The Experts below are selected from a list of 66 Experts worldwide ranked by ideXlab platform
Stephen M King - One of the best experts on this subject based on the ideXlab platform.
-
a bipartite ca2 regulated nucleoside diphosphate kinase system within the chlamydomonas flagellum the regulatory subunit P72
Journal of Biological Chemistry, 2002Co-Authors: Ramila S Patelking, Sharon E Benashski, Stephen M KingAbstract:Abstract Regulation of flagellar activity inChlamydomonas involves both Ca2+ and cAMP-mediated signaling pathways. However, Chlamydomonasand sea urchin sperm flagella also exhibit nucleoside-diphosphate kinase (NDK) activity, suggesting a requirement for GTP within this highly conserved organelle. In sea urchin sperm, the NDK catalytic subunit is an integral component of the outer dynein arm. Here we describe a modular Protein (P72) from the Chlamydomonasflagellum that consists of three domains closely related to the presumptive regulatory segment of rat NDK-7 followed by two EF-hands that are predicted to bind Ca2+. There are close homologues of P72 in both mammalian and insect genomes. The P72 Protein is tightly associated with the flagellar axoneme and is located along the entire length except at the transition zone. Cross-linking experiments suggest that P72 interacts with two or three additional axonemal polypeptides. The sensitivity of P72 to tryptic digestion differed considerably in the presence and the absence of Ca2+, suggesting that it indeed binds this ligand. These studies indicate that the flagellar NDK system is bipartite with the regulatory and catalytic components residing on different polypeptides. We propose that Ca2+regulation of flagellar motility in Chlamydomonas may be achieved in part through a downstream GTP-mediated signaling pathway.
-
a bipartite ca2 regulated nucleoside diphosphate kinase system within thechlamydomonas flagellum the regulatory subunit P72
Journal of Biological Chemistry, 2002Co-Authors: Ramila S Patelking, Sharon E Benashski, Stephen M KingAbstract:Regulation of flagellar activity inChlamydomonas involves both Ca2+ and cAMP-mediated signaling pathways. However, Chlamydomonasand sea urchin sperm flagella also exhibit nucleoside-diphosphate kinase (NDK) activity, suggesting a requirement for GTP within this highly conserved organelle. In sea urchin sperm, the NDK catalytic subunit is an integral component of the outer dynein arm. Here we describe a modular Protein (P72) from the Chlamydomonasflagellum that consists of three domains closely related to the presumptive regulatory segment of rat NDK-7 followed by two EF-hands that are predicted to bind Ca2+. There are close homologues of P72 in both mammalian and insect genomes. The P72 Protein is tightly associated with the flagellar axoneme and is located along the entire length except at the transition zone. Cross-linking experiments suggest that P72 interacts with two or three additional axonemal polypeptides. The sensitivity of P72 to tryptic digestion differed considerably in the presence and the absence of Ca2+, suggesting that it indeed binds this ligand. These studies indicate that the flagellar NDK system is bipartite with the regulatory and catalytic components residing on different polypeptides. We propose that Ca2+regulation of flagellar motility in Chlamydomonas may be achieved in part through a downstream GTP-mediated signaling pathway.
Eladio Vinuela - One of the best experts on this subject based on the ideXlab platform.
-
inducible gene expression from african swine fever virus recombinants analysis of the major capsid Protein P72
Journal of Virology, 1998Co-Authors: Ramon Garciaescudero, German Andres, Fernando Almazan, Eladio VinuelaAbstract:A method to study the function of individual African swine fever virus (ASFV) gene products utilizing the Escherichia coli lac repressor-operator system has been developed. Recombinant viruses containing both the lacI gene encoding the lac repressor and a strong virus late promoter modified by the insertion of one or two copies of the lac operator sequence at various positions were constructed. The ability of each modified promoter to regulate expression of the firefly luciferase gene was assayed in the presence and in the absence of the inducer isopropyl beta-D-thiogalactoside (IPTG). Induction and repression of gene activity were dependent on the position(s) of the operator(s) with respect to the promoter and on the number of operators inserted. The ability of this system to regulate the expression of ASFV genes was analyzed by constructing a recombinant virus inducibly expressing the major capsid Protein P72. Electron microscopy analysis revealed that under nonpermissive conditions, electron-dense membrane-like structures accumulated in the viral factories and capsid formation was inhibited. Induction of P72 expression allowed the progressive building of the capsid on these structures, leading to assembly of ASFV particles. The results of this report demonstrate that the transferred inducible expression system is a powerful tool for analyzing the function of ASFV genes.
-
characterization of the african swine fever virus structural Protein p14 5 a dna binding Protein
Virology, 1997Co-Authors: Luisa Martinezpomares, Carmen Simonmateo, Carlos Lopezotin, Eladio VinuelaAbstract:Abstract The gene encoding the structural Protein p14.5 of African swine fever virus (ASFV) has been mapped and sequenced. This gene, designated E120R, is located in theSalI H/EcoRI E restriction fragment of the ASFV genome and is predicted to encode a Protein of 120 amino acids with a molecular weight of 13.4 kDa. Northern-blot analysis showed that E120R is transcribed at late times during the viral replication cycle. The E120R gene product has been expressed inEscherichia coli,purified, and used as an antigen for antibody production. The antiserum anti-pE120R recognized a Protein in infected cell extracts with an apparent molecular mass of 14.5 kDa, named p14.5. This antiserum also detected Protein p14.5 in purified virus particles. Protein p14.5 is synthesized late in infection and is located in viral factories. Immunoprecipitation analysis and binding-assay experiments have shown that Protein p14.5 interacts with a Protein that could correspond to the major structural Protein P72. Purified Protein p14.5 interacts with DNA in a sequence-independent manner. It binds to both single-stranded and double-stranded DNA. A possible role of Protein p14.5 in the encapsidation of ASFV DNA is suggested.
-
high level expression in escherichia coli of the gene coding for the major structural Protein P72 of african swine fever virus
Gene, 1993Co-Authors: Jose M P Freije, Eladio Vinuela, M Munoz, Carlos LopezotinAbstract:Abstract The gene encoding the major structural Protein (P72) of African swine fever virus (ASFV) has been expressed in Escherichia coli using a T7 RNA polymerase system. The use of a recombinant plasmid which contains the entire gene inserted between the T7 promoter and the transcription terminator of the expression vector allowed us to obtain a high expression level of the intact viral Protein. This polypeptide, which appears in the insoluble fraction of the bacterial extracts, showed an intense reaction with the antibodies present in the sera of ASFV-infected animals, as demonstrated by Western blot and enzyme-linked immunosorbent assay. The recombinant Protein was purified by size-exclusion highperformance liquid chromatography and used to develop a serological test of the disease.
Ramila S Patelking - One of the best experts on this subject based on the ideXlab platform.
-
a bipartite ca2 regulated nucleoside diphosphate kinase system within the chlamydomonas flagellum the regulatory subunit P72
Journal of Biological Chemistry, 2002Co-Authors: Ramila S Patelking, Sharon E Benashski, Stephen M KingAbstract:Abstract Regulation of flagellar activity inChlamydomonas involves both Ca2+ and cAMP-mediated signaling pathways. However, Chlamydomonasand sea urchin sperm flagella also exhibit nucleoside-diphosphate kinase (NDK) activity, suggesting a requirement for GTP within this highly conserved organelle. In sea urchin sperm, the NDK catalytic subunit is an integral component of the outer dynein arm. Here we describe a modular Protein (P72) from the Chlamydomonasflagellum that consists of three domains closely related to the presumptive regulatory segment of rat NDK-7 followed by two EF-hands that are predicted to bind Ca2+. There are close homologues of P72 in both mammalian and insect genomes. The P72 Protein is tightly associated with the flagellar axoneme and is located along the entire length except at the transition zone. Cross-linking experiments suggest that P72 interacts with two or three additional axonemal polypeptides. The sensitivity of P72 to tryptic digestion differed considerably in the presence and the absence of Ca2+, suggesting that it indeed binds this ligand. These studies indicate that the flagellar NDK system is bipartite with the regulatory and catalytic components residing on different polypeptides. We propose that Ca2+regulation of flagellar motility in Chlamydomonas may be achieved in part through a downstream GTP-mediated signaling pathway.
-
a bipartite ca2 regulated nucleoside diphosphate kinase system within thechlamydomonas flagellum the regulatory subunit P72
Journal of Biological Chemistry, 2002Co-Authors: Ramila S Patelking, Sharon E Benashski, Stephen M KingAbstract:Regulation of flagellar activity inChlamydomonas involves both Ca2+ and cAMP-mediated signaling pathways. However, Chlamydomonasand sea urchin sperm flagella also exhibit nucleoside-diphosphate kinase (NDK) activity, suggesting a requirement for GTP within this highly conserved organelle. In sea urchin sperm, the NDK catalytic subunit is an integral component of the outer dynein arm. Here we describe a modular Protein (P72) from the Chlamydomonasflagellum that consists of three domains closely related to the presumptive regulatory segment of rat NDK-7 followed by two EF-hands that are predicted to bind Ca2+. There are close homologues of P72 in both mammalian and insect genomes. The P72 Protein is tightly associated with the flagellar axoneme and is located along the entire length except at the transition zone. Cross-linking experiments suggest that P72 interacts with two or three additional axonemal polypeptides. The sensitivity of P72 to tryptic digestion differed considerably in the presence and the absence of Ca2+, suggesting that it indeed binds this ligand. These studies indicate that the flagellar NDK system is bipartite with the regulatory and catalytic components residing on different polypeptides. We propose that Ca2+regulation of flagellar motility in Chlamydomonas may be achieved in part through a downstream GTP-mediated signaling pathway.
Sharon E Benashski - One of the best experts on this subject based on the ideXlab platform.
-
a bipartite ca2 regulated nucleoside diphosphate kinase system within the chlamydomonas flagellum the regulatory subunit P72
Journal of Biological Chemistry, 2002Co-Authors: Ramila S Patelking, Sharon E Benashski, Stephen M KingAbstract:Abstract Regulation of flagellar activity inChlamydomonas involves both Ca2+ and cAMP-mediated signaling pathways. However, Chlamydomonasand sea urchin sperm flagella also exhibit nucleoside-diphosphate kinase (NDK) activity, suggesting a requirement for GTP within this highly conserved organelle. In sea urchin sperm, the NDK catalytic subunit is an integral component of the outer dynein arm. Here we describe a modular Protein (P72) from the Chlamydomonasflagellum that consists of three domains closely related to the presumptive regulatory segment of rat NDK-7 followed by two EF-hands that are predicted to bind Ca2+. There are close homologues of P72 in both mammalian and insect genomes. The P72 Protein is tightly associated with the flagellar axoneme and is located along the entire length except at the transition zone. Cross-linking experiments suggest that P72 interacts with two or three additional axonemal polypeptides. The sensitivity of P72 to tryptic digestion differed considerably in the presence and the absence of Ca2+, suggesting that it indeed binds this ligand. These studies indicate that the flagellar NDK system is bipartite with the regulatory and catalytic components residing on different polypeptides. We propose that Ca2+regulation of flagellar motility in Chlamydomonas may be achieved in part through a downstream GTP-mediated signaling pathway.
-
a bipartite ca2 regulated nucleoside diphosphate kinase system within thechlamydomonas flagellum the regulatory subunit P72
Journal of Biological Chemistry, 2002Co-Authors: Ramila S Patelking, Sharon E Benashski, Stephen M KingAbstract:Regulation of flagellar activity inChlamydomonas involves both Ca2+ and cAMP-mediated signaling pathways. However, Chlamydomonasand sea urchin sperm flagella also exhibit nucleoside-diphosphate kinase (NDK) activity, suggesting a requirement for GTP within this highly conserved organelle. In sea urchin sperm, the NDK catalytic subunit is an integral component of the outer dynein arm. Here we describe a modular Protein (P72) from the Chlamydomonasflagellum that consists of three domains closely related to the presumptive regulatory segment of rat NDK-7 followed by two EF-hands that are predicted to bind Ca2+. There are close homologues of P72 in both mammalian and insect genomes. The P72 Protein is tightly associated with the flagellar axoneme and is located along the entire length except at the transition zone. Cross-linking experiments suggest that P72 interacts with two or three additional axonemal polypeptides. The sensitivity of P72 to tryptic digestion differed considerably in the presence and the absence of Ca2+, suggesting that it indeed binds this ligand. These studies indicate that the flagellar NDK system is bipartite with the regulatory and catalytic components residing on different polypeptides. We propose that Ca2+regulation of flagellar motility in Chlamydomonas may be achieved in part through a downstream GTP-mediated signaling pathway.
D L Rock - One of the best experts on this subject based on the ideXlab platform.
-
african swine fever virus structural Protein P72 contains a conformational neutralizing epitope
Virology, 1994Co-Authors: Manuel V Borca, P M Irusta, C Carrillo, Claudio L Afonso, T G Burrage, D L RockAbstract:Abstract We have previously described a monoclonal antibody (mAb 135D4) to an unidentified 70- to 72-kDa African swine fever virus (ASFV) Protein that exhibited high levels of neutralizing activity against various virulent ASFV isolates. Here, we identify the reactive ASFV Protein as the major virus structural Protein P72. In vitro -translated products of the P72 Protein gene were specifically immunoprecipitated by mAb 135D4. Immunoprecipitation of a nested set of truncated P72 in vitro translation products defined the region between amino acid residues 400 and 404 as necessary for mAb 135D4 reactivity. Five partially overlapping peptides (15mers) covering residues 388-446 failed to react with mAb 135D4, suggesting the conformational dependence of the epitope. Supporting this interpretation, larger in vitro translation products representing residues 56282, 159-361, 360-508, and 507-646 also failed to react with mAb 135D4. Consistent with its involvement in virus neutralization, immunoelectromicroscopy, using a rabbit antiserum against mAb 135D4-purified P72, located the Protein on the surface of unenveloped virus particles.
