic layers. The alpha 3 subunit was present in synaptic layers, Müller cells, and some cone and amacrine cells. The alpha 4 subunit was broadly distributed in the nuclear layers but was reduced in synaptic layers. The alpha 5 subunit was broadly expressed in the nuclear and synaptic layers with enriched labeling in the outer plexiform layer. Labeling for the alpha 6 subunit was restricted to the outer limiting membrane and some cone outer segments. Double-labeling studies indicated that photoreceptor terminals may exhibit alpha 1 and alpha 5 subunits, while processes from second-order neurons may exhibit alpha 1, alpha 3, and alpha 5 subunits. Integrin receptors containing the alpha 1, alpha 3, and alpha 5 subunits may have important functions at retinal synapses, in addition to roles in the nuclear layers. Integrin receptors containing alpha 2, alpha 4, and alpha 6 subunits probably serve non-synaptic functions.

  4. Respiratory and behavioral dysfunction following loss of the GABAA receptor α4 subunit

    PubMed Central

    Loria, C Jean; Stevens, Ashley M; Crummy, Ellen; Casadesus, Gemma; Jacono, Frank J; Dick, Thomas E; Siegel, Ruth E

    2013-01-01

    γ-Aminobutyric acid type A (GABAA) receptor plasticity participates in mediating adaptation to environmental change. Previous studies in rats demonstrated that extrasynaptic GABAA receptor subunits and receptors in the pons, a brainstem region involved in respiratory control, are upregulated by exposure to sustained hypobaric hypoxia. In these animals, expression of the mRNA encoding the extrasynaptic α4 subunit rose after 3 days in sustained hypoxia, while those encoding the α6 and δ subunits increased dramatically by 2 weeks. However, the participation of extrasynaptic subunits in maintaining respiration in normoxic conditions remains unknown. To examine the importance of α4 in a normal environment, respiratory function, motor and anxiety-like behaviors, and expression of other GABAA receptor subunit mRNAs were compared in wild-type (WT) and α4 subunit-deficient mice. Loss of the α4 subunit did not impact frequency, but did lead to reduced ventilatory pattern variability. In addition, mice lacking the subunit exhibited increased anxiety-like behavior. Finally, α4 subunit loss resulted in reduced expression of other extrasynaptic (α6 and δ) subunit mRNAs in the pons without altering those encoding the most prominent synaptic subunits. These findings on subunit-deficient mice maintained in normoxia, in conjunction with earlier findings on animals maintained in chronic hypoxia, suggest that the expression and regulation of extrasynaptic GABAA receptor subunits in the pons is interdependent and that their levels influence respiratory control as well as adaptation to stress. PMID:23533098

  5. Developmental aspects of a unique glutathione S-transferase subunit Yx in the liver cytosol from rats with hereditary hyperbilirubinuria. Comparison with rat fetal liver transferase subunit Yfetus.

    PubMed Central

    Igarashi, T; Tsuchiya, T; Shikata, Y; Sagami, F; Tagaya, O; Horie, T; Satoh, T

    1992-01-01

    The unique glutathione S-transferase (GST) subunit Yx, which is undetectable in normal adult rat liver cytosol, was shown to occur in the liver cytosol of rats with hereditary hyperbilirubinuria (EHB). The Yx subunit is a member of the Alpha-class GST subunits, and is immunologically closely related to the Yc subunit. The Yx subunit has an apparent M(r) of 26,400, different from those of Ya (M(r) 25,800), Yb1 and Yb2 (both M(r) 27,200) and Yc (M(r) 28,400). During postnatal development in livers of EHB rats, the Yx subunit concentration in either sex was highest during the first week post partum and declined rapidly with age. Although the concentration of subunit Yx at 8 weeks of age accounted for about 60% in females and 40% in males of that observed in 1-week-old 'neonatal' male EHB rats, concentrations in females thereafter increased gradually to almost the neonatal level and remained at this high level at least up to 37 weeks of age, whereas the concentration in males did not increase again. Thus the post-pubertal Yx subunit concentration was 2-fold higher in females than in males. In contrast, in normal Sprague-Dawley rat liver, the Yfetus subunit, with the same M(r) as the Yx subunit, had the highest concentration in 10-day-old animals, declined rapidly thereafter, and was not detectable in the post-pubertal period. The Yfetus subunit was also immunoreactive with an antibody against GST YcYc. The analysis of GST subunits by reverse-phase h.p.l.c. revealed that the Yx subunit was eluted at a retention time different from other known subunits, but coincided with that of Yfetus. The N-terminal amino acid sequence of the Yx subunit displayed a high degree of sequence similarity to that of the Yfetus subunit. These data suggest that the Yx subunit in EHB rats may be very similar to, if not identical with, the Yfetus subunit. Images Fig. 1. Fig. 2. Fig. 3. PMID:1567376

  6. Catalytic Subunit 1 of Protein Phosphatase 2A Is a Subunit of the STRIPAK Complex and Governs Fungal Sexual Development

    PubMed Central

    Beier, Anna; Krisp, Christoph; Wolters, Dirk A.

    2016-01-01

    ABSTRACT The generation of complex three-dimensional structures is a key developmental step for most eukaryotic organisms. The details of the molecular machinery controlling this step remain to be determined. An excellent model system to study this general process is the generation of three-dimensional fruiting bodies in filamentous fungi like Sordaria macrospora. Fruiting body development is controlled by subunits of the highly conserved striatin-interacting phosphatase and kinase (STRIPAK) complex, which has been described in organisms ranging from yeasts to humans. The highly conserved heterotrimeric protein phosphatase PP2A is a subunit of STRIPAK. Here, catalytic subunit 1 of PP2A was functionally characterized. The Δpp2Ac1 strain is sterile, unable to undergo hyphal fusion, and devoid of ascogonial septation. Further, PP2Ac1, together with STRIPAK subunit PRO22, governs vegetative and stress-related growth. We revealed in vitro catalytic activity of wild-type PP2Ac1, and our in vivo analysis showed that inactive PP2Ac1 blocks the complementation of the sterile deletion strain. Tandem affinity purification, followed by mass spectrometry and yeast two-hybrid analysis, verified that PP2Ac1 is a subunit of STRIPAK. Further, these data indicate links between the STRIPAK complex and other developmental signaling pathways, implying the presence of a large interconnected signaling network that controls eukaryotic developmental processes. The insights gained in our study can be transferred to higher eukaryotes and will be important for understanding eukaryotic cellular development in general. PMID:27329756

  7. Nonbridging phosphate oxygens in 16S rRNA important for 30S subunit assembly and association with the 50S ribosomal subunit.

    PubMed

    Ghosh, Srikanta; Joseph, Simpson

    2005-05-01

    Ribosomes are composed of RNA and protein molecules that associate together to form a supramolecular machine responsible for protein biosynthesis. Detailed information about the structure of the ribosome has come from the recent X-ray crystal structures of the ribosome and the ribosomal subunits. However, the molecular interactions between the rRNAs and the r-proteins that occur during the intermediate steps of ribosome assembly are poorly understood. Here we describe a modification-interference approach to identify nonbridging phosphate oxygens within 16S rRNA that are important for the in vitro assembly of the Escherichia coli 30S small ribosomal subunit and for its association with the 50S large ribosomal subunit. The 30S small subunit was reconstituted from phosphorothioate-substituted 16S rRNA and small subunit proteins. Active 30S subunits were selected by their ability to bind to the 50S large subunit and form 70S ribosomes. Analysis of the selected population shows that phosphate oxygens at specific positions in the 16S rRNA are important for either subunit assembly or for binding to the 50S subunit. The X-ray crystallographic structures of the 30S subunit suggest that some of these phosphate oxygens participate in r-protein binding, coordination of metal ions, or for the formation of intersubunit bridges in the mature 30S subunit. Interestingly, however, several of the phosphate oxygens identified in this study do not participate in any interaction in the mature 30S subunit, suggesting that they play a role in the early steps of the 30S subunit assembly.

  8. Arrangement of Kv1 alpha subunits dictates sensitivity to tetraethylammonium.

    PubMed

    Al-Sabi, Ahmed; Shamotienko, Oleg; Dhochartaigh, Sorcha Ni; Muniyappa, Nagesh; Le Berre, Marie; Shaban, Hamdy; Wang, Jiafu; Sack, Jon T; Dolly, J Oliver

    2010-09-01

    Shaker-related Kv1 channels contain four channel-forming alpha subunits. Subfamily member Kv1.1 often occurs oligomerized with Kv1.2 alpha subunits in synaptic membranes, and so information was sought on the influence of their positions within tetramers on the channels' properties. Kv1.1 and 1.2 alpha genes were tandem linked in various arrangements, followed by expression as single-chain proteins in mammalian cells. As some concatenations reported previously seemed not to reliably position Kv1 subunits in their assemblies, the identity of expressed channels was methodically evaluated. Surface protein, isolated by biotinylation of intact transiently transfected HEK-293 cells, gave Kv1.1/1.2 reactivity on immunoblots with electrophoretic mobilities corresponding to full-length concatenated tetramers. There was no evidence of protein degradation, indicating that concatemers were delivered intact to the plasmalemma. Constructs with like genes adjacent (Kv1.1-1.1-1.2-1.2 or Kv1.2-1.2-1.1-1.1) yielded delayed-rectifying, voltage-dependent K(+) currents with activation parameters and inactivation kinetics slightly different from the diagonally positioned genes (Kv1.1-1.2-1.1-1.2 or 1.2-1.1-1.2-1.1). Pore-blocking petidergic toxins, alpha dendrotoxin, agitoxin-1, tityustoxin-Kalpha, and kaliotoxin, were unable to distinguish between the adjacent and diagonal concatamers. Unprecedentedly, external application of the pore-blocker tetraethylammonium (TEA) differentially inhibited the adjacent versus diagonal subunit arrangements, with diagonal constructs having enhanced susceptibility. Concatenation did not directly alter the sensitivities of homomeric Kv1.1 or 1.2 channels to TEA or the toxins. TEA inhibition of currents generated by channels made up from dimers (Kv1.1-1.2 and/or Kv1.2-1.1) was similar to the adjacently arranged constructs. These collective findings indicate that assembly of alpha subunits can be directed by this optimized concatenation, and that subunit

  9. Mechanism of β4 Subunit Modulation of BK Channels

    PubMed Central

    Wang, Bin; Rothberg, Brad S.; Brenner, Robert

    2006-01-01

    Large-conductance (BK-type) Ca2+-activated potassium channels are activated by membrane depolarization and cytoplasmic Ca2+. BK channels are expressed in a broad variety of cells and have a corresponding diversity in properties. Underlying much of the functional diversity is a family of four tissue-specific accessory subunits (β1–β4). Biophysical characterization has shown that the β4 subunit confers properties of the so-called “type II” BK channel isotypes seen in brain. These properties include slow gating kinetics and resistance to iberiotoxin and charybdotoxin blockade. In addition, the β4 subunit reduces the apparent voltage sensitivity of channel activation and has complex effects on apparent Ca2+ sensitivity. Specifically, channel activity at low Ca2+ is inhibited, while at high Ca2+, activity is enhanced. The goal of this study is to understand the mechanism underlying β4 subunit action in the context of a dual allosteric model for BK channel gating. We observed that β4's most profound effect is a decrease in Po (at least 11-fold) in the absence of calcium binding and voltage sensor activation. However, β4 promotes channel opening by increasing voltage dependence of Po-V relations at negative membrane potentials. In the context of the dual allosteric model for BK channels, we find these properties are explained by distinct and opposing actions of β4 on BK channels. β4 reduces channel opening by decreasing the intrinsic gating equilibrium (L0), and decreasing the allosteric coupling between calcium binding and voltage sensor activation (E). However, β4 has a compensatory effect on channel opening following depolarization by shifting open channel voltage sensor activation (Vho) to more negative membrane potentials. The consequence is that β4 causes a net positive shift of the G-V relationship (relative to α subunit alone) at low calcium. At higher calcium, the contribution by Vho and an increase in allosteric coupling to Ca2+ binding (C

  10. Human Mind Maps

    ERIC Educational Resources Information Center

    Glass, Tom

    2016-01-01

    When students generate mind maps, or concept maps, the maps are usually on paper, computer screens, or a blackboard. Human Mind Maps require few resources and little preparation. The main requirements are space where students can move around and a little creativity and imagination. Mind maps can be used for a variety of purposes, and Human Mind…

  11. Regulation of expression of a soybean storage protein subunit gene. Progress report

    SciTech Connect

    Thompson, J.F.; Madison, J.T.

    1984-04-23

    We have found that the methionine repression of the ..beta..-subunit gene expression is not due to degradation of the ..beta..-subunit but is due to an effect on synthesis of the ..beta..-subunit. The effect of methionine on the synthesis of the ..beta..-is due to an inhibition of ..beta..-subunit mRNA synthesis. 3 references, 1 figure.

  12. Differential Contribution of Subunit Interfaces to α9α10 Nicotinic Acetylcholine Receptor Function.

    PubMed

    Boffi, Juan Carlos; Marcovich, Irina; Gill-Thind, JasKiran K; Corradi, Jeremías; Collins, Toby; Lipovsek, María Marcela; Moglie, Marcelo; Plazas, Paola V; Craig, Patricio O; Millar, Neil S; Bouzat, Cecilia; Elgoyhen, Ana Belén

    2017-03-01

    Nicotinic acetylcholine receptors can be assembled from either homomeric or heteromeric pentameric subunit combinations. At the interface of the extracellular domains of adjacent subunits lies the acetylcholine binding site, composed of a principal component provided by one subunit and a complementary component of the adjacent subunit. Compared with neuronal nicotinic acetylcholine cholinergic receptors (nAChRs) assembled from α and β subunits, the α9α10 receptor is an atypical member of the family. It is a heteromeric receptor composed only of α subunits. Whereas mammalian α9 subunits can form functional homomeric α9 receptors, α10 subunits do not generate functional channels when expressed heterologously. Hence, it has been proposed that α10 might serve as a structural subunit, much like a β subunit of heteromeric nAChRs, providing only complementary components to the agonist binding site. Here, we have made use of site-directed mutagenesis to examine the contribution of subunit interface domains to α9α10 receptors by a combination of electrophysiological and radioligand binding studies. Characterization of receptors containing Y190T mutations revealed unexpectedly that both α9 and α10 subunits equally contribute to the principal components of the α9α10 nAChR. In addition, we have shown that the introduction of a W55T mutation impairs receptor binding and function in the rat α9 subunit but not in the α10 subunit, indicating that the contribution of α9 and α10 subunits to complementary components of the ligand-binding site is nonequivalent. We conclude that this asymmetry, which is supported by molecular docking studies, results from adaptive amino acid changes acquired only during the evolution of mammalian α10 subunits.

  13. Bedaquiline Targets the ε Subunit of Mycobacterial F-ATP Synthase.

    PubMed

    Kundu, Subhashri; Biukovic, Goran; Grüber, Gerhard; Dick, Thomas

    2016-11-01

    The tuberculosis drug bedaquiline inhibits mycobacterial F-ATP synthase by binding to its c subunit. Using the purified ε subunit of the synthase and spectroscopy, we previously demonstrated that the drug interacts with this protein near its unique tryptophan residue. Here, we show that replacement of ε's tryptophan with alanine resulted in bedaquiline hypersusceptibility of the bacteria. Overexpression of the wild-type ε subunit caused resistance. These results suggest that the drug also targets the ε subunit.

  14. Bedaquiline Targets the ε Subunit of Mycobacterial F-ATP Synthase

    PubMed Central

    Kundu, Subhashri; Biukovic, Goran; Grüber, Gerhard

    2016-01-01

    The tuberculosis drug bedaquiline inhibits mycobacterial F-ATP synthase by binding to its c subunit. Using the purified ε subunit of the synthase and spectroscopy, we previously demonstrated that the drug interacts with this protein near its unique tryptophan residue. Here, we show that replacement of ε's tryptophan with alanine resulted in bedaquiline hypersusceptibility of the bacteria. Overexpression of the wild-type ε subunit caused resistance. These results suggest that the drug also targets the ε subunit. PMID:27620476

  15. Differential Contribution of Subunit Interfaces to α9α10 Nicotinic Acetylcholine Receptor Function

    PubMed Central

    Boffi, Juan Carlos; Marcovich, Irina; Gill-Thind, JasKiran K.; Corradi, Jeremías; Collins, Toby; Lipovsek, María Marcela; Moglie, Marcelo; Plazas, Paola V.; Craig, Patricio O.; Millar, Neil S.; Bouzat, Cecilia

    2017-01-01

    Nicotinic acetylcholine receptors can be assembled from either homomeric or heteromeric pentameric subunit combinations. At the interface of the extracellular domains of adjacent subunits lies the acetylcholine binding site, composed of a principal component provided by one subunit and a complementary component of the adjacent subunit. Compared with neuronal nicotinic acetylcholine cholinergic receptors (nAChRs) assembled from α and β subunits, the α9α10 receptor is an atypical member of the family. It is a heteromeric receptor composed only of α subunits. Whereas mammalian α9 subunits can form functional homomeric α9 receptors, α10 subunits do not generate functional channels when expressed heterologously. Hence, it has been proposed that α10 might serve as a structural subunit, much like a β subunit of heteromeric nAChRs, providing only complementary components to the agonist binding site. Here, we have made use of site-directed mutagenesis to examine the contribution of subunit interface domains to α9α10 receptors by a combination of electrophysiological and radioligand binding studies. Characterization of receptors containing Y190T mutations revealed unexpectedly that both α9 and α10 subunits equally contribute to the principal components of the α9α10 nAChR. In addition, we have shown that the introduction of a W55T mutation impairs receptor binding and function in the rat α9 subunit but not in the α10 subunit, indicating that the contribution of α9 and α10 subunits to complementary components of the ligand-binding site is nonequivalent. We conclude that this asymmetry, which is supported by molecular docking studies, results from adaptive amino acid changes acquired only during the evolution of mammalian α10 subunits. PMID:28069778

  16. Concept Mapping

    PubMed Central

    Brennan, Laura K.; Brownson, Ross C.; Kelly, Cheryl; Ivey, Melissa K.; Leviton, Laura C.

    2016-01-01

    Background From 2003 to 2008, 25 cross-sector, multidisciplinary community partnerships funded through the Active Living by Design (ALbD) national program designed, planned, and implemented policy and environmental changes, with complementary programs and promotions. This paper describes the use of concept-mapping methods to gain insights into promising active living intervention strategies based on the collective experience of community representatives implementing ALbD initiatives. Methods Using Concept Systems software, community representatives (n=43) anonymously generated actions and changes in their communities to support active living (183 original statements, 79 condensed statements). Next, respondents (n=26, from 23 partnerships) sorted the 79 statements into self-created categories, or active living intervention approaches. Respondents then rated statements based on their perceptions of the most important strategies for creating community changes (n=25, from 22 partnerships) and increasing community rates of physical activity (n=23, from 20 partnerships). Cluster analysis and multidimensional scaling were used to describe data patterns. Results ALbD community partnerships identified three active living intervention approaches with the greatest perceived importance to create community change and increase population levels of physical activity: changes to the built and natural environment, partnership and collaboration efforts, and land-use and transportation policies. The relative importance of intervention approaches varied according to subgroups of partnerships working with different populations. Conclusions Decision makers, practitioners, and community residents can incorporate what has been learned from the 25 community partnerships to prioritize active living policy, physical project, promotional, and programmatic strategies for work in different populations and settings. PMID:23079266

  17. AMPK beta subunits display isoform specific affinities for carbohydrates.

    PubMed

    Koay, Ann; Woodcroft, Ben; Petrie, Emma J; Yue, Helen; Emanuelle, Shane; Bieri, Michael; Bailey, Michael F; Hargreaves, Mark; Park, Jong-Tae; Park, Kwan-Hwa; Ralph, Stuart; Neumann, Dietbert; Stapleton, David; Gooley, Paul R

    2010-08-04

    AMP-activated protein kinase (AMPK) is a heterotrimer of catalytic (alpha) and regulatory (beta and gamma) subunits with at least two isoforms for each subunit. AMPK beta1 is widely expressed whilst AMPK beta2 is highly expressed in muscle and both beta isoforms contain a mid-molecule carbohydrate-binding module (beta-CBM). Here we show that beta2-CBM has evolved to contain a Thr insertion and increased affinity for glycogen mimetics with a preference for oligosaccharides containing a single alpha-1,6 branched residue. Deletion of Thr-101 reduces affinity for single alpha-1,6 branched oligosaccharides by 3-fold, while insertion of this residue into the equivalent position in the beta1-CBM sequence increases affinity by 3-fold, confirming the functional importance of this residue. Copyright (c) 2010. Published by Elsevier B.V.

  18. NADH dehydrogenase subunit genes in the mitochondrial DNA of yeasts.

    PubMed Central

    Nosek, J; Fukuhara, H

    1994-01-01

    The genes encoding the NADH dehydrogenase subunits of respiratory complex I have not been identified so far in the mitochondrial DNA (mtDNA) of yeasts. In the linear mtDNA of Candida parapsilosis, we found six new open reading frames whose sequences were unambiguously homologous to those of the genes known to code for NADH dehydrogenase subunit proteins of different organisms, i.e., ND1, ND2, ND3, ND4L, ND5, and ND6. The gene for ND4 also appears to be present, as judged from hybridization experiments with a Podospora gene probe. Specific transcripts from these open reading frames (ND genes) could be detected in the mitochondria. Hybridization experiments using C. parapsilosis genes as probes suggested that ND genes are present in the mtDNAs of a wide range of yeast species including Candida catenulata, Pichia guilliermondii, Clavispora lusitaniae, Debaryomyces hansenii, Hansenula polymorpha, and others. Images PMID:7521869

  19. On the specificity of antibiotics targeting the large ribosomal subunit.

    PubMed

    Wilson, Daniel N

    2011-12-01

    The peptidyltransferase center of the large ribosomal subunit is responsible for catalyzing peptide bonds. This active site is the target of a variety of diverse antibiotics, many of which are used clinically. The past decade has seen a plethora of structures of antibiotics in complex with the large ribosomal subunit, providing unprecedented insight into the mechanism of action of these inhibitors. Ten distinct antibiotics (chloramphenicol, clindamycin, linezolid, tiamulin, sparsomycin, and five macrolides) have been crystallized in complex with four distinct ribosomal species, three bacterial, and one archaeal. This review aims to compare these structures in order to provide insight into the conserved and species-specific modes of interaction for particular members of each class of antibiotics. Coupled with the wealth of biochemical data, a picture is emerging defining the specific functional states of the ribosome that antibiotics preferentially target. Such mechanistic insight into antibiotic inhibition will be important for the development of the next generation of antimicrobial agents.

  20. Maps & minds : mapping through the ages

    USGS Publications Warehouse

    ,

    1984-01-01

    Throughout time, maps have expressed our understanding of our world. Human affairs have been influenced strongly by the quality of maps available to us at the major turning points in our history. "Maps & Minds" traces the ebb and flow of a few central ideas in the mainstream of mapping. Our expanding knowledge of our cosmic neighborhood stems largely from a small number of simple but grand ideas, vigorously pursued.

  1. The role of auxiliary subunits for the functional diversity of voltage-gated calcium channels.

    PubMed

    Campiglio, Marta; Flucher, Bernhard E

    2015-09-01

    Voltage-gated calcium channels (VGCCs) represent the sole mechanism to convert membrane depolarization into cellular functions like secretion, contraction, or gene regulation. VGCCs consist of a pore-forming α(1) subunit and several auxiliary channel subunits. These subunits come in multiple isoforms and splice-variants giving rise to a stunning molecular diversity of possible subunit combinations. It is generally believed that specific auxiliary subunits differentially regulate the channels and thereby contribute to the great functional diversity of VGCCs. If auxiliary subunits can associate and dissociate from pre-existing channel complexes, this would allow dynamic regulation of channel properties. However, most auxiliary subunits modulate current properties very similarly, and proof that any cellular calcium channel function is indeed modulated by the physiological exchange of auxiliary subunits is still lacking. In this review we summarize available information supporting a differential modulation of calcium channel functions by exchange of auxiliary subunits, as well as experimental evidence in support of alternative functions of the auxiliary subunits. At the heart of the discussion is the concept that, in their native environment, VGCCs function in the context of macromolecular signaling complexes and that the auxiliary subunits help to orchestrate the diverse protein-protein interactions found in these calcium channel signalosomes. Thus, in addition to a putative differential modulation of current properties, differential subcellular targeting properties and differential protein-protein interactions of the auxiliary subunits may explain the need for their vast molecular diversity.

  2. Electrophysiology and beyond: multiple roles of Na+ channel β subunits in development and disease.

    PubMed

    Patino, Gustavo A; Isom, Lori L

    2010-12-10

    Voltage-gated Na+ channel (VGSC) β Subunits are not "auxiliary." These multi-functional molecules not only modulate Na+ current (I(Na)), but also function as cell adhesion molecules (CAMs)-playing roles in aggregation, migration, invasion, neurite outgrowth, and axonal fasciculation. β subunits are integral members of VGSC signaling complexes at nodes of Ranvier, axon initial segments, and cardiac intercalated disks, regulating action potential propagation through critical intermolecular and cell-cell communication events. At least in vitro, many β subunit cell adhesive functions occur both in the presence and absence of pore-forming VGSC α subunits, and in vivo β subunits are expressed in excitable as well as non-excitable cells, thus β subunits may play important functional roles on their own, in the absence of α subunits. VGSC β1 subunits are essential for life and appear to be especially important during brain development. Mutations in β subunit genes result in a variety of human neurological and cardiovascular diseases. Moreover, some cancer cells exhibit alterations in β subunit expression during metastasis. In short, these proteins, originally thought of as merely accessory to α subunits, are critical players in their own right in human health and disease. Here we discuss the role of VGSC β subunits in the nervous system.

  3. Mapping: A Course.

    ERIC Educational Resources Information Center

    Whitmore, Paul M.

    1988-01-01

    Reviews the history of cartography. Describes the contributions of Strabo and Ptolemy in early maps. Identifies the work of Gerhard Mercator as the most important advancement in mapping. Discusses present mapping standards from history. (CW)

  4. Mapping: A Course.

    ERIC Educational Resources Information Center

    Whitmore, Paul M.

    1988-01-01

    Reviews the history of cartography. Describes the contributions of Strabo and Ptolemy in early maps. Identifies the work of Gerhard Mercator as the most important advancement in mapping. Discusses present mapping standards from history. (CW)

  5. Database on the structure of large ribosomal subunit RNA.

    PubMed Central

    De Rijk, P; Caers, A; Van de Peer, Y; De Wachter, R

    1998-01-01

    The rRNA WWW Server at URL http://rrna.uia.ac.be/ now provides a database of 496 large subunit ribosomal RNA sequences. All these sequences are aligned, incorporate secondary structure information, and can be obtained in a number of formats. Other information about the sequences, such as literature references, accession numbers and taxonomic information is also available and searchable. If necessary, the data on the server can also be obtained by anonymous ftp. PMID:9399830

  6. Database on the structure of small ribosomal subunit RNA.

    PubMed Central

    Van de Peer, Y; Caers, A; De Rijk, P; De Wachter, R

    1998-01-01

    About 8600 complete or nearly complete sequences are now available from the Antwerp database on small ribosomal subunit RNA. All these sequences are aligned with one another on the basis of the adopted secondary structure model, which is corroborated by the observation of compensating substitutions in the alignment. Literature references, accession numbers and detailed taxonomic information are also compiled. The database can be consulted via the World Wide Web at URL http://rrna.uia.ac.be/ssu/ PMID:9399829

  7. Database on the structure of large ribosomal subunit RNA.

    PubMed Central

    De Rijk, P; Van de Peer, Y; De Wachter, R

    1996-01-01

    Our database on large ribosomal subunit RNA contained 334 sequences in July, 1995. All sequences in the database are aligned, taking into account secondary structure. The aligned sequences are provided, together with incorporated secondary structure information, in several computer-readable formats. These data can easily be obtained through the World Wide Web. The files in the database are also available via anonymous ftp. PMID:8594610

  8. Screening for AMPA receptor auxiliary subunit specific modulators

    PubMed Central

    Azumaya, Caleigh M.; Days, Emily L.; Vinson, Paige N.; Stauffer, Shaun; Sulikowski, Gary; Weaver, C. David; Nakagawa, Terunaga

    2017-01-01

    AMPA receptors (AMPAR) are ligand gated ion channels critical for synaptic transmission and plasticity. Their dysfunction is implicated in a variety of psychiatric and neurological diseases ranging from major depressive disorder to amyotrophic lateral sclerosis. Attempting to potentiate or depress AMPAR activity is an inherently difficult balancing act between effective treatments and debilitating side effects. A newly explored strategy to target subsets of AMPARs in the central nervous system is to identify compounds that affect specific AMPAR-auxiliary subunit complexes. This exploits diverse spatio-temporal expression patterns of known AMPAR auxiliary subunits, providing means for designing brain region-selective compounds. Here we report a high-throughput screening-based pipeline that can identify compounds that are selective for GluA2-CNIH3 and GluA2-stargazin complexes. These compounds will help us build upon the growing library of AMPAR-auxiliary subunit specific inhibitors, which have thus far all been targeted to TARP γ-8. We used a cell-based assay combined with a voltage-sensitive dye (VSD) to identify changes in glutamate-gated cation flow across the membranes of HEK cells co-expressing GluA2 and an auxiliary subunit. We then used a calcium flux assay to further validate hits picked from the VSD assay. VU0612951 and VU0627849 are candidate compounds from the initial screen that were identified as negative and positive allosteric modulators (NAM and PAM), respectively. They both have lower IC50/EC50s on complexes containing stargazin and CNIH3 than GSG1L or the AMPAR alone. We have also identified a candidate compound, VU0539491, that has NAM activity in GluA2(R)-CNIH3 and GluA2(Q) complexes and PAM activity in GluA2(Q)-GSG1L complexes. PMID:28358902

  9. Mcm subunits can assemble into two different active unwinding complexes.

    PubMed

    Kanter, Diane M; Bruck, Irina; Kaplan, Daniel L

    2008-11-07

    The replication fork helicase in eukaryotes is a large complex that is composed of Mcm2-7, Cdc45, and GINS. The Mcm2-7 proteins form a heterohexameric ring that hydrolyzes ATP and provide the motor function for this unwinding complex. A comprehensive study of how individual Mcm subunit biochemical activities relate to unwinding function has not been accomplished. We studied the mechanism of the Mcm4-Mcm6-Mcm7 complex, a useful model system because this complex has helicase activity in vitro. We separately purified each of three Mcm subunits until they were each nuclease-free, and we then examined the biochemical properties of different combinations of Mcm subunits. We found that Mcm4 and Mcm7 form an active unwinding assembly. The addition of Mcm6 to Mcm4/Mcm7 results in the formation of an active Mcm4/Mcm6/Mcm7 helicase assembly. The Mcm4-Mcm7 complex forms a ringed-shaped hexamer that unwinds DNA with 3' to 5' polarity by a steric exclusion mechanism, similar to Mcm4/Mcm6/Mcm7. The Mcm4-Mcm7 complex has a high level of ATPase activity that is further stimulated by DNA. The ability of different Mcm mixtures to form rings or exhibit DNA stimulation of ATPase activity correlates with the ability of these complexes to unwind DNA. The Mcm4/Mcm7 and Mcm4/Mcm6/Mcm7 assemblies can open to load onto circular DNA to initiate unwinding. We conclude that the Mcm subunits are surprisingly flexible and dynamic in their ability to interact with one another to form active unwinding complexes.

  10. Energy-driven subunit rotation at the interface between subunit a and the c oligomer in the FO sector of Escherichia coli ATP synthase

    PubMed Central

    Hutcheon, Marcus L.; Duncan, Thomas M.; Ngai, Helen; Cross, Richard L.

    2001-01-01

    Subunit rotation within the F1 catalytic sector of the ATP synthase has been well documented, identifying the synthase as the smallest known rotary motor. In the membrane-embedded FO sector, it is thought that proton transport occurs at a rotor/stator interface between the oligomeric ring of c subunits (rotor) and the single-copy a subunit (stator). Here we report evidence for an energy-dependent rotation at this interface. FOF1 was expressed with a pair of substituted cysteines positioned to allow an intersubunit disulfide crosslink between subunit a and a c subunit [aN214C/cM65C; Jiang, W. & Fillingame, R. H. (1998) Proc. Natl. Acad. Sci. USA 95, 6607–6612]. Membranes were treated with N,N′-dicyclohexyl-[14C]carbodiimide to radiolabel the D61 residue on less than 20% of the c subunits. After oxidation to form an a–c crosslink, the c subunit properly aligned to crosslink to subunit a was found to contain very little 14C label relative to other members of the c ring. However, exposure to MgATP before oxidation significantly increased the radiolabel in the a–c crosslink, indicating that a different c subunit was now aligned with subunit a. This increase was not induced by exposure to MgADP/Pi. Furthermore, preincubation with MgADP and azide to inhibit F1 or with high concentrations of N,N′-dicyclohexylcarbodiimide to label most c subunits prevented the ATP effect. These results provide evidence for an energy-dependent rotation of the c ring relative to subunit a. PMID:11438702

  11. Na(+), K(+)-ATPase β1 subunit associates with α1 subunit modulating a "higher-NKA-in-hyposmotic media" response in gills of euryhaline milkfish, Chanos chanos.

    PubMed

    Hu, Yau-Chung; Chu, Keng-Fu; Yang, Wen-Kai; Lee, Tsung-Han

    2017-03-10

    The euryhaline milkfish (Chanos chanos) is a popular aquaculture species that can be cultured in fresh water, brackish water, or seawater in Southeast Asia. In gills of the milkfish, Na(+), K(+)-ATPase (i.e., NKA; sodium pump) responds to salinity challenges including changes in mRNA abundance, protein amount, and activity. The functional pump is composed of a heterodimeric protein complex composed of α- and β-subunits. Among the NKA genes, α1-β1 isozyme comprises the major form of NKA subunits in mammalian osmoregulatory organs; however, most studies on fish gills have focused on the α1 subunit and did not verify the α1-β1 isozyme. Based on the sequenced milkfish transcriptome, an NKA β1 subunit gene was identified that had the highest amino acid homology to β233, a NKA β1 subunit paralog originally identified in the eel. Despite this high level of homology to β233, phylogenetic analysis and the fact that only a single NKA β1 subunit gene exists in the milkfish suggest that the milkfish gene should be referred to as the NKA β1 subunit gene. The results of accurate domain prediction of the β1 subunit, co-localization of α1 and β1 subunits in epithelial ionocytes, and co-immunoprecipitation of α1 and β1 subunits, indicated the formation of a α1-β1 complex in milkfish gills. Moreover, when transferred to hyposmotic media (fresh water) from seawater, parallel increases in branchial mRNA and protein expression of NKA α1 and β1 subunits suggested their roles in hypo-osmoregulation of euryhaline milkfish. This study molecularly characterized the NKA β1 subunit and provided the first evidence for an NKA α1-β1 association in gill ionocytes of euryhaline teleosts.

  12. Molecular basis of AKAP specificity for PKA regulatory subunits.

    PubMed

    Gold, Matthew G; Lygren, Birgitte; Dokurno, Pawel; Hoshi, Naoto; McConnachie, George; Taskén, Kjetil; Carlson, Cathrine R; Scott, John D; Barford, David

    2006-11-03

    Localization of cyclic AMP (cAMP)-dependent protein kinase (PKA) by A kinase-anchoring proteins (AKAPs) restricts the action of this broad specificity kinase. The high-resolution crystal structures of the docking and dimerization (D/D) domain of the RIIalpha regulatory subunit of PKA both in the apo state and in complex with the high-affinity anchoring peptide AKAP-IS explain the molecular basis for AKAP-regulatory subunit recognition. AKAP-IS folds into an amphipathic alpha helix that engages an essentially preformed shallow groove on the surface of the RII dimer D/D domains. Conserved AKAP aliphatic residues dominate interactions to RII at the predominantly hydrophobic interface, whereas polar residues are important in conferring R subunit isoform specificity. Using a peptide screening approach, we have developed SuperAKAP-IS, a peptide that is 10,000-fold more selective for the RII isoform relative to RI and can be used to assess the impact of PKA isoform-selective anchoring on cAMP-responsive events inside cells.

  13. Serotonergic modulation of muscle acetylcholine receptors of different subunit composition.

    PubMed Central

    García-Colunga, J; Miledi, R

    1996-01-01

    Modulation of muscle acetylcholine (AcCho) receptors (AcChoRs) by serotonin [5-hydroxytryptamine (5HT)] and other serotonergic compounds was studied in Xenopus laevis oocytes. Various combinations of alpha, beta, gamma, and delta subunit RNAs were injected into oocytes, and membrane currents elicited by AcCho were recorded under voltage clamp. Judging by the amplitudes of AcCho currents generated, the levels of functional receptor expression were: alpha beta gamma delta > alpha beta delta > alpha beta gamma > alpha gamma delta. The alpha beta gamma delta and alpha beta delta AcChoR Subtypes were strongly blocked by 5HT, whereas the alpha beta gamma receptor was blocked only slightly. The order of blocking potency of AcChoRs by 5HT was: alpha beta delta > alpha beta gamma delta > alpha beta gamma. 5HT receptor antagonists, such as methysergide and spiperone, were even more potent blockers of AcChoRs than 5HT but did not show much subunit selectivity. Blockage of alpha beta gamma delta and alpha beta delta receptors by 5HT was voltage-dependent, and the voltage dependence was abolished when the delta subunit was omitted. These findings may need to be taken into consideration when trying to elucidate the mode of action of many clinically important serotonergic compounds. Images Fig. 3 PMID:8633003

  14. MULTIPLE NEUROFILAMENT SUBUNITS ARE PRESENT IN LAMPREY CNS

    PubMed Central

    Jin, Li-Qing; Zhang, Guixin; Pennicooke, Brenton; Laramore, Cindy; Selzer, Michael E.

    2010-01-01

    In mammals, there are three neurofilament (NF) subunits (NF-L, NF-M, and NF-H), but it was thought that only a single NF, NF180, exists in lamprey. However, NF180 lacked the ability to self-assemble, suggesting that like mammalian NFs, lamprey NFs are heteropolymers, and that additional NF subunits may exist. The present study provides evidence for the existence of a lamprey NF-L homolog (L-NFL). Genes encoding two new NF-M isoforms (NF132 and NF95) also have been isolated and characterized. With NF180, this makes three NF-M-like isoforms. In situ hybridization showed that all three newly cloned NFs are expressed in spinal cord neurons and in spinal-projecting neurons of the brainstem. Like NF180, there were no KSP multiphosphorylation repeat motifs in the tail regions of NF132 or NF95. NF95 was highly identical to homologous parts of NF180, sharing 2 common pieces of DNA with it. Northern blots suggested that NF95 may be expressed at very low levels in older larvae. The presence of L-NFL in lamprey CNS may support the hypothesis that as in mammals, NFs in lamprey are obligate heteropolymers, in which NF-L is a required subunit. PMID:21081119

  15. Identification of multiple cyclin subunits of human P-TEFb

    PubMed Central

    Peng, Junmin; Zhu, Yuerong; Milton, Jeffrey T.; Price, David H.

    1998-01-01

    The transition from abortive into productive elongation is proposed to be controlled by a positive transcription elongation factor b (P-TEFb) through phosphorylation of the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II. Drosophila P-TEFb was identified recently as a cyclin-dependent kinase (CDK9) paired with a cyclin subunit (cyclin T). We demonstrate here the cloning of multiple cyclin subunits of human P-TEFb (T1 and T2). Cyclin T2 has two forms (T2a and T2b) because of alternative splicing. Both cyclin T1 and T2 are ubiquitously expressed. Immunoprecipitation and immunodepletion experiments carried out on HeLa nuclear extract (HNE) indicated that cyclin T1 and T2 were associated with CDK9 in a mutually exclusive manner and that almost all CDK9 was associated with either cyclin T1 or T2. Recombinant CDK9/cyclin T1, CDK9/cyclin T2a, and CDK9/cyclin T2b produced in Sf9 cells possessed DRB-sensitive kinase activity and functioned in transcription elongation in vitro. Either cyclin T1 or T2 was required to activate CDK9, and the truncation of the carboxyl terminus of the cyclin reduced, but did not eliminate, P-TEFb activity. Cotransfection experiments indicated that all three CDK9/cyclin combinations dramatically activated the CMV promoter. PMID:9499409

  16. Molecular Modeling of the Misfolded Insulin Subunit and Amyloid Fibril

    PubMed Central

    Choi, Jay H.; May, Barnaby C.H.; Wille, Holger; Cohen, Fred E.

    2009-01-01

    Abstract Insulin, a small hormone protein comprising 51 residues in two disulfide-linked polypeptide chains, adopts a predominantly α-helical conformation in its native state. It readily undergoes protein misfolding and aggregates into amyloid fibrils under a variety of conditions. Insulin is a unique model system in which to study protein fibrillization, since its three disulfide bridges are retained in the fibrillar state and thus limit the conformational space available to the polypeptide chains during misfolding and fibrillization. Taking into account this unique conformational restriction, we modeled possible monomeric subunits of the insulin amyloid fibrils using β-solenoid folds, namely, the β-helix and β-roll. Both models agreed with currently available biophysical data. We performed molecular dynamics simulations, which allowed some limited insights into the relative structural stability, suggesting that the β-roll subunit model may be more stable than the β-helix subunit model. We also constructed β-solenoid-based insulin fibril models and conducted fiber diffraction simulation to identify plausible fibril architectures of insulin amyloid. A comparison of simulated fiber diffraction patterns of the fibril models to the experimental insulin x-ray fiber diffraction data suggests that the model fibers composed of six twisted β-roll protofilaments provide the most reasonable fit to available experimental diffraction patterns and previous biophysical studies. PMID:20006956

  17. Ribosomal small subunit domains radiate from a central core

    NASA Astrophysics Data System (ADS)

    Gulen, Burak; Petrov, Anton S.; Okafor, C. Denise; Vander Wood, Drew; O'Neill, Eric B.; Hud, Nicholas V.; Williams, Loren Dean

    2016-02-01

    The domain architecture of a large RNA can help explain and/or predict folding, function, biogenesis and evolution. We offer a formal and general definition of an RNA domain and use that definition to experimentally characterize the rRNA of the ribosomal small subunit. Here the rRNA comprising a domain is compact, with a self-contained system of molecular interactions. A given rRNA helix or stem-loop must be allocated uniquely to a single domain. Local changes such as mutations can give domain-wide effects. Helices within a domain have interdependent orientations, stabilities and interactions. With these criteria we identify a core domain (domain A) of small subunit rRNA. Domain A acts as a hub, linking the four peripheral domains and imposing orientational and positional restraints on the other domains. Experimental characterization of isolated domain A, and mutations and truncations of it, by methods including selective 2‧OH acylation analyzed by primer extension and circular dichroism spectroscopy are consistent with our architectural model. The results support the utility of the concept of an RNA domain. Domain A, which exhibits structural similarity to tRNA, appears to be an essential core of the small ribosomal subunit.

  18. Cornichon proteins determine the subunit composition of synaptic AMPA receptors.

    PubMed

    Herring, Bruce E; Shi, Yun; Suh, Young Ho; Zheng, Chan-Ying; Blankenship, Sabine M; Roche, Katherine W; Nicoll, Roger A

    2013-03-20

    Cornichon-2 and cornichon-3 (CNIH-2/-3) are AMPA receptor (AMPAR) binding proteins that promote receptor trafficking and markedly slow AMPAR deactivation in heterologous cells, but their role in neurons is unclear. Using CNIH-2 and CNIH-3 conditional knockout mice, we find a profound reduction of AMPAR synaptic transmission in the hippocampus. This deficit is due to the selective loss of surface GluA1-containing AMPARs (GluA1A2 heteromers), leaving a small residual pool of synaptic GluA2A3 heteromers. The kinetics of AMPARs in neurons lacking CNIH-2/-3 are faster than those in WT neurons due to the fast kinetics of GluA2A3 heteromers. The remarkably selective effect of CNIHs on the GluA1 subunit is probably mediated by TARP γ-8, which prevents a functional association of CNIHs with non-GluA1 subunits. These results point to a sophisticated interplay between CNIHs and γ-8 that dictates subunit-specific AMPAR trafficking and the strength and kinetics of synaptic AMPAR-mediated transmission.

  19. Differential Localization of G Protein βγ Subunits

    PubMed Central

    2015-01-01

    G protein βγ subunits play essential roles in regulating cellular signaling cascades, yet little is known about their distribution in tissues or their subcellular localization. While previous studies have suggested specific isoforms may exhibit a wide range of distributions throughout the central nervous system, a thorough investigation of the expression patterns of both Gβ and Gγ isoforms within subcellular fractions has not been conducted. To address this, we applied a targeted proteomics approach known as multiple-reaction monitoring to analyze localization patterns of Gβ and Gγ isoforms in pre- and postsynaptic fractions isolated from cortex, cerebellum, hippocampus, and striatum. Particular Gβ and Gγ subunits were found to exhibit distinct regional and subcellular localization patterns throughout the brain. Significant differences in subcellular localization between pre- and postsynaptic fractions were observed within the striatum for most Gβ and Gγ isoforms, while others exhibited completely unique expression patterns in all four brain regions examined. Such differences are a prerequisite for understanding roles of individual subunits in regulating specific signaling pathways throughout the central nervous system. PMID:24568373

  20. Rescue of lethal subunits into functional K+ channels.

    PubMed Central

    Taglialatela, M; Payne, J P; Drewe, J A; Brown, A M

    1994-01-01

    In a chimeric, voltage-dependent K+ channel (CHM), the valine at position 369 and the leucine at position 374 interact within the pore or P-region to regulate ion permeation and block. Here we show that the point mutation, CHM V369L, abolished channel function whereas previous experiments showed that CHM V369 and CHM V369I are functional. Coinjection of "lethal" CHM V369L cRNA with CHM L374V cRNA but not CHM cRNA generated functional heteromultimers. The whole-cell Rb+/K+ conductance ratio was 2.98 +/- 0.43 for CHM L374V and was reduced to 0.87 +/- 0.04 for the coexpressed CHM V369L and CHM L374V subunits. When single-channel currents were recorded, a single class of CHM V369L/CHM L374V heteromultimers was identified. This class was readily distinguishable from CHM L374V homomultimers by K+ conductance, gating, and blockade by internal tetraethylammonium. Coinjection experiments at various RNA ratios suggest that the CHM V369L/CHM L374V heteromultime, assuming it to be a tetramer, was composed of three CHM L374V subunits and one CHM V369L subunit. It appears that in the critical P-region of CHM position 369 may tolerate only one leucine. Images FIGURE 7 PMID:8130337

  1. Ribosomal small subunit domains radiate from a central core

    PubMed Central

    Gulen, Burak; Petrov, Anton S.; Okafor, C. Denise; Vander Wood, Drew; O’Neill, Eric B.; Hud, Nicholas V.; Williams, Loren Dean

    2016-01-01

    The domain architecture of a large RNA can help explain and/or predict folding, function, biogenesis and evolution. We offer a formal and general definition of an RNA domain and use that definition to experimentally characterize the rRNA of the ribosomal small subunit. Here the rRNA comprising a domain is compact, with a self-contained system of molecular interactions. A given rRNA helix or stem-loop must be allocated uniquely to a single domain. Local changes such as mutations can give domain-wide effects. Helices within a domain have interdependent orientations, stabilities and interactions. With these criteria we identify a core domain (domain A) of small subunit rRNA. Domain A acts as a hub, linking the four peripheral domains and imposing orientational and positional restraints on the other domains. Experimental characterization of isolated domain A, and mutations and truncations of it, by methods including selective 2′OH acylation analyzed by primer extension and circular dichroism spectroscopy are consistent with our architectural model. The results support the utility of the concept of an RNA domain. Domain A, which exhibits structural similarity to tRNA, appears to be an essential core of the small ribosomal subunit. PMID:26876483

  2. Individual Interactions of the b Subunits within the Stator of the Escherichia coli ATP Synthase*

    PubMed Central

    Brandt, Karsten; Maiwald, Sarah; Herkenhoff-Hesselmann, Brigitte; Gnirß, Kerstin; Greie, Jörg-Christian; Dunn, Stanley D.; Deckers-Hebestreit, Gabriele

    2013-01-01

    FOF1 ATP synthases are rotary nanomotors that couple proton translocation across biological membranes to the synthesis/hydrolysis of ATP. During catalysis, the peripheral stalk, composed of two b subunits and subunit δ in Escherichia coli, counteracts the torque generated by the rotation of the central stalk. Here we characterize individual interactions of the b subunits within the stator by use of monoclonal antibodies and nearest neighbor analyses via intersubunit disulfide bond formation. Antibody binding studies revealed that the C-terminal region of one of the two b subunits is principally involved in the binding of subunit δ, whereas the other one is accessible to antibody binding without impact on the function of FOF1. Individually substituted cysteine pairs suitable for disulfide cross-linking between the b subunits and the other stator subunits (b-α, b-β, b-δ, and b-a) were screened and combined with each other to discriminate between the two b subunits (i.e. bI and bII). The results show the b dimer to be located at a non-catalytic α/β cleft, with bI close to subunit α, whereas bII is proximal to subunit β. Furthermore, bI can be linked to subunit δ as well as to subunit a. Among the subcomplexes formed were a-bI-α, bII-β, α-bI-bII-β, and a-bI-δ. Taken together, the data obtained define the different positions of the two b subunits at a non-catalytic interface and imply that each b subunit has a different role in generating stability within the stator. We suggest that bI is functionally related to the single b subunit present in mitochondrial ATP synthase. PMID:23846684

  3. Salamander rods and cones contain distinct transducin alpha subunits.

    PubMed

    Ryan, J C; Znoiko, S; Xu, L; Crouch, R K; Ma, J X

    2000-01-01

    The mammalian retina is known to contain two distinct transducins that interact with their respective rod and cone pigments. However, there are no reports of a nonmammalian species having two distinct transducins. In the present study, we report the cloning and cellular localization of two transducin a subunits (G alpha t) from the tiger salamander. Through degenerate polymerase chain reaction (PCR) and subsequent screening of a salamander retina cDNA library, we have identified two forms of G alpha t. When compared to existing sequences in GenBank, the cloned subunits showed high similarity to rod and cone transducins. The salamander G alpha t-1 has 91.2-93.7% amino acid sequence identity to mammalian rod G alpha t subunits and 79.7-80.9% to mammalian cone Gats. The salamander G alpha t-2 has 86.2-87.9% sequence identity to mammalian cone G alpha ts and 78.9-80.9% to mammalian rod G alpha ts at the amino acid level. The G alpha t-1 cDNA encodes 350 amino acids while the G alpha t-2 cDNA encodes 354 residues, which is typical for rod and cone G alpha ts, respectively, and we thus identified the G alpha t- 1 as rod and G alpha t-2 as cone G alpha t. Sequences identified as effector binding sites and GTPase activity regions are highly conserved between the two subunits. Genomic Southern blot analysis showed that rod and cone G alpha t subunits are both encoded by single-copy genes. Northern blot analysis identified retina-specific transcripts of 3.0 kb for rod G alpha t and 2.6 kb for cone G alpha t. Immunohistochemistry in the flat-mounted salamander retina demonstrated that rod G alpha t is localized to rods, predominantly in the outer segments; similarly, cone G alpha t is localized to cone outer segments. The results confirm that the two sequences encode rod and cone transducins and demonstrate that this lower vertebrate contains two distinct transducins that are localized specifically to rod and cone photoreceptors.

  4. Two-subunit DNA escort mechanism and inactive subunit bypass in an ultra-fast ring ATPase

    PubMed Central

    Liu, Ninning; Chistol, Gheorghe; Bustamante, Carlos

    2015-01-01

    SpoIIIE is a homo-hexameric dsDNA translocase responsible for completing chromosome segregation in Bacillus subtilis. Here, we use a single-molecule approach to monitor SpoIIIE translocation when challenged with neutral-backbone DNA and non-hydrolyzable ATP analogs. We show that SpoIIIE makes multiple essential contacts with phosphates on the 5'→3' strand in the direction of translocation. Using DNA constructs with two neutral-backbone segments separated by a single charged base pair, we deduce that SpoIIIE’s step size is 2 bp. Finally, experiments with non-hydrolyzable ATP analogs suggest that SpoIIIE can operate with non-consecutive inactive subunits. We propose a two-subunit escort translocation mechanism that is strict enough to enable SpoIIIE to track one DNA strand, yet sufficiently compliant to permit the motor to bypass inactive subunits without arrest. We speculate that such a flexible mechanism arose for motors that, like SpoIIIE, constitute functional bottlenecks where the inactivation of even a single motor can be lethal for the cell. DOI: http://dx.doi.org/10.7554/eLife.09224.001 PMID:26452092

  5. Two-subunit DNA escort mechanism and inactive subunit bypass in an ultra-fast ring ATPase

    DOE PAGES

    Liu, Ninning; Chistol, Gheorghe; Bustamante, Carlos

    2015-10-09

    SpoIIIE is a homo-hexameric dsDNA translocase responsible for completing chromosome segregation in Bacillus subtilis . Here, we use a single-molecule approach to monitor SpoIIIE translocation when challenged with neutral-backbone DNA and non-hydrolyzable ATP analogs. We show that SpoIIIE makes multiple essential contacts with phosphates on the 5'→3' strand in the direction of translocation. Using DNA constructs with two neutral-backbone segments separated by a single charged base pair, we deduce that SpoIIIE’s step size is 2 bp. Finally, experiments with non-hydrolyzable ATP analogs suggest that SpoIIIE can operate with non-consecutive inactive subunits. We propose a two-subunit escort translocation mechanism thatmore » is strict enough to enable SpoIIIE to track one DNA strand, yet sufficiently compliant to permit the motor to bypass inactive subunits without arrest. We speculate that such a flexible mechanism arose for motors that, like SpoIIIE, constitute functional bottlenecks where the inactivation of even a single motor can be lethal for the cell.« less

  6. Structure of subcomplex Iβ of mammalian respiratory complex I leads to new supernumerary subunit assignments

    PubMed Central

    Zhu, Jiapeng; King, Martin S.; Yu, Minmin; Klipcan, Liron; Leslie, Andrew G. W.; Hirst, Judy

    2015-01-01

    Mitochondrial complex I (proton-pumping NADH:ubiquinone oxidoreductase) is an essential respiratory enzyme. Mammalian complex I contains 45 subunits: 14 conserved “core” subunits and 31 “supernumerary” subunits. The structure of Bos taurus complex I, determined to 5-Å resolution by electron cryomicroscopy, described the structure of the mammalian core enzyme and allowed the assignment of 14 supernumerary subunits. Here, we describe the 6.8-Å resolution X-ray crystallography structure of subcomplex Iβ, a large portion of the membrane domain of B. taurus complex I that contains two core subunits and a cohort of supernumerary subunits. By comparing the structures and composition of subcomplex Iβ and complex I, supported by comparisons with Yarrowia lipolytica complex I, we propose assignments for eight further supernumerary subunits in the structure. Our new assignments include two CHCH-domain containing subunits that contain disulfide bridges between CX9C motifs; they are processed by the Mia40 oxidative-folding pathway in the intermembrane space and probably stabilize the membrane domain. We also assign subunit B22, an LYR protein, to the matrix face of the membrane domain. We reveal that subunit B22 anchors an acyl carrier protein (ACP) to the complex, replicating the LYR protein–ACP structural module that was identified previously in the hydrophilic domain. Thus, we significantly extend knowledge of how the mammalian supernumerary subunits are arranged around the core enzyme, and provide insights into their roles in biogenesis and regulation. PMID:26371297

  7. Reconstitution of thermostable ATPase capable of energy coupling from its purified subunits.

    PubMed Central

    Yoshida, M; Okamoto, H; Sone, N; Hirata, H; Kagawa, Y

    1977-01-01

    Purified dicyclohexylcarbodiimide-sensitive ATPase (TF0-F1) from thermophilic bacterium PS3 is composed of a water soluble part with ATP hydrolytic activity (TF1) and a water insoluble moiety (TF0). All of the five subunits (alpha, beta, gamma, delta, and epsilon) of TF1 were isolated. TF1 was reconstituted from the five subunits, which catalyzed an ATP-32Pi exchange and an ATP-driven enhancement of fluorescence of 1-anilinonaphthalene-8-sulfonate, when adsorbed on proteoliposome inlaid with TF0 (TF3-vesicles). Subunit epsilon and/or delta became firmly bound to TF0-vesicles and there was no preferential sequence in the binding. Both subunits were required for binding of the remaining subunits of TF1 to TF0-vesicles, but they did not modify the high H+ -permeability of TF0-vesicles. The addition of gamma but they did not modify the high H+-permeability of TFO-vesicles. The addition of gamma subunit together with epsilon and delta subunits caused a marked decrease of H+ -permeability of TF0-vesicles, similar to that induced by TF1. We conclude tentatively that the epsilon and delta subunits connect TF0 and the other subunits forming a part of a proton pathway, gamma is a gate of proton flow coupled to ATP hydrolysis (or synthesis), and alpha and beta subunits contain the active site for energy transformation. A possible model of subunit structure of TF1 is proposed. PMID:139610

  8. Isolation of hybridoma cell lines and characterization of monoclonal antibodies against cholera enterotoxin and its subunits.

    PubMed

    Robb, M; Nichols, J C; Whoriskey, S K; Murphy, J R

    1982-10-01

    Hybridoma cell lines which produced monoclonal antibodies against cholera toxin were isolated. These cell lines were detected with an enzyme-linked immunosorbent assay screening procedure with purified cholera toxin or subunit A of cholera toxin. Seven cell lines were characterized with respect to their reactivity with cholera toxin subunits by Western blot analysis. Five clones produced antibodies which were directed against subunit A, and two clones produced antibodies which reacted with subunit B. These antibodies were also characterized by Western blot analysis for reactivity with the heat-labile enterotoxin produced by porcine and human enterotoxinogenic strains of Escherichia coli. Monoclonal antibodies which reacted with subunit A of cholera toxin also reacted with subunit A of both porcine and human heat-labile enterotoxins. In contrast, monoclonal antibodies to subunit B of cholera toxin did not react with subunit B of the heat-labile enterotoxin. Antibodies directed against subunit B neutralized the biological activity of cholera toxin in vitro in the S49 mouse lymphosarcoma assay. In contrast to polyclonal anti-subunit A antisera, monoclonal anti-subunit A from four of five clones had small but measurable neutralizing capacities in vitro.

  9. Isolation of hybridoma cell lines and characterization of monoclonal antibodies against cholera enterotoxin and its subunits.

    PubMed Central

    Robb, M; Nichols, J C; Whoriskey, S K; Murphy, J R

    1982-01-01

    Hybridoma cell lines which produced monoclonal antibodies against cholera toxin were isolated. These cell lines were detected with an enzyme-linked immunosorbent assay screening procedure with purified cholera toxin or subunit A of cholera toxin. Seven cell lines were characterized with respect to their reactivity with cholera toxin subunits by Western blot analysis. Five clones produced antibodies which were directed against subunit A, and two clones produced antibodies which reacted with subunit B. These antibodies were also characterized by Western blot analysis for reactivity with the heat-labile enterotoxin produced by porcine and human enterotoxinogenic strains of Escherichia coli. Monoclonal antibodies which reacted with subunit A of cholera toxin also reacted with subunit A of both porcine and human heat-labile enterotoxins. In contrast, monoclonal antibodies to subunit B of cholera toxin did not react with subunit B of the heat-labile enterotoxin. Antibodies directed against subunit B neutralized the biological activity of cholera toxin in vitro in the S49 mouse lymphosarcoma assay. In contrast to polyclonal anti-subunit A antisera, monoclonal anti-subunit A from four of five clones had small but measurable neutralizing capacities in vitro. Images PMID:6183210

  10. Early expression of GABA(A) receptor delta subunit in the neonatal rat hippocampus.

    PubMed

    Didelon, F; Mladinic', M; Cherubini, E; Bradbury, A

    2000-12-01

    The cDNA library screening strategy was used to identify the genes encoding for GABA(A) receptor subunits in the rat hippocampus during development. With this technique, genes encoding eleven GABA(A) receptor subunits were identified. The alpha5 subunit was by far the most highly expressed, followed by the gamma2, alpha2 and alpha4 subunits respectively. The expression of the beta2, alpha1, gamma1, beta1 and beta3 subunits was moderate, although that of the alpha3 and delta subunits was weak. In situ hybridization experiments, using digoxigenin-labeled cRNA probes, confirmed that the delta subunit was expressed in the neonatal as well as in the adult hippocampus, and is likely to form functional receptors in association with other subunits of the GABA(A) receptor. When the more sensitive RT-PCR approach was used, the gamma3 subunit was also detected, suggesting that this subunit is present in the hippocampus during development but at low levels of expression. The insertion of the delta subunit into functional GABA(A) receptors may enhance the efficacy of GABA in the immediate postnatal period when this amino acid is still exerting a depolarizing and excitatory action.

  11. Ribosome Subunit Stapling for Orthogonal Translation in E.  coli.

    PubMed

    Fried, Stephen D; Schmied, Wolfgang H; Uttamapinant, Chayasith; Chin, Jason W

    2015-10-19

    The creation of orthogonal large and small ribosomal subunits, which interact with each other but not with endogenous ribosomal subunits, would extend our capacity to create new functions in the ribosome by making the large subunit evolvable. To this end, we rationally designed a ribosomal RNA that covalently links the ribosome subunits via an RNA staple. The stapled ribosome is directed to an orthogonal mRNA, allowing the introduction of mutations into the large subunit that reduce orthogonal translation, but have minimal effects on cell growth. Our approach provides a promising route towards orthogonal subunit association, which may enable the evolution of key functional centers in the large subunit, including the peptidyl-transferase center, for unnatural polymer synthesis in cells.

  12. Modulation of BK Channel Function by Auxiliary Beta and Gamma Subunits

    PubMed Central

    Li, Q.; Yan, J.

    2016-01-01

    The large-conductance, Ca2+- and voltage-activated K+ (BK) channel is ubiquitously expressed in mammalian tissues and displays diverse biophysical or pharmacological characteristics. This diversity is in part conferred by channel modulation with different regulatory auxiliary subunits. To date, two distinct classes of BK channel auxiliary subunits have been identified: β subunits and γ subunits. Modulation of BK channels by the four auxiliary β (β1–β4) subunits has been well established and intensively investigated over the past two decades. The auxiliary γ subunits, however, were identified only very recently, which adds a new dimension to BK channel regulation and improves our understanding of the physiological functions of BK channels in various tissues and cell types. This chapter will review the current understanding of BK channel modulation by auxiliary β and γ subunits, especially the latest findings. PMID:27238261

  13. Functional Diversification of Maize RNA Polymerase IV and V subtypes via Alternative Catalytic Subunits

    SciTech Connect

    Haag, Jeremy R.; Brower-Toland, Brent; Krieger, Elysia K.; Sidorenko, Lyudmila; Nicora, Carrie D.; Norbeck, Angela D.; Irsigler, Andre; LaRue, Huachun; Brzeski, Jan; Mcginnis, Karen A.; Ivashuta, Sergey; Pasa-Tolic, Ljiljana; Chandler, Vicki L.; Pikaard, Craig S.

    2014-10-01

    Unlike nuclear multisubunit RNA polymerases I, II, and III, whose subunit compositions are conserved throughout eukaryotes, plant RNA polymerases IV and V are nonessential, Pol II-related enzymes whose subunit compositions are still evolving. Whereas Arabidopsis Pols IV and V differ from Pol II in four or five of their 12 subunits, respectively, and differ from one another in three subunits, proteomic ana- lyses show that maize Pols IV and V differ from Pol II in six subunits but differ from each other only in their largest subunits. Use of alternative catalytic second subunits, which are nonredundant for development and paramutation, yields at least two sub- types of Pol IV and three subtypes of Pol V in maize. Pol IV/Pol V associations with MOP1, RMR1, AGO121, Zm_DRD1/CHR127, SHH2a, and SHH2b extend parallels between paramutation in maize and the RNA-directed DNA methylation pathway in Arabidopsis.

  14. Voltage-gated calcium channel subunits from platyhelminths: potential role in praziquantel action.

    PubMed

    Jeziorski, Michael C; Greenberg, Robert M

    2006-05-31

    Voltage-gated calcium (Ca2+) channels provide the pathway for Ca2+ influxes that underlie Ca2+ -dependent responses in muscles, nerves and other excitable cells. They are also targets of a wide variety of drugs and toxins. Ca2+ channels are multisubunit protein complexes consisting of a pore-forming alpha(1) subunit and other modulatory subunits, including the beta subunit. Here, we review the structure and function of schistosome Ca2+ channel subunits, with particular emphasis on variant Ca2+ channel beta subunits (Ca(v)betavar) found in these parasites. In particular, we examine the role these beta subunits may play in the action of praziquantel, the current drug of choice against schistosomiasis. We also present evidence that Ca(v)betavar homologs are found in other praziquantel-sensitive platyhelminths such as the pork tapeworm, Taenia solium, and that these variant beta subunits may thus represent a platyhelminth-specific gene family.

  15. Voltage-gated calcium channel subunits from platyhelminths: Potential role in praziquantel action✩

    PubMed Central

    Jeziorski, Michael C.; Greenberg, Robert M.

    2013-01-01

    Voltage-gated calcium (Ca2+) channels provide the pathway for Ca2+ influxes that underlie Ca2+-dependent responses in muscles, nerves and other excitable cells. They are also targets of a wide variety of drugs and toxins. Ca2+ channels are multisubunit protein complexes consisting of a pore-forming α1 subunit and other modulatory subunits, including the β subunit. Here, we review the structure and function of schistosome Ca2+ channel subunits, with particular emphasis on variant Ca2+ channel β subunits (Cavβvar) found in these parasites. In particular, we examine the role these β subunits may play in the action of praziquantel, the current drug of choice against schistosomiasis. We also present evidence that Cavβvar homologs are found in other praziquantel-sensitive platyhelminths such as the pork tapeworm, Taenia solium, and that these variant β subunits may thus represent a platyhelminth-specific gene family. PMID:16545816

  16. Mapping the Heart

    ERIC Educational Resources Information Center

    Hulse, Grace

    2012-01-01

    In this article, the author describes how her fourth graders made ceramic heart maps. The impetus for this project came from reading "My Map Book" by Sara Fanelli. This book is a collection of quirky, hand-drawn and collaged maps that diagram a child's world. There are maps of her stomach, her day, her family, and her heart, among others. The…

  17. Fundamentals of Physical Mapping

    USDA-ARS?s Scientific Manuscript database

    This book chapter provides an overview of physical mapping in plants and its use for map-based gene cloning. A brief overview of cytogenetics-based physical mapping strategies, and physical mapping approaches currently used and the lessons learnt from the success stories were furnished. The statisti...

  18. Mapping the Heart

    ERIC Educational Resources Information Center

    Hulse, Grace

    2012-01-01

    In this article, the author describes how her fourth graders made ceramic heart maps. The impetus for this project came from reading "My Map Book" by Sara Fanelli. This book is a collection of quirky, hand-drawn and collaged maps that diagram a child's world. There are maps of her stomach, her day, her family, and her heart, among others. The…

  19. Subunit regulation of the neuronal alpha 1A Ca2+ channel expressed in Xenopus oocytes.

    PubMed Central

    De Waard, M; Campbell, K P

    1995-01-01

    1. Voltage-dependent Ca2+ channels are multi-protein complexes composed of at least three subunits: alpha 1, alpha 2 delta and beta. Ba2+ currents were recorded in Xenopus oocytes expressing the neuronal alpha 1A Ca2+ channel, using the two-electrode voltage-clamp technique. Various subunit combinations were studied: alpha 1A, alpha 1A alpha 2 delta b, alpha 1A beta or alpha 1A alpha 2 delta b beta. 2. The alpha 1A subunit alone directs the expression of functional Ca2+ channels. It carries all the properties of the channel: gating, permeability, voltage dependence of activation and inactivation, and pharmacology. The alpha 1A channel is activated by low voltages when physiological concentrations of the permeant cation are used. Both ancillary subunits alpha 2 delta and beta induced considerable changes in the biophysical properties of the alpha 1A current. The subunit specificity of the changes in current properties was analysed for all four beta gene products by coexpressing beta 1b, beta 2a, beta 3 and beta 4. 3. All beta subunits induce a stimulation in the current amplitude, a change in inactivation kinetics, and two hyperpolarizing shifts--one in the voltage dependence of activation and a second in the voltage dependence of steady-state inactivation. The most significant difference in regulation among beta subunits is the induction of variable rate constants of current inactivation. Rates of inactivation were induced in the following order (fastest to slowest): beta 3 > beta 1b = beta 4 > beta 2a. 4. The alpha 2 delta b subunit does not modify the properties of alpha 1A Ca2+ channels in the absence of beta subunits. However, this subunit increases the beta-induced stimulation in current amplitude and also regulates the beta-induced change in inactivation kinetics. 5. Of all the subunit combinations tested, Ca2+ channels that included a beta subunit were the most prone to decrease in activity. It is concluded that beta subunits are the primary target for the

  20. Comparative immunohistochemical localisation of GABA(B1a), GABA(B1b) and GABA(B2) subunits in rat brain, spinal cord and dorsal root ganglion.

    PubMed

    Charles, K J; Evans, M L; Robbins, M J; Calver, A R; Leslie, R A; Pangalos, M N

    2001-01-01

    GABA(B) receptors are G-protein-coupled receptors mediating the slow onset and prolonged synaptic actions of GABA in the CNS. The recent cloning of two genes, GABA(B1) and GABA(B2), has revealed a novel requirement for GABA(B) receptor signalling. Studies have demonstrated that the two receptor subunits associate as a GABA(B1)/GABA(B2) heterodimer to form a functional GABA(B) receptor. In this study we have developed polyclonal antisera specific to two splice variants of the GABA(B1) subunit, GABA(B1a) and GABA(B1b), as well as an antiserum to the GABA(B2) subunit. Using affinity-purified antibodies derived from these antisera we have mapped out the distribution profile of each subunit in rat brain, spinal cord and dorsal root ganglion. In brain the highest areas of GABA(B1a), GABA(B1b) and GABA(B2) subunit expression were found in neocortex, hippocampus, thalamus, cerebellum and habenula. In spinal cord, GABA(B1) and GABA(B2) subunits were expressed in the superficial layers of the dorsal horn, as well as in motor neurones in the deeper layers of the ventral horn. GABA(B) receptor subunit immunoreactivity in dorsal root ganglion suggested that expression of GABA(B1b) was restricted to the large diameter neurones, in contrast to GABA(B1a) and GABA(B2) subunits which were expressed in both large and small diameter neurones. Although expression levels of GABA(B1) and GABA(B2) subunits varied we found no areas in which GABA(B1) was expressed in the absence of GABA(B2). This suggests that most, if not all, GABA(B1) immunoreactivity may represent functional GABA(B) receptors. Although our data are in general agreement with functional studies, some discrepancies in GABA(B1) subunit expression occurred with respect to other immunohistochemical studies. Overall our data suggest that GABA(B) receptors are widely expressed throughout the brain and spinal cord, and that GABA(B1a) and GABA(B1b) subunits can associate with GABA(B2) to form both pre- and post-synaptic receptors.

  1. National Atlas maps

    USGS Publications Warehouse

    ,

    1991-01-01

    The National Atlas of the United States of America was published by the U.S. Geological Survey in 1970. Its 765 maps and charts are on 335 14- by 19-inch pages. Many of the maps span facing pages. It's worth a quick trip to the library just to leaf through all 335 pages of this book. Rapid scanning of its thematic maps yields rich insights to the geography of issues of continuing national interest. On most maps, the geographic patterns are still valid, though the data are not current. The atlas is out of print, but many of its maps can be purchased separately. Maps that span facing pages in the atlas are printed on one sheet. The maps dated after 1970 are either revisions of original atlas maps, or new maps published in atlas format. The titles of the separate maps are listed here.

  2. Google Maps: You Are Here

    ERIC Educational Resources Information Center

    Jacobsen, Mikael

    2008-01-01

    Librarians use online mapping services such as Google Maps, MapQuest, Yahoo Maps, and others to check traffic conditions, find local businesses, and provide directions. However, few libraries are using one of Google Maps most outstanding applications, My Maps, for the creation of enhanced and interactive multimedia maps. My Maps is a simple and…

  3. Google Maps: You Are Here

    ERIC Educational Resources Information Center

    Jacobsen, Mikael

    2008-01-01

    Librarians use online mapping services such as Google Maps, MapQuest, Yahoo Maps, and others to check traffic conditions, find local businesses, and provide directions. However, few libraries are using one of Google Maps most outstanding applications, My Maps, for the creation of enhanced and interactive multimedia maps. My Maps is a simple and…

  4. The β Subunit of Voltage-Gated Ca2+ Channels

    PubMed Central

    Buraei, Zafir; Yang, Jian

    2015-01-01

    Calcium regulates a wide spectrum of physiological processes such as heartbeat, muscle contraction, neuronal communication, hormone release, cell division, and gene transcription. Major entry-ways for Ca2+ in excitable cells are high-voltage activated (HVA) Ca2+channels. These are plasma membrane proteins composed of several subunits, including α1, α2δ, β, and γ. Although the principal α1 subunit (Cavα1) contains the channel pore, gating machinery and most drug binding sites, the cytosolic auxiliary β subunit (Cavβ) plays an essential role in regulating the surface expression and gating properties of HVA Ca2+ channels. Cavβ is also crucial for the modulation of HVA Ca2+ channels by G proteins, kinases, and the Ras-related RGK GTPases. New proteins have emerged in recent years that modulate HVA Ca2+ channels by binding to Cavβ. There are also indications that Cavβ may carry out Ca2+ channel-independent functions, including directly regulating gene transcription. All four subtypes of Cavβ, encoded by different genes, have a modular organization, consisting of three variable regions, a conserved guanylate kinase (GK) domain, and a conserved Src-homology 3 (SH3) domain, placing them into the membrane-associated guanylate kinase (MAGUK) protein family. Crystal structures of Cavβs reveal how they interact with Cavα1, open new research avenues, and prompt new inquiries. In this article, we review the structure and various biological functions of Cavβ, with both a historical perspective as well as an emphasis on recent advances. PMID:20959621

  5. Properties and subunit structure of pig heart pyruvate dehydrogenase.

    PubMed

    Hamada, M; Hiraoka, T; Koike, K; Ogasahara, K; Kanzaki, T

    1976-06-01

    Pyruvate dehydrogenase [EC 1.2.4.1] was separated from the pyruvate dehydrogenase complex and its molecular weight was estimated to be about 150,000 by sedimentation equilibrium methods. The enzyme was dissociated into two subunits (alpha and beta), with estimated molecular weights of 41,000 (alpha) and 36,000 (beta), respectively, by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The subunits were separated by phosphocellulose column chromatography and their chemical properties were examined. The subunit structure of the pyruvate dehydrogenase was assigned as alpha2beta2. The content of right-handed alpha-helix in the enzyme molecule was estimated to be about 29 and 28% by optical rotatory dispersion and by circular dichroism, respectively. The enzyme contained no thiamine-PP, and its dehydrogenase activity was completely dependent on added thiamine-PP and partially dependent on added Mg2+ and Ca2+. The Km value of pyruvate dehydrogenase for thiamine diphosphate was estimated to be 6.5 X 10(-5) M in the presence of Mg2+ or Ca2+. The enzyme showed highly specific activity for thiamine-PP dependent oxidation of both pyruvate and alpha-ketobutyrate, but it also showed some activity with alpha-ketovalerate, alpha-ketoisocaproate, and alpha-ketoisovalerate. The pyruvate dehydrogenase activity was strongly inhibited by bivalent heavy metal ions and by sulfhydryl inhibitors; and the enzyme molecule contained 27 moles of 5,5'-dithiobis(2-nitrobenzoic acid)-reactive sulfhydryl groups and a total of 36 moles of sulfhydryl groups. The inhibitory effect of p-chloromercuribenzoate was prevented by preincubating the enzyme with thiamine-PP plus pyruvate. The structure of pyruvate dehydrogenase necessary for formation of the complex is also reported.

  6. Covalent dimerization of ribulose bisphosphate carboxylase subunits by UV radiation.

    PubMed

    Ferreira, R M; Franco, E; Teixeira, A R

    1996-08-15

    The effect of UV radiation (UV-A, UV-B and UV-C) on ribulose bisphosphate carboxylase from a variety of plant species was examined. The exposition of plant leaves or the pure enzyme to UV radiation produced a UV-dependent accumulation of a +5 kDa polypeptide (P65). Different approaches were utilized to elucidate the origin and structure of P65: electrophoretic and fluorographic analyses of 35S-labelled ribulose bisphosphate carboxylase exposed to UV radiation and immunological experiments using antibodies specific for P65, for the large and small subunits of ribulose bisphosphate carboxylase and for high-molecular-mass aggregates of the enzyme. These studies revealed that P65 is a dimer, formed by the covalent, non-disulphide linkage of one small subunit with one large subunit of ribulose bisphosphate carboxylase. For short periods of time (< 1 h), the amount of P65 formed increased with the duration of the exposure to the UV radiation and with the energy of the radiation applied. Prolonged exposure to UV radiation (1-6 h) resulted in the formation of high-molecular-mass aggregates of ribulose bisphosphate carboxylase. Formation of P65 was shown to depend on the native state of the protein, was stimulated by inhibitors of enzyme activity, and was inhibited by activators of enzyme activity. A UV-independent accumulation of P65 was also achieved by the in vitro incubation of plant crude extracts. However, the UV-dependent and the UV-independent formation of P65 seemed to occur by distinct molecular mechanisms. The UV-dependent accumulation of P65 was immunologically detected in all species examined, including Lemna minor, Arum italicum, Brassica oleracea, Triticum aestivum, Zea mays, Pisum sativum and Phaseolus vulgaris, suggesting that it may constitute a universal response to UV radiation, common to all photo-synthetic tissues.

  7. Spinal NMDA NR1 Subunit Expression Following Transient TNBS Colitis

    PubMed Central

    Zhou, QiQi; Price, Donald D.; Caudle, Robert M.; Verne, G. Nicholas

    2009-01-01

    Background: N-methyl-D-aspartic acid (NMDA) receptors play an important role in the development of hypersensitivity to visceral and somatic stimuli following inflammation or tissue injury. Our objective was to investigate the role of NMDA NR1 receptors in the spinal cord (T10-L1; L4-S1) of a subset of rats that remain hypersensitive following histological resolution of TNBS-induced colitis compared to saline treated rats and rats that had recovered both behaviorally and histologically. We hypothesized that NMDA NR1 subunit expression mediates hypersensitivity following transient TNBS colitis. Methods: Male Sprague-Dawley rats (150g-250g) received 20mg/rat intracolonic trinitrobenzene sulfonic acid (TNBS) in 50% ethanol or saline. Animals underwent nociceptive visceral/somatic pain testing 16 weeks after resolution of TNBS colitis. Animals were sacrificed and their spinal cord (T10-L1; L4-S1) was retrieved and 2-dimensional polyacrylamide gel electrophoresis and immunohistocytochemistry techniques were used to investigate spinal-NMDA receptor expression. Results: NR1001 was the only NMDA NR1 receptor subunit that was expressed in recovered and control rats, whereas hypersensitive animals expressed NR1011 and NR1111 as well as NR1001 subunits. Immunohistochemistry analysis demonstrated increased expression of NMDA NR1-N1, C1, and C2-plus expression in lamina I & II of the spinal cord (T10-L1; L4-S1) in hypersensitive rats but not in recovered/control rats. Conclusions: Selective increases in the expression of the NMDA NR1 splice variants occur in hypersensitive rats following resolution of TNBS colitis. This suggests that the NMDA NR1 receptor play an important role in the development of neuronal plasticity and central sensitization. The recombination of NR1 splice variants may serve as a key functional protein that maintains hypersensitivity following resolution of TNBS colitis. PMID:19406112

  8. Crystal Structure of the Cytoplasmic N-Terminal Domain of Subunit I, a Homolog of Subunit a, of V-ATPase

    SciTech Connect

    Srinivasan, Sankaranarayanan; Vyas, Nand K.; Baker, Matthew L.; Quiocho, Florante A.

    2012-02-27

    Subunit 'a' is associated with the membrane-bound (VO) complex of eukaryotic vacuolar H{sup +}-ATPase acidification machinery. It has also been shown recently to be involved in diverse membrane fusion/secretory functions independent of acidification. Here, we report the crystal structure of the N-terminal cytosolic domain from the Meiothermus ruber subunit 'I' homolog of subunit a. The structure is composed of a curved long central {alpha}-helix bundle capped on both ends by two lobes with similar {alpha}/{beta} architecture. Based on the structure, a reasonable model of its eukaryotic subunit a counterpart was obtained. The crystal structure and model fit well into reconstructions from electron microscopy of prokaryotic and eukaryotic vacuolar H{sup +}-ATPases, respectively, clarifying their orientations and interactions and revealing features that could enable subunit a to play a role in membrane fusion/secretion.

  9. Chemical synthesis of yeast mitochondrial ATP synthase membranous subunit 8.

    PubMed

    Goetz, M; Schmitter, J M; Geoffre, S; Dufourc, E J

    1999-06-01

    Chemical synthesis of highly hydrophobic peptides and proteins remains a challenging problem. Strong interchain associations within the peptide-resin matrix have to be overcome. A synthetic strategy for solid phase peptide synthesis is proposed, mainly based on prolonged coupling time using aprotic polar solvent mixtures. A tailored chromatographic purification was required to obtain a sample sufficiently pure for structural analysis. In this work, the total chemical synthesis of the membrane-embedded yeast mitochondrial ATP synthase subunit 8 is described. The quality of the synthetic protein was checked by electrospray mass spectrometry, its tendency to adopt alpha-helical secondary structure is evidenced by circular dichroism spectroscopy.

  10. Database on the structure of small ribosomal subunit RNA.

    PubMed Central

    Van de Peer, Y; Nicolaï, S; De Rijk, P; De Wachter, R

    1996-01-01

    The Antwerp database on small ribosomal subunit RNA offers over 4300 nucleotide sequences (August 1995). All these sequences are stored in the form of an alignment based on the adopted secondary structure model, which in turn is corroborated by the observation of compensating substitutions in the alignment. Besides the primary and secondary structure information, literature references, accession numbers and detailed taxonomic information are also compiled. The complete database is made available to the scientific community through anonymous ftp and World Wide Web(WWW). PMID:8594609

  11. Database on the structure of large ribosomal subunit RNA.

    PubMed Central

    De Rijk, P; Van de Peer, Y; De Wachter, R

    1997-01-01

    The latest release of the large ribosomal subunit RNA database contains 429 sequences. All these sequences are aligned, and incorporate secondary structure information. The rRNA WWW Server at URL http://rrna.uia.ac.be/ provides researchers with an easily accessible resource to obtain the data in this database in a number of computer-readable formats. A new query interface has been added to the server. If necessary, the data can also be obtained by anonymous ftp from the same site. PMID:9016517

  12. Database on the structure of small ribosomal subunit RNA.

    PubMed Central

    Van de Peer, Y; Jansen, J; De Rijk, P; De Wachter, R

    1997-01-01

    The Antwerp database on small ribosomal subunit RNA now offers more than 6000 nucleotide sequences (August 1996). All these sequences are stored in the form of an alignment based on the adopted secondary structure model, which is corroborated by the observation of compensating substitutions in the alignment. Besides the primary and secondary structure information, literature references, accession numbers and detailed taxonomic information are also compiled. For ease of use, the complete database is made available to the scientific community via World Wide Web at URL http://rrna.uia.ac.be/ssu/ . PMID:9016516

  13. Database on the structure of small ribosomal subunit RNA.

    PubMed Central

    Van de Peer, Y; Van den Broeck, I; De Rijk, P; De Wachter, R

    1994-01-01

    The database on small ribosomal subunit RNA structure contains (June 1994) 2824 nucleotide sequences. All these sequences are stored in the form of an alignment based on the adopted secondary structure model, which in turn is corroborated by the observation of compensating substitutions in the alignment. The complete database is made available to the scientific community through anonymous ftp on our server in Antwerp. A special effort was made to improve electronic retrieval and a program is supplied that allows to create different file formats. The database can also be obtained from the EMBL nucleotide sequence library. PMID:7524022

  14. Twisting and subunit rotation in single FOF1-ATP synthase

    PubMed Central

    Sielaff, Hendrik; Börsch, Michael

    2013-01-01

    FOF1-ATP synthases are ubiquitous proton- or ion-powered membrane enzymes providing ATP for all kinds of cellular processes. The mechanochemistry of catalysis is driven by two rotary nanomotors coupled within the enzyme. Their different step sizes have been observed by single-molecule microscopy including videomicroscopy of fluctuating nanobeads attached to single enzymes and single-molecule Förster resonance energy transfer. Here we review recent developments of approaches to monitor the step size of subunit rotation and the transient elastic energy storage mechanism in single FOF1-ATP synthases. PMID:23267178

  15. Association-dissociation process with aging subunits: Recursive solution

    NASA Astrophysics Data System (ADS)

    Niedermayer, Thomas; Lipowsky, Reinhard

    2015-11-01

    The coupling of stochastic growth and shrinkage of one-dimensional structures to random aging of the constituting subunits defines the simple association-dissociation-aging process which captures the essential features of the nonequilibrium assembly of cytoskeletal filaments. Because of correlations, previously employed mean-field methods fail to correctly describe filament growth. We study an alternative formulation of the full master equation of the stochastic process. An ansatz for the steady-state solution leads to a recursion relation which allows for the calculation of all emergent quantities with increasing accuracy and in excellent agreement with stochastic simulations.

  16. Interaction between 25S rRNA A loop and eukaryotic translation initiation factor 5B promotes subunit joining and ensures stringent AUG selection.

    PubMed

    Hiraishi, Hiroyuki; Shin, Byung-Sik; Udagawa, Tsuyoshi; Nemoto, Naoki; Chowdhury, Wasimul; Graham, Jymie; Cox, Christian; Reid, Megan; Brown, Susan J; Asano, Katsura

    2013-09-01

    In yeast, 25S rRNA makes up the major mass and shape of the 60S ribosomal subunit. During the last step of translation initiation, eukaryotic initiation factor 5B (eIF5B) promotes the 60S subunit joining with the 40S initiation complex (IC). Malfunctional 60S subunits produced by misfolding or mutation may disrupt the 40S IC stalling on the start codon, thereby altering the stringency of initiation. Using several point mutations isolated by random mutagenesis, here we studied the role of 25S rRNA in start codon selection. Three mutations changing bases near the ribosome surface had strong effects, allowing the initiating ribosomes to skip both AUG and non-AUG codons: C2879U and U2408C, altering the A loop and P loop, respectively, of the peptidyl transferase center, and G1735A, mapping near a Eukarya-specific bridge to the 40S subunit. Overexpression of eIF5B specifically suppressed the phenotype caused by C2879U, suggesting functional interaction between eIF5B and the A loop. In vitro reconstitution assays showed that C2879U decreased eIF5B-catalyzed 60S subunit joining with a 40S IC. Thus, eIF5B interaction with the peptidyl transferase center A loop increases the accuracy of initiation by stabilizing the overall conformation of the 80S initiation complex. This study provides an insight into the effect of ribosomal mutations on translation profiles in eukaryotes.

  17. Post-translational transformation of methionine to aspartate is catalyzed by heme iron and driven by peroxide: a novel subunit-specific mechanism in hemoglobin.

    PubMed

    Strader, Michael Brad; Hicks, Wayne A; Kassa, Tigist; Singleton, Eileen; Soman, Jayashree; Olson, John S; Weiss, Mitchell J; Mollan, Todd L; Wilson, Michael T; Alayash, Abdu I

    2014-08-08

    A pathogenic V67M mutation occurs at the E11 helical position within the heme pockets of variant human fetal and adult hemoglobins (Hb). Subsequent post-translational modification of Met to Asp was reported in γ subunits of human fetal Hb Toms River (γ67(E11)Val → Met) and β subunits of adult Hb (HbA) Bristol-Alesha (β67(E11)Val → Met) that were associated with hemolytic anemia. Using kinetic, proteomic, and crystal structural analysis, we were able to show that the Met → Asp transformation involves heme cycling through its oxoferryl state in the recombinant versions of both proteins. The conversion to Met and Asp enhanced the spontaneous autoxidation of the mutants relative to wild-type HbA and human fetal Hb, and the levels of Asp were elevated with increasing levels of hydrogen peroxide (H2O2). Using H2(18)O2, we verified incorporation of (18)O into the Asp carboxyl side chain confirming the role of H2O2 in the oxidation of the Met side chain. Under similar experimental conditions, there was no conversion to Asp at the αMet(E11) position in the corresponding HbA Evans (α62(E11)Val → Met). The crystal structures of the three recombinant Met(E11) mutants revealed similar thioether side chain orientations. However, as in the solution experiments, autoxidation of the Hb mutant crystals leads to electron density maps indicative of Asp(E11) formation in β subunits but not in α subunits. This novel post-translational modification highlights the nonequivalence of human Hb α, β, and γ subunits with respect to redox reactivity and may have direct implications to α/β hemoglobinopathies and design of oxidatively stable Hb-based oxygen therapeutics. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. Finding and using local symmetry in identifying lower domain movements in hexon subunits of the herpes simplex virus type 1 B capsid.

    PubMed

    He, J; Schmid, M F; Zhou, Z H; Rixon, F; Chiu, W

    2001-06-15

    A characteristic of virus assembly is the use of symmetry to construct a complex capsid from a limited number of different proteins. Many spherical viruses display not only icosahedral symmetry, but also local symmetries, which further increase the redundancy of their structural proteins. We have developed a computational procedure for evaluating the quality of these local symmetries that allows us to probe the extent of local structural variations among subunits. This type of analysis can also provide orientation parameters for carrying out non-icosahedral averaging of quasi-equivalent subunits during three-dimensional structural determination. We have used this procedure to analyze the three types of hexon (P, E and C) in the 8.5 A resolution map of the herpes simplex virus type 1 (HSV-1) B capsid, determined by electron cryomicroscopy. The comparison of the three hexons showed that they have good overall 6-fold symmetry and are almost identical throughout most of their lengths. The largest difference among the three lies near the inner surface in a region of about 34 A in thickness. In this region, the P hexon displays slightly lower 6-fold symmetry than the C and E hexons. More detailed analysis showed that parts of two of the P hexon subunits are displaced counterclockwise with respect to their expected 6-fold positions. The most highly displaced subunit interacts with a subunit from an adjacent P hexon (P'). Using the local 6-fold symmetry axis of the P hexon as a rotation axis, we examined the geometrical relationships among the local symmetry axes of the surrounding capsomeres. Deviations from exact symmetry are also found among these local symmetry axes. The relevance of these findings to the process of capsid assembly is considered.

  19. Map reading tools for map libraries.

    USGS Publications Warehouse

    Greenberg, G.L.

    1982-01-01

    Engineers, navigators and military strategists employ a broad array of mechanical devices to facilitate map use. A larger number of map users such as educators, students, tourists, journalists, historians, politicians, economists and librarians are unaware of the available variety of tools which can be used with maps to increase the speed and efficiency of their application and interpretation. This paper identifies map reading tools such as coordinate readers, protractors, dividers, planimeters, and symbol-templets according to a functional classification. Particularly, arrays of tools are suggested for use in determining position, direction, distance, area and form (perimeter-shape-pattern-relief). -from Author

  20. Meta-Analyses of Dehalococcoides mccartyi Strain 195 Transcriptomic Profiles Identify a Respiration Rate-Related Gene Expression Transition Point and Interoperon Recruitment of a Key Oxidoreductase Subunit

    PubMed Central

    Mansfeldt, Cresten B.; Rowe, Annette R.; Heavner, Gretchen L. W.; Zinder, Stephen H.

    2014-01-01

    A cDNA-microarray was designed and used to monitor the transcriptomic profile of Dehalococcoides mccartyi strain 195 (in a mixed community) respiring various chlorinated organics, including chloroethenes and 2,3-dichlorophenol. The cultures were continuously fed in order to establish steady-state respiration rates and substrate levels. The organization of array data into a clustered heat map revealed two major experimental partitions. This partitioning in the data set was further explored through principal component analysis. The first two principal components separated the experiments into those with slow (1.6 ± 0.6 μM Cl−/h)- and fast (22.9 ± 9.6 μM Cl−/h)-respiring cultures. Additionally, the transcripts with the highest loadings in these principal components were identified, suggesting that those transcripts were responsible for the partitioning of the experiments. By analyzing the transcriptomes (n = 53) across experiments, relationships among transcripts were identified, and hypotheses about the relationships between electron transport chain members were proposed. One hypothesis, that the hydrogenases Hup and Hym and the formate dehydrogenase-like oxidoreductase (DET0186-DET0187) form a complex (as displayed by their tight clustering in the heat map analysis), was explored using a nondenaturing protein separation technique combined with proteomic sequencing. Although these proteins did not migrate as a single complex, DET0112 (an FdhB-like protein encoded in the Hup operon) was found to comigrate with DET0187 rather than with the catalytic Hup subunit DET0110. On closer inspection of the genome annotations of all Dehalococcoides strains, the DET0185-to-DET0187 operon was found to lack a key subunit, an FdhB-like protein. Therefore, on the basis of the transcriptomic, genomic, and proteomic evidence, the place of the missing subunit in the DET0185-to-DET0187 operon is likely filled by recruiting a subunit expressed from the Hup operon (DET0112). PMID

  1. Determination of kainate receptor subunit ratios in mouse brain using novel chimeric protein standards.

    PubMed

    Watanabe-Iida, Izumi; Konno, Kohtarou; Akashi, Kaori; Abe, Manabu; Natsume, Rie; Watanabe, Masahiko; Sakimura, Kenji

    2016-01-01

    Kainate-type glutamate receptors (KARs) are tetrameric channels assembled from GluK1-5. GluK1-3 are low-affinity subunits that form homomeric and heteromeric KARs, while GluK4 and GluK5 are high-affinity subunits that require co-assembly with GluK1-3 for functional expression. Although the subunit composition is thought to be highly heterogeneous in the brain, the distribution of KAR subunits at the protein level and their relative abundance in given regions of the brain remain largely unknown. In the present study, we titrated C-terminal antibodies to each KAR subunit using chimeric GluA2-GluK fusion proteins, and measured their relative abundance in the P2 and post-synaptic density (PSD) fractions of the adult mouse hippocampus and cerebellum. Analytical western blots showed that GluK2 and GluK3 were the major KAR subunits, with additional expression of GluK5 in the hippocampus and cerebellum. In both regions, GluK4 was very low and GluK1 was below the detection threshold. The relative amount of low-affinity subunits (GluK2 plus GluK3) was several times higher than that of high-affinity subunits (GluK4 plus GluK5) in both regions. Of note, the highest ratio of high-affinity subunits to low-affinity subunits was found in the hippocampal PSD fraction (0.32), suggesting that heteromeric receptors consisting of high- and low-affinity subunits highly accumulate at hippocampal synapses. In comparison, this ratio was decreased to 0.15 in the cerebellar PSD fraction, suggesting that KARs consisting of low-affinity subunits are more prevalent in the cerebellum. Therefore, low-affinity KAR subunits are predominant in the brain, with distinct subunit combinations between the hippocampus and cerebellum. Kainate receptors, an unconventional member of the iGluR receptor family, have a tetrameric structure assembled from low-affinity (GluK1-3) and high-affinity (GluK4 and GluK5) subunits. We used a simple but novel procedure to measure the relative abundance of both low- and

  2. Phylogenetic mapping of bacterial morphology

    NASA Technical Reports Server (NTRS)

    Siefert, J. L.; Fox, G. E.

    1998-01-01

    The availability of a meaningful molecular phylogeny for bacteria provides a context for examining the historical significance of various developments in bacterial evolution. Herein, the classical morphological descriptions of selected members of the domain Bacteria are mapped upon the genealogical ancestry deduced from comparison of small-subunit rRNA sequences. For the species examined in this study, a distinct pattern emerges which indicates that the coccus shape has arisen and accumulated independently multiple times in separate lineages and typically survived as a persistent end-state morphology. At least two other morphologies persist but have evolved only once. This study demonstrates that although bacterial morphology is not useful in defining bacterial phylogeny, it is remarkably consistent with that phylogeny once it is known. An examination of the experimental evidence available for morphogenesis as well as microbial fossil evidence corroborates these findings. It is proposed that the accumulation of persistent morphologies is a result of the biophysical properties of peptidoglycan and their genetic control, and that an evolved body-plan strategy based on peptidoglycan may have been a fate-sealing step in the evolution of Bacteria. More generally, this study illustrates that significant evolutionary insights can be obtained by examining biological and biochemical data in the context of a reliable phylogenetic structure.

  3. Phylogenetic mapping of bacterial morphology

    NASA Technical Reports Server (NTRS)

    Siefert, J. L.; Fox, G. E.

    1998-01-01

    The availability of a meaningful molecular phylogeny for bacteria provides a context for examining the historical significance of various developments in bacterial evolution. Herein, the classical morphological descriptions of selected members of the domain Bacteria are mapped upon the genealogical ancestry deduced from comparison of small-subunit rRNA sequences. For the species examined in this study, a distinct pattern emerges which indicates that the coccus shape has arisen and accumulated independently multiple times in separate lineages and typically survived as a persistent end-state morphology. At least two other morphologies persist but have evolved only once. This study demonstrates that although bacterial morphology is not useful in defining bacterial phylogeny, it is remarkably consistent with that phylogeny once it is known. An examination of the experimental evidence available for morphogenesis as well as microbial fossil evidence corroborates these findings. It is proposed that the accumulation of persistent morphologies is a result of the biophysical properties of peptidoglycan and their genetic control, and that an evolved body-plan strategy based on peptidoglycan may have been a fate-sealing step in the evolution of Bacteria. More generally, this study illustrates that significant evolutionary insights can be obtained by examining biological and biochemical data in the context of a reliable phylogenetic structure.

  4. Ca(2+) channel inactivation heterogeneity reveals physiological unbinding of auxiliary beta subunits.

    PubMed Central

    Restituito, S; Cens, T; Rousset, M; Charnet, P

    2001-01-01

    Voltage gated Ca(2+) channel (VGCC) auxiliary beta subunits increase membrane expression of the main pore-forming alpha(1) subunits and finely tune channel activation and inactivation properties. In expression studies, co-expression of beta subunits also reduced neuronal Ca(2+) channel regulation by heterotrimeric G protein. Biochemical studies suggest that VGCC beta subunits and G protein betagamma can compete for overlapping interaction sites on VGCC alpha(1) subunits, suggesting a dynamic association of these subunits with alpha(1). In this work we have analyzed the stability of the alpha(1)/beta association under physiological conditions. Regulation of the alpha(1A) Ca(2+) channel inactivation properties by beta(1b) and beta(2a) subunits had two major effects: a shift in voltage-dependent inactivation (E(in)), and an increase of the non-inactivating current (R(in)). Unexpectedly, large variations in magnitude of the effects were recorded on E(in), when beta(1b) was expressed, and R(in), when beta(2a) was expressed. These variations were not proportional to the current amplitude, and occurred at similar levels of beta subunit expression. beta(2a)-induced variations of R(in) were, however, inversely proportional to the magnitude of G protein block. These data underline the two different mechanisms used by beta(1b) and beta(2a) to regulate channel inactivation, and suggest that the VGCC beta subunit can unbind the alpha1 subunit in physiological situations. PMID:11423397

  5. Cloned Shiga Toxin 2 B Subunit Induces Apoptosis in Ramos Burkitt's Lymphoma B Cells

    PubMed Central

    Marcato, Paola; Mulvey, George; Armstrong, Glen D.

    2002-01-01

    The Shiga toxins (Stx1 and Stx2), produced by Shigella dysenteriae type 1 and enterohemorrhagic Escherichia coli, consist of one A subunit and five B subunits. The Stx1 and Stx2 B subunits form a pentameric structure that binds to globotriaosylceramide (Gb3-Cer) receptors on eukaryotic cells and promotes endocytosis. The A subunit then inhibits protein biosynthesis, which triggers apoptosis in the affected cell. In addition to its Gb3-Cer binding activity, the data in the following report demonstrate that the Stx2 B pentamer induces apoptosis in Ramos Burkitt's lymphoma B cells independently of A subunit activity. Apoptosis was not observed in A subunit-free preparations of the Stx1 B pentamer which competitively inhibited Stx2 B pentamer-mediated apoptosis. The pancaspase inhibitor, Z-VAD-fmk, prevented apoptosis in Ramos cells exposed to the Stx2 B subunit, Stx1 or Stx2. Brefeldin A, an inhibitor of the Golgi transport system, also prevented Stx2 B subunit-mediated apoptosis. These observations suggest that the Stx2 B subunit must be internalized, via Gb3-Cer receptors, to induce Ramos cell apoptosis. Moreover, unlike the two holotoxins, Stx2 B subunit-mediated apoptosis does not involve inhibition of protein biosynthesis. This study provides further insight into the pathogenic potential of this family of potent bacterial exotoxins. PMID:11854211

  6. Crystal structure and molecular imaging of the Nav channel β3 subunit indicates a trimeric assembly.

    PubMed

    Namadurai, Sivakumar; Balasuriya, Dilshan; Rajappa, Rajit; Wiemhöfer, Martin; Stott, Katherine; Klingauf, Jurgen; Edwardson, J Michael; Chirgadze, Dimitri Y; Jackson, Antony P

    2014-04-11

    The vertebrate sodium (Nav) channel is composed of an ion-conducting α subunit and associated β subunits. Here, we report the crystal structure of the human β3 subunit immunoglobulin (Ig) domain, a functionally important component of Nav channels in neurons and cardiomyocytes. Surprisingly, we found that the β3 subunit Ig domain assembles as a trimer in the crystal asymmetric unit. Analytical ultracentrifugation confirmed the presence of Ig domain monomers, dimers, and trimers in free solution, and atomic force microscopy imaging also detected full-length β3 subunit monomers, dimers, and trimers. Mutation of a cysteine residue critical for maintaining the trimer interface destabilized both dimers and trimers. Using fluorescence photoactivated localization microscopy, we detected full-length β3 subunit trimers on the plasma membrane of transfected HEK293 cells. We further show that β3 subunits can bind to more than one site on the Nav 1.5 α subunit and induce the formation of α subunit oligomers, including trimers. Our results suggest a new and unexpected role for the β3 subunits in Nav channel cross-linking and provide new structural insights into some pathological Nav channel mutations.

  7. Subunit composition of mitochondrial complex I from the yeast Yarrowia lipolytica.

    PubMed

    Abdrakhmanova, Albina; Zickermann, Volker; Bostina, Mihnea; Radermacher, Michael; Schägger, Hermann; Kerscher, Stefan; Brandt, Ulrich

    2004-07-23

    Here we present a first assessment of the subunit inventory of mitochondrial complex I from the obligate aerobic yeast Yarrowia lipolytica. A total of 37 subunits were identified. In addition to the seven central, nuclear coded, and the seven mitochondrially coded subunits, 23 accessory subunits were found based on 2D electrophoretic and mass spectroscopic analysis in combination with sequence information from the Y. lipolytica genome. Nineteen of the 23 accessory subunits are clearly conserved between Y. lipolytica and mammals. The remaining four accessory subunits include NUWM, which has no apparent homologue in any other organism and is predicted to contain a single transmembrane domain bounded by highly charged extramembraneous domains. This structural organization is shared among a group of 7 subunits in the Y. lipolytica and 14 subunits in the mammalian enzyme. Because only five of these subunits display significant evolutionary conservation, their as yet unknown function is proposed to be structure- rather than sequence-specific. The NUWM subunit could be assigned to a hydrophobic subcomplex obtained by fragmentation and sucrose gradient centrifugation. Its position within the membrane arm was determined by electron microscopic single particle analysis of Y. lipolytica complex I decorated with a NUWM-specific monoclonal antibody.

  8. Forskolin Regulates L-Type Calcium Channel through Interaction between Actinin 4 and β3 Subunit in Osteoblasts.

    PubMed

    Zhang, Xuemei; Li, Fangping; Guo, Lin; Hei, Hongya; Tian, Lulu; Peng, Wen; Cai, Hui

    2015-01-01

    Voltage-dependent L-type calcium channels that permit cellular calcium influx are essential in calcium-mediated modulation of cellular signaling. Although the regulation of voltage-dependent L-type calcium channels is linked to many factors including cAMP-dependent protein kinase A (PKA) activity and actin cytoskeleton, little is known about the detailed mechanisms underlying the regulation in osteoblasts. Our present study investigated the modulation of L-type calcium channel activities through the effects of forskolin on actin reorganization and on its functional interaction with actin binding protein actinin 4. The results showed that forskolin did not significantly affect the trafficking of pore forming α1c subunit and its interaction with actin binding protein actinin 4, whereas it significantly increased the expression of β3 subunit and its interaction with actinin 4 in osteoblast cells as assessed by co-immunoprecipitation, pull-down assay, and immunostaining. Further mapping showed that the ABD and EF domains of actinin 4 were interaction sites. This interaction is independent of PKA phosphorylation. Knockdown of actinin 4 significantly decreased the activities of L-type calcium channels. Our study revealed a new aspect of the mechanisms by which the forskolin activation of adenylyl cyclase - cAMP cascade regulates the L-type calcium channel in osteoblast cells, besides the PKA mediated phosphorylation of the channel subunits. These data provide insight into the important role of interconnection among adenylyl cyclase, cAMP, PKA, the actin cytoskeleton, and the channel proteins in the regulation of voltage-dependent L-type calcium channels in osteoblast cells.

  9. Human ERAL1 is a mitochondrial RNA chaperone involved in the assembly of the 28S small mitochondrial ribosomal subunit

    PubMed Central

    Dennerlein, Sven; Rozanska, Agata; Wydro, Mateusz; Chrzanowska-Lightowlers, Zofia M. A.; Lightowlers, Robert N.

    2010-01-01

    The bacterial Ras-like protein Era has been reported previously to bind 16S rRNA within the 30S ribosomal subunit and to play a crucial role in ribosome assembly. An orthologue of this essential GTPase ERAL1 (Era G-protein-like 1) exists in higher eukaryotes and although its exact molecular function and cellular localization is unknown, its absence has been linked to apoptosis. In the present study we show that human ERAL1 is a mitochondrial protein important for the formation of the 28S small mitoribosomal subunit. We also show that ERAL1 binds in vivo to the rRNA component of the small subunit [12S mt (mitochondrial)-rRNA]. Bacterial Era associates with a 3′ unstructured nonanucleotide immediately downstream of the terminal stem–loop (helix 45) of 16S rRNA. This site contains an AUCA sequence highly conserved across all domains of life, immediately upstream of the anti-Shine–Dalgarno sequence, which is conserved in bacteria. Strikingly, this entire region is absent from 12S mt-rRNA. We have mapped the ERAL1-binding site to a 33 nucleotide section delineating the 3′ terminal stem–loop region of 12S mt-rRNA. This loop contains two adenine residues that are reported to be dimethylated on mitoribosome maturation. Furthermore, and also in contrast with the bacterial orthologue, loss of ERAL1 leads to rapid decay of nascent 12S mt-rRNA, consistent with a role as a mitochondrial RNA chaperone. Finally, whereas depletion of ERAL1 leads to apoptosis, cell death occurs prior to any appreciable loss of mitochondrial protein synthesis or reduction in the stability of mitochondrial mRNA. PMID:20604745

  10. Self-Subunit Swapping Occurs in Another Gene Type of Cobalt Nitrile Hydratase

    PubMed Central

    Xia, Yuanyuan; Cui, Youtian; Kobayashi, Michihiko; Zhou, Zhemin

    2012-01-01

    Self-subunit swapping is one of the post-translational maturation of the cobalt-containing nitrile hydratase (Co-NHase) family of enzymes. All of these NHases possess a gene organization of <β-subunit> <α-subunit> , which allows the activator protein to easily form a mediatory complex with the α-subunit of the NHase after translation. Here, we discovered that the incorporation of cobalt into another type of Co-NHase, with a gene organization of <α-subunit> <β-subunit> , was also dependent on self-subunit swapping. We successfully isolated a recombinant NHase activator protein (P14K) of Pseudomonas putida NRRL-18668 by adding a Strep-tag N-terminal to the P14K gene. P14K was found to form a complex [α(StrepP14K)2] with the α-subunit of the NHase. The incorporation of cobalt into the NHase of P. putida was confirmed to be dependent on the α-subunit substitution between the cobalt-containing α(StrepP14K)2 and the cobalt-free NHase. Cobalt was inserted into cobalt-free α(StrepP14K)2 but not into cobalt-free NHase, suggesting that P14K functions not only as a self-subunit swapping chaperone but also as a metallochaperone. In addition, NHase from P. putida was also expressed by a mutant gene that was designed with a <β-subunit> <α-subunit> order. Our findings expand the general features of self-subunit swapping maturation. PMID:23226397

  11. Removal of MAP4 from microtubules in vivo produces no observable phenotype at the cellular level

    PubMed Central

    1996-01-01

    Microtubule-associated protein 4 (MAP4) promotes MT assembly in vitro and is localized along MTs in vivo. These results and the fact that MAP4 is the major MAP in nonneuronal cells suggest that MAP4's normal functions may include the stabilization of MTs in situ. To understand MAP4 function in vivo, we produced a blocking antibody (Ab) to prevent MAP4 binding to MTs. The COOH-terminal MT binding domain of MAP4 was expressed in Escherichia coli as a glutathione transferase fusion protein and was injected into rabbits to produce an antiserum that was then affinity purified and shown to be monospecific for MAP4. This Ab blocked > 95% of MAP4 binding to MTs in an in vitro assay. Microinjection of the affinity purified Ab into human fibroblasts and monkey epithelial cells abolished MAP4 binding to MTs as assayed with a rat polyclonal antibody against the NH2-terminal projection domain of MAP4. The removal of MAP4 from MTs was accompanied by its sequestration into visible MAP4-Ab immunocomplexes. However, the MT network appeared normal. Tubulin photoactivation and nocodazole sensitivity assays indicated that MT dynamics were not altered detectably by the removal of MAP4 from the MTs. Cells progressed to mitosis with morphologically normal spindles in the absence of MAP4 binding to MTs. Depleting MAP4 from MTs also did not affect the state of posttranslational modifications of tubulin subunits. Further, no perturbations of MT- dependent organelle distribution were detected. We conclude that the association of MAP4 with MTs is not essential for MT assembly or for the MT-based functions in cultured cells that we could assay. A significant role for MAP4 is not excluded by these results, however, as MAP4 may be a component of a functionally redundant system. PMID:8636213

  12. Isolation of cDNA clones coding for the beta subunit of human beta-hexosaminidase.

    PubMed Central

    O'Dowd, B F; Quan, F; Willard, H F; Lamhonwah, A M; Korneluk, R G; Lowden, J A; Gravel, R A; Mahuran, D J

    1985-01-01

    The major forms of beta-hexosaminidase (2-acetamido-2-deoxy-beta-D-glucoside acetamidodeoxyglucohydrolase, EC 3.2.1.30) occur as multimers of alpha and beta chains--hexosaminidase A (alpha beta a beta b) and hexosaminidase B 2(beta a beta b). To facilitate the investigation of beta-chain biosynthesis and the nature of mutation in Sandhoff disease, a human hexosaminidase beta-chain cDNA clone was isolated. Hexosaminidase B (10 mg) was treated with CNBr, five peptide fragments were isolated by reverse-phase HPLC, and their amino acid sequences were determined. One of these contained a string of six amino acids from which an oligonucleotide probe was defined. The simian virus 40-transformed human fibroblast cDNA library of Okayama and Berg was screened by colony hybridization with the radiolabeled probe. Thirteen probe-binding clones were selected out of 50,000 clones screened. Four of these designated pHex were shown to be identical at their 3' ends by restriction enzyme mapping, differing only in their 5' extensions (1.4-1.7 kilobases). The nucleotide sequence of a 174-base-pair segment contained the deduced amino acid sequence of two of the five CNBr peptides, indicating that the pHex clones encode the beta subunit of hexosaminidase. In addition, pHex cDNA was found homologous to multiple bands in digests of genomic human DNA totaling 43 kilobases (kb), all of which were mapped to chromosome 5 in somatic cell hybrids, as expected of the HEXB gene. The pHex cDNA also hybridized to a 2.2-kilobase RNA that apparently codes for the pre-beta-polypeptide of hexosaminidase. This RNA species was absent in the fibroblasts of one of three patients with Sandhoff disease examined. We anticipate that these clones will be of value to diagnosis and carrier detection of Sandhoff disease in affected families. Images PMID:2579389

  13. Domain structure of HrpE, the Hrp pilus subunit of Xanthomonas campestris pv. vesicatoria.

    PubMed

    Weber, Ernst; Koebnik, Ralf

    2005-09-01

    The plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria possesses a type III secretion (TTS) system necessary for pathogenicity in susceptible hosts and induction of the hypersensitive response in resistant plants. This specialized protein transport system is encoded by a 23-kb hrp (hypersensitive response and pathogenicity) gene cluster. X. campestris pv. vesicatoria produces filamentous structures, Hrp pili, at the cell surface under hrp-inducing conditions. The Hrp pilus acts as a cell surface appendage of the TTS system and serves as a conduit for the transfer of bacterial effector proteins into the plant cell cytosol. The major pilus component, the HrpE pilin, is unique to xanthomonads and is encoded within the hrp gene cluster. In this study, functional domains of HrpE were mapped by linker-scanning mutagenesis and by reporter protein fusions to an N-terminally truncated avirulence protein (AvrBs3Delta2). Thirteen five-amino-acid peptide insertion mutants were obtained and could be grouped into six phenotypic classes. Three permissive mutations were mapped in the N-terminal half of HrpE, which is weakly conserved within the HrpE protein family. Four dominant-negative peptide insertions in the strongly conserved C-terminal region suggest that this domain is critical for oligomerization of the pilus subunits. Reporter protein fusions revealed that the N-terminal 17 amino acid residues act as an efficient TTS signal. From these results, we postulate a three-domain structure of HrpE with an N-terminal secretion signal, a surface-exposed variable region of the N-terminal half, and a C-terminal polymerization domain. Comparisons with a mutant study of HrpA, the Hrp pilin from Pseudomonas syringae pv. tomato DC3000, and hydrophobicity plot analyses of several nonhomologous Hrp pilins suggest a common architecture of Hrp pilins of different plant-pathogenic bacteria.

  14. Soybean ribulose bisphosphate carboxylase small subunit: Mechanisms and determinants of RNA turnover. Annual progress report

    SciTech Connect

    Meagher, R.B.

    1993-12-31

    An in vitro degradation system has been developed from petunia and soybean polysomes in order to investigate the mechanisms and determinants controlling RNA turnover in higher plants. This system faithfully degrades soybean ribulose-1,5-bisphosphate carboxylase small subunit (rbcS) mRNA into the same products observed in total RNA preparations. In previous years it was shown that the most stable products represent a nested constellation of fragments, which are shortened from their 3{prime} ends, and have intact 5{prime} ends. Exogenous rbcS RNA tagged with novel 5{prime} sequence 15 or 56 bp long were synthesized in vitro as Sp6 and T7 runoff transcripts, respectively. When added to the system they were degraded faithfully into constellation of products which were 15 or 56 bp longer than the endogenous products, respectively. Detailed kinetics on the appearance of these exogenous products confirmed degradation proceeds in an overall 3{prime} to 5{prime} direction but suggested that there are multiple pathways through which the RNA may be degraded. To further demonstrate a precursor product relationships, in vitro synthesized transcripts truncated at their 3{prime} ends were shown to degrade into the expected smaller fragments previously mapped in the 5{prime} portion of the rbcS RNA.

  15. Evaluation of a Subunit Vaccine to Infectious Hematopoietic Necrosis (IHN) Virus, 1984 FY Annual Report.

    SciTech Connect

    Leong, JoAnn Ching

    1985-07-01

    A prototype subunit vaccine to IHN virus is being developed by recombinant DNA techniques. The techniques involve the isolation and characterization of the glycoprotein gene, which encodes the viral protein responsible for inducing a protective immune response in fish. The viral glycoprotein gene has been cloned and a restriction map of the cloned gene has been prepared. Preliminary DNA sequence analysis of the cloned gene has been initiated so that manipulation of the gene for maximum expression in appropriate plasmid vectors is possible. A recombinant plasmid containing the viral gene inserted in the proper orientation adjacent to a very strong lambda promoter and ribosome binding site has been constructed. Evaluation of this recombinant plasmid for gene expression is being conducted. Immunization trials with purified viral glycoprotein indicate that fish are protected against lethal doses of IHNV after immersion and intraperitoneal methods of immunization. In addition, cross protection immunization trials indicate that Type 2 and Type 1 IHN virus produce glycoproteins that are cross-protective.

  16. Oligosaccharyltransferase Subunits Bind Polypeptide Substrate to Locally Enhance N-glycosylation*

    PubMed Central

    Jamaluddin, M. Fairuz B.; Bailey, Ulla-Maja; Schulz, Benjamin L.

    2014-01-01

    Oligosaccharyltransferase is a multiprotein complex that catalyzes asparagine-linked glycosylation of diverse proteins. Using yeast genetics and glycoproteomics, we found that transient interactions between nascent polypeptide and Ost3p/Ost6p, homologous subunits of oligosaccharyltransferase, were able to modulate glycosylation efficiency in a site-specific manner in vivo. These interactions were driven by hydrophobic and electrostatic complementarity between amino acids in the peptide-binding groove of Ost3p/Ost6p and the sequestered stretch of substrate polypeptide. Based on this dependence, we used in vivo scanning mutagenesis and in vitro biochemistry to map the precise interactions that affect site-specific glycosylation efficiency. We conclude that transient binding of substrate polypeptide by Ost3p/Ost6p increases glycosylation efficiency at asparagines proximal and C-terminal to sequestered sequences. We detail a novel mode of interaction between translocating nascent polypeptide and oligosaccharyltransferase in which binding to Ost3p/Ost6p segregates a short flexible loop of glycosylation-competent polypeptide substrate that is delivered to the oligosaccharyltransferase active site for efficient modification. PMID:25118247

  17. Phenotypic consequences of deletion of the {gamma}{sub 3}, {alpha}{sub 5}, or {beta}{sub 3} subunit of the type A {gamma}-aminobutyric acid receptor in mice

    SciTech Connect

    Culia, C.T.; Stubbs, L.J.; Montgomery, C.S.; Russell, L.B.; Rinchik, E.M.

    1994-03-29

    Three genes (Gabrg3, Gabra5, and Gabrb3) encoding the {gamma}{sub 3}, {alpha}{sub 5}, and {beta}{sub 3} subunits of the type A {gamma}-aminobutyric acid receptor, respectively, are known to map near the pink-eyed dilution (p) locus in mouse chromosome 7. This region shares homology with a segment of human chromosome 15 that is implicated in Angelman syndrome, an inherited neurobehavioral disorder. By mapping Gabrg3-Gabra5-Gabrb3-telomere. Like Gabrb3, neither the Gabra5 nor Gabrg3 gene is functionally imprinted in adult mouse brain. Mice deleted for all three subunits die at birth with a cleft palate, although there are rare survivors ({approximately} 5%) that do not have a cleft palate but do exhibit a neurological abnormality characterized by tremor, jerky gait, and runtiness. The authors have previously suggested that deficiency of the {beta}{sub 3} subunit may be responsible for the clefting defect. Most notably, however, in this report they describe mice carrying two overlapping, complementing p deletions that fail to express the {gamma}{sub 3} transcript, as well as mice from another line that express neither the {gamma}{sub 3} nor {alpha}{sub 5} transcripts. Surprisingly, mice from both of these lines are phenotypically normal and do not exhibit any of the neurological symptoms characteristic of the rare survivors that are deleted for all three ({gamma}{sub 3}, {alpha}{sub 5}, and {beta}{sub 3}) subunits. These mice therefore provide a whole-organism type A {gamma}-aminobutyric-acid receptor background that is devoid of any receptor subtypes that normally contain the {gamma}{sub 3} and/or {alpha}{sub 5} subunits. The absence of an overt neurological phenotype in mice lacking the {gamma}{sub 3} and/or {alpha}{sub 5} subunits also suggests that mutations in these genes are unlikely to provide useful animal models for Angelman syndrome in humans.

  18. Features of 80S mammalian ribosome and its subunits

    PubMed Central

    Budkevich, Tatyana V.; El'skaya, Anna V.; Nierhaus, Knud H.

    2008-01-01

    It is generally believed that basic features of ribosomal functions are universally valid, but a systematic test still stands out for higher eukaryotic 80S ribosomes. Here we report: (i) differences in tRNA and mRNA binding capabilities of eukaryotic and bacterial ribosomes and their subunits. Eukaryotic 40S subunits bind mRNA exclusively in the presence of cognate tRNA, whereas bacterial 30S do bind mRNA already in the absence of tRNA. 80S ribosomes bind mRNA efficiently in the absence of tRNA. In contrast, bacterial 70S interact with mRNA more productively in the presence rather than in the absence of tRNA. (ii) States of initiation (Pi), pre-translocation (PRE) and post-translocation (POST) of the ribosome were checked and no significant functional differences to the prokaryotic counterpart were observed including the reciprocal linkage between A and E sites. (iii) Eukaryotic ribosomes bind tetracycline with an affinity 15 times lower than that of bacterial ribosomes (Kd 30 μM and 1–2 μM, respectively). The drug does not effect enzymatic A-site occupation of 80S ribosomes in contrast to non-enzymatic tRNA binding to the A-site. Both observations explain the relative resistance of eukaryotic ribosomes to this antibiotic. PMID:18632761

  19. A small subunit processome protein promotes cancer by altering translation.

    PubMed

    Yang, H W; Kim, T-M; Song, S S; Menon, L; Jiang, X; Huang, W; Black, P M; Park, P J; Carroll, R S; Johnson, M D

    2015-08-20

    Dysregulation of ribosome biogenesis or translation can promote cancer, but the underlying mechanisms remain unclear. UTP18 is a component of the small subunit processome, a nucleolar multi-protein complex whose only known function is to cleave pre-ribosomal RNA to yield the 18S ribosomal RNA component of 40S ribosomal subunits. Here, we show that UTP18 also alters translation to promote stress resistance and growth, and that UTP18 is frequently gained and overexpressed in cancer. We observed that UTP18 localizes to the cytoplasm in a subset of cells, and that serum withdrawal increases cytoplasmic UTP18 localization. Cytoplasmic UTP18 associates with the translation complex and Hsp90 to upregulate the translation of IRES-containing transcripts such as HIF1a, Myc and VEGF, thereby inducing stress resistance. Hsp90 inhibition decreases cytoplasmic UTP18 and UTP18-induced increases in translation. Importantly, elevated UTP18 expression correlates with increased aggressiveness and decreased survival in numerous cancers. Enforced UTP18 overexpression promotes transformation and tumorigenesis, whereas UTP18 knockdown inhibits these processes. This stress adaptation mechanism is thus co-opted for growth by cancers, and its inhibition may represent a promising new therapeutic target.

  20. An assembly landscape for the 30S ribosomal subunit.

    PubMed

    Talkington, Megan W T; Siuzdak, Gary; Williamson, James R

    2005-12-01

    Self-assembling macromolecular machines drive fundamental cellular processes, including transcription, messenger RNA processing, translation, DNA replication and cellular transport. The ribosome, which carries out protein synthesis, is one such machine, and the 30S subunit of the bacterial ribosome is the preeminent model system for biophysical analysis of large RNA-protein complexes. Our understanding of 30S assembly is incomplete, owing to the challenges of monitoring the association of many components simultaneously. Here we have developed a method involving pulse-chase monitored by quantitative mass spectrometry (PC/QMS) to follow the assembly of the 20 ribosomal proteins with 16S ribosomal RNA during formation of the functional particle. These data represent a detailed and quantitative kinetic characterization of the assembly of a large multicomponent macromolecular complex. By measuring the protein binding rates at a range of temperatures, we find that local transformations throughout the assembling subunit have similar but distinct activation energies. Thus, the prevailing view of 30S assembly as a pathway proceeding through a global rate-limiting conformational change must give way to one in which the assembly of the complex traverses a landscape dotted with various local conformational transitions.

  1. Magnetic subunits within a single molecule-surface hybrid

    NASA Astrophysics Data System (ADS)

    Heß, Volkmar; Friedrich, Rico; Matthes, Frank; Caciuc, Vasile; Atodiresei, Nicolae; Bürgler, Daniel E.; Blügel, Stefan; Schneider, Claus M.

    2017-05-01

    Magnetic molecule-surface hybrids are ideal building blocks for molecular spintronic devices due to their appealing tailorable magnetic properties and nanoscale size. So far, assemblies of interacting molecular-surface hybrids needed for spintronic functionality were generated by depositing aromatic molecules onto transition-metal surfaces, resulting in a random arrangement of hybrid magnets due to the inherent and strong hybridization. Here, we demonstrate the formation of multiple intramolecular subunits within a single molecule-surface hybrid by means of spin-polarized scanning tunneling microscopy experiments and ab initio density functional theory calculations. This novel effect is realized by depositing a polycyclic aromatic molecule on a magnetic surface. A highly asymmetric chiral adsorption position induces different structural, electronic, and magnetic properties in each aromatic ring of the molecule. In particular, the induced molecular spin polarization near the Fermi energy varies among the rings due to site- and spin-dependent molecule-surface hybridization. Our results showcase a possible organic chemistry route of tailoring geometrically well-defined assemblies of magnetically distinguishable subunits in molecule-surface hybrids.

  2. Binding of ATP by pertussis toxin and isolated toxin subunits

    SciTech Connect

    Hausman, S.Z.; Manclark, C.R.; Burns, D.L. )

    1990-07-03

    The binding of ATP to pertussis toxin and its components, the A subunit and B oligomer, was investigated. Whereas, radiolabeled ATP bound to the B oligomer and pertussis toxin, no binding to the A subunit was observed. The binding of ({sup 3}H)ATP to pertussis toxin and the B oligomer was inhibited by nucleotides. The relative effectiveness of the nucleotides was shown to be ATP > GTP > CTP > TTP for pertussis toxin and ATP > GTP > TTP > CTP for the B oligomer. Phosphate ions inhibited the binding of ({sup 3}H)ATP to pertussis toxin in a competitive manner; however, the presence of phosphate ions was essential for binding of ATP to the B oligomer. The toxin substrate, NAD, did not affect the binding of ({sup 3}H)ATP to pertussis toxin, although the glycoprotein fetuin significantly decreased binding. These results suggest that the binding site for ATP is located on the B oligomer and is distinct from the enzymatically active site but may be located near the eukaryotic receptor binding site.

  3. Unraveling Subunit Cooperativity in Homotetrameric HCN2 Channels

    PubMed Central

    Benndorf, Klaus; Thon, Susanne; Schulz, Eckhard

    2012-01-01

    In a multimeric receptor protein, the binding of a ligand can modulate the binding of a succeeding ligand. This phenomenon, called cooperativity, is caused by the interaction of the receptor subunits. By using a complex Markovian model and a set of parameters determined previously, we analyzed how the successive binding of four ligands leads to a complex cooperative interaction of the subunits in homotetrameric HCN2 pacemaker channels. The individual steps in the model were characterized by Gibbs free energies for the equilibria and activation energies, specifying the affinity of the binding sites and the transition rates, respectively. Moreover, cooperative free energies were calculated for each binding step in both the closed and the open channel. We show that the cooperativity sequence positive-negative-positive determined for the binding affinity is generated by the combined effect of very different cooperativity sequences determined for the binding and unbinding rates, which are negative-negative-positive and no-negative-no, respectively. It is concluded that in the ligand-induced activation of HCN2 channels, the sequence of cooperativity based on the binding affinity is caused by two even qualitatively different sequences of cooperativity that are based on the rates of ligand binding and unbinding. PMID:23199914

  4. Mutations in the Drosophila Condensin Subunit dCAP-G

    PubMed Central

    Dej, Kimberley J.; Ahn, Caroline; Orr-Weaver, Terry L.

    2004-01-01

    Chromosomes are dynamic structures that are reorganized during the cell cycle to optimize them for distinct functions. SMC and non-SMC condensin proteins associate into complexes that have been implicated in the process of chromosome condensation. The roles of the individual non-SMC subunits of the complex are poorly understood, and mutations in the CAP-G subunit have not been described in metazoans. Here we elucidate a role for dCAP-G in chromosome condensation and cohesion in Drosophila. We illustrate the requirement of dCAP-G for condensation during prophase and prometaphase; however, we find that alternate mechanisms ensure that replicated chromosomes are condensed prior to metaphase. In contrast, dCAP-G is essential for chromosome condensation in metaphase of single, unreplicated sister chromatids, suggesting that there is an interplay between replicated chromatids and the condensin complex. In the dcap-g mutants, defects in sister-chromatid separation are also observed. Chromatid arms fail to resolve in prophase and are unable to separate at anaphase, whereas sister centromeres show aberrant separation in metaphase and successfully move to spindle poles at anaphase. We also identified a role for dCAP-G during interphase in regulating heterochromatic gene expression. PMID:15514062

  5. Smallpox subunit vaccine produced in planta confers protection in mice

    PubMed Central

    Golovkin, Maxim; Spitsin, Sergei; Andrianov, Vyacheslav; Smirnov, Yuriy; Xiao, Yuhong; Pogrebnyak, Natalia; Markley, Karen; Brodzik, Robert; Gleba, Yuri; Isaacs, Stuart N.; Koprowski, Hilary

    2007-01-01

    We report here the in planta production of the recombinant vaccinia virus B5 antigenic domain (pB5), an attractive component of a subunit vaccine against smallpox. The antigenic domain was expressed by using efficient transient and constitutive plant expression systems and tested by various immunization routes in two animal models. Whereas oral administration in mice or the minipig with collard-derived insoluble pB5 did not generate an anti-B5 immune response, intranasal administration of soluble pB5 led to a rise of B5-specific immunoglobulins, and parenteral immunization led to a strong anti-B5 immune response in both mice and the minipig. Mice immunized i.m. with pB5 generated an antibody response that reduced virus spread in vitro and conferred protection from challenge with a lethal dose of vaccinia virus. These results indicate the feasibility of producing safe and inexpensive subunit vaccines by using plant production systems. PMID:17428917

  6. An assembly landscape for the 30S ribosomal subunit

    PubMed Central

    Talkington, Megan W. T.; Siuzdak, Gary

    2005-01-01

    Self-assembling macromolecular machines drive fundamental cellular processes, including transcription, mRNA processing, translation, DNA replication, and cellular transport. The ribosome, which carries out protein synthesis, is one such machine, and the 30S subunit of the bacterial ribosome is the preeminent model system for biophysical analysis of large RNA-protein complexes. Our understanding of 30S assembly is incomplete, due to the challenges of monitoring the association of many components simultaneously. We have developed a new method involving pulse-chase monitored by quantitative mass spectrometry (PC/QMS) to follow the assembly of the 20 ribosomal proteins with 16S rRNA during formation of the functional particle. These data represent the first detailed and quantitative kinetic characterization of the assembly of a large multicomponent macromolecular complex. By measuring the protein binding rates at a range of temperatures, we have found that local transformations throughout the assembling subunit have similar but distinct activation energies. This observation shows that the prevailing view of 30S assembly as a pathway proceeding through a global rate-limiting conformational change must give way to a view in which the assembly of the complex traverses a landscape dotted with a variety of local conformational transitions. PMID:16319883

  7. Thermostable cross-protective subunit vaccine against Brucella species.

    PubMed

    Cherwonogrodzky, John W; Barabé, Nicole D; Grigat, Michelle L; Lee, William E; Poirier, Robert T; Jager, Scott J; Berger, Bradley J

    2014-12-01

    A subunit vaccine candidate was produced from Brucella suis 145 (biovar 4; expressing both the A antigen of Brucella abortus and the M antigen of Brucella melitensis). The preparation consisted mostly of polysaccharide (PS; >90% [wt/wt]; both cell-associated PS and exo-PS were combined) and a small amount of protein (1 to 3%) with no apparent nucleic acids. Vaccinated mice were protected (these had a statistically significant reduction in bacterial colonization compared to that of unvaccinated controls) when challenged with representative strains of three Brucella species most pathogenic for humans, i.e., B. abortus, B. melitensis, and B. suis. As little as 1 ng of the vaccine, without added adjuvant, protected mice against B. suis 145 infection (5 × 10(5) CFU), and a single injection of 1 μg of this subunit vaccine protected mice from B. suis 145 challenge for at least 14 months. A single immunization induced a serum IgG response to Brucella antigens that remained elevated for up to 9 weeks. The use of heat (i.e., boiling-water bath, autoclaving) in the vaccine preparation showed that it was thermostable. This method also ensured safety and security. The vaccine produced was immunogenic and highly protective against multiple strains of Brucella and represents a promising candidate for further evaluation.

  8. Signal transduction by the high-affinity GM-CSF receptor: two distinct cytoplasmic regions of the common beta subunit responsible for different signaling.

    PubMed Central

    Sato, N; Sakamaki, K; Terada, N; Arai, K; Miyajima, A

    1993-01-01

    The high-affinity receptors for granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin 3 (IL-3) and IL-5 consist of two subunits, alpha and beta. The alpha subunits are specific to each cytokine and the same beta subunit (beta c) is shared by these three receptors. Although none of these receptor subunits has intrinsic kinase activity, these cytokines induce protein tyrosine phosphorylation, activation of Ras, Raf-1 and MAP kinase, and transcriptional activation of nuclear proto-oncogenes such as c-myc, c-fos and c-jun. In this paper, we describe a detailed analysis of the signaling potential of the beta c subunit by using a series of cytoplasmic deletion mutants. The human beta c consists of 881 amino acid residues. A C-terminal deletion mutant of beta c at amino acid 763 (beta 763) induced phosphorylation of Shc and activation of Ras, Raf-1, MAP kinase and p70 S6 kinase, whereas a deletion at amino acid 626 (beta 626) induced none of these effects. The beta 763 mutant, as well as the full-length beta c, induced transcription of c-myc, c-fos and c-jun. Deletions at amino acid 517 (beta 517) and 626 (beta 626) induced c-myc and pim-1, but no induction of c-fos and c-jun was observed. GM-CSF increased phosphatidylinositol 3 kinase (PI3-K) activity in anti-phosphotyrosine immunoprecipitates from cells expressing beta 763 as well as beta c, whereas it was only marginally increased from cells expressing beta 517 or beta 626. Thus, there are at least two distinct regions within the cytoplasmic domain of beta c that are responsible for different signals, i.e. a membrane proximal region of approximately 60 amino acid residues upstream of Glu517 is essential for induction of c-myc and pim-1, and a distal region of approximately 140 amino acid residues (between Leu626 and Ser763) is required for activation of Ras, Raf-1, MAP kinase and p70 S6 kinase, as well as induction of c-fos and c-jun. Images PMID:8223433

  9. Membrane trafficking and mitochondrial abnormalities precede subunit c deposition in a cerebellar cell model of juvenile neuronal ceroid lipofuscinosis

    PubMed Central

    Fossale, Elisa; Wolf, Pavlina; Espinola, Janice A; Lubicz-Nawrocka, Tanya; Teed, Allison M; Gao, Hanlin; Rigamonti, Dorotea; Cattaneo, Elena; MacDonald, Marcy E; Cotman, Susan L

    2004-01-01

    Background JNCL is a recessively inherited, childhood-onset neurodegenerative disease most-commonly caused by a ~1 kb CLN3 mutation. The resulting loss of battenin activity leads to deposition of mitochondrial ATP synthase, subunit c and a specific loss of CNS neurons. We previously generated Cln3Δex7/8 knock-in mice, which replicate the common JNCL mutation, express mutant battenin and display JNCL-like pathology. Results To elucidate the consequences of the common JNCL mutation in neuronal cells, we used P4 knock-in mouse cerebella to establish conditionally immortalized CbCln3 wild-type, heterozygous, and homozygous neuronal precursor cell lines, which can be differentiated into MAP-2 and NeuN-positive, neuron-like cells. Homozygous CbCln3Δex7/8 precursor cells express low levels of mutant battenin and, when aged at confluency, accumulate ATPase subunit c. Recessive phenotypes are also observed at sub-confluent growth; cathepsin D transport and processing are altered, although enzyme activity is not significantly affected, lysosomal size and distribution are altered, and endocytosis is reduced. In addition, mitochondria are abnormally elongated, cellular ATP levels are decreased, and survival following oxidative stress is reduced. Conclusions These findings reveal that battenin is required for intracellular membrane trafficking and mitochondrial function. Moreover, these deficiencies are likely to be early events in the JNCL disease process and may particularly impact neuronal survival. PMID:15588329

  10. Troponin T3 regulates nuclear localization of the calcium channel Cavβ1a subunit in skeletal muscle.

    PubMed

    Zhang, Tan; Taylor, Jackson; Jiang, Yang; Pereyra, Andrea S; Messi, Maria Laura; Wang, Zhong-Min; Hereñú, Claudia; Delbono, Osvaldo

    2015-08-15

    The voltage-gated calcium channel (Cav) β1a subunit (Cavβ1a) plays an important role in excitation-contraction coupling (ECC), a process in the myoplasm that leads to muscle-force generation. Recently, we discovered that the Cavβ1a subunit travels to the nucleus of skeletal muscle cells where it helps to regulate gene transcription. To determine how it travels to the nucleus, we performed a yeast two-hybrid screening of the mouse fast skeletal muscle cDNA library and identified an interaction with troponin T3 (TnT3), which we subsequently confirmed by co-immunoprecipitation and co-localization assays in mouse skeletal muscle in vivo and in cultured C2C12 muscle cells. Interacting domains were mapped to the leucine zipper domain in TnT3 COOH-terminus (160-244 aa) and Cavβ1a NH2-terminus (1-99 aa), respectively. The double fluorescence assay in C2C12 cells co-expressing TnT3/DsRed and Cavβ1a/YFP shows that TnT3 facilitates Cavβ1a nuclear recruitment, suggesting that the two proteins play a heretofore unknown role during early muscle differentiation in addition to their classical role in ECC regulation. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. An S-phase specific release from a transcriptional block regulates the expression of mouse ribonucleotide reductase R2 subunit.

    PubMed Central

    Björklund, S; Skogman, E; Thelander, L

    1992-01-01

    Ribonucleotide reductase (RR) activity in mammalian cells is closely linked to DNA synthesis. The RR enzyme is composed of two non-identical subunits, proteins R1 and R2. Both proteins are required for holoenzyme activity, which is regulated by S-phase specific de novo synthesis and breakdown of the R2 subunit. In quiescent cells stimulated to proliferate and in elutriated cell populations enriched in the various cell cycle phases the R2 protein levels are correlated to R2 mRNA levels that are low in G0/G1-phase cells but increase dramatically at the G1/S border. Using an R2 promoter-luciferase reporter gene construct we demonstrate an unexpected early activation of the R2 promoter as cells pass from quiescence to proliferation. However, due to a transcriptional block, this promoter activation only results in very short R2 transcripts until cells enter the S-phase, when full-length R2 transcripts start to appear. The position for the transcriptional block was localized to a nucleotide sequence approximately 87 bp downstream from the first exon/intron boundary by S1 nuclease mapping of R2 transcripts from modified in vitro nuclear run-on experiments. These results identify blocking of transcription as a mechanism to control cell cycle regulated gene expression. Images PMID:1464320

  12. Proteolytic cleavage of the voltage-gated Ca2+ channel α2δ subunit: structural and functional features

    PubMed Central

    Andrade, Arturo; Sandoval, Alejandro; Oviedo, Norma; De Waard, Michel; Elias, David; Felix, Ricardo

    2007-01-01

    By mediating depolarization-induced Ca2+ influx high voltage-activated (HVA) Ca2+ channels control a variety of cellular events. These heteromultimeric proteins are composed of an ion-conducting (α1) and three auxiliary (α2δ, β and γ) subunits. The α2δ subunit enhances the trafficking of the channel complex to the cell surface and increases channel open probability. To exert these effects, α2δ must undergo important post-translational modifications including a proteolytic cleavage that separates the extracellular α2 from its transmembrane δ domain. After this proteolysis both domains remain linked by disulfide bonds. In spite of its central role in determining the final conformation of the fully mature α2δ almost nothing is known about the physiological implications of this structural modification. In the current report, by using site-directed mutagenesis, the proteolytic site of α2δ was mapped to amino acid residues Arg-941 and Val-946. Substitution of these residues renders the protein insensitive to proteolytic cleavage as evidenced by the lack of molecular weight shift upon treatment with a disulfide reducing agent. Interestingly, these mutations significantly decreased whole-cell patch clamp currents without affecting the voltage-dependence or kinetics of the channels, suggesting a reduction in the number of channels targeted to the plasma membrane. PMID:17408426

  13. Isolation and characterization of a novel gene from the DiGeorge chromosomal region that encodes for a mediator subunit.

    PubMed

    Berti, L; Mittler, G; Przemeck, G K; Stelzer, G; Günzler, B; Amati, F; Conti, E; Dallapiccola, B; Hrabé de Angelis, M; Novelli, G; Meisterernst, M

    2001-06-15

    Hemizygous deletions on chromosome 22q11.2 result in developmental disorders referred to as DiGeorge syndrome (DGS)/velocardiofacial syndrome (VCFS). We report the isolation of a novel gene, PCQAP (PC2 glutamine/Q-rich-associated protein), that maps to the DiGeorge typically deleted region and encodes a protein identified as a subunit of the large multiprotein complex PC2. PC2 belongs to the family of the human Mediator complexes, which exhibit coactivator function in RNA polymerase II transcription. Furthermore, we cloned the homologous mouse Pcqap cDNA. There is 83% amino acid identity between the human and the mouse predicted protein sequences, with 96% similarity at the amino- and carboxy-terminal ends. To assess the potential involvement of PCQAP in DGS/VCFS, its developmental expression pattern was analyzed. In situ hybridization of mouse embryos at different developmental stages revealed that Pcqap is ubiquitously expressed. However, higher expression was detected in the frontonasal region, pharyngeal arches, and limb buds. Moreover, analysis of subjects carrying a typical 22q11 deletion revealed that the human PCQAP gene was deleted in all patients. Many of the structures affected in DGS/VCFS evolve from Pcqap-expressing cells. Together with the observed haploinsufficiency of PCQAP in DGS/VCFS patients, this finding is consistent with a possible role for this novel Mediator subunit in the development of some of the structures affected in DGS/VCFS.

  14. Specific interaction of IP6 with human Ku70/80, the DNA-binding subunit of DNA-PK.

    PubMed

    Hanakahi, Les A; West, Stephen C

    2002-04-15

    In eukaryotic cells, DNA double-strand breaks can be repaired by non-homologous end-joining, a process dependent upon Ku70/80, XRCC4 and DNA ligase IV. In mammals, this process also requires DNA-PK(cs), the catalytic subunit of the DNA-dependent protein kinase DNA-PK. Previously, inositol hexakisphosphate (IP6) was shown to be bound by DNA-PK and to stimulate DNA-PK-dependent end-joining in vitro. Here, we localize IP6 binding to the Ku70/80 subunits of DNA- PK, and show that DNA-PK(cs) alone exhibits no detectable affinity for IP6. Moreover, proteolysis mapping of Ku70/80 in the presence and absence of IP6 indicates that binding alters the conformation of the Ku70/80 heterodimer. The yeast homologue of Ku70/80, yKu70/80, fails to bind IP6, indicating that the function of IP6 in non-homologous end-joining, like that of DNA-PK(cs), is unique to the mammalian end-joining process.

  15. Specific interaction of IP6 with human Ku70/80, the DNA-binding subunit of DNA-PK

    PubMed Central

    Hanakahi, Les A.; West, Stephen C.

    2002-01-01

    In eukaryotic cells, DNA double-strand breaks can be repaired by non-homologous end-joining, a process dependent upon Ku70/80, XRCC4 and DNA ligase IV. In mammals, this process also requires DNA-PKcs, the catalytic subunit of the DNA-dependent protein kinase DNA-PK. Previously, inositol hexakisphosphate (IP6) was shown to be bound by DNA-PK and to stimulate DNA-PK-dependent end-joining in vitro. Here, we localize IP6 binding to the Ku70/80 subunits of DNA- PK, and show that DNA-PKcs alone exhibits no detectable affinity for IP6. Moreover, proteolysis mapping of Ku70/80 in the presence and absence of IP6 indicates that binding alters the conformation of the Ku70/80 heterodimer. The yeast homologue of Ku70/80, yKu70/80, fails to bind IP6, indicating that the function of IP6 in non-homologous end-joining, like that of DNA-PKcs, is unique to the mammalian end-joining process. PMID:11953323

  16. Structural and binding studies of the C-terminal domains of yeast TFIIF subunits Tfg1 and Tfg2.

    PubMed

    Kilpatrick, Adina M; Koharudin, Leonardus M I; Calero, Guillermo A; Gronenborn, Angela M

    2012-02-01

    The general transcription factor TFIIF plays essential roles at several steps during eukaryotic transcription. While several studies have offered insights into the structure/function relationship in human TFIIF, much less is known about the yeast system. Here, we describe the first NMR structural and binding studies of the C-terminal domains (CTDs) of Tfg1 and Tfg2 subunits of Saccharomyces cerevisiae TFIIF. We used the program CS-ROSETTA to determine the three-dimensional folds of these domains in solution, and performed binding studies with DNA and protein targets. CS-ROSETTA models indicate that the Tfg1 and Tfg2 C-terminal domains have winged-helix architectures, similar to the human homologs. We showed that both Tfg1 and Tfg2 CTDs interact with double-stranded DNA oligonucleotides, and mapped the DNA binding interfaces using solution NMR. Tfg1-CTD, but not Tfg2-CTD, also binds to yeast FCP1, an RNA polymerase II-specific phosphatase, and we delineated the interaction surface with the CTD of FCP1. Our results provide insights into the structural basis of yeast TFIIF function and the differential roles of Tfg1 and Tfg2 subunits during transcription. Copyright © 2011 Wiley Periodicals, Inc.

  17. Troponin T3 regulates nuclear localization of the calcium channel Cavβ1a subunit in skeletal muscle

    PubMed Central

    Zhang, Tan; Taylor, Jackson; Jiang, Yang; Pereyra, Andrea S.; Messi, Maria Laura; Wang, Zhong-Min; Hereñú, Claudia; Delbono, Osvaldo

    2015-01-01

    The voltage-gated calcium channel (Cav) β1a subunit (Cavβ1a) plays an important role in excitation-contraction coupling (ECC), a process in the myoplasm that leads to muscle-force generation. Recently, we discovered that the Cavβ1a subunit travels to the nucleus of skeletal muscle cells where it helps to regulate gene transcription. To determine how it travels to the nucleus, we performed a yeast two-hybrid screening of the mouse fast skeletal muscle cDNA library and identified an interaction with troponin T3 (TnT3), which we subsequently confirmed by co-immunoprecipitation and co-localization assays in mouse skeletal muscle in vivo and in cultured C2C12 muscle cells. Interacting domains were mapped to the leucine zipper domain in TnT3 COOH-terminus (160-244 aa) and Cavβ1a NH2-terminus (1-99 aa), respectively. The double fluorescence assay in C2C12 cells co-expressing TnT3/DsRed and Cavβ1a/YFP shows that TnT3 facilitates Cavβ1a nuclear recruitment, suggesting that the two proteins play a heretofore unknown role during early muscle differentiation in addition to their classical role in ECC regulation. PMID:25981458

  18. The G protein β subunit controls virulence and multiple growth- and development-related traits in Verticillium dahliae.

    PubMed

    Tzima, Aliki K; Paplomatas, Epaminondas J; Tsitsigiannis, Dimitrios I; Kang, Seogchan

    2012-04-01

    To gain insight into the role of G protein-mediated signaling in virulence and development of the soilborne, wilt causing fungus Verticillium dahliae, the G protein β subunit gene (named as VGB) was disrupted in tomato race 1 strain of V. dahliae. A resulting mutant strain, 70ΔGb15, displayed drastic reduction in virulence, increased microsclerotia formation and conidiation, and decreased ethylene production compared to the corresponding wild type (wt) strain 70wt-r1. Moreover, 70ΔGb15 exhibited an elongated rather than radial growth pattern on agar media. A transformant of 70ΔGb15 (named as 70ΔGbPKAC1) that carries an extra copy of VdPKAC1, a V. dahliae gene encoding the catalytic subunit of the cAMP-dependent protein kinase A, exhibited wt growth pattern and conidiation, was unable to form microsclerotia, produced high amounts of ethylene, and exhibited virulence between that of 70ΔGb15 and 70wt-r1 on tomato plants. Phenotypical changes observed in 70ΔGb15 and 70ΔGbPKAC1 correlated with transcriptional changes in several genes involved in signaling (MAP kinase VMK1) and development (hydrophobin VDH1 and ACC synthase ACS1) of V. dahliae. Results from the present work suggest a linkage between VGB and VdPKAC1 signaling pathways in regulating virulence, hormone production and development in V. dahliae. Copyright © 2012 Elsevier Inc. All rights reserved.

  19. Gene structure, chromosomal localization, and expression pattern of Capn12, a new member of the calpain large subunit gene family.

    PubMed

    Dear, T N; Meier, N T; Hunn, M; Boehm, T

    2000-09-01

    We report the identification of mouse Capn12, a new member of the calpain large subunit gene family. It possesses potential protease and calcium-binding domains, features typical of the classical calpains. In situ hybridization and Northern blot analysis demonstrate that during the anagen phase of the hair cycle the cortex of the hair follicle is the major expression site of Capn12. The gene was sequenced in its entirety and consists of 21 exons spanning 13 kb with an exon-intron structure typical of the calpain gene family. The last exon of the mouse Actn4 gene overlaps the 3' end of Capn12 but in the opposite orientation. This overlap between the two genes is conserved in the human genome. Three versions of the Capn12 mRNA transcript were identified. They occur as a result of alternative splicing, and two of these encode a protein lacking the C-terminal calmodulin-like domain. Radiation hybrid mapping localized Capn12 to mouse chromosome 7, closely linked to a marker positioned at 10.4 cM. Refined mapping of Capn5, also previously localized to chromosome 7, indicated that it was not closely linked to Capn12, mapping tightly linked to a marker positioned at 48.5 cM.

  20. Mapping Human Epigenomes

    PubMed Central

    Rivera, Chloe M.; Ren, Bing

    2013-01-01

    As the second dimension to the genome, the epigenome contains key information specific to every type of cells. Thousands of human epigenome maps have been produced in recent years thanks to rapid development of high throughput epigenome mapping technologies. In this review, we discuss the current epigenome mapping toolkit and utilities of epigenome maps. We focus particularly on mapping of DNA methylation, chromatin modification state and chromatin structures, and emphasize the use of epigenome maps to delineate human gene regulatory sequences and developmental programs. We also provide a perspective on the progress of the epigenomics field and challenges ahead. PMID:24074860

  1. Distinct roles for ATP binding and hydrolysis at individual subunits of an archaeal clamp loader

    PubMed Central

    Seybert, Anja; Wigley, Dale B

    2004-01-01

    Circular clamps are utilised by replicative polymerases to enhance processivity. The topological problem of loading a toroidal clamp onto DNA is overcome by ATP-dependent clamp loader complexes. Different organisms use related protein machines to load clamps, but the mechanisms by which they utilise ATP are surprisingly different. Using mutant clamp loaders that are deficient in either ATP binding or hydrolysis in different subunits, we show how the different subunits of an archaeal clamp loader use ATP binding and hydrolysis in distinct ways at different steps in the loading process. Binding of nucleotide by the large subunit and three of the four small subunits is sufficient for clamp loading. However, ATP hydrolysis by the small subunits is required for release of PCNA to allow formation of the complex between PCNA and the polymerase, while hydrolysis by the large subunit is required for catalytic clamp loading. PMID:15014449

  2. The mongoose acetylcholine receptor alpha-subunit: analysis of glycosylation and alpha-bungarotoxin binding.

    PubMed

    Asher, O; Jensen, B S; Lupu-Meiri, M; Oron, Y; Fuchs, S

    1998-04-17

    The mongoose AChR alpha-subunit has been cloned and shown to be highly homologous to other AChR alpha-subunits, with only six differences in amino acid residues at positions that are conserved in animal species that bind alpha-bungarotoxin (alpha-BTX). Four of these six substitutions cluster in the ligand binding site, and one of them, Asn-187, forms a consensus N-glycosylation site. The mongoose glycosylated alpha-subunit has a higher apparent molecular mass than that of the rat glycosylated alpha-subunit, probably resulting from the additional glycosylation at Asn-187 of the mongoose subunit. The in vitro translated mongoose alpha-subunit, in a glycosylated or non-glycosylated form, does not bind alpha-BTX, indicating that lack of alpha-BTX binding can be achieved also in the absence of glycosylation.

  3. Crystal structure of yeast V-ATPase subunit C reveals its stator function

    PubMed Central

    Drory, Omri; Frolow, Felix; Nelson, Nathan

    2004-01-01

    Vacuolar H+-ATPase (V-ATPase) has a crucial role in the vacuolar system of eukaryotic cells. It provides most of the energy required for transport systems that utilize the proton-motive force that is generated by ATP hydrolysis. Some, but not all, of the V-ATPase subunits are homologous to those of F-ATPase and the nonhomologous subunits determine the unique features of V-ATPase. We determined the crystal structure of V-ATPase subunit C (Vma5p), which does not show any homology with F-ATPase subunits, at 1.75 Å resolution. The structural features suggest that subunit C functions as a flexible stator that holds together the catalytic and membrane sectors of the enzyme. A second crystal form that was solved at 2.9 Å resolution supports the flexible nature of subunit C. These structures provide a framework for exploring the unique mechanistic features of V-ATPases. PMID:15540116

  4. Operon structure and cotranslational subunit association direct protein assembly in bacteria.

    PubMed

    Shieh, Yu-Wei; Minguez, Pablo; Bork, Peer; Auburger, Josef J; Guilbride, D Lys; Kramer, Günter; Bukau, Bernd

    2015-11-06

    Assembly of protein complexes is considered a posttranslational process involving random collision of subunits. We show that within the Escherichia coli cytosol, bacterial luciferase subunits LuxA and LuxB assemble into complexes close to the site of subunit synthesis. Assembly efficiency decreases markedly if subunits are synthesized on separate messenger RNAs from genes integrated at distant chromosomal sites. Subunit assembly initiates cotranslationally on nascent LuxB in vivo. The ribosome-associated chaperone trigger factor delays the onset of cotranslational interactions until the LuxB dimer interface is fully exposed. Protein assembly is thus directly coupled to the translation process and involves spatially confined, actively chaperoned cotranslational subunit interactions. Bacterial gene organization into operons therefore reflects a fundamental cotranslational mechanism for spatial and temporal regulation that is vital to effective assembly of protein complexes. Copyright © 2015, American Association for the Advancement of Science.

  5. [Chromatographic and spectroscopic characterization of phycocyanin and its subunits purified from Anabaena variabilis CCC421].

    PubMed

    Chakdar, N; Sakha, S; Pabbi, S

    2014-01-01

    Phycocyanin, a high value pigment was purified from diazotrophic cyanobacteria Anabaena variabilis CCC421 using a strategy involving ammonium sulfate precipitation, dialysis and anion exchange chromatography using DEAE-cellulose column. 36% phycocyanin with a purity of 2.75 was recovered finally after anion exchange chromatography. Purified phycocyanin was found to contain 2 subunits of 17 and 18 kDa which were identified as a-and (3 subunits by SDS-PAGE and MALDI-TOE HPLC method using a C5 column coupled with fluorescence or photodiode-based detection was also developed to separate and detect the A. variabilis CCC421 phycocyanin subunits. The fluorescence method was more sensitive than photodiode one. The purified phycocyanin from A. variabilis CCC421 as well as its subunits was characterized with respect to absorption and IR spectra. Spectral characterization of the subunits revealed that alpha and beta subunits contained one and two phycocyanobilin groups as chromophores, respectively.

  6. Redox-sensitive extracellular gates formed by auxiliary beta subunits of calcium-activated potassium channels.

    PubMed

    Zeng, Xu-Hui; Xia, Xiao-Ming; Lingle, Christopher J

    2003-06-01

    An important step to understanding ion channels is identifying the structural components that act as the gates to ion movement. Here we describe a new channel gating mechanism, produced by the beta3 auxiliary subunits of Ca2+-activated, large-conductance BK-type K+ channels when expressed with their pore-forming alpha subunits. BK beta subunits have a cysteine-rich extracellular segment connecting two transmembrane segments, with small cytosolic N and C termini. The extracellular segments of the beta3 subunits form gates to block ion permeation, providing a mechanism by which current can be rapidly diminished upon cellular repolarization. Furthermore, this gating mechanism is abolished by reduction of extracellular disulfide linkages, suggesting that endogenous mechanisms may regulate this gating behavior. The results indicate that auxiliary beta subunits of BK channels reside sufficiently close to the ion permeation pathway defined by the alpha subunits to influence or block access of small molecules to the permeation pathway.

  7. Palmitoylation of the β4-Subunit Regulates Surface Expression of Large Conductance Calcium-activated Potassium Channel Splice Variants*

    PubMed Central

    Chen, Lie; Bi, Danlei; Tian, Lijun; McClafferty, Heather; Steeb, Franziska; Ruth, Peter; Knaus, Hans Guenther; Shipston, Michael J.

    2013-01-01

    Regulatory β-subunits of large conductance calcium- and voltage-activated potassium (BK) channels play an important role in generating functional diversity and control of cell surface expression of the pore forming α-subunits. However, in contrast to α-subunits, the role of reversible post-translational modification of intracellular residues on β-subunit function is largely unknown. Here we demonstrate that the human β4-subunit is S-acylated (palmitoylated) on a juxtamembrane cysteine residue (Cys-193) in the intracellular C terminus of the regulatory β-subunit. β4-Subunit palmitoylation is important for cell surface expression and endoplasmic reticulum (ER) exit of the β4-subunit alone. Importantly, palmitoylated β4-subunits promote the ER exit and surface expression of the pore-forming α-subunit, whereas β4-subunits that cannot be palmitoylated do not increase ER exit or surface expression of α-subunits. Strikingly, however, this palmitoylation- and β4-dependent enhancement of α-subunit surface expression was only observed in α-subunits that contain a putative trafficking motif (… REVEDEC) at the very C terminus of the α-subunit. Engineering this trafficking motif to other C-terminal α-subunit splice variants results in α-subunits with reduced surface expression that can be rescued by palmitoylated, but not depalmitoylated, β4-subunits. Our data reveal a novel mechanism by which palmitoylated β4-subunit controls surface expression of BK channels through masking of a trafficking motif in the C terminus of the α-subunit. As palmitoylation is dynamic, this mechanism would allow precise control of specific splice variants to the cell surface. Our data provide new insights into how complex interplay between the repertoire of post-transcriptional and post-translational mechanisms controls cell surface expression of BK channels. PMID:23504458

  8. Mutations in the embryonal subunit of the acetylcholine receptor (CHRNG) cause lethal and Escobar variants of multiple pterygium syndrome.

    PubMed

    Morgan, Neil V; Brueton, Louise A; Cox, Phillip; Greally, Marie T; Tolmie, John; Pasha, Shanaz; Aligianis, Irene A; van Bokhoven, Hans; Marton, Tamas; Al-Gazali, Lihadh; Morton, Jenny E V; Oley, Christine; Johnson, Colin A; Trembath, Richard C; Brunner, Han G; Maher, Eamonn R

    2006-08-01

    Multiple pterygium syndromes (MPSs) comprise a group of multiple-congenital-anomaly disorders characterized by webbing (pterygia) of the neck, elbows, and/or knees and joint contractures (arthrogryposis). In addition, a variety of developmental defects (e.g., vertebral anomalies) may occur. MPSs are phenotypically and genetically heterogeneous but are traditionally divided into prenatally lethal and nonlethal (Escobar) types. To elucidate the pathogenesis of MPS, we undertook a genomewide linkage scan of a large consanguineous family and mapped a locus to 2q36-37. We then identified germline-inactivating mutations in the embryonal acetylcholine receptor gamma subunit (CHRNG) in families with both lethal and nonlethal MPSs. These findings extend the role of acetylcholine receptor dysfunction in human disease and provide new insights into the pathogenesis and management of fetal akinesia syndromes.

  9. Conditionally lethal ribosomal protein mutants: characterization of a locus required for modification of 50S subunit proteins.

    PubMed Central

    Kushner, S R; Maples, V F; Champney, W S

    1977-01-01

    Mutagenized P1 bacteriophage were used to transduce a marker (aroE) adjacent to the cluster or ribosomal protein genes located at 72 min on the Escherichia coli chromosome. Linked temperature-sensitive transductants were isolated and characterized. A mutant unable to grow at 44 degrees was found to be defective in protein synthesis both in vivo and in vitro. At the restrictive temperature mutant cells lost all polyribosomes. Analysis of the ribosomal proteins revealed alterations in at least four 50S subunit proteins. The mutation (called rimE1, ribosomal protein modification) mapped between rpsE and aroE. It is suggested that the rimE locus is the structural gene for an activity that modifies a selected number of ribosomal proteins. Images PMID:322127

  10. Efficient expression of functional (α6β2)2β3 AChRs in Xenopus oocytes from free subunits using slightly modified α6 subunits.

    PubMed

    Ley, Carson Kai-Kwong; Kuryatov, Alexander; Wang, Jingyi; Lindstrom, Jon Martin

    2014-01-01

    Human (α6β2)(α4β2)β3 nicotinic acetylcholine receptors (AChRs) are essential for addiction to nicotine and a target for drug development for smoking cessation. Expressing this complex AChR is difficult, but has been achieved using subunit concatamers. In order to determine what limits expression of α6* AChRs and to efficiently express α6* AChRs using free subunits, we investigated expression of the simpler (α6β2)2β3 AChR. The concatameric form of this AChR assembles well, but is transported to the cell surface inefficiently. Various chimeras of α6 with the closely related α3 subunit increased expression efficiency with free subunits and produced pharmacologically equivalent functional AChRs. A chimera in which the large cytoplasmic domain of α6 was replaced with that of α3 increased assembly with β2 subunits and transport of AChRs to the oocyte surface. Another chimera replacing the unique methionine 211 of α6 with leucine found at this position in transmembrane domain 1 of α3 and other α subunits increased assembly of mature subunits containing β3 subunits within oocytes. Combining both α3 sequences in an α6 chimera increased expression of functional (α6β2)2β3 AChRs to 12-fold more than with concatamers. This is pragmatically useful, and provides insights on features of α6 subunit structure that limit its expression in transfected cells.

  11. Efficient Expression of Functional (α6β2)2β3 AChRs in Xenopus Oocytes from Free Subunits Using Slightly Modified α6 Subunits

    PubMed Central

    Ley, Carson Kai-Kwong; Kuryatov, Alexander; Wang, Jingyi; Lindstrom, Jon Martin

    2014-01-01

    Human (α6β2)(α4β2)β3 nicotinic acetylcholine receptors (AChRs) are essential for addiction to nicotine and a target for drug development for smoking cessation. Expressing this complex AChR is difficult, but has been achieved using subunit concatamers. In order to determine what limits expression of α6* AChRs and to efficiently express α6* AChRs using free subunits, we investigated expression of the simpler (α6β2)2β3 AChR. The concatameric form of this AChR assembles well, but is transported to the cell surface inefficiently. Various chimeras of α6 with the closely related α3 subunit increased expression efficiency with free subunits and produced pharmacologically equivalent functional AChRs. A chimera in which the large cytoplasmic domain of α6 was replaced with that of α3 increased assembly with β2 subunits and transport of AChRs to the oocyte surface. Another chimera replacing the unique methionine 211 of α6 with leucine found at this position in transmembrane domain 1 of α3 and other α subunits increased assembly of mature subunits containing β3 subunits within oocytes. Combining both α3 sequences in an α6 chimera increased expression of functional (α6β2)2β3 AChRs to 12-fold more than with concatamers. This is pragmatically useful, and provides insights on features of α6 subunit structure that limit its expression in transfected cells. PMID:25068303

  12. Restriction mapping of synthetic thyroglobulin structural gene as a means of investigating thyroglobulin structure.

    PubMed

    Vassart, G; Brocas, H

    1980-11-14

    Bovine 33 S thyroglobulin mRNA was reverse transcribed into double-stranded DNA under conditions allowing the synthesis of a complete 8 kilobase pair copy. A physical map of the resulting synthetic thyroglobulin structural gene was constructed using six restriction endonucleases. The following conclusions could be drawn: (i) the polypeptide chains in thyroglobulin subunits are identical; (ii) thyroglobulin is composed of a major class of molecules sharing the same primary structure; (iii) there is no evidence for precise internal repetition in the structure of thyroglobulin subunits.

  13. The NH2-terminal php domain of the alpha subunit of the Escherichia coli replicase binds the epsilon proofreading subunit.

    PubMed

    Wieczorek, Anna; McHenry, Charles S

    2006-05-05

    The alpha subunit of the replicase of all bacteria contains a php domain, initially identified by its similarity to histidinol phosphatase but of otherwise unknown function (Aravind, L., and Koonin, E. V. (1998) Nucleic Acids Res. 26, 3746-3752). Deletion of 60 residues from the NH2 terminus of the alpha php domain destroys epsilon binding. The minimal 255-residue php domain, estimated by sequence alignment with homolog YcdX, is insufficient for epsilon binding. However, a 320-residue segment including sequences that immediately precede the polymerase domain binds epsilon with the same affinity as the 1160-residue full-length alpha subunit. A subset of mutations of a conserved acidic residue (Asp43 in Escherichia coli alpha) present in the php domain of all bacterial replicases resulted in defects in epsilon binding. Using sequence alignments, we show that the prototypical gram+ Pol C, which contains the polymerase and proofreading activities within the same polypeptide chain, has an epsilon-like sequence inserted in a surface loop near the center of the homologous YcdX protein. These findings suggest that the php domain serves as a platform to enable coordination of proofreading and polymerase activities during chromosomal replication.

  14. The Role of Auxiliary Subunits for the Functional Diversity of Voltage-Gated Calcium Channels

    PubMed Central

    Campiglio, Marta; Flucher, Bernhard E

    2015-01-01

    Voltage-gated calcium channels (VGCCs) represent the sole mechanism to convert membrane depolarization into cellular functions like secretion, contraction, or gene regulation. VGCCs consist of a pore-forming α1 subunit and several auxiliary channel subunits. These subunits come in multiple isoforms and splice-variants giving rise to a stunning molecular diversity of possible subunit combinations. It is generally believed that specific auxiliary subunits differentially regulate the channels and thereby contribute to the great functional diversity of VGCCs. If auxiliary subunits can associate and dissociate from pre-existing channel complexes, this would allow dynamic regulation of channel properties. However, most auxiliary subunits modulate current properties very similarly, and proof that any cellular calcium channel function is indeed modulated by the physiological exchange of auxiliary subunits is still lacking. In this review we summarize available information supporting a differential modulation of calcium channel functions by exchange of auxiliary subunits, as well as experimental evidence in support of alternative functions of the auxiliary subunits. At the heart of the discussion is the concept that, in their native environment, VGCCs function in the context of macromolecular signaling complexes and that the auxiliary subunits help to orchestrate the diverse protein–protein interactions found in these calcium channel signalosomes. Thus, in addition to a putative differential modulation of current properties, differential subcellular targeting properties and differential protein–protein interactions of the auxiliary subunits may explain the need for their vast molecular diversity. J. Cell. Physiol. 999: 00–00, 2015. © 2015 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc. J. Cell. Physiol. 230: 2019–2031, 2015. © 2015 Wiley Periodicals, Inc. PMID:25820299

  15. Interspecific luciferase beta subunit hybrids between Vibrio harveyi, Vibrio fischeri and Photobacterium leiognathi.

    PubMed

    Almashanu, S; Gendler, I; Hadar, R; Kuhn, J

    1996-09-01

    Bacterial luciferase (EC 1.14.14.3) is a heterodimer composed of alpha- and beta-chains encoded by luxA and luxB, respectively. Although some interspecific combinations of these subunits lead to active enzyme, others do not. The beta subunits of Vibrio fischeri and Photobacterium leiognathi form active enzyme with the alpha subunits of V.fischeri, P.leiognathi and Vibrio harveyi, while the beta subunit from V.harveyi only complements the alpha subunit of V.harveyi. Inactivity is caused by a lack of dimerization of the beta subunit of V.harveyi with the alpha subunits of V.fischeri and P.leiognathi. These observations served as the basis for a search to discover which segment of the beta polypeptide confers the ability to dimerize with the alpha subunits of V.fischeri and P.leiognathi. Intragenic beta subunit hybrids were made between V.harveyi, V.fischeri and P.leiognathi. Unique restriction sites were introduced into the respective luxB genes to divide them into four roughly equal segments. In all, 78 hybrids were constructed by in vitro techniques. The N-terminal segment of the peptide contains the signals that differentiate between the beta subunits of V.fischeri and P. leiognathi and the beta subunit of V. harveyi, and allow the former to dimerize with their alpha subunits. The second segment has no major effect on enzyme activity but does exhibit some context effects. Important interactions were found between the third and fourth segments of the polypeptide with respect to enzymatic activity.

  16. Accessory subunits are integral for assembly and function of human mitochondrial complex I.

    PubMed

    Stroud, David A; Surgenor, Elliot E; Formosa, Luke E; Reljic, Boris; Frazier, Ann E; Dibley, Marris G; Osellame, Laura D; Stait, Tegan; Beilharz, Traude H; Thorburn, David R; Salim, Agus; Ryan, Michael T

    2016-10-06

    Complex I (NADH:ubiquinone oxidoreductase) is the first enzyme of the mitochondrial respiratory chain and is composed of 45 subunits in humans, making it one of the largest known multi-subunit membrane protein complexes. Complex I exists in supercomplex forms with respiratory chain complexes III and IV, which are together required for the generation of a transmembrane proton gradient used for the synthesis of ATP. Complex I is also a major source of damaging reactive oxygen species and its dysfunction is associated with mitochondrial disease, Parkinson's disease and ageing. Bacterial and human complex I share 14 core subunits that are essential for enzymatic function; however, the role and necessity of the remaining 31 human accessory subunits is unclear. The incorporation of accessory subunits into the complex increases the cellular energetic cost and has necessitated the involvement of numerous assembly factors for complex I biogenesis. Here we use gene editing to generate human knockout cell lines for each accessory subunit. We show that 25 subunits are strictly required for assembly of a functional complex and 1 subunit is essential for cell viability. Quantitative proteomic analysis of cell lines revealed that loss of each subunit affects the stability of other subunits residing in the same structural module. Analysis of proteomic changes after the loss of specific modules revealed that ATP5SL and DMAC1 are required for assembly of the distal portion of the complex I membrane arm. Our results demonstrate the broad importance of accessory subunits in the structure and function of human complex I. Coupling gene-editing technology with proteomics represents a powerful tool for dissecting large multi-subunit complexes and enables the study of complex dysfunction at a cellular level.

  17. Evidence for inhibition mediated by coassembly of GABAA and GABAC receptor subunits in native central neurons.

    PubMed

    Milligan, Carol J; Buckley, Noel J; Garret, Maurice; Deuchars, Jim; Deuchars, Susan A

    2004-08-18

    Fast inhibition in the nervous system is commonly mediated by GABA(A) receptors comprised of 2alpha/2beta/1gamma subunits. In contrast, GABA(C) receptors containing only rho subunits (rho1-rho3) have been predominantly detected in the retina. However, here using reverse transcription-PCR and in situ hybridization we show that mRNA encoding the rho1 subunit is highly expressed in brainstem neurons. Immunohistochemistry localized the rho1 subunit to neurons at light and electron microscopic levels, where it was detected at synaptic junctions. Application of the GABA(C) receptor agonist cis-4-aminocrotonic acid (100-800 microM) requires the rho1 subunit to elicit responses, which surprisingly are blocked independently by antagonists to GABA(A) (bicuculline, 10 microM) and GABA(C) [(1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA); 40-160 microM] receptors. Responses to GABA(C) agonists were also enhanced by the GABA(A) receptor modulator pentobarbitone (300 microM). Spontaneous and evoked IPSPs were reduced in amplitude but never abolished by TPMPA, but were completely blocked by bicuculline. We therefore tested the hypothesis that GABA(A) and GABA(C) subunits formed a heteromeric receptor. Immunohistochemistry indicated that rho1 and alpha1 subunits were colocalized at light and electron microscopic levels. Electrophysiology revealed that responses to GABA(C) receptor agonists were enhanced by the GABA(A) receptor modulator zolpidem (500 nm), which acts on the alpha1 subunit when the gamma2 subunit is also present. Finally, coimmunoprecipitation indicated that the rho1 subunit formed complexes that also containedalpha1 and gamma2 subunits. Taken together these separate lines of evidence suggest that the effects of GABA in central neurons can be mediated by heteromeric complexes of GABA(A) and GABA(C) receptor subunits.

  18. The NMDA receptor NR2A subunit regulates proliferation of MKN45 human gastric cancer cells

    SciTech Connect

    Watanabe, Kanako; Kanno, Takeshi; Oshima, Tadayuki; Miwa, Hiroto; Tashiro, Chikara; Nishizaki, Tomoyuki

    2008-03-07

    The present study investigated proliferation of MKN28 and MKN45 human gastric cancer cells regulated by the N-methyl-D-aspartate (NMDA) receptor subunit. The NMDA receptor antagonist DL-2-amino-5-phosphonovaleric acid (AP5) inhibited proliferation of MKN45 cells, but not MKN28 cells. Of the NMDA subunits such as NR1, NR2 (2A, 2B, 2C, and 2D), and NR3 (3A and 3B), all the NMDA subunit mRNAs except for the NR2B subunit mRNA were expressed in both MKN28 and MKN45 cells. MKN45 cells were characterized by higher expression of the NR2A subunit mRNA and lower expression of the NR1 subunit mRNA, but MKN28 otherwise by higher expression of the NR1 subunit mRNA and lower expression of the NR2A subunit mRNA. MKN45 cell proliferation was also inhibited by silencing the NR2A subunit-targeted gene. For MKN45 cells, AP5 or knocking-down the NR2A subunit increased the proportion of cells in the G{sub 1} phase of cell cycling and decreased the proportion in the S/G{sub 2} phase. The results of the present study, thus, suggest that blockage of NMDA receptors including the NR2A subunit suppresses MKN45 cell proliferation due to cell cycle arrest at the G{sub 1} phase; in other words, the NR2A subunit promotes MKN45 cell proliferation by accelerating cell cycling.

  19. Subunits of the Snf1 kinase heterotrimer show interdependence for association and activity.

    PubMed

    Elbing, Karin; Rubenstein, Eric M; McCartney, Rhonda R; Schmidt, Martin C

    2006-09-08

    The Snf1 kinase and its mammalian orthologue, the AMP-activated protein kinase (AMPK), function as heterotrimers composed of a catalytic alpha-subunit and two non-catalytic subunits, beta and gamma. The beta-subunit is thought to hold the complex together and control subcellular localization whereas the gamma-subunit plays a regulatory role by binding to and blocking the function of an auto-inhibitory domain (AID) present in the alpha-subunit. In addition, catalytic activity requires phosphorylation by a distinct upstream kinase. In yeast, any one of three Snf1-activating kinases, Sak1, Tos3, or Elm1, can fulfill this role. We have previously shown that Sak1 is the only Snf1-activating kinase that forms a stable complex with Snf1. Here we show that the formation of the Sak1.Snf1 complex requires the beta- and gamma-subunits in vivo. However, formation of the Sak1.Snf1 complex is not necessary for glucose-regulated phosphorylation of the Snf1 activation loop. Snf1 kinase purified from cells lacking the beta-subunits do not contain any gamma-subunit, indicating that the Snf1 kinase does not form a stable alphagamma dimer in vivo. In vitro kinase assays using purified full-length and truncated Snf1 proteins demonstrate that the kinase domain, which lacks the AID, is significantly more active than the full-length Snf1 protein. Addition of purified beta- and gamma-subunits could stimulate the kinase activity of the full-length alpha-subunit but only when all three subunits were present, suggesting an interdependence of all three subunits for assembly of a functional complex.

  20. Antibodies to the. cap alpha. -subunit of insulin receptor from eggs of immunized hens

    SciTech Connect

    Song, C.; Yu, J.; Bai, D.H.; Hester, P.Y.; Kim, K.

    1985-11-01

    Simple methods for the generation, purification, and assay of antibodies to the ..cap alpha..-subunit of insulin receptor from eggs of immunized hen have been described. Chicken antibodies against the ..cap alpha..-subunit inhibit insulin binding to the receptor and stimulate glucose oxidation as well as autophosphorylation of the ..beta..-subunit. Thus the properties of chicken antibodies are very similar to those of antibodies found in human autoimmune diseases and different from rabbit antibodies obtained against the same antigen.

  1. GABAA receptors are differentially sensitive to zinc: dependence on subunit composition.

    PubMed Central

    Smart, T. G.; Moss, S. J.; Xie, X.; Huganir, R. L.

    1991-01-01

    GABAA receptors with different subunit composition, were expressed in kidney cells and studied by whole cell recording. Expressed GABAA receptors were differentially sensitive to inhibition by zinc; receptors which lacked the gamma subunit were inhibited by zinc. Embryonic neurons also exhibited zinc-sensitive GABA responses, in contrast to adult neurones. This developmentally-sensitive aspect of GABAA receptor pharmacology may be partly dependent on expression of the gamma subunit. PMID:1655141

  2. Analyses of Subnanometer Resolution Cryo-EM Density Maps

    PubMed Central

    Baker, Matthew L.; Baker, Mariah R.; Hryc, Corey F.; DiMaio, Frank

    2011-01-01

    Today, electron cryomicroscopy (cryo-EM) can routinely achieve subnanometer resolutions of complex macromolecular assemblies. From a density map, one can extract key structural and functional information using a variety of computational analysis tools. At subnanometer resolution, these tools make it possible to isolate individual subunits, identify secondary structures, and accurately fit atomic models. With several cryo-EM studies achieving resolutions beyond 5 Å, computational modeling and feature recognition tools have been employed to construct backbone and atomic models of the protein components directly from a density map. In this chapter, we describe several common classes of computational tools that can be used to analyze and model subnanometer resolution reconstructions from cryo-EM. A general protocol for analyzing subnanometer resolution density maps is presented along with a full description of steps used in analyzing the 4.3 Å resolution structure of Mm-cpn. PMID:20888467

  3. Mapping of Daedalia Planum Lava Field

    NASA Astrophysics Data System (ADS)

    Giacomini, Lorenza; Carli, Cristian; Massironi, Matteo; Pasquarè, Giorgio; Sgavetti, Maria

    2010-05-01

    Daedalia Planum is one of the Tharsis volcanic plains and is located southwest of the Arsia Mons. MOLA, THEMIS, MOC and OMEGA data have been analysed, providing a multi-scale characterisation of this Martian lava field. According to Mars Global Surveyor's MOLA data, the flanks of Arsia have an average slope <5°, while the surrounding regions, including Daedalia Planum, have slopes <0,5° and commonly <0,1°. Mars Odyssey/THEMIS VIS and IR images show a plain covered by a huge number of lava flows. Older and larger lava flows on the field have a length greater than ~1500 km. Moreover most of the Daedalia flows are associated to wrinkly and ropy surfaces, typical of pahoehoe lavas. On the base of the morphology differences among the flows and through stratigraphic relationships we performed a geological map of the area. MEX/OMEGA spectra were collected in different areas of the lava field. Besides the similar absorption bands OMEGA spectra showed also some differences in reflectance and spectral slope. The spectral map created using the SAM classification reveals that these spectral variations are generally in agreement with the lava flows mapped previously on the base of the flows morphology and stratigraphy. This suggested that such variability is related with different surface textures of the lava flow. Moreover in some cases spectral map highlighted the presence of spectral subunits inside the same stratigraphic unit, due likely to a different mineralogy or rock textures. Therefore spectral analysis revealed useful to improve the geological mapping of the Daedalia Planum region.

  4. RadMap

    EPA Pesticide Factsheets

    RadMap is an interactive desktop tool featuring a nationwide geographic information systems (GIS) map of long-term radiation monitoring locations across the United States with access to key information about the monitor and the area surrounding it.

  5. Creative Concept Mapping.

    ERIC Educational Resources Information Center

    Brown, David S.

    2002-01-01

    Recommends the use of concept mapping in science teaching and proposes that it be presented as a creative activity. Includes a sample lesson plan of a potato stamp concept mapping activity for astronomy. (DDR)

  6. Riparian Wetlands: Mapping

    EPA Science Inventory

    Riparian wetlands are critical systems that perform functions and provide services disproportionate to their extent in the landscape. Mapping wetlands allows for better planning, management, and modeling, but riparian wetlands present several challenges to effective mapping due t...

  7. Using maps in genealogy

    USGS Publications Warehouse

    ,

    1994-01-01

    In genealogy, maps are most often used as clues to where public or other records about an ancestor are likely to be found. Searching for maps seldom begins until a newcomer to genealogy has mastered basic genealogical routines

  8. Creative Concept Mapping.

    ERIC Educational Resources Information Center

    Brown, David S.

    2002-01-01

    Recommends the use of concept mapping in science teaching and proposes that it be presented as a creative activity. Includes a sample lesson plan of a potato stamp concept mapping activity for astronomy. (DDR)

  9. Riparian Wetlands: Mapping

    EPA Science Inventory

    Riparian wetlands are critical systems that perform functions and provide services disproportionate to their extent in the landscape. Mapping wetlands allows for better planning, management, and modeling, but riparian wetlands present several challenges to effective mapping due t...

  10. Baby Brain Map

    MedlinePlus

    ... a Member Home Resources & Services Professional Resource Baby Brain Map Mar 17, 2016 The Brain Map was adapted in 2006 by ZERO TO ... on the age range, different hotspots on the brain will appear. Click on a hotspot to reveal ...

  11. New map data catalog

    NASA Astrophysics Data System (ADS)

    Map byproducts, including aerial photographs, color separations, map data in computer form, and other materials used in or produced during mapmaking, are described in a new catalog published by the U.S. Geological Survey.The 48-page hardcover catalog is the first listing of the unpublished USGS civilian cartographic holdings. It covers such items as mapping photographs, computer-enhanced LANDSAT pictures of Earth, cartographic data in computer form, microfilm and microfiche records, and a variety of features, including color separations, made in compiling and printing maps. The catalog also describes out-of-print maps available from USGS, along with land-use and land-cover maps, and other unusual items, such as slope maps and orthophotoquads. The catalog explains how to order advance copies of maps before they are published.

  12. Structure–Function Relationships in Fungal Large-Subunit Catalases

    SciTech Connect

    Diaz, A.; Valdez, V; Rudino-Pinera, E; Horjales, E; Hansberg, W

    2009-01-01

    Neurospora crassa has two large-subunit catalases, CAT-1 and CAT-3. CAT-1 is associated with non-growing cells and accumulates particularly in asexual spores; CAT-3 is associated with growing cells and is induced under different stress conditions. It is our interest to elucidate the structure-function relationships in large-subunit catalases. Here we have determined the CAT-3 crystal structure and compared it with the previously determined CAT-1 structure. Similar to CAT-1, CAT-3 hydrogen peroxide (H{sub 2}O{sub 2}) saturation kinetics exhibited two components, consistent with the existence of two active sites: one saturated in the millimolar range and the other in the molar range. In the CAT-1 structure, we found three interesting features related to its unusual kinetics: (a) a constriction in the channel that conveys H{sub 2}O{sub 2} to the active site; (b) a covalent bond between the tyrosine, which forms the fifth coordination bound to the iron of the heme, and a vicinal cysteine; (c) oxidation of the pyrrole ring III to form a cis-hydroxyl group in C5 and a cis-{gamma}-spirolactone in C6. The site of heme oxidation marks the starts of the central channel that communicates to the central cavity and the shortest way products can exit the active site. CAT-3 has a similar constriction in its major channel, which could function as a gating system regulated by the H{sub 2}O{sub 2} concentration before the gate. CAT-3 functional tyrosine is not covalently bonded, but has instead the electron relay mechanism described for the human catalase to divert electrons from it. Pyrrole ring III in CAT-3 is not oxidized as it is in other large-subunit catalases whose structure has been determined. Different in CAT-3 from these enzymes is an occupied central cavity. Results presented here indicate that CAT-3 and CAT-1 enzymes represent a functional group of catalases with distinctive structural characteristics that determine similar kinetics.

  13. Analysis of an N-terminal deletion in subunit a of the Escherichia coli ATP synthase.

    PubMed

    Ishmukhametov, Robert R; DeLeon-Rangel, Jessica; Zhu, Shaotong; Vik, Steven B

    2017-04-01

    Subunit a is a membrane-bound stator subunit of the ATP synthase and is essential for proton translocation. The N-terminus of subunit a in E. coli is localized to the periplasm, and contains a sequence motif that is conserved among some bacteria. Previous work has identified mutations in this region that impair enzyme activity. Here, an internal deletion was constructed in subunit a in which residues 6-20 were replaced by a single lysine residue, and this mutant was unable to grow on succinate minimal medium. Membrane vesicles prepared from this mutant lacked ATP synthesis and ATP-driven proton translocation, even though immunoblots showed a significant level of subunit a. Similar results were obtained after purification and reconstitution of the mutant ATP synthase into liposomes. The location of subunit a with respect to its neighboring subunits b and c was probed by introducing cysteine substitutions that were known to promote cross-linking: a_L207C + c_I55C, a_L121C + b_N4C, and a_T107C + b_V18C. The last pair was unable to form cross-links in the background of the deletion mutant. The results indicate that loss of the N-terminal region of subunit a does not generally disrupt its structure, but does alter interactions with subunit b.

  14. Distinct forms of the. beta. subunit of GTP-binding regulatory proteins identified by molecular cloning

    SciTech Connect

    Fong, H.K.W.; Amatruda, T.T. III; Birren, B.W.; Simon, M.I.

    1987-06-01

    Two distinct ..beta.. subunits of guanine nucleotide-binding regulatory proteins have been identified by cDNA cloning and are referred to as ..beta../sub 1/ and ..beta../sub 1/ subunits. The bovine transducin ..beta.. subunit (..beta../sub 1/) has been cloned previously. The author now isolated and analyzed cDNA clones that encode the ..beta../sub 2/ subunit from bovine adrenal, bovine brain, and a human myeloid leukemia cell line, HL-60. The 340-residue M/sub r/ 37,329 BETA/sub 2/ protein is 90% identical with ..beta../sub 1/ in predicted amino acid sequence, and it is also organized as a series of repetitive homologous segments. The major mRNA that encodes the bovine ..beta../sub 2/ subunit is 1.7 kilobases in length. It is expressed at lower levels than ..beta../sub 1/ subunit mRNA in all tissues examined. The ..beta../sub 1/ and ..beta../sub 2/ messages are expressed in cloned human cell lines. Hybridization of cDNA probes to bovine DNA showed that ..beta../sub 1/ and ..beta../sub 2/ are encoded by separate genes. The amino acid sequences for the bovine and human ..beta../sub 2/ subunit are identical, as are the amino acid sequences for the bovine and human ..beta../sub 1/ subunit. This evolutionary conservation suggests that the two ..beta.. subunits have different roles in the signal transduction process.

  15. Molecular Pathology of Genetic Epilepsies Associated with GABAA Receptor Subunit Mutations

    PubMed Central

    Macdonald, Robert L; Kang, Jing-Qiong

    2009-01-01

    Mutations in ligand-gated ion channel genes associated with idiopathic generalized epilepsies have been reported in excitatory acetylcholine receptor α4 and β2 subunit genes linked to autosomal dominant nocturnal frontal lobe epilepsy and in inhibitory GABAA receptor α1, β3, γ2, and δ subunit genes associated with childhood absence epilepsy, juvenile myoclonic epilepsy, pure febrile seizures, generalized epilepsy with febrile seizures plus, and generalized epilepsy with tonic–clonic seizures. Recent studies suggest that these mutations alter receptor function or biogenesis, including impaired receptor subunit messenger RNA stability, receptor subunit protein folding and stability, receptor assembly, and receptor trafficking. PMID:19396344

  16. The α2δ subunits of voltage-gated calcium channels.

    PubMed

    Dolphin, Annette C

    2013-07-01

    Voltage-gated calcium channels consist of the main pore-forming α1 subunit, together, except in the case of the T-type channels, with β and α2δ and sometimes γ subunits, which are collectively termed auxiliary or accessory subunits. This review will concentrate on the properties and role of the α2δ subunits of these channels. These proteins are largely extracellular, membrane-associated proteins which influence the trafficking, localization, and biophysical properties of the channels. This article is part of a Special Issue entitled: Calcium channels.

  17. Differential expression of the protein kinase A subunits in normal adrenal glands and adrenocortical adenomas.

    PubMed

    Weigand, Isabel; Ronchi, Cristina L; Rizk-Rabin, Marthe; Dalmazi, Guido Di; Wild, Vanessa; Bathon, Kerstin; Rubin, Beatrice; Calebiro, Davide; Beuschlein, Felix; Bertherat, Jérôme; Fassnacht, Martin; Sbiera, Silviu

    2017-12-01

    Somatic mutations in protein kinase A catalytic α subunit (PRKACA) were found to be causative for 30-40% of cortisol-producing adenomas (CPA) of the adrenal gland, rendering PKA signalling constitutively active. In its resting state, PKA is a stable and inactive heterotetramer, consisting of two catalytic and two regulatory subunits with the latter inhibiting PKA activity. The human genome encodes three different PKA catalytic subunits and four different regulatory subunits that are preferentially expressed in different organs. In normal adrenal glands all regulatory subunits are expressed, while CPA exhibit reduced protein levels of the regulatory subunit IIβ. In this study, we linked for the first time the loss of RIIβ protein levels to the PRKACA mutation status and found the down-regulation of RIIβ to arise post-transcriptionally. We further found the PKA subunit expression pattern of different tumours is also present in the zones of the normal adrenal cortex and demonstrate that the different PKA subunits have a differential expression pattern in each zone of the normal adrenal gland, indicating potential specific roles of these subunits in the regulation of different hormones secretion.

  18. Enrichment of GABAA Receptor α-Subunits on the Axonal Initial Segment Shows Regional Differences

    PubMed Central

    Gao, Yudong; Heldt, Scott A.

    2016-01-01

    Although it is generally recognized that certain α-subunits of γ-aminobutyric acid type A receptors (GABAARs) form enriched clusters on the axonal initial segment (AIS), the degree to which these clusters vary in different brain areas is not well known. In the current study, we quantified the density, size, and enrichment ratio of fluorescently labeled α1-, α2-, or α3-subunits aggregates co-localized with the AIS-marker ankyrin G and compared them to aggregates in non-AIS locations among different brain areas including hippocampal subfields, basal lateral amygdala (BLA), prefrontal cortex (PFC), and sensory cortex (CTX). We found regional differences in the enrichment of GABAAR α-subunits on the AIS. Significant enrichment was identified in the CA3 of hippocampus for α1-subunits, in the CA1, CA3, and BLA for α2-subunits, and in the BLA for α3-subunits. Using α-subunit knock-out (KO) mice, we found that BLA enrichment of α2- and α3-subunits were physiologically independent of each other, as the enrichment of one subunit was unaffected by the genomic deletion of the other. To further investigate the unique pattern of α-subunit enrichment in the BLA, we examined the association of α2- and α3-subunits with the presynaptic vesicular GABA transporter (vGAT) and the anchoring protein gephyrin (Geph). As expected, both α2- and α3-subunits on the AIS within the BLA received prominent GABAergic innervation from vGAT-positive terminals. Further, we found that the association of α2- and α3-subunits with Geph was weaker in AIS versus non-AIS locations, suggesting that Geph might be playing a lesser role in the enrichment of α2- and α3-subunits on the AIS. Overall, these observations suggest that GABAARs on the AIS differ in subunit composition across brain regions. As with somatodendritic GABAARs, the distinctive expression pattern of AIS-located GABAAR α-subunits in the BLA, and other brain areas, likely contribute to unique forms of GABAergic inhibitory

  19. beta-subunits of Snf1 kinase are required for kinase function and substrate definition.

    PubMed

    Schmidt, M C; McCartney, R R

    2000-09-15

    The Snf1 kinase and its mammalian homolog, the AMP-activated protein kinase, are heterotrimeric enzymes composed of a catalytic alpha-subunit, a regulatory gamma-subunit and a beta-subunit that mediates heterotrimer formation. Saccharomyces cerevisiae encodes three beta-subunit genes, SIP1, SIP2 and GAL83. Earlier studies suggested that these subunits may not be required for Snf1 kinase function. We show here that complete and precise deletion of all three beta-subunit genes inactivates the Snf1 kinase. The sip1Delta sip2Delta gal83Delta strain is unable to derepress invertase, grows poorly on alternative carbon sources and fails to direct the phosphorylation of the Mig1 and Sip4 proteins in vivo. The SIP1 sip2Delta gal83Delta strain manifests a subset of Snf phenotypes (Raf(+), Gly(-)) observed in the snf1Delta 10 strain (Raf(-), Gly(-)), suggesting that individual beta-subunits direct the Snf1 kinase to a subset of its targets in vivo. Indeed, deletion of individual beta-subunit genes causes distinct differences in the induction and phosphorylation of Sip4, strongly suggesting that the beta-subunits play an important role in substrate definition.

  20. Stage and Tissue Specific Expression of Four TCR Subunits in Olive Flounder (Paralichthys olivaceus).

    PubMed

    Lee, Young Mee; Lee, Jeong-Ho; Noh, Jae Koo; Kim, Hyun Chul; Park, Choul-Ji; Park, Jong-Won; Hwang, In Joon; Kim, Sung Yeon

    2013-12-01

    TCR subunits are members of membrane-bound receptors which allow the fast and efficient elimination of the specific fish pathogens have regulated function in adaptive immunity. Sequence structure of TCR subunits have been reported for various teleosts, but the information of each TCR subunit functional characterization through expression analysis in fish was unknown. In this study, we examined the gene expression of TCR subunits in the early developmental stages and observed transcript levels in various tissues from healthy adult olive flounder by RT-PCR. The mRNA expression of alpha subunit was already detected in the previous hatching step. But the transcripts of another TCR subunit were not observed during embryo development and increased after hatching and maintained until metamorphosis at the same level. It was found that all TCR subunits mRNAs are commonly expressed in the immune-related organ such as spleen, kidney and gill, also weak expressed in fin and eye. TCR alpha and beta subunit were expressed in brain, whereas gamma and delta were not expressed same tissue. The sequence alignment analysis shows that there are more than 80% sequence homology between TCR subunits. Because it has a high similarity of amino acid sequence to expect similar in function, but expression analysis show that will have may functional diversity due to different time and place of expression.

  1. The separation of glutathione transferase subunits by using reverse-phase high-pressure liquid chromatography.

    PubMed Central

    Ostlund Farrants, A K; Meyer, D J; Coles, B; Southan, C; Aitken, A; Johnson, P J; Ketterer, B

    1987-01-01

    A simple method is described for the separation and quantification of the subunits of GSH transferases present in rat tissue extracts. This method, involving GSH-agarose affinity chromatography followed by reverse-phase h.p.l.c., is rapid and sufficiently sensitive to measure 5 micrograms of each subunit in a mixture. Examples are given of its application to extracts of rat kidney, adrenal, testicular interstitial cells and seminiferous tubules. The analysis of seminiferous tubules indicates that the technique may be of value for the identification of novel subunits. Preliminary separations of subunits from human GSH transferases are also described. PMID:3663168

  2. Exploring Maps Teaching Packet.

    ERIC Educational Resources Information Center

    Geological Survey (Dept. of Interior), Reston, VA.

    This interdisciplinary packet of materials on mapping is intended for grades 7-12. The lessons are organized around themes: location, navigation, information, and exploration. Each lesson has an introductory text and two main activities. Students learn basic mapmaking and map-reading skills and see how maps help answer fundamental geographic…

  3. Mapping a Changing World.

    ERIC Educational Resources Information Center

    Stoltman, Joseph P.

    1992-01-01

    Addresses the importance of maps for instruction in both history and geography. Suggests that maps have gotten recent attention because of the rapid political changes occurring in Europe and the quincentenary of Columbus' voyage. Discusses different map projections and the importance of media and satellite display of real pictures of the world.…

  4. Mapping Sociological Concepts.

    ERIC Educational Resources Information Center

    Trepagnier, Barbara

    2002-01-01

    Focuses on the use of cognitive mapping within sociology. Describes an assignment where students created a cognitive map that focused on names of theorists and concepts related to them. Discusses sociological imagination in relation to cognitive mapping and the assessment of the assignment. (CMK)

  5. Oil Exploration Mapping

    NASA Technical Reports Server (NTRS)

    1994-01-01

    After concluding an oil exploration agreement with the Republic of Yemen, Chevron International needed detailed geologic and topographic maps of the area. Chevron's remote sensing team used imagery from Landsat and SPOT, combining images into composite views. The project was successfully concluded and resulted in greatly improved base maps and unique topographic maps.

  6. Reading Angles in Maps

    ERIC Educational Resources Information Center

    Izard, Véronique; O'Donnell, Evan; Spelke, Elizabeth S.

    2014-01-01

    Preschool children can navigate by simple geometric maps of the environment, but the nature of the geometric relations they use in map reading remains unclear. Here, children were tested specifically on their sensitivity to angle. Forty-eight children (age 47:15-53:30 months) were presented with fragments of geometric maps, in which angle sections…

  7. Statistical Mapping by Computer.

    ERIC Educational Resources Information Center

    Utano, Jack J.

    The function of a statistical map is to provide readers with a visual impression of the data so that they may be able to identify any geographic characteristics of the displayed phenomena. The increasingly important role played by the computer in the production of statistical maps is manifested by the varied examples of computer maps in recent…

  8. Quantitative DNA fiber mapping

    DOEpatents

    Gray, Joe W.; Weier, Heinz-Ulrich G.

    1998-01-01

    The present invention relates generally to the DNA mapping and sequencing technologies. In particular, the present invention provides enhanced methods and compositions for the physical mapping and positional cloning of genomic DNA. The present invention also provides a useful analytical technique to directly map cloned DNA sequences onto individual stretched DNA molecules.

  9. Using maps in genealogy

    USGS Publications Warehouse

    ,

    1999-01-01

    Maps are one of many sources you may need to complete a family tree. In genealogical research, maps can provide clues to where our ancestors may have lived and where to look for written records about them. Beginners should master basic genealogical research techniques before starting to use topographic maps.

  10. Surface Chemistry Maps

    NASA Image and Video Library

    2015-03-13

    Maps of magnesium/silicon (left) and thermal neutron absorption (right) across Mercury's surface (red indicates high values, blue low) are shown. These maps, together with maps of other elemental abundances, reveal the presence of distinct geochemical terranes. Volcanic smooth plains deposits are outlined in white. Read the mission news story to learn more! http://photojournal.jpl.nasa.gov/catalog/PIA19242

  11. Adventures with Maps.

    ERIC Educational Resources Information Center

    Hofferber, Michael

    1989-01-01

    Orienteering--the game of following a map to find predetermined locations--can spark interest and develop skills in map making and map reading. This article gives background on orienteering; describes indoor and outdoor orienteering activities; offers suggestions for incorporating orienteering into science, math, and language arts; and provides a…

  12. Reading Angles in Maps

    ERIC Educational Resources Information Center

    Izard, Véronique; O'Donnell, Evan; Spelke, Elizabeth S.

    2014-01-01

    Preschool children can navigate by simple geometric maps of the environment, but the nature of the geometric relations they use in map reading remains unclear. Here, children were tested specifically on their sensitivity to angle. Forty-eight children (age 47:15-53:30 months) were presented with fragments of geometric maps, in which angle sections…

  13. Mapping with Young Children.

    ERIC Educational Resources Information Center

    Sunal, Cynthia Szymanski; Warash, Bobbi Gibson

    Techniques for encouraging young children to discover the purpose and use of maps are discussed. Motor activity and topological studies form a base from which the teacher and children can build a mapping program of progressive sophistication. Concepts important to mapping include boundaries, regions, exteriors, interiors, holes, order, point of…

  14. Mapping landscape corridors

    Treesearch

    Peter Vogt; Kurt H. Riitters; Marcin Iwanowski; Christine Estreguil; Jacek Kozak; Pierre Soille

    2007-01-01

    Corridors are important geographic features for biological conservation and biodiversity assessment. The identification and mapping of corridors is usually based on visual interpretations of movement patterns (functional corridors) or habitat maps (structural corridors). We present a method for automated corridor mapping with morphological image processing, and...

  15. What Do Maps Show?

    ERIC Educational Resources Information Center

    Geological Survey (Dept. of Interior), Reston, VA.

    This curriculum packet, appropriate for grades 4-8, features a teaching poster which shows different types of maps (different views of Salt Lake City, Utah), as well as three reproducible maps and reproducible activity sheets which complement the maps. The poster provides teacher background, including step-by-step lesson plans for four geography…

  16. Adaptive Composite Map Projections.

    PubMed

    Jenny, B

    2012-12-01

    All major web mapping services use the web Mercator projection. This is a poor choice for maps of the entire globe or areas of the size of continents or larger countries because the Mercator projection shows medium and higher latitudes with extreme areal distortion and provides an erroneous impression of distances and relative areas. The web Mercator projection is also not able to show the entire globe, as polar latitudes cannot be mapped. When selecting an alternative projection for information visualization, rivaling factors have to be taken into account, such as map scale, the geographic area shown, the map's height-to-width ratio, and the type of cartographic visualization. It is impossible for a single map projection to meet the requirements for all these factors. The proposed composite map projection combines several projections that are recommended in cartographic literature and seamlessly morphs map space as the user changes map scale or the geographic region displayed. The composite projection adapts the map's geometry to scale, to the map's height-to-width ratio, and to the central latitude of the displayed area by replacing projections and adjusting their parameters. The composite projection shows the entire globe including poles; it portrays continents or larger countries with less distortion (optionally without areal distortion); and it can morph to the web Mercator projection for maps showing small regions.

  17. Structure of the large ribosomal subunit from human mitochondria.

    PubMed

    Brown, Alan; Amunts, Alexey; Bai, Xiao-chen; Sugimoto, Yoichiro; Edwards, Patricia C; Murshudov, Garib; Scheres, Sjors H W; Ramakrishnan, V

    2014-11-07

    Human mitochondrial ribosomes are highly divergent from all other known ribosomes and are specialized to exclusively translate membrane proteins. They are linked with hereditary mitochondrial diseases and are often the unintended targets of various clinically useful antibiotics. Using single-particle cryogenic electron microscopy, we have determined the structure of its large subunit to 3.4 angstrom resolution, revealing 48 proteins, 21 of which are specific to mitochondria. The structure unveils an adaptation of the exit tunnel for hydrophobic nascent peptides, extensive remodeling of the central protuberance, including recruitment of mitochondrial valine transfer RNA (tRNA(Val)) to play an integral structural role, and changes in the tRNA binding sites related to the unusual characteristics of mitochondrial tRNAs.

  18. Bacterial ribosomal subunit assembly is an antibiotic target.

    PubMed

    Champney, W Scott

    2003-01-01

    A substantial number of antimicrobial agents target some activity of the bacterial ribosome for inhibition. Mechanistic studies and recent structural investigations of the ribosome have identified the binding sites and presumed mechanism of inhibitory activity for some compounds. A second target for many of these antibiotics has recently been examined. Formation of both 30S and 50S ribosomal subunits in bacterial cells is impaired by translational inhibitors. For many antimicrobial agents, inhibition of this target is equivalent to inhibition of translation in preventing cell growth. This review will describe features of this new target including the types of compounds which affect particle assembly and differences in the process in different microorganisms. The characteristics of this new target will be identified and aspects of a model to explain this new inhibitory activity will be explored.

  19. Structure of the large ribosomal subunit from human mitochondria

    PubMed Central

    Bai, Xiao-chen; Sugimoto, Yoichiro; Edwards, Patricia C.; Murshudov, Garib; Scheres, Sjors H. W.; Ramakrishnan, V.

    2014-01-01

    Human mitochondrial ribosomes are highly divergent from all other known ribosomes and are specialized to exclusively translate membrane proteins. They are linked with hereditary mitochondrial diseases, and are often the unintended targets of various clinically useful antibiotics. Using single-particle electron cryo-microscopy we have determined the structure of its large subunit to 3.4 angstrom resolution, revealing 48 proteins, 21 of which are specific to mitochondria. The structure unveils an adaptation of the exit tunnel for hydrophobic nascent peptides, extensive remodeling of the central protuberance including recruitment of mitochondrial tRNAVal to play an integral structural role, and changes in the tRNA binding sites related to the unusual characteristics of mitochondrial tRNAs. PMID:25278503

  20. Catalytic subunit stoichiometry within the cellulose synthase complex.

    PubMed

    Gonneau, Martine; Desprez, Thierry; Guillot, Alain; Vernhettes, Samantha; Höfte, Herman

    2014-12-01

    Cellulose synthesis is driven by large plasma membrane-inserted protein complexes, which in plants have 6-fold symmetry. In Arabidopsis (Arabidopsis thaliana), functional cellulose synthesis complexes (CSCs) are composed of at least three different cellulose synthase catalytic subunits (CESAs), but the actual ratio of the CESA isoforms within the CSCs remains unresolved. In this work, the stoichiometry of the CESAs in the primary cell wall CSC was determined, after elimination of CESA redundancy in a mutant background, by coimmunoprecipitation and mass spectrometry using label-free quantitative methods. Based on spectral counting, we show that CESA1, CESA3, and CESA6 are present in a 1:1:1 molecular ratio. © 2014 American Society of Plant Biologists. All Rights Reserved.

  1. hERG subunit composition determines differential drug sensitivity

    PubMed Central

    Abi-Gerges, N; Holkham, H; Jones, EMC; Pollard, CE; Valentin, J-P; Robertson, GA

    2011-01-01

    BACKGROUND AND PURPOSE The majority of human ether-a-go-go-related gene (hERG) screens aiming to minimize the risk of drug-induced long QT syndrome have been conducted using heterologous systems expressing the hERG 1a subunit, although both hERG 1a and 1b subunits contribute to the K+ channels producing the repolarizing current IKr. We tested a range of compounds selected for their diversity to determine whether hERG 1a and 1a/1b channels exhibit different sensitivities that may influence safety margins or contribute to a stratified risk analysis. EXPERIMENTAL APPROACH We used the IonWorks™ plate-based electrophysiology device to compare sensitivity of hERG 1a and 1a/1b channels stably expressed in HEK293 cells to 50 compounds previously shown to target hERG channels. Potency was determined as IC50 values (µM) obtained from non-cumulative, eight-point concentration–effect curves of normalized data, fitted to the Hill equation. To minimize possible sources of variability, compound potency was assessed using test plates arranged in alternating columns of cells expressing hERG 1a and 1a/1b. KEY RESULTS Although the potency of most compounds was similar for the two targets, some surprising differences were observed. Fluoxetine (Prozac) was more potent at blocking hERG 1a/1b than 1a channels, yielding a corresponding reduction in the safety margin. In contrast, E-4031 was a more potent blocker of hERG 1a compared with 1a/1b channels, as previously reported, as was dofetilide, another high-affinity blocker. CONCLUSIONS AND IMPLICATIONS The current assays may underestimate the risk of some drugs to cause torsades de pointes arrhythmia, and overestimate the risk of others. PMID:21449979

  2. Effective polymer adjuvants for sustained delivery of protein subunit vaccines.

    PubMed

    Adams, Justin R; Haughney, Shannon L; Mallapragada, Surya K

    2015-03-01

    We have synthesized thermogelling cationic amphiphilic pentablock copolymers that have the potential to act as injectable vaccine carriers and adjuvants that can simultaneously provide sustained delivery and enhance the immunogenicity of released antigen. While these pentablock copolymers have shown efficacy in DNA delivery in past studies, the ability to deliver both DNA and protein for subunit vaccines using the same polymeric carrier can provide greater flexibility and efficacy. We demonstrate the ability of these pentablock copolymers, and the parent triblock Pluronic copolymers to slowly release structurally intact and antigenically stable protein antigens in vitro, create an antigen depot through long-term injection-site persistence and enhance the in vivo immune response to these antigens. We show release of the model protein antigen ovalbumin in vitro from the thermogelling block copolymers with the primary, secondary and tertiary structures of the released protein unchanged compared to the native protein, and its antigenicity preserved upon release. The block copolymers form a gel at physiological temperatures that serves as an antigenic depot and persists in vivo at the site of injection for over 50days. The pentablock copolymers show a significant fivefold enhancement in the immune response compared to soluble protein alone, even 6weeks after the administration, based on measurement of antibody titers. These results demonstrate the potential of these block copolymers hydrogels to persist for several weeks and sustain the release of antigen with minimal effects on protein stability and antigenicity; and their ability to be used simultaneously as a sustained delivery device as well as a subunit vaccine adjuvant platform.

  3. The subunit interfaces of weakly associated homodimeric proteins.

    PubMed

    Dey, Sucharita; Pal, Arumay; Chakrabarti, Pinak; Janin, Joël

    2010-04-23

    We analyzed subunit interfaces in 315 homodimers with an X-ray structure in the Protein Data Bank, validated by checking the literature for data that indicate that the proteins are dimeric in solution and that, in the case of the "weak" dimers, the homodimer is in equilibrium with the monomer. The interfaces of the 42 weak dimers, which are smaller by a factor of 2.4 on average than in the remainder of the set, are comparable in size with antibody-antigen or protease-inhibitor interfaces. Nevertheless, they are more hydrophobic than in the average transient protein-protein complex and similar in amino acid composition to the other homodimer interfaces. The mean numbers of interface hydrogen bonds and hydration water molecules per unit area are also similar in homodimers and transient complexes. Parameters related to the atomic packing suggest that many of the weak dimer interfaces are loosely packed, and we suggest that this contributes to their low stability. To evaluate the evolutionary selection pressure on interface residues, we calculated the Shannon entropy of homologous amino acid sequences at 60% sequence identity. In 93% of the homodimers, the interface residues are better conserved than the residues on the protein surface. The weak dimers display the same high degree of interface conservation as other homodimers, but their homologs may be heterodimers as well as homodimers. Their interfaces may be good models in terms of their size, composition, and evolutionary conservation for the labile subunit contacts that allow protein assemblies to share and exchange components, allosteric proteins to undergo quaternary structure transitions, and molecular machines to operate in the cell.

  4. Subunit interface mutants of rabbit muscle aldolase form active dimers.

    PubMed Central

    Beernink, P. T.; Tolan, D. R.

    1994-01-01

    We report the construction of subunit interface mutants of rabbit muscle aldolase A with altered quaternary structure. A mutation has been described that causes nonspherocytic hemolytic anemia and produces a thermolabile aldolase (Kishi H et al., 1987, Proc Natl Acad Sci USA 84:8623-8627). The disease arises from substitution of Gly for Asp-128, a residue at the subunit interface of human aldolase A. To elucidate the role of this residue in the highly homologous rabbit aldolase A, site-directed mutagenesis is used to replace Asp-128 with Gly, Ala, Asn, Gln, or Val. Rabbit aldolase D128G purified from Escherichia coli is found to be similar to human D128G by kinetic analysis, CD, and thermal inactivation assays. All of the mutant rabbit aldolases are similar to the wild-type rabbit enzyme in secondary structure and kinetic properties. In contrast, whereas the wild-type enzyme is a tetramer, chemical crosslinking and gel filtration indicate that a new dimeric species exists for the mutants. In sedimentation velocity experiments, the mutant enzymes as mixtures of dimer and tetramer at 4 degrees C. Sedimentation at 20 degrees C shows that the mutant enzymes are > 99.5% dimeric and, in the presence of substrate, that the dimeric species is active. Differential scanning calorimetry demonstrates that Tm values of the mutant enzymes are decreased by 12 degrees C compared to wild-type enzyme. The results indicate that Asp-128 is important for interface stability and suggest that 1 role of the quaternary structure of aldolase is to provide thermostability. PMID:7833800

  5. A G protein γ subunit-like domain shared between RGS11 and other RGS proteins specifies binding to Gβ5 subunits

    PubMed Central

    Snow, Bryan E.; Krumins, Andrejs M.; Brothers, Greg M.; Lee, Sheu-Fen; Wall, Mark A.; Chung, Stephen; Mangion, Joan; Arya, Sudha; Gilman, Alfred G.; Siderovski, David P.

    1998-01-01

    Regulators of G protein signaling (RGS) proteins act as GTPase-activating proteins (GAPs) toward the α subunits of heterotrimeric, signal-transducing G proteins. RGS11 contains a G protein γ subunit-like (GGL) domain between its Dishevelled/Egl-10/Pleckstrin and RGS domains. GGL domains are also found in RGS6, RGS7, RGS9, and the Caenorhabditis elegans protein EGL-10. Coexpression of RGS11 with different Gβ subunits reveals specific interaction between RGS11 and Gβ5. The expression of mRNA for RGS11 and Gβ5 in human tissues overlaps. The Gβ5/RGS11 heterodimer acts as a GAP on Gαo, apparently selectively. RGS proteins that contain GGL domains appear to act as GAPs for Gα proteins and form complexes with specific Gβ subunits, adding to the combinatorial complexity of G protein-mediated signaling pathways. PMID:9789084

  6. Global Map of Pluto

    NASA Image and Video Library

    2015-07-27

    The science team of NASA's New Horizons mission has produced an updated global map of the dwarf planet Pluto. The map includes all resolved images of the surface acquired between July 7-14, 2015, at pixel resolutions ranging from 40 kilometers (24 miles) on the Charon-facing hemisphere (left and right sides of the map) to 400 meters (1,250 feet) on the anti-Charon facing hemisphere (map center). Many additional images are expected in fall of 2015 and these will be used to complete the global map. http://photojournal.jpl.nasa.gov/catalog/PIA19858

  7. Map projections for larger-scale mapping

    NASA Technical Reports Server (NTRS)

    Snyder, J. P.

    1982-01-01

    For the U.S. Geological Survey maps at 1:1,000,000-scale and larger, the most common projections are conformal, such as the Transverse Mercator and Lambert Conformal Conic. Projections for these scales should treat the Earth as an ellipsoid. In addition, the USGS has conceived and designed some new projections, including the Space Oblique Mercator, the first map projection designed to permit low-distortion mapping of the Earth from satellite imagery, continuously following the groundtrack. The USGS has programmed nearly all pertinent projection equations for inverse and forward calculations. These are used to plot maps or to transform coordinates from one projection to another. The projections in current use are described.

  8. Reduction of tomato polygalacturonase beta subunit expression affects pectin solubilization and degradation during fruit ripening.

    PubMed Central

    Watson, C F; Zheng, L; DellaPenna, D

    1994-01-01

    The developmental changes that accompany tomato fruit ripening include increased solubilization and depolymerization of pectins due to the action of polygalacturonase (PG). Two PG isoenzymes can be extracted from ripe fruit: PG2, which is a single catalytic PG polypeptide, and PG1, which is composed of PG2 tightly associated with a second noncatalytic protein, the beta subunit. Previous studies have correlated ripening-associated increases in pectin solubilization and depolymerization with the presence of extractable PG1 activity, prior to the appearance of PG2, suggesting a functional role for the beta subunit and PG1 in pectin metabolism. To assess the function of the beta subunit, we produced and characterized transgenic tomatoes constitutively expressing a beta subunit antisense gene. Fruit from antisense lines had greatly reduced levels of beta subunit mRNA and protein and accumulated < 1% of their total extractable PG activity in ripe fruit as PG1, as compared with 25% for wild type. Inhibition of beta subunit expression resulted in significantly elevated levels of EDTA-soluble polyuronides at all stages of fruit ripening and a significantly higher degree of depolymerization at later ripening stages. Decreased beta subunit protein and extractable PG1 enzyme activity and increased pectin solubility and depolymerization all cosegregated with the beta subunit antisense transgene in T2 progeny. These results indicate (1) that PG2 is responsible for pectin solubilization and depolymerization in vivo and (2) that the beta subunit protein is not required for PG2 activity in vivo but (3) does play a significant role in regulating pectin metabolism in wild-type fruit by limiting the extent of pectin solubilization and depolymerization that can occur during ripening. Whether this occurs by direct interaction of the beta subunit with PG2 or indirectly by interaction of the beta subunit with the pectic substrate remains to be determined. PMID:7827495

  9. Molecular cloning and functional analysis of three subunits of yeast proteasome.

    PubMed Central

    Emori, Y; Tsukahara, T; Kawasaki, H; Ishiura, S; Sugita, H; Suzuki, K

    1991-01-01

    The genes encoding three subunits of Saccharomyces cerevisiae proteasome were cloned and sequenced. The deduced amino acid sequences were homologous not only to each other (30 to 40% identity) but also to those of rat and Drosophila proteasomes (25 to 65% identity). However, none of these sequences showed any similarity to any other known sequences, including various proteases, suggesting that these proteasome subunits may constitute a unique gene family. Gene disruption analyses revealed that two of the three subunits (subunits Y7 and Y8) are essential for growth, indicating that the proteasome and its individual subunits play an indispensable role in fundamental biological processes. On the other hand, subunit Y13 is not essential; haploid cells with a disrupted Y13 gene can proliferate, although the doubling time is longer than that of cells with nondisrupted genes. In addition, biochemical analysis revealed that proteasome prepared from the Y13 disrupted cells contains tryptic and chymotryptic activities equivalent to those of nondisrupted cells, indicating that the Y13 subunit is not essential for tryptic or chymotryptic activity. However, the chymotryptic activity of the Y13 disrupted cells is not dependent on sodium dodecyl sulfate (SDS), an activator of proteasome, since nearly full activity was observed in the absence of SDS. Thus, the activity in proteasome of the Y13 disrupted cells might result in unregulated intracellular proteolysis, thus leading to the prolonged cell cycle. These results indicate that cloned proteasome subunits having similar sequences to the yeast Y13 subunit are structural, but not catalytic, components of proteasome. It is also suggested that two subunits (Y7 and Y8) might occupy positions essential to proteasome structure or activity, whereas subunit Y13 is in a nonessential but important position. Images PMID:1898763

  10. Trends in Nasal Subunit Reconstruction by Facial Plastic and Reconstructive Surgeons.

    PubMed

    Larrabee, Yuna C; Phillips, David J; Sclafani, Anthony P

    2017-02-01

    To determine if facial plastic and reconstructive surgeons still adhere to the classic nasal subunit principle as described by Burget and Menick. Observational survey. A Weill Cornell Medicine institutional review board approved electronic survey that was sent via e-mail to active members of the American Academy of Facial Plastic and Reconstructive Surgery (AAFPRS). The survey consisted of 32 multiple-choice questions pertaining to the operative management of small (22-30%), medium (50-58%), and large (75-81%) defects of each subunit of the nose, as well as demographic, provider, and practice characteristics. There were 111 responses to the survey (10.1% response rate). Ninety-eight percent of respondents reported familiarity with the subunit principle, and 59.6% considered the subunit principle in greater than 90% of cases. Almost three-quarters (70.4%) of respondents felt the subunit principle should be applied but could be modified based on the particular nasal defect, whereas 28.7% felt it was only sometimes helpful and was not mandatory for successful nasal reconstruction. Large defects of the tip and ala are generally treated by excision of the remaining subunit (79.4 and 80.6%, respectively). Fewer surgeons would excise the remaining subunit for large defects of the dorsum (39.8%), sidewall (38.8%), and soft tissue facet (18.4%). Simple repair without additional excision was the treatment of choice for small defects of the tip (58.2%), ala (59.2%), sidewall (65%), dorsum (68%), and soft tissue facet (71.8%). However, in many small- (up to 32%) and medium- (up to 51%) sized defects of the tip, ala, sidewall, and dorsum, respondents reported partial subunit excision. The majority of AAFPRS members abide to the classical subunit principle by completely excising the remaining subunit for large defects of the tip and ala. Many surgeons modify the subunit principle in small and medium defects.

  11. Differential regulation of thyrotropin subunit apoprotein and carbohydrate biosynthesis by thyroid hormone

    SciTech Connect

    Taylor, T.; Weintraub, B.D.

    1985-04-01

    The regulation of TSH apoprotein and carbohydrate biosynthesis by thyroid hormone was studied by incubating pituitaries from normal and hypothyroid (3 weeks post-thyroidectomy) rats in medium containing (/sup 14/C)alanine and (/sup 3/H) glucosamine. After 6 h, samples were sequentially treated with anti-TSH beta to precipitate TSH and free TSH beta, anti-LH beta to clear the sample of LH and free LH beta, then anti-LH alpha to precipitate free alpha-subunit. Total proteins were acid precipitated. All precipitates were subjected to electrophoresis on sodium dodecyl sulfate-polyacrylamide gels, which were then sliced and assayed by scintillation spectrometry. In hypothyroid pituitaries plus medium, (/sup 14/C)alanine incorporation in combined and free beta-subunits was 26 times normal and considerably greater than the 3.4-fold increase seen in total protein; combined and free alpha-subunits showed no specific increase in apoprotein synthesis. (/sup 3/H)Glucosamine incorporation in combined alpha- and beta-subunits in hypothyroid samples was 13 and 21 times normal, respectively, and was greater than the 1.9-fold increase in total protein; free alpha-subunit showed no specific increase in carbohydrate synthesis. The glucosamine to alanine ratio, reflecting relative glycosylation of newly synthesized molecules, was increased in hypothyroidism for combined alpha-subunits, but not for combined beta-subunits, free alpha-subunits, or total proteins. In summary, short term hypothyroidism selectively stimulated TSH beta apoprotein synthesis and carbohydrate synthesis of combined alpha- and beta-subunits. Hypothyroidism also increased the relative glycosylation of combined alpha-subunit. Thus, thyroid hormone deficiency appears to alter the rate-limiting step in TSH assembly (i.e. beta-subunit synthesis) as well as the carbohydrate structure of TSH, which may play important roles in its biological function.

  12. Methionine adenosyltransferase II beta subunit gene expression provides a proliferative advantage in human hepatoma.

    PubMed

    Martínez-Chantar, Maria L; García-Trevijano, Elena R; Latasa, M Ujue; Martín-Duce, Antonio; Fortes, Puri; Caballería, Juan; Avila, Matías A; Mato, José M

    2003-04-01

    Of the 2 genes (MAT1A, MAT2A) encoding methionine adenosyltransferase, the enzyme that synthesizes S-adenosylmethionine, MAT1A, is expressed in liver, whereas MAT2A is expressed in extrahepatic tissues. In liver, MAT2A expression associates with growth, dedifferentiation, and cancer. Here, we identified the beta subunit as a regulator of proliferation in human hepatoma cell lines. The beta subunit has been cloned and shown to lower the K(m) of methionine adenosyltransferase II alpha2 (the MAT2A product) for methionine and to render the enzyme more susceptible to S-adenosylmethionine inhibition. Methionine adenosyltransferase II alpha2 and beta subunit expression was analyzed in human and rat liver and hepatoma cell lines and their interaction studied in HuH7 cells. beta Subunit expression was up- and down-regulated in human hepatoma cell lines and the effect on DNA synthesis determined. We found that beta subunit is expressed in rat extrahepatic tissues but not in normal liver. In human liver, beta subunit expression associates with cirrhosis and hepatoma. beta Subunit is expressed in most (HepG2, PLC, and Hep3B) but not all (HuH7) hepatoma cell lines. Transfection of beta subunit reduced S-adenosylmethionine content and stimulated DNA synthesis in HuH7 cells, whereas down-regulation of beta subunit expression diminished DNA synthesis in HepG2. The interaction between methionine adenosyltransferase II alpha2 and beta subunit was demonstrated in HuH7 cells. Our findings indicate that beta subunit associates with cirrhosis and cancer providing a proliferative advantage in hepatoma cells through its interaction with methionine adenosyltransferase II alpha2 and down-regulation of S-adenosylmethionine levels.

  13. Species-specific Differences among KCNMB3 BK β3 Auxiliary Subunits: Some β3 N-terminal Variants May Be Primate-specific Subunits

    PubMed Central

    Zeng, Xuhui; Xia, Xiao-Ming; Lingle, Christopher J.

    2008-01-01

    The KCNMB3 gene encodes one of a family of four auxiliary β subunits found in the mammalian genome that associate with Slo1 α subunits and regulate BK channel function. In humans, the KCNMB3 gene contains four N-terminal alternative exons that produce four functionally distinct β3 subunits, β3a–d. Three variants, β3a–c, exhibit kinetically distinct inactivation behaviors. Since investigation of the physiological roles of BK auxiliary subunits will depend on studies in rodents, here we have determined the identity and functional properties of mouse β3 variants. Whereas β1, β2, and β4 subunits exhibit 83.2%, 95.3%, and 93.8% identity between mouse and human, the mouse β3 subunit, excluding N-terminal splice variants, shares only 62.8% amino acid identity with its human counterpart. Based on an examination of the mouse genome and screening of mouse cDNA libraries, here we have identified only two N-terminal candidates, β3a and β3b, of the four found in humans. Both human and mouse β3a subunits produce a characteristic use-dependent inactivation. Surprisingly, whereas the hβ3b exhibits rapid inactivation, the putative mβ3b does not inactivate. Furthermore, unlike hβ3, the mβ3 subunit, irrespective of the N terminus, mediates a shift in gating to more negative potentials at a given Ca2+ concentration. The shift in gating gradually is lost following patch excision, suggesting that the gating shift involves some regulatory process dependent on the cytosolic milieu. Examination of additional genomes to assess conservation among splice variants suggests that the putative mβ3b N terminus may not be a true orthologue of the hβ3b N terminus and that both β3c and β3d appear likely to be primate-specific N-terminal variants. These results have three key implications: first, functional properties of homologous β3 subunits may differ among mammalian species; second, the specific physiological roles of homologous β3 subunits may differ among mammalian

  14. Copolymer semiconductors comprising thiazolothiazole or benzobisthiazole, or benzobisoxazole electron acceptor subunits, and electron donor subunits, and their uses in transistors and solar cells

    DOEpatents

    Jenekhe, Samson A; Subramaniyan, Selvam; Ahmed, Eilaf; Xin, Hao; Kim, Felix Sunjoo

    2014-10-28

    The inventions disclosed, described, and/or claimed herein relate to copolymers comprising copolymers comprising electron accepting A subunits that comprise thiazolothiazole, benzobisthiazole, or benzobisoxazoles rings, and electron donating subunits that comprise certain heterocyclic groups. The copolymers are useful for manufacturing organic electronic devices, including transistors and solar cells. The invention also relates to certain synthetic precursors of the copolymers. Methods for making the copolymers and the derivative electronic devices are also described.

  15. Functional hybrid rubisco enzymes with plant small subunits and algal large subunits: engineered rbcS cDNA for expression in chlamydomonas.

    PubMed

    Genkov, Todor; Meyer, Moritz; Griffiths, Howard; Spreitzer, Robert J

    2010-06-25

    There has been much interest in the chloroplast-encoded large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) as a target for engineering an increase in net CO(2) fixation in photosynthesis. Improvements in the enzyme would lead to an increase in the production of food, fiber, and renewable energy. Although the large subunit contains the active site, a family of rbcS nuclear genes encodes the Rubisco small subunits, which can also influence the carboxylation catalytic efficiency and CO(2)/O(2) specificity of the enzyme. To further define the role of the small subunit in Rubisco function, small subunits from spinach, Arabidopsis, and sunflower were assembled with algal large subunits by transformation of a Chlamydomonas reinhardtii mutant that lacks the rbcS gene family. Foreign rbcS cDNAs were successfully expressed in Chlamydomonas by fusing them to a Chlamydomonas rbcS transit peptide sequence engineered to contain rbcS introns. Although plant Rubisco generally has greater CO(2)/O(2) specificity but a lower carboxylation V(max) than Chlamydomonas Rubisco, the hybrid enzymes have 3-11% increases in CO(2)/O(2) specificity and retain near normal V(max) values. Thus, small subunits may make a significant contribution to the overall catalytic performance of Rubisco. Despite having normal amounts of catalytically proficient Rubisco, the hybrid mutant strains display reduced levels of photosynthetic growth and lack chloroplast pyrenoids. It appears that small subunits contain the structural elements responsible for targeting Rubisco to the algal pyrenoid, which is the site where CO(2) is concentrated for optimal photosynthesis.

  16. Cartographic mapping study

    NASA Tec