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Sample records for catalytic subunit gene

  1. Gene targeting of CK2 catalytic subunits

    Science.gov (United States)

    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

  2. Gene targeting of CK2 catalytic subunits.

    Science.gov (United States)

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

    2008-09-01

    Protein kinase CK2 is a highly conserved and ubiquitous serine-threonine kinase. It is a tetrameric enzyme that is made up of two regulatory CK2beta subunits and two catalytic subunits, either CK2alpha/CK2alpha, CK2alpha/CK2alpha', or CK2alpha'/CK2alpha'. 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 CK2alpha' is essential for male germ cell development, and we now demonstrate that CK2alpha has an essential role in embryogenesis, as mice lacking CK2alpha die in mid-embryogenesis, with cardiac and neural tube defects.

  3. Gene for the catalytic subunit of mouse DNA-dependent protein kinase maps to the scid locus.

    Science.gov (United States)

    Miller, R D; Hogg, J; Ozaki, J H; Gell, D; Jackson, S P; Riblet, R

    1995-01-01

    The gene encoding the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) has been proposed recently as a candidate gene for the mouse severe combined immune deficiency (scid) locus. We have used a partial cDNA clone for human DNA-PKcs to map the mouse homologue using a large interspecific backcross panel. We found that the mouse gene for DNA-PKcs does not recombine with scid, consistent with the hypothesis that scid is a mutation in the mouse gene for DNA-PKcs. Images Fig. 3 PMID:7479885

  4. The catalytic subunit of cAMP-dependent protein kinase induces expression of genes containing cAMP-responsive enhancer elements.

    Science.gov (United States)

    Riabowol, K T; Fink, J S; Gilman, M Z; Walsh, D A; Goodman, R H; Feramisco, J R

    1988-11-03

    Transcriptional regulation of eukaryotic genes by cyclic AMP requires a cAMP-dependent protein kinase (A kinase). Two hypotheses have been proposed to explain how the holoenzyme of the A kinase induces transcription. The regulatory subunits of the A kinase, which bind cAMP and DNA, and have amino-acid homology with the Escherichia coli catabolite activator protein could directly stimulate gene expression. Alternatively, phosphorylation by the catalytic subunits could induce transcription by activating proteins involved in gene transcription. To distinguish between these models, we microinjected purified preparations of the catalytic and regulatory subunits of A kinase into tissue culture cells and monitored expression of a stably integrated fusion gene containing a cAMP-responsive human promoter fused to a bacterial reporter gene, or of the endogenous c-fos gene. The catalytic subunit stimulated expression of these genes, whereas the regulatory subunit did not. These results indicate that the catalytic subunit of A kinase is sufficient to induce expression of two cAMP-responsive genes, without increasing levels of cAMP.

  5. Elevated breast cancer risk in irradiated BALB/c mice associates with unique functional polymorphism of the Prkdc (DNA-dependent protein kinase catalytic subunit) gene

    Science.gov (United States)

    Yu, Y.; Okayasu, R.; Weil, M. M.; Silver, A.; McCarthy, M.; Zabriskie, R.; Long, S.; Cox, R.; Ullrich, R. L.

    2001-01-01

    Female BALB/c mice are unusually radiosensitive and more susceptible than C57BL/6 and other tested inbred mice to ionizing radiation (IR)-induced mammary tumors. This breast cancer susceptibility is correlated with elevated susceptibility for mammary cell transformation and genomic instability following irradiation. In this study, we report the identification of two BALB/c strain-specific polymorphisms in the coding region of Prkdc, the gene encoding the DNA-dependent protein kinase catalytic subunit, which is known to be involved in DNA double-stranded break repair and post-IR signal transduction. First, we identified an A --> G transition at base 11530 resulting in a Met --> Val conversion at codon 3844 (M3844V) in the phosphatidylinositol 3-kinase domain upstream of the scid mutation (Y4046X). Second, we identified a C --> T transition at base 6418 resulting in an Arg --> Cys conversion at codon 2140 (R2140C) downstream of the putative leucine zipper domain. This unique PrkdcBALB variant gene is shown to be associated with decreased DNA-dependent protein kinase catalytic subunit activity and with increased susceptibility to IR-induced genomic instability in primary mammary epithelial cells. The data provide the first evidence that naturally arising allelic variation in a mouse DNA damage response gene may associate with IR response and breast cancer risk.

  6. INCURVATA2 Encodes the Catalytic Subunit of DNA Polymerase α and Interacts with Genes Involved in Chromatin-Mediated Cellular Memory in Arabidopsis thaliana

    Science.gov (United States)

    Barrero, José María; González-Bayón, Rebeca; del Pozo, Juan Carlos; Ponce, María Rosa; Micol, José Luis

    2007-01-01

    Cell type–specific gene expression patterns are maintained by the stable inheritance of transcriptional states through mitosis, requiring the action of multiprotein complexes that remodel chromatin structure. Genetic and molecular interactions between chromatin remodeling factors and components of the DNA replication machinery have been identified in Schizosaccharomyces pombe, indicating that some epigenetic marks are replicated simultaneously to DNA with the participation of the DNA replication complexes. This model of epigenetic inheritance might be extended to the plant kingdom, as we report here with the positional cloning and characterization of INCURVATA2 (ICU2), which encodes the putative catalytic subunit of the DNA polymerase α of Arabidopsis thaliana. The strong icu2-2 and icu2-3 insertional alleles caused fully penetrant zygotic lethality when homozygous and incompletely penetrant gametophytic lethality, probably because of loss of DNA polymerase activity. The weak icu2-1 allele carried a point mutation and caused early flowering, leaf incurvature, and homeotic transformations of sepals into carpels and of petals into stamens. Further genetic analyses indicated that ICU2 interacts with TERMINAL FLOWER2, the ortholog of HETEROCHROMATIN PROTEIN1 of animals and yeasts, and with the Polycomb group (PcG) gene CURLY LEAF. Another PcG gene, EMBRYONIC FLOWER2, was found to be epistatic to ICU2. Quantitative RT-PCR analyses indicated that a number of regulatory genes were derepressed in the icu2-1 mutant, including genes associated with flowering time, floral meristem, and floral organ identity. PMID:17873092

  7. INCURVATA2 encodes the catalytic subunit of DNA Polymerase alpha and interacts with genes involved in chromatin-mediated cellular memory in Arabidopsis thaliana.

    Science.gov (United States)

    Barrero, José María; González-Bayón, Rebeca; del Pozo, Juan Carlos; Ponce, María Rosa; Micol, José Luis

    2007-09-01

    Cell type-specific gene expression patterns are maintained by the stable inheritance of transcriptional states through mitosis, requiring the action of multiprotein complexes that remodel chromatin structure. Genetic and molecular interactions between chromatin remodeling factors and components of the DNA replication machinery have been identified in Schizosaccharomyces pombe, indicating that some epigenetic marks are replicated simultaneously to DNA with the participation of the DNA replication complexes. This model of epigenetic inheritance might be extended to the plant kingdom, as we report here with the positional cloning and characterization of INCURVATA2 (ICU2), which encodes the putative catalytic subunit of the DNA polymerase alpha of Arabidopsis thaliana. The strong icu2-2 and icu2-3 insertional alleles caused fully penetrant zygotic lethality when homozygous and incompletely penetrant gametophytic lethality, probably because of loss of DNA polymerase activity. The weak icu2-1 allele carried a point mutation and caused early flowering, leaf incurvature, and homeotic transformations of sepals into carpels and of petals into stamens. Further genetic analyses indicated that ICU2 interacts with TERMINAL FLOWER2, the ortholog of HETEROCHROMATIN PROTEIN1 of animals and yeasts, and with the Polycomb group (PcG) gene CURLY LEAF. Another PcG gene, EMBRYONIC FLOWER2, was found to be epistatic to ICU2. Quantitative RT-PCR analyses indicated that a number of regulatory genes were derepressed in the icu2-1 mutant, including genes associated with flowering time, floral meristem, and floral organ identity.

  8. Functional expression and characterization of the Epstein-Barr virus DNA polymerase catalytic subunit.

    OpenAIRE

    1993-01-01

    A recombinant baculovirus containing the complete sequence for the Epstein-Barr virus (EBV) DNA polymerase catalytic subunit, BALF5 gene product, under the control of the baculovirus polyhedrin promoter was constructed. Insect cells infected with the recombinant virus produced a protein of 110 kDa, recognized by anti-BALF5 protein-specific polyclonal antibody. The expressed EBV DNA polymerase catalytic polypeptide was purified from the cytosolic fraction of the recombinant virus-infected inse...

  9. Two Arabidopsis ADP-glucose pyrophosphorylase large subunits (APL1 and APL2) are catalytic.

    Science.gov (United States)

    Ventriglia, Tiziana; Kuhn, Misty L; Ruiz, Ma Teresa; Ribeiro-Pedro, Marina; Valverde, Federico; Ballicora, Miguel A; Preiss, Jack; Romero, José M

    2008-09-01

    ADP-glucose (Glc) pyrophosphorylase (ADP-Glc PPase) catalyzes the first committed step in starch biosynthesis. Higher plant ADP-Glc PPase is a heterotetramer (alpha(2)beta(2)) consisting of two small and two large subunits. There is increasing evidence that suggests that catalytic and regulatory properties of the enzyme from higher plants result from the synergy of both types of subunits. In Arabidopsis (Arabidopsis thaliana), two genes encode small subunits (APS1 and APS2) and four large subunits (APL1-APL4). Here, we show that in Arabidopsis, APL1 and APL2, besides their regulatory role, have catalytic activity. Heterotetramers formed by combinations of a noncatalytic APS1 and the four large subunits showed that APL1 and APL2 exhibited ADP-Glc PPase activity with distinctive sensitivities to the allosteric activator (3-phosphoglycerate). Mutation of the Glc-1-P binding site of Arabidopsis and potato (Solanum tuberosum) isoforms confirmed these observations. To determine the relevance of these activities in planta, a T-DNA mutant of APS1 (aps1) was characterized. aps1 is starchless, lacks ADP-Glc PPase activity, APS1 mRNA, and APS1 protein, and is late flowering in long days. Transgenic lines of the aps1 mutant, expressing an inactivated form of APS1, recovered the wild-type phenotype, indicating that APL1 and APL2 have catalytic activity and may contribute to ADP-Glc synthesis in planta.

  10. Two Arabidopsis ADP-Glucose Pyrophosphorylase Large Subunits (APL1 and APL2) Are Catalytic1

    Science.gov (United States)

    Ventriglia, Tiziana; Kuhn, Misty L.; Ruiz, Ma Teresa; Ribeiro-Pedro, Marina; Valverde, Federico; Ballicora, Miguel A.; Preiss, Jack; Romero, José M.

    2008-01-01

    ADP-glucose (Glc) pyrophosphorylase (ADP-Glc PPase) catalyzes the first committed step in starch biosynthesis. Higher plant ADP-Glc PPase is a heterotetramer (α2β2) consisting of two small and two large subunits. There is increasing evidence that suggests that catalytic and regulatory properties of the enzyme from higher plants result from the synergy of both types of subunits. In Arabidopsis (Arabidopsis thaliana), two genes encode small subunits (APS1 and APS2) and four large subunits (APL1–APL4). Here, we show that in Arabidopsis, APL1 and APL2, besides their regulatory role, have catalytic activity. Heterotetramers formed by combinations of a noncatalytic APS1 and the four large subunits showed that APL1 and APL2 exhibited ADP-Glc PPase activity with distinctive sensitivities to the allosteric activator (3-phosphoglycerate). Mutation of the Glc-1-P binding site of Arabidopsis and potato (Solanum tuberosum) isoforms confirmed these observations. To determine the relevance of these activities in planta, a T-DNA mutant of APS1 (aps1) was characterized. aps1 is starchless, lacks ADP-Glc PPase activity, APS1 mRNA, and APS1 protein, and is late flowering in long days. Transgenic lines of the aps1 mutant, expressing an inactivated form of APS1, recovered the wild-type phenotype, indicating that APL1 and APL2 have catalytic activity and may contribute to ADP-Glc synthesis in planta. PMID:18614708

  11. The Alpha Catalytic Subunit of Protein Kinase CK2 Is Required for Mouse Embryonic Development▿

    Science.gov (United States)

    Lou, David Y.; Dominguez, Isabel; Toselli, Paul; Landesman-Bollag, Esther; O'Brien, Conor; Seldin, David C.

    2008-01-01

    Protein kinase CK2 (formerly casein kinase II) is a highly conserved and ubiquitous serine/threonine kinase that is composed of two catalytic subunits (CK2α and/or CK2α′) and two CK2β regulatory subunits. CK2 has many substrates in cells, and key roles in yeast cell physiology have been uncovered by introducing subunit mutations. Gene-targeting experiments have demonstrated that in mice, the CK2β gene is required for early embryonic development, while the CK2α′ subunit appears to be essential only for normal spermatogenesis. We have used homologous recombination to disrupt the CK2α gene in the mouse germ line. Embryos lacking CK2α have a marked reduction in CK2 activity in spite of the presence of the CK2α′ subunit. CK2α−/− embryos die in mid-gestation, with abnormalities including open neural tubes and reductions in the branchial arches. Defects in the formation of the heart lead to hydrops fetalis and are likely the cause of embryonic lethality. Thus, CK2α appears to play an essential and uncompensated role in mammalian development. PMID:17954558

  12. The upr-1 gene encodes a catalytic subunit of the DNA polymerase zeta which is involved in damage-induced mutagenesis in Neurospora crassa.

    Science.gov (United States)

    Sakai, W; Ishii, C; Inoue, H

    2002-05-01

    The upr-1 mutant was one of the first mutagen-sensitive mutants to be isolated in Neurospora crassa. However, the function of the upr-1 gene has not yet been elucidated, although some genetic and biochemical data have been accumulated. In order to clone the upr-1 gene, we performed a chromosome walk from the mat locus, the closest genetic marker to upr-1 for which a molecular probe was available, towards the centromere, and a chromosomal contig of about 300-400 kb was constructed. Some of these clones complemented the temperature sensitivity of the un-16 mutation, which is located between mat and upr-1. The un-16 gene was sequenced, and localized in the MIPS Neurospora crassa genome database. We then searched the regions flanking un-16 for homologs of known DNA repair genes, and found a gene homologous to the REV3 gene of budding yeast. The phenotype of the upr-1 mutant is similar to that of the yeast rev3 mutant. An ncrev3 mutant carrying mutations in the N. crassa REV3 homolog was constructed using the RIP (repeat-induced point mutation) process. The spectrum of mutagen sensitivity of the ncrev3 mutant was similar to that of the upr-1 mutant. Complementation tests between the upr-1 and ncrev3 mutations indicated that the upr-1 gene is in fact identical to the ncrev3 gene. To clarify the role of the upr-1 gene in DNA repair, the frequency of MMS and 4NQO-induced mutations was assayed using the ad-8 reversion test. The upr-1 mutant was about 10 times less sensitive to both chemicals than the wild type. The expression level of the upr-1 gene is increased on exposure to UV irradiation in the uvs-2 and mus-8 mutants, which belong to postreplication repair group, as well as in the wild type. All these results suggest that the product of the upr-1 gene functions in damage-induced mutagenesis and DNA translesion synthesis in N. crassa.

  13. trt-1 is the Caenorhabditis elegans catalytic subunit of telomerase.

    Directory of Open Access Journals (Sweden)

    Bettina Meier

    2006-02-01

    Full Text Available Mutants of trt-1, the Caenorhabditis elegans telomerase reverse transcriptase, reproduce normally for several generations but eventually become sterile as a consequence of telomere erosion and end-to-end chromosome fusions. Telomere erosion and uncapping do not cause an increase in apoptosis in the germlines of trt-1 mutants. Instead, late-generation trt-1 mutants display chromosome segregation defects that are likely to be the direct cause of sterility. trt-1 functions in the same telomere replication pathway as mrt-2, a component of the Rad9/Rad1/Hus1 (9-1-1 proliferating cell nuclear antigen-like sliding clamp. Thus, the 9-1-1 complex may be required for telomerase to act at chromosome ends in C. elegans. Although telomere erosion limits replicative life span in human somatic cells, neither trt-1 nor telomere shortening affects postmitotic aging in C. elegans. These findings illustrate effects of telomere dysfunction in C. elegans mutants lacking the catalytic subunit of telomerase, trt-1.

  14. Structure of the Tribolium castaneum Telomerase Catalytic Subunit TERT

    Energy Technology Data Exchange (ETDEWEB)

    Gillis,A.; Schuller, A.; Skordalakes, E.

    2008-01-01

    A common hallmark of human cancers is the overexpression of telomerase, a ribonucleoprotein complex that is responsible for maintaining the length and integrity of chromosome ends. Telomere length deregulation and telomerase activation is an early, and perhaps necessary, step in cancer cell evolution. Here we present the high-resolution structure of the Tribolium castaneum catalytic subunit of telomerase, TERT. The protein consists of three highly conserved domains, organized into a ring-like structure that shares common features with retroviral reverse transcriptases, viral RNA polymerases and B-family DNA polymerases. Domain organization places motifs implicated in substrate binding and catalysis in the interior of the ring, which can accommodate seven to eight bases of double-stranded nucleic acid. Modelling of an RNA-DNA heteroduplex in the interior of this ring demonstrates a perfect fit between the protein and the nucleic acid substrate, and positions the 3'-end of the DNA primer at the active site of the enzyme, providing evidence for the formation of an active telomerase elongation complex.

  15. Cadmium treatment suppresses DNA polymerase δ catalytic subunit gene expression by acting on the p53 and Sp1 regulatory axis.

    Science.gov (United States)

    Antoniali, Giulia; Marcuzzi, Federica; Casarano, Elena; Tell, Gianluca

    2015-11-01

    Cadmium (Cd) is a carcinogenic and neurotoxic environmental pollutant. Among the proposed mechanisms for Cd toxic effects, its ability to promote oxidative stress and to inhibit, in vitro, the activities of some Base Excision DNA Repair (BER) enzymes, such as hOGG1, XRCC1 and APE1, have been already established. However, the molecular mechanisms at the basis of these processes are largely unknown especially at sub-lethal doses of Cd and no information is available on the effect of Cd on the expression levels of BER enzymes. Here, we show that non-toxic treatment of neuronal cell lines, with pro-mitogenic doses of Cd, promotes a significant time- and dose-dependent down-regulation of DNA polymerase δ (POLD1) expression through a transcriptional mechanism with a modest effect on Polβ, XRCC1 and APE1. We further elucidated that the observed transcriptional repression on Polδ is acted by through competition by activated p53 on Sp1 at POLD1 promoter and by a squelching effect. We further proved the positive effect of Sp1 not only on POLD1 expression but also on Polβ, XRCC1 and APE1 expression, suggesting that Sp1 has pleiotropic effects on the whole BER pathway. Our results indicated that Cd-mediated impairment of BER pathway, besides acting on the enzymatic functions of some key proteins, is also exerted at the gene expression level of Polδ by acting on the p53-Sp1 regulatory axis. These data may explain not only the Cd-induced neurotoxic effects but also the potential carcinogenicity of this heavy metal.

  16. Functional Diversification of Maize RNA Polymerase IV and V subtypes via Alternative Catalytic Subunits

    Energy Technology Data Exchange (ETDEWEB)

    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.

  17. First inactive conformation of CK2 alpha, the catalytic subunit of protein kinase CK2

    DEFF Research Database (Denmark)

    Raaf, Jennifer; Issinger, Olaf-Georg; Niefind, Karsten

    2009-01-01

    (EPKs). To function as regulatory key components, EPKs normally exist in inactive ground states and are activated only upon specific signals. Typically, this activation is accompanied by large conformational changes in helix alpha C and in the activation segment, leading to a characteristic arrangement......The Ser/Thr kinase casein kinase 2 (CK2) is a heterotetrameric enzyme composed of two catalytic chains (CK2alpha, catalytic subunit of CK2) attached to a dimer of two noncatalytic subunits (CK2beta, noncatalytic subunit of CK2). CK2alpha belongs to the superfamily of eukaryotic protein kinases...... of catalytic key elements. For CK2alpha, however, no strict physiological control of activity is known. Accordingly, CK2alpha was found so far exclusively in the characteristic conformation of active EPKs, which is, in this case, additionally stabilized by a unique intramolecular contact between the N...

  18. Structure of the human protein kinase CK2 catalytic subunit CK2α' and interaction thermodynamics with the regulatory subunit CK2β

    DEFF Research Database (Denmark)

    Bischoff, Nils; Olsen, Birgitte; Raaf, Jennifer;

    2011-01-01

    the limited biochemical knowledge about the second paralog (CK2α'), we developed a well-soluble catalytically active full-length mutant of human CK2α', characterized it by Michaelis-Menten kinetics and isothermal titration calorimetry, and determined its crystal structure to a resolution of 2 Å. The affinity......Protein kinase CK2 (formerly "casein kinase 2") is composed of a central dimer of noncatalytic subunits (CK2β) binding two catalytic subunits. In humans, there are two isoforms of the catalytic subunit (and an additional splicing variant), one of which (CK2α) is well characterized. To supplement...

  19. In situ structure of trypanosomal ATP synthase dimer reveals a unique arrangement of catalytic subunits

    Science.gov (United States)

    Mühleip, Alexander W.; Dewar, Caroline E.; Schnaufer, Achim; Kühlbrandt, Werner; Davies, Karen M.

    2017-01-01

    We used electron cryotomography and subtomogram averaging to determine the in situ structures of mitochondrial ATP synthase dimers from two organisms belonging to the phylum euglenozoa: Trypanosoma brucei, a lethal human parasite, and Euglena gracilis, a photosynthetic protist. At a resolution of 32.5 Å and 27.5 Å, respectively, the two structures clearly exhibit a noncanonical F1 head, in which the catalytic (αβ)3 assembly forms a triangular pyramid rather than the pseudo-sixfold ring arrangement typical of all other ATP synthases investigated so far. Fitting of known X-ray structures reveals that this unusual geometry results from a phylum-specific cleavage of the α subunit, in which the C-terminal αC fragments are displaced by ∼20 Å and rotated by ∼30° from their expected positions. In this location, the αC fragment is unable to form the conserved catalytic interface that was thought to be essential for ATP synthesis, and cannot convert γ-subunit rotation into the conformational changes implicit in rotary catalysis. The new arrangement of catalytic subunits suggests that the mechanism of ATP generation by rotary ATPases is less strictly conserved than has been generally assumed. The ATP synthases of these organisms present a unique model system for discerning the individual contributions of the α and β subunits to the fundamental process of ATP synthesis. PMID:28096380

  20. Functional Diversification of Maize RNA Polymerase IV and V Subtypes via Alternative Catalytic Subunits

    Directory of Open Access Journals (Sweden)

    Jeremy R. Haag

    2014-10-01

    Full Text Available 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 analyses 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 subtypes 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.

  1. Cyclic AMP-dependent protein kinase I: Cyclic nucleotide binding, structural changes, and release of the catalytic subunits

    OpenAIRE

    Smith, Stephen B.; White, Hillary D.; Siegel, Jeffrey B.; Krebs, Edwin G.

    1981-01-01

    Type I cyclic AMP (cAMP)-dependent protein kinase is composed of a dimeric regulatory subunit (R2) and two catalytic subunits (C subunits). The R2 dimer binds four cAMP molecules to release the two C subunits. To characterize the cAMP binding sites and elucidate their role in the release of the C subunits, the R2 dimer has been studied by equilibrium methods. The cAMP titration of R2 was monitored by endogenous tryptophan fluorescence, and the results suggest one class of binding sites. The t...

  2. The catalytic and the RNA subunits of human telomerase are required to immortalize equid primary fibroblasts.

    Science.gov (United States)

    Vidale, Pamela; Magnani, Elisa; Nergadze, Solomon G; Santagostino, Marco; Cristofari, Gael; Smirnova, Alexandra; Mondello, Chiara; Giulotto, Elena

    2012-10-01

    Many human primary somatic cells can be immortalized by inducing telomerase activity through the exogenous expression of the human telomerase catalytic subunit (hTERT). This approach has been extended to the immortalization of cell lines from several mammals. Here, we show that hTERT expression is not sufficient to immortalize primary fibroblasts from three equid species, namely donkey, Burchelli's zebra and Grevy's zebra. In vitro analysis of a reconstituted telomerase composed by hTERT and an equid RNA component of telomerase (TERC) revealed a low activity of this enzyme compared to human telomerase, suggesting a low compatibility of equid and human telomerase subunits. This conclusion was also strengthened by comparison of human and equid TERC sequences, which revealed nucleotide differences in key regions for TERC and TERT interaction. We then succeeded in immortalizing equid fibroblasts by expressing hTERT and hTERC concomitantly. Expression of both human telomerase subunits led to telomerase activity and telomere elongation, indicating that human telomerase is compatible with the other equid telomerase subunits and proteins involved in telomere metabolism. The immortalization procedure described herein could be extended to primary cells from other mammals. The availability of immortal cells from endangered species could be particularly useful for obtaining new information on the organization and function of their genomes, which is relevant for their preservation.

  3. Effect of antisense oligonucleotides targeting telomerase catalytic subunit on tumor cell proliferationin vitro

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    To screen specific antitumor drugs targeting telomerase catalytic subunit (hEST2), 12 different hEST2 antisense oligonucleotides were designed based on hEST2 mRNA second structure and transfected into tumor cell lines by the lipofectin-mediated method. Cell growth activity was evaluated by MTT assay. hEST212 was picked out and its specificity, antitumor tree and continuous effect were analyzed. The results showed that hEST212 had promising antitumor activity in vitro, hEST2 can be used as a pratical target and an antisense drug candidate for cancer.

  4. Structural Basis for Telomerase Catalytic Subunit TERT Binding to RNA Template and Telomeric DNA

    Energy Technology Data Exchange (ETDEWEB)

    Mitchell, M.; Gillis, A; Futahashi, M; Fujiwara, H; Skordalakes, E

    2010-01-01

    Telomerase is a specialized DNA polymerase that extends the 3{prime} ends of eukaryotic linear chromosomes, a process required for genomic stability and cell viability. Here we present the crystal structure of the active Tribolium castaneum telomerase catalytic subunit, TERT, bound to an RNA-DNA hairpin designed to resemble the putative RNA-templating region and telomeric DNA. The RNA-DNA hybrid adopts a helical structure, docked in the interior cavity of the TERT ring. Contacts between the RNA template and motifs 2 and B{prime} position the solvent-accessible RNA bases close to the enzyme active site for nucleotide binding and selectivity. Nucleic acid binding induces rigid TERT conformational changes to form a tight catalytic complex. Overall, TERT-RNA template and TERT-telomeric DNA associations are remarkably similar to those observed for retroviral reverse transcriptases, suggesting common mechanistic aspects of DNA replication between the two families of enzymes.

  5. Dissecting structural basis of the unique substrate selectivity of human enteropeptidase catalytic subunit.

    Science.gov (United States)

    Ostapchenko, Valeriy G; Gasparian, Marine E; Kosinsky, Yurij A; Efremov, Roman G; Dolgikh, Dmitry A; Kirpichnikov, Mikhail P

    2012-01-01

    Enteropeptidase is a key enzyme in the digestion system of higher animals. It initiates enzymatic cascade cleaving trypsinogen activation peptide after a unique sequence DDDDK. Recently, we have found specific activity of human enteropeptidase catalytic subunit (L-HEP) being significantly higher than that of its bovine ortholog (L-BEP). Moreover, we have discovered that L-HEP hydrolyzed several nonspecific peptidic substrates. In this work, we aimed to further characterize species-specific enteropeptidase activities and to reveal their structural basis. First, we compared hydrolysis of peptides and proteins lacking DDDDK sequence by L-HEP and L-BEP. In each case human enzyme was more efficient, with the highest hydrolysis rate observed for substrates with a large hydrophobic residue in P2-position. Computer modeling suggested enzyme exosite residues 96 (Arg in L-HEP, Lys in L-BEP) and 219 (Lys in L-HEP, Gln in L-BEP) to be responsible for these differences in enteropeptidase catalytic activity. Indeed, human-to-bovine mutations Arg96Lys, Lys219Gln shifted catalytic properties of L-HEP toward those of L-BEP. This effect was amplified in case of the double mutation Arg96Lys/Lys219Gln, but still did not cover the full difference in catalytic activities of human and bovine enzymes. To find a missing link, we studied monopeptide benzyl-arginine-β-naphthylamide hydrolysis. L-HEP catalyzed it with an order lower K (m) than L-BEP, suggesting the monopeptide-binding S1 site input into catalytic distinction between two enteropeptidase species. Together, our findings suggest structural basis of the unique catalytic properties of human enteropeptidase and instigate further studies of its tentative physiological and pathological roles.

  6. Human liver phosphatase 2A: cDNA and amino acid sequence of two catalytic subunit isotypes

    Energy Technology Data Exchange (ETDEWEB)

    Arino, J.; Woon, Chee Wai; Brautigan, D.L.; Miller, T.B. Jr.; Johnson, G.L. (Univ. of Massachusetts Medical School, Worcester (USA))

    1988-06-01

    Two cDNA clones were isolated from a human liver library that encode two phosphatase 2A catalytic subunits. The two cDNAs differed in eight amino acids (97% identity) with three nonconservative substitutions. All of the amino acid substitutions were clustered in the amino-terminal domain of the protein. Amino acid sequence of one human liver clone (HL-14) was identical to the rabbit skeletal muscle phosphatase 2A cDNA (with 97% nucleotide identity). The second human liver clone (HL-1) is encoded by a separate gene, and RNA gel blot analysis indicates that both mRNAs are expressed similarly in several human clonal cell lines. Sequence comparison with phosphatase 1 and 2A indicates highly divergent amino acid sequences at the amino and carboxyl termini of the proteins and identifies six highly conserved regions between the two proteins that are predicted to be important for phosphatase enzymatic activity.

  7. Expression of a nonmyristylated variant of the catalytic subunit of protein kinase A during male germ-cell development.

    Science.gov (United States)

    Desseyn, J L; Burton, K A; McKnight, G S

    2000-06-06

    The catalytic subunits of protein kinase A are transcribed in all mouse tissues from two distinct genes that code for the Calpha and Cbeta isoforms. Alternative promoters exist for the Cbeta gene that are used in a tissue-specific fashion and give rise to variants that differ in their amino-terminal sequences. We have characterized an alternative promoter that is present in the first intron of the Calpha gene and is transcriptionally active in male germ cells. Transcription from this promoter is coincident with the appearance of pachytene spermatocytes and leads to a Calpha protein (Calpha2) that contains a distinctive 7 amino acid amino-terminus differing from the 14 amino acid amino-terminus of Calpha1. The Calpha2 protein does not contain the myristylation signal present on Calpha1 and migrates at a lower molecular weight on SDS/PAGE gels. By Western blotting, we estimate that most or all of the Calpha protein present in mature sperm is Calpha2. The amino-terminal sequence of Calpha2 is similar to that of ovine sperm C as previously reported [San Agustin, J. T., Leszyk, J. D., Nuwaysir, L. M. & Witman, G. B. (1998) J. Biol. Chem. 273, 24874-24883], and we show by cDNA cloning that human sperm also express a highly related Calpha2 homolog. The Calpha2 subunit forms holoenzymes with either RIIalpha or RIalpha, and both activate at the same concentration of cyclic nucleotide. Because protein kinase A is thought to play a pivotal role in sperm motility and capacitation, the distinctive biochemical properties of the unmyristylated Calpha2 may be essential for fertility in the male.

  8. Expression, purification and crystallization of the catalytic subunit of protein kinase CK2 from Zea mays

    DEFF Research Database (Denmark)

    Guerra, B; Niefind, K; Pinna, L A

    1998-01-01

    a = 142.6, b = 61.3, c = 45.6 A, beta = 103.3 degrees and diffract X-rays to at least 2.0 A resolution. The calculated crystal packing parameter is Vm = 2.47 A3 Da-1 suggesting that one CK2alpha molecule is contained in the asymmetric unit and that the solvent content of the unit cell is 50%.......The catalytic (alpha) subunit of protein kinase CK2 (CK2alpha) was originally cloned and overexpressed in the Escherichia coli strain pT7-7/BL21(DE3). The protein has been purified to homogeneity and crystallized. The crystals belong to the monoclinic space group C2, they have unit-cell parameters...

  9. Functional analysis of Trichoderma reesei CKIIα2, a catalytic subunit of casein kinase II.

    Science.gov (United States)

    Wang, Mingyu; Yang, Hui; Zhang, Meiling; Liu, Kuimei; Wang, Hanbin; Luo, Yi; Fang, Xu

    2015-07-01

    Trichoderma reesei is the most important industrial cellulase-producing filamentous fungus. Although its molecular physiology has been investigated, the signal transduction pathways are not fully understood. In particular, the role of casein kinase II (CKII) is not yet clear. In this work, we carried out functional investigations on a catalytic subunit of CKII, CKIIα2. Comparison of the phenotypic features of T. reesei parent and Δck2α2 strains showed significant changes following ck2α2 disruption. T. reesei Δck2α2 form significantly smaller mycelial pellets in glucose-containing liquid minimum media, have shorter and fewer branch hyphae, produce smaller amounts of chitinases, produce more spores, show more robust growth on glucose-containing agar plates, and consume glucose at a significantly higher rate. Suggestions can be made that CKIIα2 governs chitinase expression, and the disruption of ck2α2 results in lower levels of chitinase production, leading to a weaker cell wall disruption capability, further resulting in weaker hyphal branching, which eventually leads to smaller mycelial pellets in liquid media. Further conclusions can be made that CKIIα2 is involved in repression of sporulation and glucose metabolism, which is consistent with the proposal that CKIIα2 represses global metabolism. These observations make the deletion of ck2α2 a potentially beneficial genetic disruption for T. reesei during industrial applications, as smaller mycelial pellets, more spores and more robust glucose metabolism are all desired traits for industrial fermentation. This work reports novel unique functions of a CKII catalytic subunit and is also the first genetic and physiological investigation on CKII in T. reesei.

  10. Mapping of the Mouse Actin Capping Protein Beta Subunit Gene

    Directory of Open Access Journals (Sweden)

    Cooper John A

    2000-07-01

    Full Text Available Abstract Background Capping protein (CP, a heterodimer of α and β subunits, is found in all eukaryotes. CP binds to the barbed ends of actin filaments in vitro and controls actin assembly and cell motility in vivo. Vertebrates have three isoforms of CPβ produced by alternatively splicing from one gene; lower organisms have one gene and one isoform. Results We isolated genomic clones corresponding to the β subunit of mouse CP and identified its chromosomal location by interspecies backcross mapping. Conclusions The CPβ gene (Cappb1 mapped to Chromosome 4 between Cdc42 and D4Mit312. Three mouse mutations, snubnose, curly tail, and cribriform degeneration, map in the vicinity of the β gene.

  11. Crystal structures of catalytic and regulatory subunits of rat protein kinase CK2

    Institute of Scientific and Technical Information of China (English)

    ZHOU WeiHong; SHEN YueQuan; QIN XiaoHong; YAN XiaoJie; XIE XingQiao; LI Liang; FANG ShaSha; LONG JiaFu; ADELMAN John; TANG Wei-Jen

    2009-01-01

    Protein kinase CK2 consists of two catalytic subunits (CK2α) and two regulatory subunits (CK2β). Here,we report the crystal structures of rat CK2α mutant (rCK2α-△C, 1-335) and CK2β(rCK2β). The overall topology of rCK2α-△C and rCK2βare very similar to the human enzyme, although large structural dif-ferences could be observed in the N-terminal domain of rCK2α-△C. Our reported structure of rCK2α-△C is in the close conformation state while the counterpart hCK2α is in the open conformation state, indi-cating the conformation of CK2α molecule has high plasticity. The structure of rCK2β represents the conformation of free CK2β. Upon CK2α binding, the C-terminal region undergoes a drastic conforma-tional change. The major region of interaction within the interface of CK2αCK2β may serve as a bridge to transmit the conformational change and thus regulate the activity of CK2α.

  12. An Fe-S cluster in the conserved Cys-rich region in the catalytic subunit of FAD-dependent dehydrogenase complexes.

    Science.gov (United States)

    Shiota, Masaki; Yamazaki, Tomohiko; Yoshimatsu, Keiichi; Kojima, Katsuhiro; Tsugawa, Wakako; Ferri, Stefano; Sode, Koji

    2016-12-01

    Several bacterial flavin adenine dinucleotide (FAD)-harboring dehydrogenase complexes comprise three distinct subunits: a catalytic subunit with FAD, a cytochrome c subunit containing three hemes, and a small subunit. Owing to the cytochrome c subunit, these dehydrogenase complexes have the potential to transfer electrons directly to an electrode. Despite various electrochemical applications and engineering studies of FAD-dependent dehydrogenase complexes, the intra/inter-molecular electron transfer pathway has not yet been revealed. In this study, we focused on the conserved Cys-rich region in the catalytic subunits using the catalytic subunit of FAD dependent glucose dehydrogenase complex (FADGDH) as a model, and site-directed mutagenesis and electron paramagnetic resonance (EPR) were performed. By co-expressing a hitch-hiker protein (γ-subunit) and a catalytic subunit (α-subunit), FADGDH γα complexes were prepared, and the properties of the catalytic subunit of both wild type and mutant FADGDHs were investigated. Substitution of the conserved Cys residues with Ser resulted in the loss of dye-mediated glucose dehydrogenase activity. ICP-AEM and EPR analyses of the wild-type FADGDH catalytic subunit revealed the presence of a 3Fe-4S-type iron-sulfur cluster, whereas none of the Ser-substituted mutants showed the EPR spectrum characteristic for this cluster. The results suggested that three Cys residues in the Cys-rich region constitute an iron-sulfur cluster that may play an important role in the electron transfer from FAD (intra-molecular) to the multi-heme cytochrome c subunit (inter-molecular) electron transfer pathway. These features appear to be conserved in the other three-subunit dehydrogenases having an FAD cofactor.

  13. Crystal structure of catalytic domain of the initiation factor 2B epsilon subunit

    DEFF Research Database (Denmark)

    Boesen, Thomas; Mohammad, Sarah S.; Pavitt, Graham D.;

    this motif is involved in binding to the N-terminal part of the eIF2β subunit The aliphatic residues in the AA box motifs are involved in specific contacts in the hydrophobic core of the C-terminal helices important for maintaining the overall structure, whereas acidic residues in the motifs form a clustered......-terminal two helices contain the catalytic part of the domain, whereas the C-terminal six helices harbor the two Aromatic Acidic (AA) box motifs. This motif is also found in initiation factor 5, the GTPase activator protein of eIF2, and furthermore in mammalian initiation factor 4G. In eIF2B and eIF5......, surface exposed acidic patch which might interact with the lysine boxes of eIF2β. Interestingly, Tryptophan 699 was found to be solvent exposed and involved in crystal packing. This residue could possibly be important for the specific interaction with eIF2β. Furthermore, the structure shows the location...

  14. Loss of the catalytic subunit of the DNA-dependent protein kinase in DNA double-strand-break-repair mutant mammalian cells

    Energy Technology Data Exchange (ETDEWEB)

    Peterson, S.R. [Los Alamos National Lab., NM (United States)]|[Tottori Univ., Yonago (Japan); Kurimasa, Akihiro; Oshimura, Mitsuo [Tottori Univ., Yonago (Japan); Dynan, W.S. [Univ. of Colorado, Boulder, CO (United States); Bradbury, E.M. [Los Alamos National Lab., NM (United States)]|[Univ. of California, Davis, CA (United States); Chen, D.J. [Los Alamos National Lab., NM (United States)

    1995-04-11

    The DNA-dependent protein kinase (DNA-PK) consists of three polypeptide components: Ku-70, Ku-80, and an {approx}350-kDa catalytic subunit (p350). The gene encoding the Ku-80 subunit is identical to the x-ray-sensitive group 5 complementing gene XRCC5. Expression of the Ku-80 cDNA rescues both DNA double-strand break (DSB) repair and V(D)J recombination in group 5 mutant cells. The involvement of Ku-80 in these processes suggests that the underlying defect in these mutant cells may be disruption of the DNA-PK holoenzyme. In this report we show that the p350 kinase subunit is deleted in cells derived from the severe combined immunodeficiency mouse and in the Chinese hamster ovary cell line V-3, both of which are defective in DSB repair and V(D)J recombination. A centromeric fragment of human chromosome 8 that complements the scid defect also restores p350 protein expression and rescues in vitro DNA-PK activity. These data suggest the scid gene may encode the p350 protein or regulate its expression and are consistent with a model whereby DNA-PK is a critical component of the DSB-repair pathway. 38 refs., 3 figs.

  15. Protein kinase CK2: evidence for a protein kinase CK2beta subunit fraction, devoid of the catalytic CK2alpha subunit, in mouse brain and testicles

    DEFF Research Database (Denmark)

    Guerra, B; Siemer, S; Boldyreff, B;

    1999-01-01

    The highest CK2 activity was found in mouse testicles and brain, followed by spleen, liver, lung, kidney and heart. The activity values were directly correlated with the protein expression level of the CK2 subunits alpha (catalytic) and beta (regulatory). The alpha' subunit was only detected...... found for testicles and brain. The amount of CK2beta protein in brain in comparison to the other organs (except testicles) was estimated to be ca. 2-3-fold higher whereas the ratio of CK2beta between testicles and brain was estimated to be 3-4-fold. Results from the immunoprecipitation experiments...... support the notion for the existence of free CK2beta population and/or CK2beta in complex with other protein(s) present in brain and testicles. In all other mouse organs investigated, i.e. heart, lung, liver, kidney and spleen, no comparable amount of free CK2beta was observed. This is the first...

  16. Differential expression of G protein alpha and ß subunit genes during development of Phytophthora infestans

    NARCIS (Netherlands)

    Laxalt, A.M.; Latijnhouwers, M.; Hulten, van M.; Govers, F.

    2002-01-01

    A G protein subunit gene (pigpa1) and a G protein subunit gene (pigpb1) were isolated from the oomycete Phytophthora infestans, the causal agent of potato late blight. Heterotrimeric G proteins are evolutionary conserved GTP-binding proteins that are composed of ,, and subunits and participate in di

  17. daf-31 encodes the catalytic subunit of N alpha-acetyltransferase that regulates Caenorhabditis elegans development, metabolism and adult lifespan.

    Science.gov (United States)

    Chen, Di; Zhang, Jiuli; Minnerly, Justin; Kaul, Tiffany; Riddle, Donald L; Jia, Kailiang

    2014-10-01

    The Caenorhabditis elegans dauer larva is a facultative state of diapause. Mutations affecting dauer signal transduction and morphogenesis have been reported. Of these, most that result in constitutive formation of dauer larvae are temperature-sensitive (ts). The daf-31 mutant was isolated in genetic screens looking for novel and underrepresented classes of mutants that form dauer and dauer-like larvae non-conditionally. Dauer-like larvae are arrested in development and have some, but not all, of the normal dauer characteristics. We show here that daf-31 mutants form dauer-like larvae under starvation conditions but are sensitive to SDS treatment. Moreover, metabolism is shifted to fat accumulation in daf-31 mutants. We cloned the daf-31 gene and it encodes an ortholog of the arrest-defective-1 protein (ARD1) that is the catalytic subunit of the major N alpha-acetyltransferase (NatA). A daf-31 promoter::GFP reporter gene indicates daf-31 is expressed in multiple tissues including neurons, pharynx, intestine and hypodermal cells. Interestingly, overexpression of daf-31 enhances the longevity phenotype of daf-2 mutants, which is dependent on the forkhead transcription factor (FOXO) DAF-16. We demonstrate that overexpression of daf-31 stimulates the transcriptional activity of DAF-16 without influencing its subcellular localization. These data reveal an essential role of NatA in controlling C. elegans life history and also a novel interaction between ARD1 and FOXO transcription factors, which may contribute to understanding the function of ARD1 in mammals.

  18. Interaction of the regulatory and catalytic subunits of cAMP-dependent protein kinase. Electrostatic sites on the type Ialpha regulatory subunit.

    Science.gov (United States)

    Gibson, R M; Ji-Buechler, Y; Taylor, S S

    1997-06-27

    Since a basic surface on the catalytic (C) subunit of cAMP-dependent protein kinase is important for binding to the regulatory (R) subunit, acidic residues in R were sought that might contribute to R-C interaction. Using differential labeling by a water-soluble carbodiimide (Buechler, T. A., and Taylor, S. S. (1990) Biochemistry 29, 1937-1943), seven specific carboxylates in RIalpha were identified that were protected from chemical modification in the holoenzyme; each was then replaced with Ala. Of these, rRI(E15A/E106A/D107A)), rRI(E105A), rRI(D140A), rRI(E143A), and rRI(D258A) all were defective in holoenzyme formation and define negative electrostatic surfaces on RIalpha. An additional conserved carboxylate, Glu101 in RIalpha and the equivalent, Glu99 in RIIalpha were mutated to Ala. Replacement of Glu101 had no effect while rRII(E99A) was very defective. RIalpha and RIIalpha thus differ in the molecular details of how they recognize C. Unlike wild-type RI, two additional mutants, rRI(D170A) and rRI(K242A), inhibited C-subunit stoichiometrically in the presence of cAMP and show increases in both on- and off-rates. Asp170, which contributes directly to the hydrogen bonding network in cAMP-binding site A, thus contributes also to holoenzyme stability.

  19. Molecular characterisation of cAMP-dependent protein kinase (PK-A) catalytic subunit isoforms in the male tick, Amblyomma hebraeum.

    Science.gov (United States)

    Tabish, Mohammad; Clegg, Roger A; Turner, Philip C; Jonczy, Jan; Rees, Huw H; Fisher, Michael J

    2006-12-01

    The cAMP-dependent protein kinase (protein kinase A, PK-A) plays a central role in the regulation of diverse aspects of cellular activity. Specifically, PK-A appears to play a key controlling role in the maturation of spermatids. Using a PCR-based approach, with degenerate primers from the highly conserved regions of the PK-A catalytic (C) subunit in combination with 5' and 3' RACE, we have cloned three cDNAs for the PK-A C-subunit of the male tick, Amblyomma hebraeum. The three cDNAs have open reading frames of 1059, 1275 and 1404bp which encode proteins of 40.6, 48.2 and 52.5kDa, respectively. These transcripts appear to arise from 5' alternative splicing of RNA derived from a single gene for the PK-A C-subunit. One isoform (AH-PK-A C1), in common with PK-A C-subunits from a range of species, contains a consensus sequence for N-myristoylation. RT-PCR and Western blot experiments suggest that the three splice variants are expressed ubiquitously; however, expression of the myristoylatable AH-PK-A C1 isoform is predominant in all investigated tissues (accessory gland, midgut, Malpighian tubules, salivary gland, testis and immature spermatids). There is no evidence for a sperm-specific PK-A C-subunit (Cs) in tick sperm; however, tyrosine protein phosphorylation, previously shown to be modulated by PK-A activity during mammalian sperm maturation, was observed in tick sperm.

  20. Expansion of transducin subunit gene families in early vertebrate tetraploidizations.

    Science.gov (United States)

    Lagman, David; Sundström, Görel; Ocampo Daza, Daniel; Abalo, Xesús M; Larhammar, Dan

    2012-10-01

    Hundreds of gene families expanded in the early vertebrate tetraploidizations including many gene families in the phototransduction cascade. We have investigated the evolution of the heterotrimeric G-proteins of photoreceptors, the transducins, in relation to these events using both phylogenetic analyses and synteny comparisons. Three alpha subunit genes were identified in amniotes and the coelacanth, GNAT1-3; two of these were identified in amphibians and teleost fish, GNAT1 and GNAT2. Most tetrapods have four beta genes, GNB1-4, and teleosts have additional duplicates. Finally, three gamma genes were identified in mammals, GNGT1, GNG11 and GNGT2. Of these, GNGT1 and GNGT2 were found in the other vertebrates. In frog and zebrafish additional duplicates of GNGT2 were identified. Our analyses show all three transducin families expanded during the early vertebrate tetraploidizations and the beta and gamma families gained additional copies in the teleost-specific genome duplication. This suggests that the tetraploidizations contributed to visual specialisations.

  1. Epidermal growth factor receptor levels are reduced in mice with targeted disruption of the protein kinase A catalytic subunit

    Directory of Open Access Journals (Sweden)

    Huitfeldt Henrik S

    2008-04-01

    Full Text Available Abstract Background Epidermal Growth Factor Receptor (EGFR is a key target molecule in current treatment of several neoplastic diseases. Hence, in order to develop and improve current drugs targeting EGFR signalling, an accurate understanding of how this signalling pathway is regulated is required. It has recently been demonstrated that inhibition of cAMP-dependent protein kinase (PKA induces a ligand-independent internalization of EGFR. Cyclic-AMP-dependent protein kinase consists of a regulatory dimer bound to two catalytic subunits. Results We have investigated the effect on EGFR levels after ablating the two catalytic subunits, Cα and Cβ in two different models. The first model used targeted disruption of either Cα or Cβ in mice whereas the second model used Cα and Cβ RNA interference in HeLa cells. In both models we observed a significant reduction of EGFR expression at the protein but not mRNA level. Conclusion Our results suggest that PKA may represent a target that when manipulated can maintain EGFR protein levels at the single cell level as well as in intact animals.

  2. Conserved Residues in the Subunit Interface of tau Glutathione S-transferase Affect Catalytic and Structural Functions

    Institute of Scientific and Technical Information of China (English)

    Cai-Ling Wang; Hai-Ling Yang

    2011-01-01

    The tau class glutathione S-transferases(GSTs)have important roles in stress tolerance and the detoxification of herbicides in crops and weeds.Structural investigations of a wheat tau GST(TaGSTU4) show two subunit interactions:a hydrogen bond between the Tyr93 and Pro65 from another subunit of the dimer,and two salt bridges between residues Glu78 and side chains of Arg95 and Arg99 in the opposite subunit.By investigating enzyme activities,kinetic parameters and structural characterizations,this study showed the following results:(i)the hydrogen bond interaction between the Tyr93 and Pro65 was not essential for dimerization,but contributed to the enzyme's catalytic activity,thermal stability and affinity towards substrates glutathione and 1-chloro-2,4-dinitrobenzene;and(ii)two salt bridges mainly contributed to the protein structure stability and catalysis.The results of this study form a structural and functional basis for rational design of more selective and environmentally friendly herbicides.

  3. 2',6'-Dihalostyrylanilines, pyridines, and pyrimidines for the inhibition of the catalytic subunit of methionine S-adenosyltransferase-2.

    Science.gov (United States)

    Sviripa, Vitaliy M; Zhang, Wen; Balia, Andrii G; Tsodikov, Oleg V; Nickell, Justin R; Gizard, Florence; Yu, Tianxin; Lee, Eun Y; Dwoskin, Linda P; Liu, Chunming; Watt, David S

    2014-07-24

    Inhibition of the catalytic subunit of the heterodimeric methionine S-adenosyl transferase-2 (MAT2A) with fluorinated N,N-dialkylaminostilbenes (FIDAS agents) offers a potential avenue for the treatment of liver and colorectal cancers where upregulation of this enzyme occurs. A study of structure-activity relationships led to the identification of the most active compounds as those with (1) either a 2,6-difluorostyryl or 2-chloro-6-fluorostyryl subunit, (2) either an N-methylamino or N,N-dimethylamino group attached in a para orientation relative to the 2,6-dihalostyryl subunit, and (3) either an N-methylaniline or a 2-(N,N-dimethylamino)pyridine ring. These modifications led to FIDAS agents that were active in the low nanomolar range, that formed water-soluble hydrochloride salts, and that possessed the desired property of not inhibiting the human hERG potassium ion channel at concentrations at which the FIDAS agents inhibit MAT2A. The active FIDAS agents may inhibit cancer cells through alterations of methylation reactions essential for cancer cell survival and growth.

  4. Crystal Structure of the Human Pol α B Subunit in Complex with the C-terminal Domain of the Catalytic Subunit.

    Science.gov (United States)

    Suwa, Yoshiaki; Gu, Jianyou; Baranovskiy, Andrey G; Babayeva, Nigar D; Pavlov, Youri I; Tahirov, Tahir H

    2015-06-05

    In eukaryotic DNA replication, short RNA-DNA hybrid primers synthesized by primase-DNA polymerase α (Prim-Pol α) are needed to start DNA replication by the replicative DNA polymerases, Pol δ and Pol ϵ. The C terminus of the Pol α catalytic subunit (p180C) in complex with the B subunit (p70) regulates the RNA priming and DNA polymerizing activities of Prim-Pol α. It tethers Pol α and primase, facilitating RNA primer handover from primase to Pol α. To understand these regulatory mechanisms and to reveal the details of human Pol α organization, we determined the crystal structure of p70 in complex with p180C. The structured portion of p70 includes a phosphodiesterase (PDE) domain and an oligonucleotide/oligosaccharide binding (OB) domain. The N-terminal domain and the linker connecting it to the PDE domain are disordered in the reported crystal structure. The p180C adopts an elongated asymmetric saddle shape, with a three-helix bundle in the middle and zinc-binding modules (Zn1 and Zn2) on each side. The extensive p180C-p70 interactions involve 20 hydrogen bonds and a number of hydrophobic interactions resulting in an extended buried surface of 4080 Å(2). Importantly, in the structure of the p180C-p70 complex with full-length p70, the residues from the N-terminal to the OB domain contribute to interactions with p180C. The comparative structural analysis revealed both the conserved features and the differences between the human and yeast Pol α complexes.

  5. Cloning and sequence analysis of a full-length cDNA of SmPP1cb encoding turbot protein phosphatase 1 beta catalytic subunit

    Science.gov (United States)

    Qi, Fei; Guo, Huarong; Wang, Jian

    2008-02-01

    Reversible protein phosphorylation, catalyzed by protein kinases and phosphatases, is an important and versatile mechanism by which eukaryotic cells regulate almost all the signaling processes. Protein phosphatase 1 (PP1) is the first and well-characterized member of the protein serine/threonine phosphatase family. In the present study, a full-length cDNA encoding the beta isoform of the catalytic subunit of protein phosphatase 1(PP1cb), was for the first time isolated and sequenced from the skin tissue of flatfish turbot Scophthalmus maximus, designated SmPP1cb, by the rapid amplification of cDNA ends (RACE) technique. The cDNA sequence of SmPP1cb we obtained contains a 984 bp open reading frame (ORF), flanked by a complete 39 bp 5' untranslated region and 462 bp 3' untranslated region. The ORF encodes a putative 327 amino acid protein, and the N-terminal section of this protein is highly acidic, Met-Ala-Glu-Gly-Glu-Leu-Asp-Val-Asp, a common feature for PP1 catalytic subunit but absent in protein phosphatase 2B (PP2B). And its calculated molecular mass is 37 193 Da and pI 5.8. Sequence analysis indicated that, SmPP1cb is extremely conserved in both amino acid and nucleotide acid levels compared with the PP1cb of other vertebrates and invertebrates, and its Kozak motif contained in the 5'UTR around ATG start codon is GXXAXXGXX ATGG, which is different from mammalian in two positions A-6 and G-3, indicating the possibility of different initiation of translation in turbot, and also the 3'UTR of SmPP1cb is highly diverse in the sequence similarity and length compared with other animals, especially zebrafish. The cloning and sequencing of SmPP1cb gene lays a good foundation for the future work on the biological functions of PP1 in the flatfish turbot.

  6. Improved crystallization of Escherichia coli ATP synthase catalytic complex (F1) by introducing a phosphomimetic mutation in subunit ε.

    Science.gov (United States)

    Roy, Ankoor; Hutcheon, Marcus L; Duncan, Thomas M; Cingolani, Gino

    2012-10-01

    The bacterial ATP synthase (F(O)F(1)) of Escherichia coli has been the prominent model system for genetics, biochemical and more recently single-molecule studies on F-type ATP synthases. With 22 total polypeptide chains (total mass of ∼529 kDa), E. coli F(O)F(1) represents nature's smallest rotary motor, composed of a membrane-embedded proton transporter (F(O)) and a peripheral catalytic complex (F(1)). The ATPase activity of isolated F(1) is fully expressed by the α(3)β(3)γ 'core', whereas single δ and ε subunits are required for structural and functional coupling of E. coli F(1) to F(O). In contrast to mitochondrial F(1)-ATPases that have been determined to atomic resolution, the bacterial homologues have proven very difficult to crystallize. In this paper, we describe a biochemical strategy that led us to improve the crystallogenesis of the E. coli F(1)-ATPase catalytic core. Destabilizing the compact conformation of ε's C-terminal domain with a phosphomimetic mutation (εS65D) dramatically increased crystallization success and reproducibility, yielding crystals of E. coli F(1) that diffract to ∼3.15 Å resolution.

  7. Human intestinal maltase-glucoamylase: crystal structure of the N-terminal catalytic subunit and basis of inhibition and substrate specificity

    Science.gov (United States)

    Human maltase-glucoamylase (MGAM) is one of the two enzymes responsible for catalyzing the last glucose-releasing step in starch digestion. MGAM is anchored to the small-intestinal brush-border epithelial cells and contains two homologous glycosyl hydrolase family 31 catalytic subunits: an N-termina...

  8. Significant prognostic values of nuclear genes encoding mitochondrial complex I subunits in tumor patients.

    Science.gov (United States)

    Li, L D; Sun, H F; Bai, Y; Gao, S P; Jiang, H L; Jin, W

    2016-01-01

    In cancer biology, it remains still open question concerning the oncogenic versus oncosuppressor behavior of metabolic genes, which includes those encoding mitochondrial complex I (CI) subunits. The prognostic value of nuclear genome mRNAs expression of CI subunits is to be evaluated in the tumor patients. We used the Kaplan Meier plotter database, the cBio Cancer Genomics Portal, and the Oncomine in which gene expression data and survival information were from thousands of tumor patients to assess the relevance of nuclear genome mRNAs level of CI subunits to patients' survival, as well as their alterations in gene and expression level in tumors. We presented that the relative expression level of overwhelming majority of the nuclear genes of CI subunits with survival significance (overall survival, relapse free survival, progression free survival, distant metastasis free survival, post progression survival, and first progression), had consistent effects for patients in each type of four tumors separately, including breast cancer, ovarian cancer, lung cancer, and gastric cancer. However, in gene level, frequent cumulative or individual alteration of these genes could not significantly affect patients' survival and the overexpression of the individual gene was not ubiquitous in tumors versus normal tissues. Given that reprogrammed energy metabolism was viewed as an emerging hallmark of tumor, thus tumor patients' survival might potentially to be evaluated by certain threshold for overall expression of CI subunits. Comprehensive understanding of the nuclear genome encoded CI subunits may have guiding significance for the diagnosis and prognosis in tumor patients.

  9. Mapping of a liver phosphorylase kinase [alpha]-subunit gene on the mouse x chromosome

    Energy Technology Data Exchange (ETDEWEB)

    Geng, Yan; Derry, J.M.J.; Barnard, P.J. (MRC Molecular Neurobiology Unit, Cambridge (United Kingdom)); Hendrickx, J.; Coucke, P.; Willems, P.R. (Univ. of Antwerp (Belgium))

    1993-01-01

    Phosphorylase kinase (PHK) is a regulatory enzyme of the glycogenolytic pathway composed of a complex of four subunits. We recently mapped the muscle [alpha]-subunit gene (Phka) to the mouse X chromosome in a region syntenic with the proximal long arm of the human X chromosome and containing the human homologue of this gene, PHKA. We now report the mapping of the liver [alpha]-subunit gene to the telomeric end of the mouse X chromosome. This mapping position would suggest a location for the human liver [alpha]-subunit gene on the proximal short arm of the X chromosome, a region recently implicated in X-linked liver glycogenosis (XLG). 20 refs., 2 figs.

  10. Structure of a catalytic dimer of the α- and β-subunits of the F-ATPase from Paracoccus denitrificans at 2.3 Å resolution

    Energy Technology Data Exchange (ETDEWEB)

    Morales-Ríos, Edgar; Montgomery, Martin G. [The Medical Research Council Mitochondrial Biology Unit, Cambridge Biomedical Campus, Hills Road, Cambridge CB2 0XY (United Kingdom); Leslie, Andrew G. W. [The Medical Research Council Laboratory of Molecular Biology, Cambridge Biomedical Campus, Francis Crick Avenue, Cambridge CB2 0QH (United Kingdom); García-Trejo, José J. [Universidad Nacional Autónoma de México, Mexico City (Mexico); Walker, John E., E-mail: walker@mrc-mbu.cam.ac.uk [The Medical Research Council Mitochondrial Biology Unit, Cambridge Biomedical Campus, Hills Road, Cambridge CB2 0XY (United Kingdom)

    2015-09-23

    The structure of the αβ heterodimer of the F-ATPase from the α-proteobacterium P. denitrificans has been determined at 2.3 Å resolution. It corresponds to the ‘open’ or ‘empty’ catalytic interface found in other F-ATPases. The structures of F-ATPases have predominantly been determined from mitochondrial enzymes, and those of the enzymes in eubacteria have been less studied. Paracoccus denitrificans is a member of the α-proteobacteria and is related to the extinct protomitochondrion that became engulfed by the ancestor of eukaryotic cells. The P. denitrificans F-ATPase is an example of a eubacterial F-ATPase that can carry out ATP synthesis only, whereas many others can catalyse both the synthesis and the hydrolysis of ATP. Inhibition of the ATP hydrolytic activity of the P. denitrificans F-ATPase involves the ζ inhibitor protein, an α-helical protein that binds to the catalytic F{sub 1} domain of the enzyme. This domain is a complex of three α-subunits and three β-subunits, and one copy of each of the γ-, δ- and ∊-subunits. Attempts to crystallize the F{sub 1}–ζ inhibitor complex yielded crystals of a subcomplex of the catalytic domain containing the α- and β-subunits only. Its structure was determined to 2.3 Å resolution and consists of a heterodimer of one α-subunit and one β-subunit. It has no bound nucleotides, and it corresponds to the ‘open’ or ‘empty’ catalytic interface found in other F-ATPases. The main significance of this structure is that it aids in the determination of the structure of the intact membrane-bound F-ATPase, which has been crystallized.

  11. Identification of a nonsense mutation in the carboxyl-terminal region of DNA-dependent protein kinase catalytic subunit in the scid mouse.

    Science.gov (United States)

    Blunt, T; Gell, D; Fox, M; Taccioli, G E; Lehmann, A R; Jackson, S P; Jeggo, P A

    1996-01-01

    DNA-dependent protein kinase (DNA-PK) consists of a heterodimeric protein (Ku) and a large catalytic subunit (DNA-PKcs). The Ku protein has double-stranded DNA end-binding activity that serves to recruit the complex to DNA ends. Despite having serine/threonine protein kinase activity, DNA-PKcs falls into the phosphatidylinositol 3-kinase superfamily. DNA-PK functions in DNA double-strand break repair and V(D)J recombination, and recent evidence has shown that mouse scid cells are defective in DNA-PKcs. In this study we have cloned the cDNA for the carboxyl-terminal region of DNA-PKcs in rodent cells and identified the existence of two differently spliced products in human cells. We show that DNA-PKcs maps to the same chromosomal region as the mouse scid gene. scid cells contain approximately wild-type levels of DNA-PKcs transcripts, whereas the V-3 cell line, which is also defective in DNA-PKcs, contains very reduced transcript levels. Sequence comparison of the carboxyl-terminal region of scid and wild-type mouse cells enabled us to identify a nonsense mutation within a highly conserved region of the gene in mouse scid cells. This represents a strong candidate for the inactivating mutation in DNA-PKcs in the scid mouse. Images Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8816792

  12. Adaptation of HepG2 cells to a steady-state reduction in the content of protein phosphatase 6 (PP6) catalytic subunit.

    Science.gov (United States)

    Boylan, Joan M; Salomon, Arthur R; Tantravahi, Umadevi; Gruppuso, Philip A

    2015-07-15

    Protein phosphatase 6 (PP6) is a ubiquitous Ser/Thr phosphatase involved in an array of cellular processes. To assess the potential of PP6 as a therapeutic target in liver disorders, we attenuated expression of the PP6 catalytic subunit in HepG2 cells using lentiviral-transduced shRNA. Two PP6 knock-down (PP6KD) cell lines (90% reduction of PP6-C protein content) were studied in depth. Both proliferated at a rate similar to control cells. However, flow cytometry indicated G2/M cell cycle arrest that was accounted for by a shift of the cells from a diploid to tetraploid state. PP6KD cells did not show an increase in apoptosis, nor did they exhibit reduced viability in the presence of bleomycin or taxol. Gene expression analysis by microarray showed attenuated anti-inflammatory signaling. Genes associated with DNA replication were downregulated. Mass spectrometry-based phosphoproteomic analysis yielded 80 phosphopeptides representing 56 proteins that were significantly affected by a stable reduction in PP6-C. Proteins involved in DNA replication, DNA damage repair and pre-mRNA splicing were overrepresented among these. PP6KD cells showed intact mTOR signaling. Our studies demonstrated involvement of PP6 in a diverse set of biological pathways and an adaptive response that may limit the effectiveness of targeting PP6 in liver disorders.

  13. Evasion of the innate immune response: the Old World alphavirus nsP2 protein induces rapid degradation of Rpb1, a catalytic subunit of RNA polymerase II.

    Science.gov (United States)

    Akhrymuk, Ivan; Kulemzin, Sergey V; Frolova, Elena I

    2012-07-01

    The Old World alphaviruses are emerging human pathogens with an ability to cause widespread epidemics. The latest epidemic of Chikungunya virus, from 2005 to 2007, affected over 40 countries in Africa, Asia, and Europe. The Old World alphaviruses are highly cytopathic and known to evade the cellular antiviral response by inducing global inhibition of transcription in vertebrate cells. This function was shown to be mediated by their nonstructural nsP2 protein; however, the detailed mechanism of this phenomenon has remained unknown. Here, we report that nsP2 proteins of Sindbis, Semliki Forest, and Chikungunya viruses inhibit cellular transcription by inducing rapid degradation of Rpb1, a catalytic subunit of the RNAPII complex. This degradation of Rpb1 is independent of the nsP2-associated protease activity, but, instead, it proceeds through nsP2-mediated Rpb1 ubiquitination. This function of nsP2 depends on the integrity of the helicase and S-adenosylmethionine (SAM)-dependent methyltransferase-like domains, and point mutations in either of these domains abolish Rpb1 degradation. We go on to show that complete degradation of Rpb1 in alphavirus-infected cells occurs within 6 h postinfection, before other previously described virus-induced changes in cell physiology, such as apoptosis, autophagy, and inhibition of STAT1 phosphorylation, are detected. Since Rpb1 is a subunit that catalyzes the polymerase reaction during RNA transcription, degradation of Rpb1 plays an indispensable role in blocking the activation of cellular genes and downregulating cellular antiviral response. This indicates that the nsP2-induced degradation of Rpb1 is a critical mechanism utilized by the Old World alphaviruses to subvert the cellular antiviral response.

  14. Cloning and sequence analysis of a full-length cDNA of SmPP1cb encoding turbot protein phosphatase 1 beta catalytic subunit

    Institute of Scientific and Technical Information of China (English)

    QI Fei; GUO Huarong; WANG Jian

    2008-01-01

    Reversible protein phosphorylation,catalyzed by protein kinases and phosphatases,is an important and versatile mechanism by which eukaryotic cells regulate almost all the signaling processes.Protein phosphatase 1(PP1) is the first and well-characterized member of the protein serine/threoninephosphatase family.In the present study.a full-length cDNA encoding the beta isolorm of the catalytic subunit of protein phosphatase 1(PP1cb).was for the first time isolated and sequenced from the skin tissue of flatfish turbot Scophthalmus maximus,designated SmPP1cb,by the rapid amplification of cDNA ends (RACE) technique.The cDNA sequence of SmPP1cb we obtained contains a 984 bp open reading frame(ORF),flanked by a complete 39 bp 5' untranslated region and 462 bp 3' untranslated region.The ORF encodes a putative 327 amino acid protein.and the N-terminal section of this protein iS highly acidic,Met-Ala-Glu-Gly-Glu-Leu-Asp-Val-Asp.a common feature for PP1 catalytic subunit but absent in protein phosphatase 2B(PP2B).And its calculated molecular mass is 37 193 Da and pI 5.8.Sequence analysis indicated that,SmPP1cb is extremely conserved in both amino acid and nucleotide acid levels compared with the PP1cb of other vertebrates and invertebrates.and its Kozak motif contained in the 5'UTR around ATG start codon is GXXAXXGXXATGG,which is different from mammalian in two positions A-6 and G-3,indicating the possibility of different initiation of translation in turbot,and also the 3'UTR of SmPP1cb is highly diverse in the sequence similarity and length compared with other animals.especially zebrafish.The cloning and sequencing of SmPP1cb gene lays a good foundation for the future work on the biological functions of PP1 in the flatfish turbot.

  15. miR-502 inhibits cell proliferation and tumor growth in hepatocellular carcinoma through suppressing phosphoinositide 3-kinase catalytic subunit gamma

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Suling, E-mail: suling_chen86@163.com [Department of Infectious Disease, Heping Hospital Attached to Changzhi Medical College, Changzhi 046000 (China); Li, Fang; Chai, Haiyun; Tao, Xin [Department of Infectious Disease, Heping Hospital Attached to Changzhi Medical College, Changzhi 046000 (China); Wang, Haili [Department of Hematology, Heping Hospital Attached to Changzhi Medical College, Changzhi 046000 (China); Ji, Aifang [Central Laboratory, Heping Hospital Attached to Changzhi Medical College, Changzhi 046000 (China)

    2015-08-21

    MicroRNAs (miRNAs) play a key role in carcinogenesis and tumor progression in hepatocellular carcinoma (HCC). In the present study, we demonstrated that miR-502 significantly inhibits HCC cell proliferation in vitro and tumor growth in vivo. G1/S cell cycle arrest and apoptosis of HCC cells were induced by miR-502. Phosphoinositide 3-kinase catalytic subunit gamma (PIK3CG) was identified as a direct downstream target of miR-502 in HCC cells. Notably, overexpression of PIK3CG reversed the inhibitory effects of miR-502 in HCC cells. Our findings suggest that miR-502 functions as a tumor suppressor in HCC via inhibition of PI3KCG, supporting its utility as a promising therapeutic gene target for this tumor type. - Highlights: • miR-502 suppresses HCC cell proliferation in vitro and tumorigenicity in vivo. • miR-502 regulates cell cycle and apoptosis in HCC cells. • PIK3CG is a direct target of miR-502. • miR-502 and PIK3CG expression patterns are inversely correlated in HCC tissues.

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

    Science.gov (United States)

    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

  17. A model of 3D-structure of H+,K+-ATPase catalytic subunit derived by homology modeling

    Institute of Scientific and Technical Information of China (English)

    Dong YAN; Yuan-dong HU; Song LI; Mao-sheng CHENG

    2004-01-01

    AIM: To build a model of 3D-structure of H+, K+-ATPase catalytic subunit for theoretical study and anti-ulcer drug design. METHODS: The model was built on the basis of structural data from the Ca2+-ATPase. Structurally conserved regions were defined by amino acid sequence comparisons, optimum interconnecting loops were selected from the protein databank, and amino (N)- and carboxyl (C)-terminal ends were generated as random coil structures. Applying molecular mechanics method then minimized the model energy. Molecular dynamics technique was used to do further structural optimization. RESULTS: The model of 3D-structure of H+, K+-ATPase was derived. The model is reasonable according to several validation criteria. There were ten transmembrane helices (TM1-TM 10) in the model and inhibitor-binding site was identified on the TM5-8 riched negatively charged residues.CONCLUSION: The 3D-structure model from our study is informative to guide future molecular biology study about H+, K+-ATPase and drug design based on database searching.

  18. The Catalytic Subunit of DNA-Dependent Protein Kinase Coordinates with Polo-Like Kinase 1 to Facilitate Mitotic Entry.

    Science.gov (United States)

    Lee, Kyung-Jong; Shang, Zeng-Fu; Lin, Yu-Fen; Sun, Jingxin; Morotomi-Yano, Keiko; Saha, Debabrata; Chen, Benjamin P C

    2015-04-01

    DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is the key regulator of the non-homologous end joining pathway of DNA double-strand break repair. We have previously reported that DNA-PKcs is required for maintaining chromosomal stability and mitosis progression. Our further investigations reveal that deficiency in DNA-PKcs activity caused a delay in mitotic entry due to dysregulation of cyclin-dependent kinase 1 (Cdk1), the key driving force for cell cycle progression through G2/M transition. Timely activation of Cdk1 requires polo-like kinase 1 (Plk1), which affects modulators of Cdk1. We found that DNA-PKcs physically interacts with Plk1 and could facilitate Plk1 activation both in vitro and in vivo. Further, DNA-PKcs-deficient cells are highly sensitive to Plk1 inhibitor BI2536, suggesting that the coordination between DNA-PKcs and Plk1 is not only crucial to ensure normal cell cycle progression through G2/M phases but also required for cellular resistance to mitotic stress. On the basis of the current study, it is predictable that combined inhibition of DNA-PKcs and Plk1 can be employed in cancer therapy strategy for synthetic lethality.

  19. The Catalytic Subunit of DNA-Dependent Protein Kinase Coordinates with Polo-Like Kinase 1 to Facilitate Mitotic Entry

    Directory of Open Access Journals (Sweden)

    Kyung-Jong Lee

    2015-04-01

    Full Text Available DNA-dependent protein kinase catalytic subunit (DNA-PKcs is the key regulator of the non-homologous end joining pathway of DNA double-strand break repair. We have previously reported that DNA-PKcs is required for maintaining chromosomal stability and mitosis progression. Our further investigations reveal that deficiency in DNA-PKcs activity caused a delay in mitotic entry due to dysregulation of cyclin-dependent kinase 1 (Cdk1, the key driving force for cell cycle progression through G2/M transition. Timely activation of Cdk1 requires polo-like kinase 1 (Plk1, which affects modulators of Cdk1. We found that DNA-PKcs physically interacts with Plk1 and could facilitate Plk1 activation both in vitro and in vivo. Further, DNA-PKcs–deficient cells are highly sensitive to Plk1 inhibitor BI2536, suggesting that the coordination between DNA-PKcs and Plk1 is not only crucial to ensure normal cell cycle progression through G2/M phases but also required for cellular resistance to mitotic stress. On the basis of the current study, it is predictable that combined inhibition of DNA-PKcs and Plk1 can be employed in cancer therapy strategy for synthetic lethality.

  20. Adaptation of HepG2 cells to a steady-state reduction in the content of protein phosphatase 6 (PP6) catalytic subunit

    Energy Technology Data Exchange (ETDEWEB)

    Boylan, Joan M. [Department of Pediatrics, Brown University and Rhode Island Hospital, Providence, RI (United States); Salomon, Arthur R. [Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, RI (United States); Department of Chemistry, Brown University, Providence, RI (United States); Tantravahi, Umadevi [Division of Genetics, Department of Pathology, Brown University and Women and Infants Hospital, Providence, RI (United States); Gruppuso, Philip A., E-mail: philip_gruppuso@brown.edu [Department of Pediatrics, Brown University and Rhode Island Hospital, Providence, RI (United States); Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, RI (United States)

    2015-07-15

    Protein phosphatase 6 (PP6) is a ubiquitous Ser/Thr phosphatase involved in an array of cellular processes. To assess the potential of PP6 as a therapeutic target in liver disorders, we attenuated expression of the PP6 catalytic subunit in HepG2 cells using lentiviral-transduced shRNA. Two PP6 knock-down (PP6KD) cell lines (90% reduction of PP6-C protein content) were studied in depth. Both proliferated at a rate similar to control cells. However, flow cytometry indicated G2/M cell cycle arrest that was accounted for by a shift of the cells from a diploid to tetraploid state. PP6KD cells did not show an increase in apoptosis, nor did they exhibit reduced viability in the presence of bleomycin or taxol. Gene expression analysis by microarray showed attenuated anti-inflammatory signaling. Genes associated with DNA replication were downregulated. Mass spectrometry-based phosphoproteomic analysis yielded 80 phosphopeptides representing 56 proteins that were significantly affected by a stable reduction in PP6-C. Proteins involved in DNA replication, DNA damage repair and pre-mRNA splicing were overrepresented among these. PP6KD cells showed intact mTOR signaling. Our studies demonstrated involvement of PP6 in a diverse set of biological pathways and an adaptive response that may limit the effectiveness of targeting PP6 in liver disorders. - Highlights: • Lentiviral-transduced shRNA was used to generate a stable knockdown of PP6 in HepG2 cells. • Cells adapted to reduced PP6; cell proliferation was unaffected, and cell survival was normal. • However, PP6 knockdown was associated with a transition to a tetraploid state. • Genomic profiling showed downregulated anti-inflammatory signaling and DNA replication. • Phosphoproteomic profiling showed changes in proteins associated with DNA replication and repair.

  1. Identification of DNA-dependent protein kinase catalytic subunit (DNA-PKcs as a novel target of bisphenol A.

    Directory of Open Access Journals (Sweden)

    Yuki Ito

    Full Text Available Bisphenol A (BPA forms the backbone of plastics and epoxy resins used to produce packaging for various foods and beverages. BPA is also an estrogenic disruptor, interacting with human estrogen receptors (ER and other related nuclear receptors. Nevertheless, the effects of BPA on human health remain unclear. The present study identified DNA-dependent protein kinase catalytic subunit (DNA-PKcs as a novel BPA-binding protein. DNA-PKcs, in association with the Ku heterodimer (Ku70/80, is a critical enzyme involved in the repair of DNA double-strand breaks. Low levels of DNA-PK activity are previously reported to be associated with an increased risk of certain types of cancer. Although the Kd for the interaction between BPA and a drug-binding mutant of DNA-PKcs was comparatively low (137 nM, high doses of BPA were required before cellular effects were observed (100-300 μM. The results of an in vitro kinase assay showed that BPA inhibited DNA-PK kinase activity in a concentration-dependent manner. In M059K cells, BPA inhibited the phosphorylation of DNA-PKcs at Ser2056 and H2AX at Ser139 in response to ionizing radiation (IR-irradiation. BPA also disrupted DNA-PKcs binding to Ku70/80 and increased the radiosensitivity of M059K cells, but not M059J cells (which are DNA-PKcs-deficient. Taken together, these results provide new evidence of the effects of BPA on DNA repair in mammalian cells, which are mediated via inhibition of DNA-PK activity. This study may warrant the consideration of the possible carcinogenic effects of high doses of BPA, which are mediated through its action on DNA-PK.

  2. The Over-expression of the β2 Catalytic Subunit of the Proteasome Decreases Homologous Recombination and Impairs DNA Double-Strand Break Repair in Human Cells

    Directory of Open Access Journals (Sweden)

    Anita Collavoli

    2011-01-01

    Full Text Available By a human cDNA library screening, we have previously identified two sequences coding two different catalytic subunits of the proteasome which increase homologous recombination (HR when overexpressed in the yeast Saccharomyces cerevisiae. Here, we investigated the effect of proteasome on spontaneous HR and DNA repair in human cells. To determine if the proteasome has a role in the occurrence of spontaneous HR in human cells, we overexpressed the β2 subunit of the proteasome in HeLa cells and determined the effect on intrachromosomal HR. Results showed that the overexpression of β2 subunit decreased HR in human cells without altering the cell proteasome activity and the Rad51p level. Moreover, exposure to MG132 that inhibits the proteasome activity reduced HR in human cells. We also found that the expression of the β2 subunit increases the sensitivity to the camptothecin that induces DNA double-strand break (DSB. This suggests that the β2 subunit has an active role in HR and DSB repair but does not alter the intracellular level of the Rad51p.

  3. Early transcription factor subunits are encoded by vaccinia virus late genes.

    Science.gov (United States)

    Gershon, P D; Moss, B

    1990-06-01

    The vaccinia virus early transcription factor (VETF) was shown to be a virus-encoded heterodimer. The gene for the 82-kDa subunit was identified as open reading frame (ORF) A8L, based on the N-terminal sequence of factor purified by using DNA-affinity magnetic beads. The 70-kDa subunit of VETF was refractory to N-terminal analysis, and so N-terminal sequences were obtained for three internal tryptic peptides. All three peptides matched sequences within ORF D6R. ORFs A8L and D6R are located within the central region of the vaccinia virus genome and are separated by about 13,600 base pairs. Proteins corresponding to the 3' ends of ORFs A8L and D6R were overexpressed in Escherichia coli and used to prepare antisera that bound to the larger and smaller subunits, respectively, of affinity-purified VETF. Immunoblot analysis of proteins from infected cells indicated that both subunits are expressed exclusively in the late phase of infection, just prior to their packaging in virus particles. The two subunits of VETF have no significant local or overall amino acid sequence homology to one another, to other entries in biological sequence data bases including bacterial sigma factors, or to recently determined sequences of some eukaryotic transcription factors. The 70-kDa subunit, however, has motifs in common with a super-family of established and putative DNA and RNA helicases.

  4. Mutations in genes encoding subunits of RNA polymerases I and III cause Treacher Collins syndrome.

    NARCIS (Netherlands)

    Dauwerse, J.G.; Dixon, J.; Seland, S.; Ruivenkamp, C.A.; Haeringen, A. van; Hoefsloot, L.H.; Peters, D.J.; Boers, A.C.; Daumer-Haas, C.; Maiwald, R.; Zweier, C.; Kerr, B.; Cobo, A.M.; Toral, J.F.; Hoogeboom, A.J.M.; Lohmann, D.R.; Hehr, U.; Dixon, M.J.; Breuning, M.H.; Wieczorek, D.

    2011-01-01

    We identified a deletion of a gene encoding a subunit of RNA polymerases I and III, POLR1D, in an individual with Treacher Collins syndrome (TCS). Subsequently, we detected 20 additional heterozygous mutations of POLR1D in 252 individuals with TCS. Furthermore, we discovered mutations in both allele

  5. Structure of the gene encoding the murine protein kinase CK2 beta subunit

    DEFF Research Database (Denmark)

    Boldyreff, B; Issinger, O G

    1995-01-01

    The mouse protein kinase CK2 beta subunit gene (Csnk2b) is composed of seven exons contained within 7874 bp. The exon and intron lengths extend from 76 to 321 and 111 to 1272 bp, respectively. The lengths of the murine coding exons correspond exactly to the lengths of the exons in the human CK2...

  6. STEADY-STATE TRANSCRIPT LEVELS OF CYTOCHROME-C-OXIDASE GENES DURING HUMAN MYOGENESIS INDICATE SUBUNIT SWITCHING OF SUBUNIT VIA AND COEXPRESSION OF SUBUNIT VIIA ISOFORMS

    NARCIS (Netherlands)

    TAANMAN, JW; HERZBERG, NH; DEVRIES, H; BOLHUIS, PA; VANDENBOGERT, C

    1992-01-01

    Steady-state levels of the mitochondrial rRNAs, of mRNAs for mitochondrially and nuclear-encoded subunits of cytochrome c oxidase and for the beta-subunit of ATP synthase were assessed by Northern blot hybridizations during the in vitro differentiation of human myoblasts. Transcript levels of the so

  7. Genetic variations of glycinin subunit genes among cultivated and wild type soybean species

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Glycinin is a predominant storage protein in most soybean accessions. It is a hexamer constituted by five major subunits, which can be classified into two groups. Group Ⅰ contains Gl, G2 and G3, and Group Ⅱ contains G4 and G5. The genes encoding these subunits have been designated from Gyl to Gy5, respectively. In the present study, Gyl genomic fragments were cloned from wild accessions of subgenera Glycine glycine, Glycine soja and a cultivar of Glycine max. Their sequences and the deduced amino acid sequences were compared. The residues critical for assembling of G1 subunits from the wild perennial accession were conservative. The Gy4 fragments were cloned from two wild perennial accessions and compared with that from subgenus Soja. The intron 3 of Gy4 had abundant variations between the subgenera G. Soja and G. Glycine as well as within the subgenus G. Glycine. Abundant variations existed in the disordered regions 3 and 4 of G4 subunits from two wild perennial accessions. The genomic organization of glycinin genes was analyzed in 19 accessions from subgenera Soja and Glycine. The hybridization patterns were identical among the accessions of subgenus Soja. On the contrary, abundant polymorphisms existed between the accessions from subgenus Glycine. These results indicated that glycinin genes have high degree of conservation within subgenus Soja but more variations within subgenus Glycine.

  8. Glutathione S-transferase Ya subunit gene: identification of regulatory elements required for basal level and inducible expression.

    OpenAIRE

    Telakowski-Hopkins, C A; King, R. G.; Pickett, C B

    1988-01-01

    The function of the 5'-flanking region of a rat glutathione S-transferase Ya subunit structural gene has been examined in homologous and heterologous cells. By using the 5'-flanking region of the Ya subunit gene fused to the structural gene encoding chloramphenicol acetyltransferase, we have identified two cis-acting regulatory elements in the upstream region of the Ya gene. One element is required for maximum basal level expression in homologous cells, whereas maximum basal level expression ...

  9. A remarkably stable TipE gene cluster: evolution of insect Para sodium channel auxiliary subunits

    Directory of Open Access Journals (Sweden)

    Li Jia

    2011-11-01

    Full Text Available Abstract Background First identified in fruit flies with temperature-sensitive paralysis phenotypes, the Drosophila melanogaster TipE locus encodes four voltage-gated sodium (NaV channel auxiliary subunits. This cluster of TipE-like genes on chromosome 3L, and a fifth family member on chromosome 3R, are important for the optional expression and functionality of the Para NaV channel but appear quite distinct from auxiliary subunits in vertebrates. Here, we exploited available arthropod genomic resources to trace the origin of TipE-like genes by mapping their evolutionary histories and examining their genomic architectures. Results We identified a remarkably conserved synteny block of TipE-like orthologues with well-maintained local gene arrangements from 21 insect species. Homologues in the water flea, Daphnia pulex, suggest an ancestral pancrustacean repertoire of four TipE-like genes; a subsequent gene duplication may have generated functional redundancy allowing gene losses in the silk moth and mosquitoes. Intronic nesting of the insect TipE gene cluster probably occurred following the divergence from crustaceans, but in the flour beetle and silk moth genomes the clusters apparently escaped from nesting. Across Pancrustacea, TipE gene family members have experienced intronic nesting, escape from nesting, retrotransposition, translocation, and gene loss events while generally maintaining their local gene neighbourhoods. D. melanogaster TipE-like genes exhibit coordinated spatial and temporal regulation of expression distinct from their host gene but well-correlated with their regulatory target, the Para NaV channel, suggesting that functional constraints may preserve the TipE gene cluster. We identified homology between TipE-like NaV channel regulators and vertebrate Slo-beta auxiliary subunits of big-conductance calcium-activated potassium (BKCa channels, which suggests that ion channel regulatory partners have evolved distinct lineage

  10. Gyrase activity and number of copies of the gyrase B subunit gene in Haemophilus influenzae

    Energy Technology Data Exchange (ETDEWEB)

    Cabrera-Juarez, E.; Setlow, J.K.

    1985-11-01

    Gyrase activities in extracts of various strains of Haemophilus influenzae can differ by more than an order of magnitude. Measurements of in vitro activity and copy number indicated that most of these differences arose from variations in the number of copies of the gene for the gyrase B subunit, with some strains containing multicopy plasmids coding for that subunit. The quantitative relationship between gyrase and copy number depended on the mutations in the plasmids and in the host. The possibility that the in vivo gyrase activity did not reflect the in vitro data was explored by measurement of alkaline phosphatase and ATPase activity in the extracts. Alkaline phosphatase activity increased with increasing gyrase activity measured in vitro, but ATPase activity did not. The authors conclude that extra supercoiling enhanced transcription of the alkaline phosphatase gene but not the ATPase gene and that it is unlikely that there is much discrepancy between gyrase activity assayed in vitro and the activity in the cell.

  11. Uncovering the stoichiometry of Pyrococcus furiosus RNase P, a multi-subunit catalytic ribonucleoprotein complex, by surface-induced dissociation and ion mobility mass spectrometry.

    Science.gov (United States)

    Ma, Xin; Lai, Lien B; Lai, Stella M; Tanimoto, Akiko; Foster, Mark P; Wysocki, Vicki H; Gopalan, Venkat

    2014-10-20

    We demonstrate that surface-induced dissociation (SID) coupled with ion mobility mass spectrometry (IM-MS) is a powerful tool for determining the stoichiometry of a multi-subunit ribonucleoprotein (RNP) complex assembled in a solution containing Mg(2+). We investigated Pyrococcus furiosus (Pfu) RNase P, an archaeal RNP that catalyzes tRNA 5' maturation. Previous step-wise, Mg(2+)-dependent reconstitutions of Pfu RNase P with its catalytic RNA subunit and two interacting protein cofactor pairs (RPP21⋅RPP29 and POP5⋅RPP30) revealed functional RNP intermediates en route to the RNase P enzyme, but provided no information on subunit stoichiometry. Our native MS studies with the proteins showed RPP21⋅RPP29 and (POP5⋅RPP30)2 complexes, but indicated a 1:1 composition for all subunits when either one or both protein complexes bind the cognate RNA. These results highlight the utility of SID and IM-MS in resolving conformational heterogeneity and yielding insights on RNP assembly.

  12. Conservation of the Nrf2-Mediated Gene Regulation of Proteasome Subunits and Glucose Metabolism in Zebrafish

    Directory of Open Access Journals (Sweden)

    Vu Thanh Nguyen

    2016-01-01

    Full Text Available The Keap1-Nrf2 system is an evolutionarily conserved defense mechanism against oxidative and xenobiotic stress. Besides the exogenous stress response, Nrf2 has been found to regulate numerous cellular functions, including protein turnover and glucose metabolism; however, the evolutionary origins of these functions remain unknown. In the present study, we searched for novel target genes associated with the zebrafish Nrf2 to answer this question. A microarray analysis of zebrafish embryos that overexpressed Nrf2 revealed that 115 candidate genes were targets of Nrf2, including genes encoding proteasome subunits and enzymes involved in glucose metabolism. A real-time quantitative PCR suggested that the expression of 3 proteasome subunits (psma3, psma5, and psmb7 and 2 enzymes involved in glucose metabolism (pgd and fbp1a were regulated by zebrafish Nrf2. We thus next examined the upregulation of these genes by an Nrf2 activator, diethyl maleate, using Nrf2 mutant zebrafish larvae. The results of real-time quantitative PCR and whole-mount in situ hybridization showed that all of these 5 genes were upregulated by diethyl maleate treatment in an Nrf2-dependent manner, especially in the liver. These findings implied that the Nrf2-mediated regulation of the proteasome subunits and glucose metabolism is evolutionarily conserved among vertebrates.

  13. The C-terminal domain of human grp94 protects the catalytic subunit of protein kinase CK2 (CK2alpha) against thermal aggregation. Role of disulfide bonds

    DEFF Research Database (Denmark)

    Roher, N; Miró, F; Boldyreff, B

    2001-01-01

    The C-terminal domain (residues 518-803) of the 94 kDa glucose regulated protein (grp94) was expressed in Escherichia coli as a fusion protein with a His6-N-terminal tag (grp94-CT). This truncated form of grp94 formed dimers and oligomers that could be dissociated into monomers by treatment...... with dithiothreitol. Grp94-CT conferred protection against aggregation on the catalytic subunit of protein kinase CK2 (CK2alpha), although it did not protect against thermal inactivation. This anti-aggregation effect of grp94-CT was concentration dependent, with full protection achieved at grp94-CT/CK2alpha molar...

  14. GABAA receptor subunit gene expression in human prefrontal cortex: comparison of schizophrenics and controls

    Science.gov (United States)

    Akbarian, S.; Huntsman, M. M.; Kim, J. J.; Tafazzoli, A.; Potkin, S. G.; Bunney, W. E. Jr; Jones, E. G.; Bloom, F. E. (Principal Investigator)

    1995-01-01

    The prefrontal cortex of schizophrenics is hypoactive and displays changes related to inhibitory, GABAergic neurons, and GABAergic synapses. These changes include decreased levels of glutamic acid decarboxylase (GAD), the enzyme for GABA synthesis, upregulation of muscimol binding, and downregulation of benzodiazepine binding to GABAA receptors. Studies in the visual cortex of nonhuman primates have demonstrated that gene expression for GAD and for several GABAA receptor subunit polypeptides is under control of neuronal activity, raising the possibility that similar mechanisms in the hypoactive prefrontal cortex of schizophrenics may explain the abnormalities in GAD and in GABAA receptor regulation. In the present study, which is the first of its type on human cerebral cortex, levels of mRNAs for six GABAA receptor subunits (alpha 1, alpha 2, alpha 5, beta 1, beta 2, gamma 2) and their laminar expression patterns were analyzed in the prefrontal cortex of schizophrenics and matched controls, using in situ hybridization histochemistry and densitometry. Three types of laminar expression pattern were observed: mRNAs for the alpha 1, beta 2, and gamma 2 subunits, which are the predominant receptor subunits expressed in the mature cortex, were expressed at comparatively high levels by cells of all six cortical layers, but most intensely by cells in lower layer III and layer IV. mRNAs for the alpha 2, alpha 5, and beta 1 subunits were expressed at lower levels; alpha 2 and beta 1 were expressed predominantly by cells in layers II, III, and IV; alpha 5 was expressed predominantly in layers IV, V, and VI. There were no significant changes in overall mRNA levels for any of the receptor subunits in the prefrontal cortex of schizophrenics, and the laminar expression pattern of all six receptor subunit mRNAs did not differ between schizophrenics and controls. Because gene expression for GABAA receptor subunits is not consistently altered in the prefrontal cortex of

  15. The human thyrotropin beta-subunit gene differs in 5' structure from murine TSH-beta genes.

    Science.gov (United States)

    Guidon, P T; Whitfield, G K; Porti, D; Kourides, I A

    1988-12-01

    The gene encoding the beta-subunit of human thyrotropin (hTSH-beta) was isolated, and its nucleotide sequence was determined. The gene is 4.3 kb in length, consists of three exons and two introns, and is present as a single copy as determined by Southern blot analysis of total genomic DNA. The protein coding portion of the gene, which includes exons 2 and 3, was isolated from a human genomic phage library, while exon 1, which encodes only 5' untranslated mRNA sequence, was isolated from a plasmid library of size-selected genomic DNA fragments. Here we describe the isolation of the 5' untranslated exon of the hTSH-beta subunit and 5'-flanking region. The structure of the hTSH-beta gene is very similar to the previously characterized TSH-beta genes from mouse and rat. The genes from all three species have two distinct promoter regions, but while both promoters are utilized by the murine TSH-beta genes, the human TSH-beta gene apparently utilizes only the proximal promoter for transcription initiation. A striking difference in hTSH-beta gene structure compared to the murine genes is that exon 1 of the human gene is 36 nucleotides. An analysis of the mouse, rat, and human exon 1 and 5'-flanking region shows a high percentage of sequence homology, with the exception of a 9-nucleotide insertion 13 bases 3' from the proximal TATA box found in the human gene but not found in the other two species. We propose that this insertion results in the additional length of human exon 1 compared to the mouse and rat genes. By isolating the promoter region of the hTSH-beta gene, we can begin to identify specific sequences involved in the regulation of hTSH gene expression.

  16. Expression of five acetylcholine receptor subunit genes in Brugia malayi adult worms.

    Science.gov (United States)

    Li, Ben-Wen; Rush, Amy C; Weil, Gary J

    2015-12-01

    Acetylcholine receptors (AChRs) are required for body movement in parasitic nematodes and are targets of "classical" anthelmintic drugs such as levamisole and pyrantel and of newer drugs such as tribendimidine and derquantel. While neurotransmission explains the effects of these drugs on nematode movement, their effects on parasite reproduction are unexplained. The levamisole AChR type (L-AChRs) in Caenorhabditis elegans is comprised of five subunits: Cel-UNC-29, Cel-UNC-38, Cel-UNC-63, Cel-LEV-1 and Cel-LEV-8. The genome of the filarial parasite Brugia malayi contains nine AChRs subunits including orthologues of Cel-unc-29, Cel-unc-38, and Cel-unc-63. We performed in situ hybridization with RNA probes to localize the expression of five AChR genes (Bm1_35890-Bma-unc-29, Bm1_20330-Bma-unc-38, Bm1_38195-Bma-unc-63, Bm1_48815-Bma-acr-26 and Bm1_40515-Bma-acr-12) in B. malayi adult worms. Four of these genes had similar expression patterns with signals in body muscle, developing embryos, spermatogonia, uterine wall adjacent to stretched microfilariae, wall of V as deferens, and lateral cord. Three L-AChR subunit genes (Bma-unc-29, Bma-unc-38 and Bma-unc-63) were expressed in body muscle, which is a known target of levamisole. Bma-acr-12 was co-expressed with these levamisole subunit genes in muscle, and this suggests that its protein product may form receptors with other alpha subunits. Bma-acr-26 was expressed in male muscle but not in female muscle. Strong expression signals of these genes in early embryos and gametes in uterus and testis suggest that AChRs may have a role in nervous system development of embryogenesis and spermatogenesis. This would be consistent with embryotoxic effects of drugs that target these receptors in filarial worms. Our data show that the expression of these receptor genes is tightly regulated with regard to localization in adult worms and developmental stage in embryos and gametes. These results may help to explain the broad effects of

  17. Genetic differentiation of the mitochondrial cytochrome oxidase C subunit I gene in genus Paramecium (Protista, Ciliophora.

    Directory of Open Access Journals (Sweden)

    Yan Zhao

    Full Text Available BACKGROUND: The mitochondrial cytochrome c oxidase subunit I (COI gene is being used increasingly for evaluating inter- and intra-specific genetic diversity of ciliated protists. However, very few studies focus on assessing genetic divergence of the COI gene within individuals and how its presence might affect species identification and population structure analyses. METHODOLOGY/PRINCIPAL FINDINGS: We evaluated the genetic variation of the COI gene in five Paramecium species for a total of 147 clones derived from 21 individuals and 7 populations. We identified a total of 90 haplotypes with several individuals carrying more than one haplotype. Parsimony network and phylogenetic tree analyses revealed that intra-individual diversity had no effect in species identification and only a minor effect on population structure. CONCLUSIONS: Our results suggest that the COI gene is a suitable marker for resolving inter- and intra-specific relationships of Paramecium spp.

  18. Suppressor of MEK null (SMEK)/protein phosphatase 4 catalytic subunit (PP4C) is a key regulator of hepatic gluconeogenesis

    OpenAIRE

    Yoon, Young-Sil; Lee, Min-Woo; Ryu, Dongryeol; Kim, Jeong Ho; MA, Hui; Seo, Woo-Young; Kim, Yo-Na; Kim, Su Sung; Lee, Chul Ho; Hunter, Tony; Choi, Cheol Soo; Montminy, Marc R.; Koo, Seung-Hoi

    2010-01-01

    Fasting promotes hepatic gluconeogenesis to maintain glucose homeostasis. The cAMP-response element binding protein (CREB)-regulated transcriptional coactivator 2 (CRTC2) is responsible for transcriptional activation of gluconeogenic genes and is critical for conveying the opposing hormonal signals of glucagon and insulin in the liver. Here, we show that suppressor of MEK null 1 (SMEK1) and SMEK2 [protein phosphatase 4 (PP4) regulatory subunits 3a and 3b, respectively] are directly involved i...

  19. Multiple thyrotropin β-subunit and thyrotropin receptor-related genes arose during vertebrate evolution.

    Directory of Open Access Journals (Sweden)

    Gersende Maugars

    Full Text Available Thyroid-stimulating hormone (TSH is composed of a specific β subunit and an α subunit that is shared with the two pituitary gonadotropins. The three β subunits derive from a common ancestral gene through two genome duplications (1R and 2R that took place before the radiation of vertebrates. Analysis of genomic data from phylogenetically relevant species allowed us to identify an additional Tshβ subunit-related gene that was generated through 2R. This gene, named Tshβ2, present in cartilaginous fish, little skate and elephant shark, and in early lobe-finned fish, coelacanth and lungfish, was lost in ray-finned fish and tetrapods. The absence of a second type of TSH receptor (Tshr gene in these species suggests that both TSHs act through the same receptor. A novel Tshβ sister gene, named Tshβ3, was generated through the third genomic duplication (3R that occurred early in the teleost lineage. Tshβ3 is present in most teleost groups but was lostin tedraodontiforms. The 3R also generated a second Tshr, named Tshrb. Interestingly, the new Tshrb was translocated from its original chromosomic position after the emergence of eels and was then maintained in its new position. Tshrb was lost in tetraodontiforms and in ostariophysians including zebrafish although the latter species have two TSHs, suggesting that TSHRb may be dispensable. The tissue distribution of duplicated Tshβs and Tshrs was studied in the European eel. The endocrine thyrotropic function in the eel would be essentially mediated by the classical Tshβ and Tshra, which are mainly expressed in the pituitary and thyroid, respectively. Tshβ3 and Tshrb showed a similar distribution pattern in the brain, pituitary, ovary and adipose tissue, suggesting a possible paracrine/autocrine mode of action in these non-thyroidal tissues. Further studies will be needed to determine the binding specificity of the two receptors and how these two TSH systems are interrelated.

  20. Fragmentation of the large subunit ribosomal RNA gene in oyster mitochondrial genomes

    Directory of Open Access Journals (Sweden)

    Milbury Coren A

    2010-09-01

    Full Text Available Abstract Background Discontinuous genes have been observed in bacteria, archaea, and eukaryotic nuclei, mitochondria and chloroplasts. Gene discontinuity occurs in multiple forms: the two most frequent forms result from introns that are spliced out of the RNA and the resulting exons are spliced together to form a single transcript, and fragmented gene transcripts that are not covalently attached post-transcriptionally. Within the past few years, fragmented ribosomal RNA (rRNA genes have been discovered in bilateral metazoan mitochondria, all within a group of related oysters. Results In this study, we have characterized this fragmentation with comparative analysis and experimentation. We present secondary structures, modeled using comparative sequence analysis of the discontinuous mitochondrial large subunit rRNA genes of the cupped oysters C. virginica, C. gigas, and C. hongkongensis. Comparative structure models for the large subunit rRNA in each of the three oyster species are generally similar to those for other bilateral metazoans. We also used RT-PCR and analyzed ESTs to determine if the two fragmented LSU rRNAs are spliced together. The two segments are transcribed separately, and not spliced together although they still form functional rRNAs and ribosomes. Conclusions Although many examples of discontinuous ribosomal genes have been documented in bacteria and archaea, as well as the nuclei, chloroplasts, and mitochondria of eukaryotes, oysters are some of the first characterized examples of fragmented bilateral animal mitochondrial rRNA genes. The secondary structures of the oyster LSU rRNA fragments have been predicted on the basis of previous comparative metazoan mitochondrial LSU rRNA structure models.

  1. 体重指数与谷氨酰半胱氨酸合成酶催化亚基基因多态性的交互作用对女性乳腺癌风险的影响%Interaction of body mass index and a polymorphism in gene of catalytic subunit of glutamate cysteine ligase on breast cancer risk among Chinese women

    Institute of Scientific and Technical Information of China (English)

    林威; 唐录英; 岑玉玲; 林颖; 苏逢锡; 贾卫华; 任泽舫

    2013-01-01

    目的 探讨谷氨酰半胱氨酸合成酶催化亚基(GCLC)rs17883901多态性位点对BMI与乳腺癌关联的影响.方法 于2008年10月至2010年6月对中山大学3所附属医院新诊断的839例乳腺癌患者(病例组)在接受治疗前及同时期863名年龄频数匹配的对照(对照组)进行问卷调查和收集血样;采用基质辅助激光解吸-飞行时间质谱仪(MALDI-TOF-MS),在Sequenom平台检测rs17883901位点基因型;采用非条件logistic回归分析计算BMI和GCLC与乳腺癌关联的OR值及其95%CI.结果 (1)病例组和对照组接受调查时当前的BMI、20岁时的BMI和GCLCrs17883901位点分布的差异均无统计学意义(P=0.44、0.52和0.47);(2)未发现当前的BMI与绝经前及绝经后乳腺癌风险相关,20岁时BMI为18.5~22.9 kg/m2可降低绝经前乳腺癌风险(OR=0.69,95%CI:0.48~1.00),而未发现其与绝经后乳腺癌风险相关;(3)在GCLC rs17883901位点突变型CT/TT人群中,当前的BMI≥25 kg/m2显著增加乳腺癌风险(OR=1.91,95%CI:1.09~ 3.36),而20岁时BMI为18.5 ~ 22.9 kg/m2与降低乳腺癌风险有关(OR=0.56,95%CI:0.31 ~ 0.99).当前的BMI与GCLC基因多态性对乳腺癌发生风险存在交互作用(P=0.043),而20岁时的BMI与GCLC交互项无统计学意义(P=0.15).结论 20岁时增加BMI可能是绝经前乳腺癌的保护因素;GCLCrs17883901位点本身与乳腺癌发生风险无显著关联,但其变异基因型可使当前的肥胖状态(BMI≥25 kg/m2)显著增加乳腺癌发生风险.%Objective To investigate the interaction of body mass index (BMI) and a single nucleotide polymorphism (SNP,rs17883901) in catalytic subunit of glutamate-cysteine ligase (GCLC) on breast cancer risk.Methods A total of 839 women with incident breast cancer and 863 age-matched controls without cancer were recruited at the same period in three affiliated hospitals of Sun Yat-sen University in Guangzhou from October 2008 to June 2010.GCLC rs17883901 was genotyped

  2. Apoptosis Gene Hunting Using Retroviral Expression Cloning: Identification of Vacuolar ATPase Subunit E

    Directory of Open Access Journals (Sweden)

    Claire L. Anderson

    2003-01-01

    Full Text Available Over the past 10-15 years there has been an explosion of interest in apoptosis. The delayed realisation that cell death is an essential part of life for any multicellular organism has meant that, despite the recent and rapid developments of the last decade, the precise biochemical pathways involved in apoptosis remain incomplete and potentially novel genes may, as yet, remain undiscovered. The hunt is therefore on to bridge the remaining gaps in our knowledge. Our contribution to this research effort utilises a functional cloning approach to isolate important regulatory genes involved in apoptosis. This mini-review focuses on the use and advantages of a retroviral expression cloning strategy and describes the isolation and identification of one such potential apoptosis regulatory gene, namely that encoding vacuolar ATPase subunit E.

  3. Structural analysis of ARC-type inhibitor (ARC-1034) binding to protein kinase A catalytic subunit and rational design of bisubstrate analogue inhibitors of basophilic protein kinases.

    Science.gov (United States)

    Lavogina, Darja; Lust, Marje; Viil, Indrek; König, Norbert; Raidaru, Gerda; Rogozina, Jevgenia; Enkvist, Erki; Uri, Asko; Bossemeyer, Dirk

    2009-01-22

    The crystal structure of a complex of the catalytic subunit (type alpha) of cAMP-dependent protein kinase (PKA C alpha) with ARC-type inhibitor (ARC-1034), the presumed lead scaffold of previously reported adenosine-oligo-arginine conjugate-based (ARC-type) inhibitors, was solved. Structural elements important for interaction with the kinase were established with specifically modified derivatives of the lead compound. On the basis of this knowledge, a new generation of inhibitors, conjugates of adenosine-4'-dehydroxymethyl-4'-carboxylic acid moiety and oligo(D-arginine), was developed with inhibitory constants well into the subnanomolar range. The structural determinants of selectivity of the new compounds were established in assays with ROCK-II and PKBgamma.

  4. Knockdown of glutamate cysteine ligase catalytic subunit by siRNA causes the gold nanoparticles-induced cytotoxicity in lung cancer cells.

    Directory of Open Access Journals (Sweden)

    Min Liu

    Full Text Available Gold nanoparticles (GNPs have shown promising medical applications in cancer treatment involved in the regulation of intracellular redox balance. Previously, we have reported that GNPs can trigger apoptosis and necrosis in human lung cancer cells (A549 when L-buthionine-sulfoximine (BSO was used to decrease the expression of intracellular glutathione (GSH. Herein, we investigated the cytotoxicity of GNPs toward lung cancer cells under the glutamate cysteine ligase catalytic subunit (GCLC was silenced by siRNA. Our results showed that GNPs cause apoptosis and necrosis in cells transfected with GCLC siRNA by elevating intracellular reactive oxygen species (ROS. These findings demonstrated that the regulation of glutathione synthesis by GCLC siRNA in A549 cells can initiate the gold nanoparticles-induced cytotoxicity.

  5. Construction of Recombinant Plasmid Containing S. Mutans F-ATPase β Subunit Gene

    Institute of Scientific and Technical Information of China (English)

    YU Dan-ni; JIANG Li

    2005-01-01

    objective: construct a homologous recombinant plasmid which was expected to be transformed into S. mutans Methods: a region at the 5' terminus of the S. mutans F-ATPase β subunit gene was amplified by PCR, the PCR product was inserted into vector pVA891, yielding recombinant plasmid. Results: the DNA sequence of the recombinant plasmid was identified correct in whole by restriction endonuclease and DNA sequence techniques. Conclusion: the recombinant plasmid of S. mutans DNA was cloned in effect ,it may assist in construction of homologues recombinant mutant.

  6. Structural bases of dimerization of yeast telomere protein Cdc13 and its interaction with the catalytic subunit of DNA polymerase α

    Institute of Scientific and Technical Information of China (English)

    Jia Sun; Neal F Lue; Ming Lei; Yuting Yang; Ke Wan; Ninghui Mao; Tai-Yuan Yu; Yi-Chien Lin; Diane C DeZwaan; Brian C Freeman; Jing-Jer Lin

    2011-01-01

    Budding yeast Cdc13-Stnl-Tenl (CST) complex plays an essential role in telomere protection and maintenance, and has been proposed to be a telomere-specific replication protein A (RPA)-like complex. Previous genetic and structural studies revealed a close resemblance between Stn1-Ten1 and RPA32-RPA14. However, the relationship between Cdc13 and RPA70, the largest subunit of RPA, has remained unclear. Here, we report the crystal structure of the N-terminal OB (oligonucleotide/oligosaccharide binding) fold of Cdc13. Although Cdc13 has an RPA70-like domain organization, the structures of Cdc13 OB folds are significantly different from their counterparts in RPA70, suggesting that they have distinct evolutionary origins. Furthermore, our structural and biochemical analyses revealed unexpected dimerization by the N-terminal OB fold and showed that homodimerization is probably a conserved feature of all Cdc13 proteins. We also uncovered the structural basis of the interaction between the Cdc13 N-terminal OB ' fold and the catalytic subunit of DNA polymerase a (Pol1), and demonstrated a role for Cdc13 dimerization in Pol1 binding. Analysis of the phenotypes of mutants defective in Cdc13 dimerization and Cdc13-Pol1 interaction revealed multiple mechanisms by which dimerization regulates telomere lengths in vivo. Collectively, our findings provide novel insights into the mechanisms and evolution of Cdc13.

  7. Structural bases of dimerization of yeast telomere protein Cdc13 and its interaction with the catalytic subunit of DNA polymerase [alpha

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Jia; Yang, Yuting; Wan, Ke; Mao, Ninghui; Yu, Tai-Yuan; Lin, Yi-Chien; DeZwaan, Diane C.; Freeman, Brian C.; Lin, Jing-Jer; Lue, Neal F.; Lei, Ming

    2011-08-24

    Budding yeast Cdc13-Stn1-Ten1 (CST) complex plays an essential role in telomere protection and maintenance, and has been proposed to be a telomere-specific replication protein A (RPA)-like complex. Previous genetic and structural studies revealed a close resemblance between Stn1-Ten1 and RPA32-RPA14. However, the relationship between Cdc13 and RPA70, the largest subunit of RPA, has remained unclear. Here, we report the crystal structure of the N-terminal OB (oligonucleotide/oligosaccharide binding) fold of Cdc13. Although Cdc13 has an RPA70-like domain organization, the structures of Cdc13 OB folds are significantly different from their counterparts in RPA70, suggesting that they have distinct evolutionary origins. Furthermore, our structural and biochemical analyses revealed unexpected dimerization by the N-terminal OB fold and showed that homodimerization is probably a conserved feature of all Cdc13 proteins. We also uncovered the structural basis of the interaction between the Cdc13 N-terminal OB fold and the catalytic subunit of DNA polymerase {alpha} (Pol1), and demonstrated a role for Cdc13 dimerization in Pol1 binding. Analysis of the phenotypes of mutants defective in Cdc13 dimerization and Cdc13-Pol1 interaction revealed multiple mechanisms by which dimerization regulates telomere lengths in vivo. Collectively, our findings provide novel insights into the mechanisms and evolution of Cdc13.

  8. Sequence analysis of dolphin ferritin H and L subunits and possible iron-dependent translational control of dolphin ferritin gene

    Directory of Open Access Journals (Sweden)

    Sasaki Yukako

    2008-10-01

    Full Text Available Abstract Background Iron-storage protein, ferritin plays a central role in iron metabolism. Ferritin has dual function to store iron and segregate iron for protection of iron-catalyzed reactive oxygen species. Tissue ferritin is composed of two kinds of subunits (H: heavy chain or heart-type subunit; L: light chain or liver-type subunit. Ferritin gene expression is controlled at translational level in iron-dependent manner or at transcriptional level in iron-independent manner. However, sequencing analysis of marine mammalian ferritin subunits has not yet been performed fully. The purpose of this study is to reveal cDNA-derived amino acid sequences of cetacean ferritin H and L subunits, and demonstrate the possibility of expression of these subunits, especially H subunit, by iron. Methods Sequence analyses of cetacean ferritin H and L subunits were performed by direct sequencing of polymerase chain reaction (PCR fragments from cDNAs generated via reverse transcription-PCR of leukocyte total RNA prepared from blood samples of six different dolphin species (Pseudorca crassidens, Lagenorhynchus obliquidens, Grampus griseus, Globicephala macrorhynchus, Tursiops truncatus, and Delphinapterus leucas. The putative iron-responsive element sequence in the 5'-untranslated region of the six different dolphin species was revealed by direct sequencing of PCR fragments obtained using leukocyte genomic DNA. Results Dolphin H and L subunits consist of 182 and 174 amino acids, respectively, and amino acid sequence identities of ferritin subunits among these dolphins are highly conserved (H: 99–100%, (99→98 ; L: 98–100%. The conserved 28 bp IRE sequence was located -144 bp upstream from the initiation codon in the six different dolphin species. Conclusion These results indicate that six different dolphin species have conserved ferritin sequences, and suggest that these genes are iron-dependently expressed.

  9. The dnaN gene codes for the beta subunit of DNA polymerase III holoenzyme of escherichia coli.

    Science.gov (United States)

    Burgers, P M; Kornberg, A; Sakakibara, Y

    1981-09-01

    An Escherichia coli mutant, dnaN59, stops DNA synthesis promptly upon a shift to a high temperature; the wild-type dnaN gene carried in a transducing phage encodes a polypeptide of about 41,000 daltons [Sakakibara, Y. & Mizukami, T. (1980) Mol. Gen. Genet. 178, 541-553; Yuasa, S. & Sakakibara, Y. (1980) Mol. Gen. Genet. 180, 267-273]. We now find that the product of dnaN gene is the beta subunit of DNA polymerase III holoenzyme, the principal DNA synthetic multipolypeptide complex in E. coli. The conclusion is based on the following observations: (i) Extracts from dnaN59 cells were defective in phage phi X174 and G4 DNA synthesis after the mutant cells had been exposed to the increased temperature. (ii) The enzymatic defect was overcome by addition of purified beta subunit but not by other subunits of DNA polymerase III holoenzyme or by other replication proteins required for phi X174 DNA synthesis. (iii) Partially purified beta subunit from the dnaN mutant, unlike that from the wild type, was inactive in reconstituting the holoenzyme when mixed with the other purified subunits. (iv) Increased dosage of the dnaN gene provided by a plasmid carrying the gene raised cellular levels of the beta subunit 5- to 6-fold.

  10. Crystal structure of the catalytic domain of the initiation factor 2B epsilon subunit from saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Boesen, Thomas; Pavitt, Graham D.; Andersen, Gregers Rom

    -terminal domain harbors the two aa-box motifs involved in binding to the N-terminal part of the initiation factor 2 β subunit. Aliphatic residues in the aa-box motifs are involved in specific contacts in the hydrophobic core of the C-terminal bundle important for maintaining the overall structure, whereas, acidic...... residues in the motifs form a surface exposed acidic patch which might interact with the lysine boxes of initiation factor 2 β. Interestingly, tryptophan 699 was found to be solvent exposed and involved in crystal packing. This residue could possibly be important for the specific interaction...

  11. Characterization of the gene for the a subunit of human factor XIII (plasma transglutaminase), a blood coagulation factor

    Energy Technology Data Exchange (ETDEWEB)

    Ichinose, A.; Davie, E.W. (Univ. of Washington, Seattle (USA))

    1988-08-01

    Factor XIII (plasma transglutaminase, fibrin stabilizing factor) is a glycoprotein that circulates in blood as a tetramer (a{sub 2}b{sub 2}) consisting of two a and two b subunits. The primary structures of the a and b subunits of human factor XIII have been reported by a combination of cDNA cloning and amino acid sequence analysis. To establish the gene structure of the a subunit for factor XIII, several human genomic libraries were screened by using the cDNA encoding the a subunit as a probe. Among {approx}5 {times} 10{sup 7} recombinant phage, 121 have been shown to contain an insert encoding a portion of the a subunit. Twenty-five unique clones were than characterized by restriction mapping, Southern blotting, and DNA sequencing. Overlapping clones encoding the a subunit of factor XIII span >160 kilobases. DNA sequence analysis revealed that the activation peptide released by thrombin, the active site cysteine region, the two putative calcium-binding regions, and the thrombin cleavage site leading to inactivation are encoded by separate exons. This suggest that the introns may separate the a subunit into functional and structural domains. A comparison of the amino acid sequence deduced from the genomic DNA sequence with those deduced from cDNA or determined by amino acid sequence analysis of the plasma and placental proteins revealed apparent amino acid polymorphisms in six positions of the polypeptide chain of the a subunit.

  12. Low-density crystal packing of human protein kinase CK2 catalytic subunit in complex with resorufin or other ligands

    DEFF Research Database (Denmark)

    Klopffleisch, Karsten; Issinger, Olaf Georg; Niefind, Karsten

    2012-01-01

    adopts a closed conformation correlating to a canonically established catalytic spine as is typical for eukaryotic protein kinases. In the corresponding crystal packing the hinge/helix αD region is nearly unaffected by crystal contacts, so that largely unbiased conformational adaptions are possible....... This is documented by published human CK2α structures with the same crystal packing but with an open hinge/helix αD region, one of which has been redetermined here with a higher symmetry. An overview of all published human CK2α crystal packings serves as the basis for a discussion of the factors that determine...

  13. Gene regulation, alternative splicing, and posttranslational modification of troponin subunits in cardiac development and adaptation: a focused review.

    Science.gov (United States)

    Sheng, Juan-Juan; Jin, Jian-Ping

    2014-01-01

    Troponin plays a central role in regulating the contraction and relaxation of vertebrate striated muscles. This review focuses on the isoform gene regulation, alternative RNA splicing, and posttranslational modifications of troponin subunits in cardiac development and adaptation. Transcriptional and posttranscriptional regulations such as phosphorylation and proteolysis modifications, and structure-function relationships of troponin subunit proteins are summarized. The physiological and pathophysiological significances are discussed for impacts on cardiac muscle contractility, heart function, and adaptations in health and diseases.

  14. Agrobacterium-mediated transformation of the β-subunit gene in 7S globulin protein in soybean using RNAi technology.

    Science.gov (United States)

    Qu, J; Liu, S Y; Wang, P W; Guan, S Y; Fan, Y G; Yao, D; Zhang, L; Dai, J L

    2016-04-26

    The objective of this study was to use RNA interference (RNAi) to improve protein quality and decrease anti-nutritional effects in soybean. Agrobacterium tumefaciens-mediated transformation was conducted using RNAi and an expression vector containing the 7S globulin β-subunit gene. The BAR gene was used as the selective marker and cotyledonary nodes of soybean genotype Jinong 27 were chosen as explant material. Regenerated plants were detected by molecular biology techniques. Transformation of the β-subunit gene in the 7S protein was detected by PCR, Southern blot, and q-PCR. Positive plants (10 T0, and 6 T1, and 13 T2) were tested by PCR. Hybridization bands were detected by Southern blot analysis in two of the T1 transgenic plants. RNAi expression vectors containing the soybean 7S protein β-subunit gene were successfully integrated into the genome of transgenic plants. qRT-PCR analysis in soybean seeds showed a clear decrease in expression of the soybean β-subunit gene. The level of 7S protein β-subunit expression in transgenic plants decreased by 77.5% as compared to that of the wild-type plants. This study has established a basis for the application of RNAi to improve the anti-nutritional effects of soybean.

  15. Silencing expression of the catalytic subunit of DNA-dependent protein kinase by small interfering RNA sensitizes human cells for radiation-induced chromosome damage, cell killing, and mutation

    Science.gov (United States)

    Peng, Yuanlin; Zhang, Qinming; Nagasawa, Hatsumi; Okayasu, Ryuichi; Liber, Howard L.; Bedford, Joel S.

    2002-01-01

    Targeted gene silencing in mammalian cells by RNA interference (RNAi) using small interfering RNAs (siRNAs) was recently described by Elbashir et al. (S. M. Elbashir et al., Nature (Lond.), 411: 494-498, 2001). We have used this methodology in several human cell strains to reduce expression of the Prkdc (DNA-PKcs) gene coding for the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) that is involved in the nonhomologous end joining of DNA double-strand breaks. We have also demonstrated a radiosensitization for several phenotypic endpoints of radiation damage. In low-passage normal human fibroblasts, siRNA knock-down of DNA-PKcs resulted in a reduced capacity for restitution of radiation-induced interphase chromosome breaks as measured by premature chromosome condensation, an increased yield of acentric chromosome fragments at the first postirradiation mitosis, and an increased radiosensitivity for cell killing. For three strains of related human lymphoblasts, DNA-PKcs-targeted siRNA transfection resulted in little or no increase in radiosensitivity with respect to cell killing, a 1.5-fold decrease in induced mutant yield in TK6- and p53-null NH32 cells, but about a 2-fold increase in induced mutant yield in p53-mutant WTK1 cells at both the hypoxanthine quanine phosphoribosyl transferase (hprt) and the thymidine kinase loci.

  16. Molecular cloning and analysis of zebrafish voltage-gated sodium channel beta subunit genes: implications for the evolution of electrical signaling in vertebrates

    Directory of Open Access Journals (Sweden)

    Zhong Tao P

    2007-07-01

    Full Text Available Abstract Background Action potential generation in excitable cells such as myocytes and neurons critically depends on voltage-gated sodium channels. In mammals, sodium channels exist as macromolecular complexes that include a pore-forming alpha subunit and 1 or more modulatory beta subunits. Although alpha subunit genes have been cloned from diverse metazoans including flies, jellyfish, and humans, beta subunits have not previously been identified in any non-mammalian species. To gain further insight into the evolution of electrical signaling in vertebrates, we investigated beta subunit genes in the teleost Danio rerio (zebrafish. Results We identified and cloned single zebrafish gene homologs for beta1-beta3 (zbeta1-zbeta3 and duplicate genes for beta4 (zbeta4.1, zbeta4.2. Sodium channel beta subunit loci are similarly organized in fish and mammalian genomes. Unlike their mammalian counterparts, zbeta1 and zbeta2 subunit genes display extensive alternative splicing. Zebrafish beta subunit genes and their splice variants are differentially-expressed in excitable tissues, indicating tissue-specific regulation of zbeta1-4 expression and splicing. Co-expression of the genes encoding zbeta1 and the zebrafish sodium channel alpha subunit Nav1.5 in Chinese Hamster Ovary cells increased sodium current and altered channel gating, demonstrating functional interactions between zebrafish alpha and beta subunits. Analysis of the synteny and phylogeny of mammalian, teleost, amphibian, and avian beta subunit and related genes indicated that all extant vertebrate beta subunits are orthologous, that beta2/beta4 and beta1/beta3 share common ancestry, and that beta subunits are closely related to other proteins sharing the V-type immunoglobulin domain structure. Vertebrate sodium channel beta subunit genes were not identified in the genomes of invertebrate chordates and are unrelated to known subunits of the para sodium channel in Drosophila. Conclusion The

  17. MUSCLE-SPECIFIC OVEREXPRESSION OF THE CATALYTIC SUBUNIT OF DNA POLYMERASE γ INDUCES PUPAL LETHALITY IN Drosophila melanogaster

    Science.gov (United States)

    Martínez-Azorín, Francisco; Calleja, Manuel; Hernández-Sierra, Rosana; Farr, Carol L.; Kaguni, Laurie S.; Garesse, Rafael

    2015-01-01

    We show the physiological effects and molecular characterization of overexpression of the catalytic core of mitochondrial DNA (mtDNA) polymerase (pol γ-α) in muscle of Drosophila melanogaster. Muscle-specific overexpression of pol γ-α using the UAS/GAL4 (where UAS is upstream activation sequence) system produced more than 90% of lethality at the end of pupal stage at 25°C, and the survivor adult flies showed a significant reduction in life span. The survivor flies displayed a decreased mtDNA level that is accompanied by a corresponding decrease in the levels of the nucleoid-binding protein mitochondrial transcription factor A (mtTFA). Furthermore, an increase in apoptosis is detected in larvae and adults overexpressing pol γ-α. We suggest that the pupal lethality and reduced life span of survivor adult flies are both caused mainly by massive apoptosis of muscle cells induced by mtDNA depletion. PMID:23729397

  18. Mutations in the Gene Encoding the Ancillary Pilin Subunit of the Streptococcus suis srtF Cluster Result in Pili Formed by the Major Subunit Only

    Science.gov (United States)

    Fittipaldi, Nahuel; Takamatsu, Daisuke; la Cruz Domínguez-Punaro, María de; Lecours, Marie-Pier; Montpetit, Diane; Osaki, Makoto; Sekizaki, Tsutomu; Gottschalk, Marcelo

    2010-01-01

    Pili have been shown to contribute to the virulence of different Gram-positive pathogenic species. Among other critical steps of bacterial pathogenesis, these structures participate in adherence to host cells, colonization and systemic virulence. Recently, the presence of at least four discrete gene clusters encoding putative pili has been revealed in the major swine pathogen and emerging zoonotic agent Streptococcus suis. However, pili production by this species has not yet been demonstrated. In this study, we investigated the functionality of one of these pili clusters, known as the srtF pilus cluster, by the construction of mutant strains for each of the four genes of the cluster as well as by the generation of antibodies against the putative pilin subunits. Results revealed that the S. suis serotype 2 strain P1/7, as well as several other highly virulent invasive S. suis serotype 2 isolates express pili from this cluster. However, in most cases tested, and as a result of nonsense mutations at the 5′ end of the gene encoding the minor pilin subunit (a putative adhesin), pili were formed by the major pilin subunit only. We then evaluated the role these pili play in S. suis virulence. Abolishment of the expression of srtF cluster-encoded pili did not result in impaired interactions of S. suis with porcine brain microvascular endothelial cells. Furthermore, non-piliated mutants were as virulent as the wild type strain when evaluated in a murine model of S. suis sepsis. Our results show that srtF cluster-encoded, S. suis pili are atypical compared to other Gram-positive pili. In addition, since the highly virulent strains under investigation are unlikely to produce other pili, our results suggest that pili might be dispensable for critical steps of the S. suis pathogenesis of infection. PMID:20052283

  19. NUCLEOTIDE-SEQUENCE OF THE LAST EXON OF THE GENE FOR HUMAN CYTOCHROME-C-OXIDASE SUBUNIT-VIB AND ITS FLANKING REGIONS

    NARCIS (Netherlands)

    TAANMAN, JW; SCHRAGE, C; BOKMA, E; REUVEKAMP, P; AGSTERIBBE, E; DEVRIES, H

    1991-01-01

    A human genomic clone encompassing the last exon of the gene for cytochrome c oxidase subunit VIb and a human genomic clone containing the most distal end of this gene were characterized. The last exon of the gene codes for the 17 C-terminal amino acid residues of the subunit and the 3' noncoding re

  20. Analysis of the cytochrome c oxidase subunit II (COX2) gene in giant panda, Ailuropoda melanoleuca.

    Science.gov (United States)

    Ling, S S; Zhu, Y; Lan, D; Li, D S; Pang, H Z; Wang, Y; Li, D Y; Wei, R P; Zhang, H M; Wang, C D; Hu, Y D

    2017-01-23

    The giant panda, Ailuropoda melanoleuca (Ursidae), has a unique bamboo-based diet; however, this low-energy intake has been sufficient to maintain the metabolic processes of this species since the fourth ice age. As mitochondria are the main sites for energy metabolism in animals, the protein-coding genes involved in mitochondrial respiratory chains, particularly cytochrome c oxidase subunit II (COX2), which is the rate-limiting enzyme in electron transfer, could play an important role in giant panda metabolism. Therefore, the present study aimed to isolate, sequence, and analyze the COX2 DNA from individuals kept at the Giant Panda Protection and Research Center, China, and compare these sequences with those of the other Ursidae family members. Multiple sequence alignment showed that the COX2 gene had three point mutations that defined three haplotypes, with 60% of the sequences corresponding to haplotype I. The neutrality tests revealed that the COX2 gene was conserved throughout evolution, and the maximum likelihood phylogenetic analysis, using homologous sequences from other Ursidae species, showed clustering of the COX2 sequences of giant pandas, suggesting that this gene evolved differently in them.

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

    Science.gov (United States)

    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.

  2. A novel mutation in the sodium channel α1 subunit gene in a child with Dravet syndrome in Turkey

    Institute of Scientific and Technical Information of China (English)

    Mutluay Arslan; Ulu(c) Yi(s); Hande (C)a(g)layan; R1dvan Akin

    2013-01-01

    Dravet syndrome is a rare epileptic encephalopathy characterized by frequent seizures beginning in the first year of life and behavioral disorders. Mutations in the sodium channel α1 subunit gene are the main cause of this disease. We report two patients with refractory seizures and psychomotor retardation in whom the final diagnosis was Dravet syndrome with confirmed mutations in the sodium channel α1 subunit gene. The mutation identified in the second patient was a novel frame shift mutation, which resulted from the deletion of five nucleotides in exon 24.

  3. Nicotinic Acetylcholine Receptor α4 Subunit Gene Variation Associated with Attention Deficit Hyperactivity Disorder

    Institute of Scientific and Technical Information of China (English)

    HUANG Xuezhu; XU Yong; LI Qianqian; LIU Pozi; YANG Yuan; ZHANG Fuquan; GUO Tianyou; YANG Chuang; GUO Lanting

    2009-01-01

    Previous pharmacological, human genetics, and animal models have implicated the nicotinic ace-tylcholine receptor a4 subunit (CHRNA4) gene in the pathogenesis of attention deficit/hyperactivity disorder (ADHD). The objective of this study is to examine the genetic association between single nucleotide poly-morphisms in the CHRNA4 gene (rs2273502, rs1044396, rs1044397, and rs3827020 loci) and ADHD. Both case-control and family-based designs are used. Children aged 6 to 16 years were interviewed and as-sessed with the children behavior checklist and the revised conner' parent rating scale to identify probands. No significant differences in the frequency distribution of genotypes or alleles were found between the case and control groups. However, further haplotype analyses showed the CCGG haplotype on dsk for ADHD in 164 case-control samples and the standard transmission disequilibrium test analyses suggest that the allele C of rs2273502 was over-transferred in 98 ADHD parent-offspring tdos. These findings suggest that the CHRNA4 gene may play a role in the pathogenesis of ADHD.

  4. Molecular cloning and expression analysis of a novel BCCP subunit gene from Aleurites moluccana.

    Science.gov (United States)

    Xuan, W Y; Zhang, Y; Liu, Z Q; Feng, D; Luo, M Y

    2015-08-19

    Aleurites moluccana L. is grown as a roadside tree in southern China and the oil content of its seed is higher than other oil plants, such as Jatropha curcas and Camellia oleifera. A. moluccana is considered a promising energy plant because its seed oil could be used to produce biodiesel and bio-jet fuel. In addition, the bark, leaves, and kernels of A. moluccana have various medical and commercial uses. Here, a novel gene coding the biotin carboxyl carrier protein subunit (BCCP) was cloned from A. moluccana L. using the homology cloning method combined with rapid amplification of cDNA end (RACE) technology. The isolated full-length cDNA sequence (designated AM-accB) was 1188 bp, containing a 795-bp open reading frame coding for 265 amino acids. The deduced amino acid sequence of AM-accB contained a biotinylated domain located between amino acids 190 and 263. A. moluccana BCCP shows high identity at the amino acid level to its homologues in other higher plants, such as Vernicia fordii, J. curcas, and Ricinus communis (86, 77, and 70%, respectively), which all contain conserved domains for ACCase activity. The expression of the AM-accB gene during the middle stage of development and maturation in A. moluccana seeds was higher than that in early and later stages. The expression pattern of the AM-accB gene is very similar to that of the oil accumulation rate.

  5. Recognition of DNA Termini by the C-Terminal Region of the Ku80 and the DNA-Dependent Protein Kinase Catalytic Subunit.

    Directory of Open Access Journals (Sweden)

    Derek S Woods

    Full Text Available DNA double strand breaks (DSBs can be generated by endogenous cellular processes or exogenous agents in mammalian cells. These breaks are highly variable with respect to DNA sequence and structure and all are recognized in some context by the DNA-dependent protein kinase (DNA-PK. DNA-PK is a critical component necessary for the recognition and repair of DSBs via non-homologous end joining (NHEJ. Previously studies have shown that DNA-PK responds differentially to variations in DSB structure, but how DNA-PK senses differences in DNA substrate sequence and structure is unknown. Here we explore the enzymatic mechanisms by which DNA-PK is activated by various DNA substrates and provide evidence that the DNA-PK is differentially activated by DNA structural variations as a function of the C-terminal region of Ku80. Discrimination based on terminal DNA sequence variations, on the other hand, is independent of the Ku80 C-terminal interactions and likely results exclusively from DNA-dependent protein kinase catalytic subunit interactions with the DNA. We also show that sequence differences in DNA termini can drastically influence DNA repair through altered DNA-PK activation. These results indicate that even subtle differences in DNA substrates influence DNA-PK activation and ultimately the efficiency of DSB repair.

  6. Inhibition of CK2 Activity by TCDD via Binding to ATP-competitive Binding Site of Catalytic Subunit:Insight from Computational Studies

    Institute of Scientific and Technical Information of China (English)

    XU Xian-jin; CANNISTRARO Salvatore; BIZZARRI Anna-rita; ZENG Yi; CHEN Wei-zu; WANG Cun-xin

    2013-01-01

    Alternative mechanisms of toxic effects induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin(TCDD),instead of the binding to aryl hydrocarbon receptor(AhR),have been taken into consideration.It has been recently shown that TCDD reduces rapidly the activity of CK2(casein kinase Ⅱ) both in vivo and in vitro.It is found that TCDD has high molecular similarities to the known inhibitors of CK2 catalytic subunit(CK2α).This suggests that TCDD could also be an ATP-competitive inhibitor of CK2α.In this work,docking TCDD to CK2 was carried out based on the two structures of CK2α from maize and human,respectively.The binding free energies of the predicted CK2α-TCDD complexes estimated by the molecular mechanics/Poisson-Boltzmann surface area(MM/PBSA) method are from -85.1 kJ/mol to-114.3 kJ/mol for maize and are from-96.1 kJ/mol to-118.2 kJ/mol for human,which are comparable to those estimated for the known inhibitor and also ATP with CK2α.The energetic analysis also reveals that the van der Waals interaction is the dominant contribution to the binding free energy.These results are also useful for designing new drugs for a target of overexpressing CK2 in cancers.

  7. Expression of gp91phox and p22phox, catalytic subunits of NADPH oxidase, on microglia in Nasu-Hakola disease brains

    Science.gov (United States)

    Satoh, Jun-ichi; Kino, Yoshihiro; Yanaizu, Motoaki; Tosaki, Youhei; Sakai, Kenji; Ishida, Tusyoshi; Saito, Yuko

    2016-01-01

    Summary The superoxide-producing nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex of phagocytes (phox) plays a key role in production of reactive oxygen species (ROS) by microglia. The catalytic subunits of the NADPH oxidase are composed of p22phox and gp91phox. Nasu-Hakola disease (NHD) is a rare autosomal recessive disorder caused by a loss-of-function mutation of either TYROBP (DAP12) or TREM2. Pathologically, the brains of NHD patients exhibit extensive demyelination designated leukoencephalopathy, astrogliosis, accumulation of axonal spheroids, and remarkable activation of microglia predominantly in the white matter of frontal and temporal lobes. However, a pathological role of the gp91phox-p22phox complex in generation of leukoencephalopathy in NHD remains unknown. We clarified the expression of gp91phox and p22phox in the white matter of the frontal cortex derived from five NHD and eight control subjects. We identified the expression of p22phox and gp91phox immunoreactivity almost exclusively on microglia. Microglia overexpressed gp91phox in NHD brains and p22phox in myotonic dystrophy (MD) brains, when compared with non-neurological control (NC) brains. These results suggest that the enhanced expression of gp91phox by microglia might contribute to overproduction of ROS highly toxic to myelinating oligodendrocytes, resulting in oligodendrocyte cell death that induces leukoencephalopathy in NHD brains. PMID:27904823

  8. Nuclear life of the voltage-gated Cacnb4 subunit and its role in gene transcription regulation.

    Science.gov (United States)

    Ronjat, Michel; Kiyonaka, Shigeki; Barbado, Maud; De Waard, Michel; Mori, Yasuo

    2013-01-01

    The pore-forming subunit of voltage-gated calcium channels is associated to auxiliary subunits among which the cytoplasmic β subunit. The different isoforms of this subunit control both the plasma membrane targeting and the biophysical properties of the channel moiety. In a recent study, we demonstrated that the Cacnb4 (β 4) isoform is at the center of a new signaling pathway that connects neuronal excitability and gene transcription. This mechanism relies on nuclear targeting of β 4 triggered by neuronal electrical stimulation. This re-localization of β 4 is promoted by its interaction with Ppp2r5d a regulatory subunit of PP2A in complex with PP2A itself. The formation, as well as the nuclear translocation, of the β 4/ Ppp2r5d/ PP2A complex is totally impaired by the premature R482X stops mutation of β 4 that has been previously associated with juvenile epilepsy. Taking as a case study the tyrosine hydroxylase gene that is strongly upregulated in brain of lethargic mice, deficient for β 4 expression, we deciphered the molecular steps presiding to this signaling pathway. Here we show that expression of wild-type β 4 in HEK293 cells results in the regulation of several genes, while expression of the mutated β 4 (β 1-481) produces a different set of gene regulation. Several genes regulated by β 4 in HEK293 cells were also regulated upon neuronal differentiation of NG108-15 cells that induces nuclear translocation of β 4 suggesting a link between β 4 nuclear targeting and gene regulation.

  9. Frequent germ-line succinate dehydrogenase subunit D gene mutations in patients with apparently sporadic parasympathetic paraganglioma

    NARCIS (Netherlands)

    H. Dannenberg (Hilde); W.N.M. Dinjens (Winand); M. Abbou; H. van Urk (Hero); B.K. Pauw; D. Mouwen; W.J. Mooi (Wolter); R.R. de Krijger (Ronald)

    2002-01-01

    textabstractPURPOSE: Recently, familial paraganglioma (PGL) was shown to be caused bymutations in the gene encoding succinate dehydrogenase subunit D (SDHD). However, the prevalence of SDHD mutations in apparently sporadic PGL is unknown. We studied the frequency and spectrum of ge

  10. A novel mutation in the succinate dehydrogenase subunit D gene in siblings with the hereditary paraganglioma-pheochromocytoma syndrome.

    Science.gov (United States)

    Prasad, Chaithra; Oakley, Gerard J; Yip, Linwah; Coyne, Christopher; Rangaswamy, Balasubramanya; Dixit, Sanjay B

    2014-01-01

    Germline mutations in the succinate dehydrogenase complex subunit D gene are now known to be associated with hereditary paraganglioma-pheochromocytoma syndromes. Since the initial succinate dehydrogenase complex subunit D gene mutation was identified about a decade ago, more than 131 unique variants have been reported. We report the case of two siblings presenting with multiple paragangliomas and pheochromocytomas; they were both found to carry a mutation in the succinate dehydrogenase complex subunit D gene involving a substitution of thymine to guanine at nucleotide 236 in exon 3. This particular mutation of the succinate dehydrogenase complex subunit D gene has only been reported in one previous patient in Japan; this is, therefore, the first report of this pathogenic mutation in siblings and the first report of this mutation in North America. With continued screening of more individuals, we will be able to create a robust mutation database that can help us understand disease patterns associated with particular variants and may be a starting point in the development of new therapies for familial paraganglioma syndromes.

  11. A novel mutation in the succinate dehydrogenase subunit D gene in siblings with the hereditary paraganglioma–pheochromocytoma syndrome

    Directory of Open Access Journals (Sweden)

    Chaithra Prasad

    2014-10-01

    Full Text Available Germline mutations in the succinate dehydrogenase complex subunit D gene are now known to be associated with hereditary paraganglioma–pheochromocytoma syndromes. Since the initial succinate dehydrogenase complex subunit D gene mutation was identified about a decade ago, more than 131 unique variants have been reported. We report the case of two siblings presenting with multiple paragangliomas and pheochromocytomas; they were both found to carry a mutation in the succinate dehydrogenase complex subunit D gene involving a substitution of thymine to guanine at nucleotide 236 in exon 3. This particular mutation of the succinate dehydrogenase complex subunit D gene has only been reported in one previous patient in Japan; this is, therefore, the first report of this pathogenic mutation in siblings and the first report of this mutation in North America. With continued screening of more individuals, we will be able to create a robust mutation database that can help us understand disease patterns associated with particular variants and may be a starting point in the development of new therapies for familial paraganglioma syndromes.

  12. A novel mutation in the succinate dehydrogenase subunit D gene in siblings with the hereditary paraganglioma–pheochromocytoma syndrome

    Science.gov (United States)

    Oakley, Gerard J; Yip, Linwah; Coyne, Christopher; Rangaswamy, Balasubramanya; Dixit, Sanjay B

    2014-01-01

    Germline mutations in the succinate dehydrogenase complex subunit D gene are now known to be associated with hereditary paraganglioma–pheochromocytoma syndromes. Since the initial succinate dehydrogenase complex subunit D gene mutation was identified about a decade ago, more than 131 unique variants have been reported. We report the case of two siblings presenting with multiple paragangliomas and pheochromocytomas; they were both found to carry a mutation in the succinate dehydrogenase complex subunit D gene involving a substitution of thymine to guanine at nucleotide 236 in exon 3. This particular mutation of the succinate dehydrogenase complex subunit D gene has only been reported in one previous patient in Japan; this is, therefore, the first report of this pathogenic mutation in siblings and the first report of this mutation in North America. With continued screening of more individuals, we will be able to create a robust mutation database that can help us understand disease patterns associated with particular variants and may be a starting point in the development of new therapies for familial paraganglioma syndromes. PMID:27489656

  13. Characterization of the low-molecular-weight glutenin subunit gene family members using a PCR-based marker approach

    Science.gov (United States)

    Low-molecular-weight glutenin subunits (LMW-GS) are a class of seed storage proteins that play a major role in the determination of the processing quality of wheat flour. The LMW-GS are encoded by multi-gene families located on the short arms of the homoeologous group 1 chromosomes, at the Glu-A3, G...

  14. Molecular evolution of the cytochrome c oxidase subunit 5A gene in primates

    Directory of Open Access Journals (Sweden)

    Wildman Derek E

    2008-01-01

    Full Text Available Abstract Background Many electron transport chain (ETC genes show accelerated rates of nonsynonymous nucleotide substitutions in anthropoid primate lineages, yet in non-anthropoid lineages the ETC proteins are typically highly conserved. Here, we test the hypothesis that COX5A, the ETC gene that encodes cytochrome c oxidase subunit 5A, shows a pattern of anthropoid-specific adaptive evolution, and investigate the distribution of this protein in catarrhine brains. Results In a dataset comprising 29 vertebrate taxa, including representatives from all major groups of primates, there is nearly 100% conservation of the COX5A amino acid sequence among extant, non-anthropoid placental mammals. The most recent common ancestor of these species lived about 100 million years (MY ago. In contrast, anthropoid primates show markedly elevated rates of nonsynonymous evolution. In particular, branch site tests identify five positively selected codons in anthropoids, and ancestral reconstructions infer that substitutions in these codons occurred predominantly on stem lineages (anthropoid, ape and New World monkey and on the human terminal branch. Examination of catarrhine brain samples by immunohistochemistry characterizes for the first time COX5A protein distribution in the primate neocortex, and suggests that the protein is most abundant in the mitochondria of large-size projection neurons. Real time quantitative PCR supports previous microarray results showing COX5A is expressed in cerebral cortical tissue at a higher level in human than in chimpanzee or gorilla. Conclusion Taken together, these results suggest that both protein structural and gene regulatory changes contributed to COX5A evolution during humankind's ancestry. Furthermore, these findings are consistent with the hypothesis that adaptations in ETC genes contributed to the emergence of the energetically expensive anthropoid neocortex.

  15. An ancient repeat sequence in the ATP synthase beta-subunit gene of forcipulate sea stars.

    Science.gov (United States)

    Foltz, David W

    2007-11-01

    A novel repeat sequence with a conserved secondary structure is described from two nonadjacent introns of the ATP synthase beta-subunit gene in sea stars of the order Forcipulatida (Echinodermata: Asteroidea). The repeat is present in both introns of all forcipulate sea stars examined, which suggests that it is an ancient feature of this gene (with an approximate age of 200 Mya). Both stem and loop regions show high levels of sequence constraint when compared to flanking nonrepetitive intronic regions. The repeat was also detected in (1) the family Pterasteridae, order Velatida and (2) the family Korethrasteridae, order Velatida. The repeat was not detected in (1) the family Echinasteridae, order Spinulosida, (2) the family Astropectinidae, order Paxillosida, (3) the family Solasteridae, order Velatida, or (4) the family Goniasteridae, order Valvatida. The repeat lacks similarity to published sequences in unrestricted GenBank searches, and there are no significant open reading frames in the repeat or in the flanking intron sequences. Comparison via parametric bootstrapping to a published phylogeny based on 4.2 kb of nuclear and mitochondrial sequence for a subset of these species allowed the null hypothesis of a congruent phylogeny to be rejected for each repeat, when compared separately to the published phylogeny. In contrast, the flanking nonrepetitive sequences in each intron yielded separate phylogenies that were each congruent with the published phylogeny. In four species, the repeat in one or both introns has apparently experienced gene conversion. The two introns also show a correlated pattern of nucleotide substitutions, even after excluding the putative cases of gene conversion.

  16. Function of Partially Duplicated Human α7 Nicotinic Receptor Subunit CHRFAM7A Gene

    Science.gov (United States)

    de Lucas-Cerrillo, Ana M.; Maldifassi, M. Constanza; Arnalich, Francisco; Renart, Jaime; Atienza, Gema; Serantes, Rocío; Cruces, Jesús; Sánchez-Pacheco, Aurora; Andrés-Mateos, Eva; Montiel, Carmen

    2011-01-01

    The neuronal α7 nicotinic receptor subunit gene (CHRNA7) is partially duplicated in the human genome forming a hybrid gene (CHRFAM7A) with the novel FAM7A gene. The hybrid gene transcript, dupα7, has been identified in brain, immune cells, and the HL-60 cell line, although its translation and function are still unknown. In this study, dupα7 cDNA has been cloned and expressed in GH4C1 cells and Xenopus oocytes to study the pattern and functional role of the expressed protein. Our results reveal that dupα7 transcript was natively translated in HL-60 cells and heterologously expressed in GH4C1 cells and oocytes. Injection of dupα7 mRNA into oocytes failed to generate functional receptors, but when co-injected with α7 mRNA at α7/dupα7 ratios of 5:1, 2:1, 1:1, 1:5, and 1:10, it reduced the nicotine-elicited α7 current generated in control oocytes (α7 alone) by 26, 53, 75, 93, and 94%, respectively. This effect is mainly due to a reduction in the number of functional α7 receptors reaching the oocyte membrane, as deduced from α-bungarotoxin binding and fluorescent confocal assays. Two additional findings open the possibility that the dominant negative effect of dupα7 on α7 receptor activity observed in vitro could be extrapolated to in vivo situations. (i) Compared with α7 mRNA, basal dupα7 mRNA levels are substantial in human cerebral cortex and higher in macrophages. (ii) dupα7 mRNA levels in macrophages are down-regulated by IL-1β, LPS, and nicotine. Thus, dupα7 could modulate α7 receptor-mediated synaptic transmission and cholinergic anti-inflammatory response. PMID:21047781

  17. ASSIGNMENT OF THE GENE CODING FOR HUMAN CYTOCHROME-C-OXIDASE SUBUNIT-VIB TO CHROMOSOME-19, BAND-Q13.1, BY FLUORESCENCE INSITU HYBRIDIZATION

    NARCIS (Netherlands)

    TAANMAN, JW; VANDERVEEN, AY; SCHRAGE, C; DEVRIES, H; BUYS, CHCM

    1991-01-01

    A cloned, 40 kb, genomic DNA fragment, containing the last exon of the gene for human cytochrome c oxidase subunit VIb and its flanking sequences, was used as a probe to localize the subunit VIb gene on human metaphase chromosomes. The probe was labelled with Bio-11-dUTP and detected by fluorescence

  18. Molecular evolution at the cytochrome oxidase subunit 2 gene among divergent populations of the intertidal copepod, Tigriopus californicus.

    Science.gov (United States)

    Rawson, Paul D; Burton, Ronald S

    2006-06-01

    The cytochrome c oxidase subunit 2 gene (COII) encodes a highly conserved protein that is directly responsible for the initial transfer of electrons from cytochrome c to cytochrome c oxidase (COX) crucial to the production of ATP during cellular respiration. Despite its integral role in electron transport, we have observed extensive intraspecific nucleotide and amino acid variation among 26 full-length COII sequences sampled from seven populations of the marine copepod, Tigriopus californicus. Although intrapopulation divergence was virtually nonexistent, interpopulation divergence at the COII locus was nearly 20% at the nucleotide level, including 38 nonsynonymous substitutions. Given the high degree of interaction between the cytochrome c oxidase subunit 2 protein (COX2) and the nuclear-encoded subunits of COX and cytochrome c (CYC), we hypothesized that some codons in the COII gene are likely to be under positive selection in order to compensate for amino acid substitutions in other subunits. Estimates of the ratio of nonsynonymous to synonymous substitution (omega), obtained using a series of maximum likelihood models of codon substitution, indicated that the majority of codons in T. californicus COII are under strong purifying selection (omega < 1), while approximately 4% of the sites in this gene appear to evolve under relaxed selective constraint (omega = 1). A branch-site maximum likelihood model identified three sites that may have experienced positive selection within the central California sequence clade in our COII phylogeny; these results are consistent with previous studies showing functional and fitness consequences among interpopulation hybrids between central and northern California populations.

  19. Phosphatidylinositol 3-kinase p85 regulatory subunit gene and spinal muscular atrophy disease

    Directory of Open Access Journals (Sweden)

    Monica STAVARACHI

    2009-11-01

    Full Text Available Spinal muscular atrophy (SMA is a frequent neuromuscular disorder caused by motoneuronal apoptosis, as a result of SMN (Survival Motor Neuron protein deficiency. Although the SMA determining gene was identified, the molecular mechanism of the disease is not clearly understood, due to the heterogeneity of clinical manifestations. Trying to complete the molecular describing SMA picture, by identifying potential modulators factors, we investigated the relationship between phosphatidylinositol 3-kinase p85 regulatory subunit gene (PIK3R1 and SMA pathology. As IGF signaling pathway has been reported to play an important role in motoneurons survival and PIK3 is a key element of this cascade signaling, we focused on the relationship between PIK3R1 gene Met326Ile polymorphism and SMA type I, the most severe form of the disease. A total of 80 subjects (40 SMA type I patients and 40 unrelated healthy controls were included in the study. The statistical analyzes performed consequently to the genotyping by mismatch PCR-RFLP method, revealed that Met326Ile polymorphism is not associated with SMA type I disease: ORMet/Met = 0.398 with a p = 0.072 meanwhile ORMet = 0.495, p = 0.063. However, the Cochrane – Armitage test indicated that there is a statistically association trend between the analyzed polymorphism and SMA type I pathology: ORMet = 0.438, p = 0.032. We concluded that additional researches with an increased subjects number and replicates studies in other populations will clarify the investigated relationship and it may contribute to the SMA molecular mechanism understanding.

  20. Differential expression of genes encoding neuronal ion-channel subunits in major depression, bipolar disorder and schizophrenia: implications for pathophysiology.

    Science.gov (United States)

    Smolin, Bella; Karry, Rachel; Gal-Ben-Ari, Shunit; Ben-Shachar, Dorit

    2012-08-01

    Evidence concerning ion-channel abnormalities in the pathophysiology of common psychiatric disorders is still limited. Given the significance of ion channels in neuronal activity, neurotransmission and neuronal plasticity we hypothesized that the expression patterns of genes encoding different ion channels may be altered in schizophrenia, bipolar and unipolar disorders. Frozen samples of striatum including the nucleus accumbens (Str-NAc) and the lateral cerebellar hemisphere of 60 brains from depressed (MDD), bipolar (BD), schizophrenic and normal subjects, obtained from the Stanley Foundation Brain Collection, were assayed. mRNA of 72 different ion-channel subunits were determined by qRT-PCR and alteration in four genes were verified by immunoblotting. In the Str-NAc the prominent change was observed in the MDD group, in which there was a significant up-regulation in genes encoding voltage-gated potassium-channel subunits. However, in the lateral cerebellar hemisphere (cerebellum), the main change was observed in schizophrenia specimens, as multiple genes encoding various ion-channel subunits were significantly down-regulated. The impaired expression of genes encoding ion channels demonstrates a disease-related neuroanatomical pattern. The alterations observed in Str-NAc of MDD may imply electrical hypo-activity of this region that could be of relevance to MDD symptoms and treatment. The robust unidirectional alteration of both excitatory and inhibitory ion channels in the cerebellum may suggests cerebellar general hypo-transcriptional activity in schizophrenia.

  1. Familial Congenital Hypothyroidism Caused by Abnormal and Bioinactive TSH due to Mutations in the beta-Subunit Gene.

    Science.gov (United States)

    Medeiros-Neto, G; de Lacerda, L; Wondisford, F E

    1997-01-01

    Hereditary TSH deficiency is a rare autosomal recessive disease described in inbred Japanese families and in Greek and Brazilian kindreds. The TSH-beta-subunit gene has been shown to be the site of mutations that will give rise to truncated proteins that cannot dimerize with the alpha subunit or, alternatively, will produce a mutated TSH that is present in the circulation of the affected patients, but it is biologically inactive. Characteristically, the patients with TSH-beta-subunit-defects are born with congenital hypothyroidism, with very low levels of serum thyroid hormones and serum thyroglobulin and, paradoxically, with serum TSH levels that are consistently undetectable or at very low levels. Goiter is not present at birth, but the low radioactive thyroid uptake will increase after bovine TSH stimulation. Other pituitary hormones responses to provocative tests are normal. The subunit levels are at high concentration and are significantly increased following TRH stimulation. In two kindreds, molecular biological studies have indicated mutations in two different sites of exon 2, generating a peptide that would not dimerize with subunits to synthesize TSH molecules. In one kindred, a truncated TSH-beta protein was translated that generated a biologically inactive but detectable serum TSH molecule. (c) 1997, Elsevier Science Inc. (Trends Endocrinol Metab 1997;8:15-20).

  2. O-GlcNAcylation of protein kinase A catalytic subunits enhances its activity: a mechanism linked to learning and memory deficits in Alzheimer's disease.

    Science.gov (United States)

    Xie, Shutao; Jin, Nana; Gu, Jianlan; Shi, Jianhua; Sun, Jianming; Chu, Dandan; Zhang, Liang; Dai, Chun-Ling; Gu, Jin-Hua; Gong, Cheng-Xin; Iqbal, Khalid; Liu, Fei

    2016-06-01

    Alzheimer's disease (AD) is characterized clinically by memory loss and cognitive decline. Protein kinase A (PKA)-CREB signaling plays a critical role in learning and memory. It is known that glucose uptake and O-GlcNAcylation are reduced in AD brain. In this study, we found that PKA catalytic subunits (PKAcs) were posttranslationally modified by O-linked N-acetylglucosamine (O-GlcNAc). O-GlcNAcylation regulated the subcellular location of PKAcα and PKAcβ and enhanced their kinase activity. Upregulation of O-GlcNAcylation in metabolically active rat brain slices by O-(2-acetamido-2-deoxy-d-glucopyranosylidenamino) N-phenylcarbamate (PUGNAc), an inhibitor of N-acetylglucosaminidase, increased the phosphorylation of tau at the PKA site, Ser214, but not at the non-PKA site, Thr205. In contrast, in rat and mouse brains, downregulation of O-GlcNAcylation caused decreases in the phosphorylation of CREB at Ser133 and of tau at Ser214, but not at Thr205. Reduction in O-GlcNAcylation through intracerebroventricular injection of 6-diazo-5-oxo-l-norleucine (DON), the inhibitor of glutamine fructose-6-phosphate amidotransferase, suppressed PKA-CREB signaling and impaired learning and memory in mice. These results indicate that in addition to cAMP and phosphorylation, O-GlcNAcylation is a novel mechanism that regulates PKA-CREB signaling. Downregulation of O-GlcNAcylation suppresses PKA-CREB signaling and consequently causes learning and memory deficits in AD.

  3. Hepatocyte-Specific Ablation of PP2A Catalytic Subunit α Attenuates Liver Fibrosis Progression via TGF-β1/Smad Signaling

    Directory of Open Access Journals (Sweden)

    Na Lu

    2015-01-01

    Full Text Available Protein phosphatase 2A (PP2A, a family of the major serine/threonine phosphatases in cells, regulates many aspects of physiological processes. However, isoform-specific substrates and the biological role of each specific member of the PP2A family remain largely unknown. In this study, we investigated whether PP2A catalytic subunit Cα (PP2Acα is involved in chronic hepatic injury and fibrosis. A hepatocyte-specific PP2Acα ablation mice model was established to examine the effect of PP2Acα on carbon tetrachloride- (CCl4- induced chronic hepatic injury and fibrosis. Our results showed that PP2Acα knockout mice were less susceptible to chronic CCl4-induced liver injury as evidenced by lower levels of serum alanine aminotransferase and aspartate aminotransferase, decreased hepatocyte proliferation, and increased rate of apoptotic removal of the injured hepatocytes. PP2Acα knockout mice also displayed a lesser extent of liver fibrosis as a significant decrease in the proportion of α-smooth muscle actin-expressing cells and collagen deposition was observed in their liver tissues. Furthermore, the levels of serum TGF-β1 and hepatocytic Smad phosphorylation were reduced in the PP2Acα knockout mice. These data suggest that hepatocyte-specific ablation of PP2Acα protects against CCl4-induced chronic hepatic injury and fibrogenesis and the protective effect is mediated at least partially through the impaired TGF-β1/Smad signaling.

  4. Polo-like kinase 1 (PLK1) and protein phosphatase 6 (PP6) regulate DNA-dependent protein kinase catalytic subunit (DNA-PKcs) phosphorylation in mitosis.

    Science.gov (United States)

    Douglas, Pauline; Ye, Ruiqiong; Trinkle-Mulcahy, Laura; Neal, Jessica A; De Wever, Veerle; Morrice, Nick A; Meek, Katheryn; Lees-Miller, Susan P

    2014-06-25

    The protein kinase activity of the DNA-PKcs (DNA-dependent protein kinase catalytic subunit) and its autophosphorylation are critical for DBS (DNA double-strand break) repair via NHEJ (non-homologous end-joining). Recent studies have shown that depletion or inactivation of DNA-PKcs kinase activity also results in mitotic defects. DNA-PKcs is autophosphorylated on Ser2056, Thr2647 and Thr2609 in mitosis and phosphorylated DNA-PKcs localize to centrosomes, mitotic spindles and the midbody. DNA-PKcs also interacts with PP6 (protein phosphatase 6), and PP6 has been shown to dephosphorylate Aurora A kinase in mitosis. Here we report that DNA-PKcs is phosphorylated on Ser3205 and Thr3950 in mitosis. Phosphorylation of Thr3950 is DNA-PK-dependent, whereas phosphorylation of Ser3205 requires PLK1 (polo-like kinase 1). Moreover, PLK1 phosphorylates DNA-PKcs on Ser3205 in vitro and interacts with DNA-PKcs in mitosis. In addition, PP6 dephosphorylates DNA-PKcs at Ser3205 in mitosis and after IR (ionizing radiation). DNA-PKcs also phosphorylates Chk2 on Thr68 in mitosis and both phosphorylation of Chk2 and autophosphorylation of DNA-PKcs in mitosis occur in the apparent absence of Ku and DNA damage. Our findings provide mechanistic insight into the roles of DNA-PKcs and PP6 in mitosis and suggest that DNA-PKcs' role in mitosis may be mechanistically distinct from its well-established role in NHEJ.

  5. Phosphatidyl-inositol-3-kinase alpha catalytic subunit mutation and response to neoadjuvant endocrine therapy for estrogen receptor positive breast cancer

    Science.gov (United States)

    Ellis, Matthew J; Lin, Li; Crowder, Robert; Tao, Yu; Hoog, Jeremy; Snider, Jacqueline; Davies, Sherri; DeSchryver, Katherine; Evans, Dean B; Steinseifer, Jutta; Bandaru, Raj; Liu, WeiHua; Gardner, Humphrey; Semiglazov, Vladimir; Watson, Mark; Hunt, Kelly; Olson, John; Baselga, José

    2010-01-01

    Background Mutations in the alpha catalytic subunit of phosphoinositol-3-kinase (PIK3CA) occur in ~30% of ER positive breast cancers. We therefore sought to determine the impact of PIK3CA mutation on response to neoadjuvant endocrine therapy. Methods Exon 9 (helical domain - HD) and Exon 20 (kinase domain- KD) mutations in PIK3CA were determined samples from four neoadjuvant endocrine therapy trials. Interactions with clinical, pathological and biomarker response parameters were examined. Results A weak negative interaction between PIK3CA mutation status and clinical response to neoadjuvant endocrine treatment was detected (N=235 P=<0.05), but not with treatment-induced changes in Ki67-based proliferation index (N=418). Despite these findings, PIK3CA KD mutation was a favorable prognostic factor for relapse-free survival (RFS log rank P=0.02) in the P024 trial (N=153). The favorable prognostic effect was maintained in a multivariable analysis (N=125) that included the preoperative prognostic index (PEPI), an approach to predicting RFS based on post neoadjuvant endocrine therapy pathological stage, ER and Ki67 levels (HR for no PIK3CA KD mutation, 14, CI 1.9–105 P=0.01). Conclusion PIK3CA mutation status did not strongly interact with neoadjuvant endocrine therapy responsiveness in estrogen receptor positive breast cancer. Nonetheless, as with other recent studies, a favorable interaction between PIK3CA kinase domain mutation and prognosis was detected. The mechanism for the favorable prognostic impact of PIK3CA mutation status therefore remains unexplained. PMID:19844788

  6. PHYLOGENY OF ANGIOSTRONGYLUS CANTONENSIS IN THAILAND BASED ON CYTOCHROME C OXIDASE SUBUNIT I GENE SEQUENCE.

    Science.gov (United States)

    Apichat, Vitta; Narongrit, Srisongcram; Jittranuch, Thiproaj; Anucha, Wongma; Wilaiwan, Polsut; Chamaiporn, Fukruksa; Thatcha, Yimthin; Bandid, Mangkit; Aunchalee, Thanwisai; Paron, Dekumyoy

    2016-05-01

    Angiostrongylus cantonensis is an emerging infectious agent causing eosinophilic meningitis or meningoencephalitis in humans with clinical manifestation of severe headache. Molecular genetic studies on classification and phylogeny of A. cantonensis in Thailand are limited. This study surveyed A. cantonensis larvae prevalence in natural intermediate hosts across Thailand and analyzed their phylogenetic relationships. A total of 14,032 freshwater and land snails were collected from 19 provinces of Thailand. None of Filopaludina sp, Pomacea sp, and Cyclophorus sp were infected with Angiostrongylus larvae, whereas Achatina fulica, Cryptozona siamensis, and Megaustenia siamensis collected from Kalasin, Kamphaeng Phet, Phetchabun, Phitsanulok, and Tak Provinces were infected, with C. siamensis being the common intermediate host. Based on morphology, larvae isolated from 11 samples of these naturally infected snails preliminarily were identified as A. cantonensis. Comparison of partial nucleotide sequences of cytochrome c oxidase subunit I gene revealed that four sequences are identical to A. cantonensis haplotype ac4 from Bangkok and the other seven to that of A. cantonensis isolate AC Thai, indicating two independent lineages of A. cantonensis in Thailand.

  7. Cloning of the Gene Encoding Urease Subunit A in Helicobacter Pylori

    Institute of Scientific and Technical Information of China (English)

    施理; 张宜俊; 陈劼; 候晓华

    2004-01-01

    Summary: The gene encoding urease subunit A (ureA) of Helicobacter pylori (H. pylori) was cloned from H. pylori isolate by polymerase chain reaction (PCR). Sterile distilled water instead of DNA served as negative control. The nucleotide sequence of the amplified product was determined.Homologous analysis of the ureA against that reported by Clayton CL and the GenBank and SwissProt databases were performed with the BLAST program at the Genome Net through the Internet.0.8 kb PCR product was amplified from all H. pylori clinical isolators. The nucleotide sequence of the ureA was determined. The nucleotide sequence of the ureA began with ATG as the initiation codon and terminated in TAA as stop codon. The coding regions had a 44 % G+ C content. The DNA sequence was 98 % homologous to that reported by Clayton CL (688 out of 702 residues were identical). The derived amino-acid sequences of the ureA were 99 % homologous to that reported by Clayton CL (232 out of 234 residues were identical). The nucleotide sequence and the predicted protein showed significant homology to ureA of H. pylori in the NCBI Entrez database.

  8. Amplification of TLO Mediator Subunit Genes Facilitate Filamentous Growth in Candida Spp.

    Science.gov (United States)

    Liu, Zhongle; Moran, Gary P.; Myers, Lawrence C.

    2016-01-01

    Filamentous growth is a hallmark of C. albicans pathogenicity compared to less-virulent ascomycetes. A multitude of transcription factors regulate filamentous growth in response to specific environmental cues. Our work, however, suggests the evolutionary history of C. albicans that resulted in its filamentous growth plasticity may be tied to a change in the general transcription machinery rather than transcription factors and their specific targets. A key genomic difference between C. albicans and its less-virulent relatives, including its closest relative C. dubliniensis, is the unique expansion of the TLO (TeLOmere-associated) gene family in C. albicans. Individual Tlo proteins are fungal-specific subunits of Mediator, a large multi-subunit eukaryotic transcriptional co-activator complex. This amplification results in a large pool of ‘free,’ non-Mediator associated, Tlo protein present in C. albicans, but not in C. dubliniensis or other ascomycetes with attenuated virulence. We show that engineering a large ‘free’ pool of the C. dubliniensis Tlo2 (CdTlo2) protein in C. dubliniensis, through overexpression, results in a number of filamentation phenotypes typically associated only with C. albicans. The amplitude of these phenotypes is proportional to the amount of overexpressed CdTlo2 protein. Overexpression of other C. dubliniensis and C. albicans Tlo proteins do result in these phenotypes. Tlo proteins and their orthologs contain a Mediator interaction domain, and a potent transcriptional activation domain. Nuclear localization of the CdTlo2 activation domain, facilitated naturally by the Tlo Mediator binding domain or artificially through an appended nuclear localization signal, is sufficient for the CdTlo2 overexpression phenotypes. A C. albicans med3 null mutant causes multiple defects including the inability to localize Tlo proteins to the nucleus and reduced virulence in a murine systemic infection model. Our data supports a model in which the

  9. Sequential mutations in the interleukin-3 (IL3)/granulocyte-macrophage colony-stimulating factor/IL5 receptor beta-subunit genes are necessary for the complete conversion to growth autonomy mediated by a truncated beta C subunit.

    Science.gov (United States)

    Hannemann, J; Hara, T; Kawai, M; Miyajima, A; Ostertag, W; Stocking, C

    1995-05-01

    An amino-terminally truncated beta C receptor (beta C-R) subunit of the interleukin-3 (IL3)/granulocyte-macrophage colony-stimulating factor/IL5 receptor complex mediates factor-independent and tumorigenic growth in two spontaneous mutants of a promyelocytic cell line. The constitutive activation of the JAK2 protein kinase in these mutants confirms that signaling occurs through the truncated receptor protein. Noteworthily, in addition to a 10-kb deletion in the beta C-R subunit gene encoding the truncated receptor, several secondary and independent mutations that result in the deletion or functional inactivation of the allelic beta C-R subunit and the closely related beta IL3-R subunit genes were observed in both mutants, suggesting that such mutations are necessary for the full oncogenic penetrance of the truncated beta C-R subunit. Reversion of these mutations by the expression of the wild-type beta C-R in the two mutants resulted in a fivefold decrease in cloning efficiency of the mutants in the absence of IL3, confirming a functional interaction between the wild-type and truncated proteins. Furthermore, expression of the truncated beta C-R subunit in factor-dependent myeloid cells did not immediately render the cells autonomous but increased the spontaneous frequency to factor-independent growth by 4 orders of magnitude. Implications for both leukemogenic progression and receptor-subunit interaction and signaling are discussed.

  10. Structure and expression analysis of genes encoding ADP-glucose pyrophosphorylase large subunit in wheat and its relatives.

    Science.gov (United States)

    Zhang, Xiao-Wei; Li, Si-Yu; Zhang, Ling-Ling; Yang, Qiang; Jiang, Qian-Tao; Ma, Jian; Qi, Peng-Fei; Li, Wei; Chen, Guo-Yue; Lan, Xiu-Jin; Deng, Mei; Lu, Zhen-Xiang; Liu, Chunji; Wei, Yu-Ming; Zheng, You-Liang

    2016-07-01

    ADP-glucose pyrophosphorylase (AGP), which consists of two large subunits (AGP-L) and two small subunits (AGP-S), controls the rate-limiting step in the starch biosynthetic pathway. In this study, a full-length open reading frame (ORF) of AGP-L gene (named as Agp2) in wheat and a series of Agp2 gene sequences in wheat relatives were isolated. The coding region of Agp2 contained 15 exons and 14 introns including a full-length ORF of 1566 nucleotides, and the deduced protein contained 522 amino acids (57.8 kDa). Generally, the phylogenetic tree of Agp2 indicated that sequences from A- and D-genome donor species were most similar to each other and sequences from B-genome donor species contained more variation. Starch accumulation and Agp2 expression in wheat grains reached their peak at 21 and 15 days post anthesis (DPA), respectively.

  11. Evolution, expression differentiation and interaction specificity of heterotrimeric G-protein subunit gene family in the mesohexaploid Brassica rapa.

    Directory of Open Access Journals (Sweden)

    Gulab C Arya

    Full Text Available Heterotrimeric G-proteins, comprising of Gα, Gβ, and Gγ subunits, are important signal transducers which regulate many aspects of fundamental growth and developmental processes in all eukaryotes. Initial studies in model plants Arabidopsis and rice suggest that the repertoire of plant G-protein is much simpler than that observed in metazoans. In order to assess the consequence of whole genome triplication events within Brassicaceae family, we investigated the multiplicity of G-protein subunit genes in mesohexaploid Brassica rapa, a globally important vegetable and oilseed crop. We identified one Gα (BraA.Gα1, three Gβ (BraA.Gβ1, BraA.Gβ2, and BraA.Gβ3, and five Gγ (BraA.Gγ1, BraA.Gγ2, BraA.Gγ3, BraA.Gγ4, and BraA.Gγ5 genes from B. rapa, with a possibility of 15 Gαβγ heterotrimer combinations. Our analysis suggested that the process of genome triplication coupled with gene-loss (gene-fractionation phenomenon have shaped the quantitative and sequence diversity of G-protein subunit genes in the extant B. rapa genome. Detailed expression analysis using qRT-PCR assays revealed that the G-protein genes have retained ubiquitous but distinct expression profiles across plant development. The expression of multiple G-protein genes was differentially regulated during seed-maturation and germination stages, and in response to various phytohormone treatments and stress conditions. Yeast-based interaction analysis showed that G-protein subunits interacted in most of the possible combinations, with some degree of subunit-specific interaction specificity, to control the functional selectivity of G-protein heterotrimer in different cell and tissue-types or in response to different environmental conditions. Taken together, this research identifies a highly diverse G-protein signaling network known to date from B. rapa, and provides a clue about the possible complexity of G-protein signaling networks present across globally important Brassica

  12. Cloning and expression in Escherichia coli of a new gene of Schistosoma japonicum encoding casein kinase Ⅱ beta subunit

    Institute of Scientific and Technical Information of China (English)

    彭寨玉; 余新炳; 吴忠道; 徐劲; 吴德; 李孜

    2004-01-01

    Background Nowadays it is now a focus topic in schistosomiasis research to find ideal vaccine candidates and new drug targets for developing anti-schistosomiasis vaccine. We cloned a new gene, casein kinase Ⅱ beta subunit, of Schistosoma japonicum (S. japonicum) and express it in Escherichia coli (E.coli).Methods The ESTs obtained in our laboratory were analyzed by homologous searching, and a new gene was recognized. The full-length cDNA of the new gene was obtained by joining the 3'RACE PCR fragment and the EST clone. To express the new gene, the cDNA was cloned into pGEX-4T-1 vector and then transformed into E.coli JM109. The recombinant protein was analyzed by SDS-PAGE and Western-blot. Results A 908 bp cDNA was isolated from S. japonicum and identified to be casein kinase Ⅱ beta subunit gene by sequence analysis. The open reading frame of the gene encodes a protein of 217 amino acids exhibiting 75.8%, 75.8%, 73.9%, 68.2%, 51.6% identity to the amino acids sequence of the corresponding genes of Homo sapiens (H. sapiens), Xenopus laevi (X. laevi), Drosophila melanogaster (D. melanogaster), Caenorhabditis elegan (C. elegan), and Schizosaccharomyces pombe (S. promber) respectively. The predicted molecular weight of the protein was 24.921 kDa. The new cDNA sequence had been submitted to GenBank, and its accession number is AY241391. This cDNA was subcloned into the pGEX-4T-1 vector and expressed in E.coli JM109.The recombinant protein could be recognized by the S. japonicum infected rabbit serum. Conclusion The full-length cDNA sequences encoding S. japonicum casein kinase Ⅱ beta subunit were firstly sequenced, cloned, and expressed in E.coli.

  13. Rational design of glycerol dehydratase: Swapping the genes encoding the subunits of glycerol dehydratase to improve enzymatic properties

    Institute of Scientific and Technical Information of China (English)

    QI Xianghui; SUN Liang; LUO Zhaofei; WU Jiequn; MENG Xiaolei; TANG Yue; WEI Yutuo; HUANG Ribo

    2006-01-01

    1,3-propanediol (1,3-PD) is an important material for chemical industry, and there has been always much interest in the production of 1,3-PD using all possible routes. The genes encoding glycerol dehydratase (GDHt) from Citrobacter freundii,Klebsiella pneumoniae and metagenome were cloned and expressed in E. coli. All glycerol dehydratases but the one from metagenome could be detected to show enzyme activities. In order to improve the enzymatic properties of GDHts, the genes encoding α and β-γ subunits were cloned, and the enzyme characteristics were evolved by rational design based on their 3D structures which were constructed by homology modeling. Six heteroenzymes were obtained by swapping the α subunit genes of these three different-source-derived GDHts. The pH,thermal stability and Vmax of some heteroenzymes were dramatically improved by 2-5 times compared with the wild one (GDHtKP). The GDHt cloned from metagenome, originally proved to be with no enzyme activity, was converted into active enzyme by swapping its subunits with other different GDHts. In addition, the effect of subtle 3D structural changes on the properties of the enzyme was also observed.

  14. Transgene inheritance and quality improvement by expressing novel HMW glutenin subunit (HMW-GS) genes in winter wheat

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    The expression vector pBPC30, which carries the high molecular weight glutenin subunit (HMW-GS) 1Dx5 and 1Dy10 genes, was transferred into hexaploid winter wheat cv. Jinghua No. 1, Jing411 and Jingdong No. 6 explants of immature embryos and immature inflorescence by particle bombardment. A large number of resistant transgenic plants were obtained under the selection of herbicide bialaphos or phosphinothricin (PPT). Confirmed transgenic plants of T0 generation showed successful integration of HMW-GS genes and bar gene into the wheat genome. T1 generation of transgenic plants can resist 20-150 mg/L PPT. Protein analysis of T2 seed by SDS-PAGE showed that HMW-GS 1Dx5 and 1Dy10 genes were well expressed in offspring seed of transgenic lines by co-expression with or substitution of endogenous 1Dx2 or 1Dy10. In one transgenic line, TG3-74, a new protein band between endogenous protein subunits 7 and 8 (marked as 8*) of glutenin appeared, but endogenous subunit 8 (encoded by 1By8 gene) was absent. Analysis of gluten rheological quality on seed proteins of 102 T3 plants showed that the sedimentation value of 5 transgenic lines (44.2-49.0 mL) was remarkably improved, 59.6%-64.3% higher than that of wild type Jinghua No. 1 and Jingdong No. 6, similar to bread wheat Cheyenne (48.0 mL). Analysis of dough rheological properties of transgenic lines showed that the dough stable time of 5 transgenic lines range from 16 to 30 min, whereas the dough stable time of wild type was only between 3-7 min. Our research suggests that introducing novel HMW-GS genes into wheat is an efficient way to improve its bread-making quality.

  15. Isolation and Sequence Analysis of HMW Glutenin Subunit 1Dy10.1 Ecoding Gene from Xinjiang Wheat (Triticum petropavlovskyi Udacz.et Migusch)

    Institute of Scientific and Technical Information of China (English)

    JIANG Qian-tao; WEI Yu-ming; WANG Ji-rui; YAN Ze-hong; ZHENG You-liang

    2006-01-01

    A novel HMW glutenin subunit gene 1Dy10.1 was isolated and characterized from Xinjiang wheat (Triticum petropavlovskyi. Udacz. et Migusch) accession Daomai 2. The complete open reading frame (ORF) of 1Dy10.1 was 1965 bp, encoding 655 amino acids. The numbers and distribution of cysteines in 1Dy10.1 were similar to those of 1Dy10 and other y-type subunits. In the N-terminal of 1Dy10.1, an amino acid was changed from L (leucine) to P (proline) at position 55. The repetitive domain of 1Dy10.1 differed from those of known HMW subunits by substitutions, insertions or/and deletions involving single or more amino acid residues. In the repetitive domain of subunit 1Dy10.1, the deletion of tripeptide GQQ in the consensus unit PGQGQQ resulted in the appearance of the motif PGQ that have not been observed in other known y-type HMW subunits. In comparison with the subunit 1Dy12, a deletion of dipeptide GQ, which occurred in subunit 1Dy10, was also observed in subunit 1Dy10.1. The cloned 1Dyl0.1 gene had been successfully expressed in Escherichia coli, and the expressed protein had the identical mobility with the endogenous subunit 1Dyl0.1 from seed.

  16. Complementation of Escherichia coli unc mutant strains by chloroplast and cyanobacterial F1-ATPase subunits.

    Science.gov (United States)

    Lill, H; Burkovski, A; Altendorf, K; Junge, W; Engelbrecht, S

    1993-10-04

    The genes encoding the five subunits of the F1 portion of the ATPases from both spinach chloroplasts and the cyanobacterium Synechocystis sp. PCC 6803 were cloned into expression vectors and expressed in Escherichia coli. The recombinant subunits formed inclusion bodies within the cells. Each particular subunit was expressed in the respective unc mutant, each unable to grow on non-fermentable carbon sources. The following subunits restored growth under conditions of oxidative phosphorylation: alpha (both sources, cyanobacterial subunit more than spinach subunit), beta (cyanobacterial subunit only), delta (both spinach and Synechocystis), and epsilon (both sources), whereas no growth was achieved with the gamma subunits from both sources. Despite a high degree of sequence homology the large subunits alpha and beta of spinach and cyanobacterial F1 were not as effective in the substitution of their E. coli counterparts. On the other hand, the two smallest subunits of the E. coli ATPase could be more effectively replaced by their cyanobacterial or chloroplast counterparts, although the sequence identity or even similarity is very low. We attribute these findings to the different roles of these subunits in F1: The large alpha and beta subunits contribute to the catalytic centers of the enzyme, a function rendering them very sensitive to even minor changes. For the smaller delta and epsilon subunits it was sufficient to maintain a certain tertiary structure during evolution, with little emphasis on the conservation of particular amino acids.

  17. Ribosomal protein S3: a KH domain subunit in NF-kappaB complexes that mediates selective gene regulation.

    Science.gov (United States)

    Wan, Fengyi; Anderson, D Eric; Barnitz, Robert A; Snow, Andrew; Bidere, Nicolas; Zheng, Lixin; Hegde, Vijay; Lam, Lloyd T; Staudt, Louis M; Levens, David; Deutsch, Walter A; Lenardo, Michael J

    2007-11-30

    NF-kappaB is a DNA-binding protein complex that transduces a variety of activating signals from the cytoplasm to specific sets of target genes. To understand the preferential recruitment of NF-kappaB to specific gene regulatory sites, we used NF-kappaB p65 in a tandem affinity purification and mass spectrometry proteomic screen. We identified ribosomal protein S3 (RPS3), a KH domain protein, as a non-Rel subunit of p65 homodimer and p65-p50 heterodimer DNA-binding complexes that synergistically enhances DNA binding. RPS3 knockdown impaired NF-kappaB-mediated transcription of selected p65 target genes but not nuclear shuttling or global protein translation. Rather, lymphocyte-activating stimuli caused nuclear translocation of RPS3, parallel to p65, to form part of NF-kappaB bound to specific regulatory sites in chromatin. Thus, RPS3 is an essential but previously unknown subunit of NF-kappaB involved in the regulation of key genes in rapid cellular activation responses. Our observations provide insight into how NF-kappaB selectively controls gene expression.

  18. Switch in glutamate receptor subunit gene expression in CA1 subfield of hippocampus following global ischemia in rats.

    Science.gov (United States)

    Pellegrini-Giampietro, D E; Zukin, R S; Bennett, M V; Cho, S; Pulsinelli, W A

    1992-11-01

    Severe, transient global ischemia of the brain induces delayed damage to specific neuronal populations. Sustained Ca2+ influx through glutamate receptor channels is thought to play a critical role in postischemic cell death. Although most kainate-type glutamate receptors are Ca(2+)-impermeable, Ca(2+)-permeable kainate receptors have been reported in specific kinds of neurons and glia. Recombinant receptors assembled from GluR1 and/or GluR3 subunits in exogenous expression systems are permeable to Ca2+; heteromeric channels containing GluR2 subunits are Ca(2+)-impermeable. Thus, altered expression of GluR2 in development or following a neurological insult or injury to the brain can act as a switch to modify Ca2+ permeability. To investigate the molecular mechanism underlying delayed postischemic cell death, GluR1, GluR2, and GluR3 gene expression was examined by in situ hybridization in postischemic rats. Following severe, transient forebrain ischemia GluR2 gene expression was preferentially reduced in CA1 hippocampal neurons at a time point that preceded their degeneration. The switch in expression of kainate/AMPA receptor subunits coincided with the previously reported increase in Ca2+ influx into CA1 cells. Timing of the switch indicates that it may play a causal role in postischemic cell death.

  19. Developmental and growth defects in mice with combined deficiency of CK2 catalytic genes.

    Science.gov (United States)

    Landesman-Bollag, Esther; Belkina, Anna; Hovey, Beth; Connors, Edward; Cox, Charles; Seldin, David C

    2011-10-01

    The CK2 α and α' catalytic gene products have overlapping biochemical activity, but in vivo, their functions are very different. Deletion of both alleles of CK2α leads to mid-gestational embryonic lethality, while deletion of both alleles of CK2α' does not interfere with viability or development of embryos; however, adult CK2α'-/-males are infertile. To further elucidate developmental roles of CK2, and analyze functional overlap between the two catalytic genes, mice with combined knockouts were bred. Mice bearing any two CK2 catalytic alleles were phenotypically normal. However, inheritance of a single CK2α allele, without either CK2α' allele, resulted in partial embryonic lethality. Such mice that survived through embryogenesis were smaller at birth than littermate controls, and weighed less throughout life. However, their cardiac function and lifespan were normal. Fibroblasts derived from CK2α+/-CK2α'-/- embryos grew poorly in culture. These experiments demonstrate that combined loss of one CK2α allele and both CK2α' alleles leads to unique abnormalities of growth and development.

  20. Nuclear factor YY1 activates the mammalian F0F1 ATP synthase alpha-subunit gene.

    Science.gov (United States)

    Breen, G A; Vander Zee, C A; Jordan, E M

    1996-01-01

    Analysis of the promoters of the bovine and human nuclear-encoded mitochondrial F0F1 ATP synthase alpha-subunit genes (ATPA) has identified several positive cis-acting regulatory regions that are important for basal promoter activity in human HeLa cells. We have previously determined that the binding of a protein factor, termed ATPF1, to an E-box sequence (CANNTG) located within one of these cis-acting regions is critical for transcriptional activation of the ATPA gene. In this article, we describe a second positive cis-acting regulatory element of the ATPA gene that is important for expression of the ATPA gene. We show that this cis-acting element also contains a binding site for a protein present in HeLa cells. On the basis of electrophoretic mobility shift patterns, oligonucleotide competition assays, and immunological cross-reactivity, we conclude that this protein factor is Yin-Yang 1 (YY1). Experiments carried out to examine the functional role of YY1 within the context of the ATPA promoter demonstrated that YY1 acts as a positive regulator of the ATPA gene. For example, when the YY1 binding site of the ATPA promoter was placed upstream of a reporter gene it was found to activate transcription in transient transfection assays. In addition, disruption of the YY1 binding site in the ATPA gene resulted in a loss of transcriptional activity. Furthermore, in cotransfection experiments overexpression of YY1 in trans was found to activate transcription of ATPA promoter-CAT constructs. Thus, at least two positive trans-acting regulatory factors, ATPF1 and YY1, are important for expression of the bovine and human F0F1 ATP synthase alpha-subunit genes.

  1. Basic residues in the 74-83 and 191-198 segments of protein kinase CK2 catalytic subunit are implicated in negative but not in positive regulation by the beta-subunit

    DEFF Research Database (Denmark)

    Sarno, S; Vaglio, P; Marin, O;

    1997-01-01

    by the beta-subunit many fold more than that of alpha wild type, while extrastimulation by beta mutant D55L56E57A, observable with alpha wild type, is abolished with these mutants. These data support the conclusion that down regulation by the acidic residues clustered in the N-terminal moiety of beta...... sensitive to inhibition by either beta-subunit or its N-terminal, CK2beta-(1-77)-peptide, while its stimulation by the C-terminal peptide, CK2beta-(155-215), is comparable to that of alpha wild type. These observations suggest an indirect role of Val66 in conferring to the alpha-subunit a conformation less...

  2. Conserved cis-regulatory modules in promoters of genes encoding wheat high-molecular-weight glutenin subunits.

    Science.gov (United States)

    Ravel, Catherine; Fiquet, Samuel; Boudet, Julie; Dardevet, Mireille; Vincent, Jonathan; Merlino, Marielle; Michard, Robin; Martre, Pierre

    2014-01-01

    The concentration and composition of the gliadin and glutenin seed storage proteins (SSPs) in wheat flour are the most important determinants of its end-use value. In cereals, the synthesis of SSPs is predominantly regulated at the transcriptional level by a complex network involving at least five cis-elements in gene promoters. The high-molecular-weight glutenin subunits (HMW-GS) are encoded by two tightly linked genes located on the long arms of group 1 chromosomes. Here, we sequenced and annotated the HMW-GS gene promoters of 22 electrophoretic wheat alleles to identify putative cis-regulatory motifs. We focused on 24 motifs known to be involved in SSP gene regulation. Most of them were identified in at least one HMW-GS gene promoter sequence. A common regulatory framework was observed in all the HMW-GS gene promoters, as they shared conserved cis-regulatory modules (CCRMs) including all the five motifs known to regulate the transcription of SSP genes. This common regulatory framework comprises a composite box made of the GATA motifs and GCN4-like Motifs (GLMs) and was shown to be functional as the GLMs are able to bind a bZIP transcriptional factor SPA (Storage Protein Activator). In addition to this regulatory framework, each HMW-GS gene promoter had additional motifs organized differently. The promoters of most highly expressed x-type HMW-GS genes contain an additional box predicted to bind R2R3-MYB transcriptional factors. However, the differences in annotation between promoter alleles could not be related to their level of expression. In summary, we identified a common modular organization of HMW-GS gene promoters but the lack of correlation between the cis-motifs of each HMW-GS gene promoter and their level of expression suggests that other cis-elements or other mechanisms regulate HMW-GS gene expression.

  3. Nucleotide sequence of a crustacean 18S ribosomal RNA gene and secondary structure of eukaryotic small subunit ribosomal RNAs.

    Science.gov (United States)

    Nelles, L; Fang, B L; Volckaert, G; Vandenberghe, A; De Wachter, R

    1984-12-11

    The primary structure of the gene for 18 S rRNA of the crustacean Artemia salina was determined. The sequence has been aligned with 13 other small ribosomal subunit RNA sequences of eukaryotic, archaebacterial, eubacterial, chloroplastic and plant mitochondrial origin. Secondary structure models for these RNAs were derived on the basis of previously proposed models and additional comparative evidence found in the alignment. Although there is a general similarity in the secondary structure models for eukaryotes and prokaryotes, the evidence seems to indicate a different topology in a central area of the structures.

  4. Cloning, expression and molecular analysis of Iranian Brucella melitensis Omp25 gene for designing a subunit vaccine

    Science.gov (United States)

    Yousefi, Soheil; Tahmoorespur, Mojtaba; Sekhavati, Mohammad Hadi

    2016-01-01

    Brucellosis is a well-known domestic animal infectious disease, which is caused by Brucella bacterium. The outer membrane protein 25 kDa (Omp25) gene plays an important role in simulating of TNF-α, IFN-α, macrophage, and cytokines cells. In the current study molecular cloning and expression analysis of Omp25 gene for designing a subunit vaccine against Brucella was investigated. Amplifying the full length of candidate gene was performed using specific primers. Sub-cloning of this gene conducted using pTZ57R/T vector in TOP10F strain of Escherichia coli(E.coli) as the host. Also, pET32(a)+ vector used for expression in BL21 (DE3) strain of E.coli. Omp25 gene with 642 bp size was amplified and cloned successfully. The expression results were confirmed by sequencing and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analyses which showed 42 kDa protein band correctly. Also, phylogenic analysis showed this gene has a near genetic relation with other Brucella strains. According to our results we can propose this gene as a candidate useful for stimulation of cell-mediated and humoral immunity system in future study. PMID:27920824

  5. hELP3 Subunit of the Elongator Complex Regulates the Transcription of HSP70 Gene in Human Cells

    Institute of Scientific and Technical Information of China (English)

    Qiuju HAN; Xiaozhe HOU; Dongmei SU; Lina PAN; Jizhou DUAN; Liguo CUI; Baiqu HUANG; Jun LU

    2007-01-01

    The human Elongator complex is remarkably similar to its yeast counterpart in several aspects.In a previous study, we analyzed the functions of the human elongation protein 3 (hELP3) subunit of the human Elongator by using an in vivo yeast complementation system. However, direct evidence for hELP3 functions in regulating gene expression in human cells was not obtained. In this study, we used hELP3 antisense oligonucleotide inhibitors to knock down hELP3 gene expression to investigate its function in human 293T cells. The results showed that specific reduction of hELP3 mRNA and protein caused a significant suppression of HSP70-2 gene expression, and this was accompanied by histone H3 hypoacetylation and decreased RNA polymerase Ⅱ density at the HSP70-2 gene. Moreover, the data also showed that hELP3 exerted the transcriptional regulatory function directly through its presence on the HSP70-2 gene. Data presented in this report provide further insight and direct evidence of the functions of hELP3 in HSP70-2 gene transcriptional elongation in human cells.

  6. Structural organization, sequence, and expression of the mouse HEXA gene encoding the alpha subunit of hexosaminidase A.

    Science.gov (United States)

    Wakamatsu, N; Benoit, G; Lamhonwah, A M; Zhang, Z X; Trasler, J M; Triggs-Raine, B L; Gravel, R A

    1994-11-01

    Genomic clones of the mouse HEXA gene encoding the alpha subunit of lysosomal beta-hexosaminidase A have been isolated, analyzed, and sequenced. The HEXA gene spans approximately 26 kb and consists of 14 exons and 13 introns. The 5' flanking region of the gene has three candidate GC boxes and a number of potential promoter and regulatory elements. Promoter analysis using deletion constructs of 5' flanking sequence fused to the bacterial chloramphenicol acetyltransferase (CAT) gene showed that 150 bp of 5' sequence was sufficient for expression in transfected monkey kidney COS cells. Determination of the sequence of the 5' end of the Hex alpha mRNA by an "anchor-ligation PCR" procedure showed that transcription is initiated from a cluster of sites centered -42, -32, and -21 bp from the first in-frame ATG. Northern blot analysis from 11 different tissues showed over five times the steady-state level of Hex alpha mRNA in testis as compared to that found in three different brain regions; the lowest level (about 1/3 of brain) was found in liver. Comparison of the 5' flanking sequence with that of the human HEXA gene revealed 78% identity within the first 100 bp. These data suggest that the mouse HEXA gene is controlled mainly by sequences located within 150 bp of the 5' flanking region, and we speculate that it may have a role, not only in brain and other tissues, but also in reproductive function in the adult male mouse.

  7. Serotonin Transporter (5-HTT) and gamma-Aminobutyric Acid Receptor Subunit beta3 (GABRB3) Gene Polymorphisms are not Associated with Autism in the IMGSA Families

    DEFF Research Database (Denmark)

    Maestrini, E.; Lai, C.; Marlow, A.;

    1999-01-01

    Previous studies have suggested that the serotonin transporter (5-HTT) gene and the gamma-aminobutyric acid receptor subunit beta3 (GABRB3) gene, or other genes in the 15q11-q13 region, are possibly involved in susceptibility to autism. To test this hypothesis we performed an association study...... and the GABRB3 genes are unlikely to play a major role in the aetiology of autism in our family data set....

  8. CLONING, SEQUENCING AND EXPRESSION STUDIES OF THE GENES ENCODING AMICYANIN AND THE BETA-SUBUNIT OF METHYLAMINE DEHYDROGENASE FROM THIOBACILLUS-VERSUTUS

    NARCIS (Netherlands)

    UBBINK, M; VANKLEEF, MAG; KLEINJAN, DJ; HOITINK, CWG; HUITEMA, F; BEINTEMA, JJ; DUINE, JA; CANTERS, GW

    1991-01-01

    The genes encoding amicyanin and the beta-subunit of methylamine dehydrogenase (MADH) from Thiobacillus versutus have been cloned and sequenced. The organization of these genes makes it likely that they are coordinately expressed and it supports earlier findings that the blue copper protein amicyani

  9. Sequence analysis of β-subunit genes of the 20S proteasome in patients with relapsed multiple myeloma treated with bortezomib or dexamethasone

    NARCIS (Netherlands)

    D.I. Lichter (David); H. Danaee (Hadi); M.D. Pickard (Michael); O. Tayber (Olga); M. Sintchak (Michael); H. Shi (Hongliang); P.G. Richardson (Paul Gerard); J. Cavenagh (Jamie); J. Bladé (Joan); T. Facon (Thierry); R. Niesvizky; M. Alsina (Melissa); W. Dalton (William); P. Sonneveld (Pieter); S. Lonial (Sagar); H. van de Velde (Helgi); D. Ricci (Deborah); D.-L. Esseltine (Dixie-Lee); W.L. Trepicchio (William); G. Mulligan (George); K.C. Anderson (Kenneth Carl)

    2012-01-01

    textabstractVariations within proteasome β (PSMB) genes, which encode the β subunits of the 20S proteasome, may affect proteasome function, assembly, and/or binding of proteasome inhibitors. To investigate the potential association between PSMB gene variants and treatment-emergent resistance to bort

  10. Mouse mutants for the nicotinic acetylcholine receptor ß2 subunit display changes in cell adhesion and neurodegeneration response genes.

    Directory of Open Access Journals (Sweden)

    Carol M Rubin

    Full Text Available Mice lacking expression of the ß2 subunit of the neuronal nicotinic acetylcholine receptor (CHRNB2 display abnormal retinal waves and a dispersed projection of retinal ganglion cell (RGC axons to their dorsal lateral geniculate nuclei (dLGNs. Transcriptomes of LGN tissue from two independently generated Chrnb2-/- mutants and from wildtype mice were obtained at postnatal day 4 (P4, during the normal period of segregation of eye-specific afferents to the LGN. Microarray analysis reveals reduced expression of genes located on the cell membrane or in extracellular space, and of genes active in cell adhesion and calcium signaling. In particular, mRNA for cadherin 1 (Cdh1, a known axon growth regulator, is reduced to nearly undetectable levels in the LGN of P4 mutant mice and Lypd2 mRNA is similarly suppressed. Similar analysis of retinal tissue shows increased expression of crumbs 1 (Crb1 and chemokine (C-C motif ligand 21 (Ccl21 mRNAs in Chrnb2-/- mutant animals. Mutations in these genes are associated with retinal neuronal degeneration. The retinas of Chrnb2-/- mutants are normal in appearance, but the increased expression of these genes may also be involved in the abnormal projection patterns of RGC to the LGN. These data may provide the tools to distinguish the interplay between neural activity and molecular expression. Finally, comparison of the transcriptomes of the two different Chrnb2-/- mutant strains reveals the effects of genetic background upon gene expression.

  11. Transcritption regulation of soybean ribulose-1,5-bisphos-phate carboxylase small sub-unit gene by external factors

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Ribulose-1,5-bisphosphate carboxylase small subunit gene (rbcS) is present with multi-gene family in plant genome. In Glycine max, the rbcS polypeptide (EC4.1.1.39) is encoded by a gene family containing 4-8 members. Three full-length rbcS cDNA clones were isolated and characterized from soybean seedlings, and both of their nucleotide and amino acid sequences showed high similarity. Differential accumulation of the rbcS mRNA was observed among roots, hypocotyls, cotyledons, epicotyls and leaves. The rbcS genes were up-regulated by various external factors such as salicylic acid (SA), salt stress and drought stress. The expression level of rbcS genes after being treated by 2.0 mmol/L SA and 0.4% NaCl, respectively, is 2.5-3.0-fold as high as that of control sample. Moreover, soybean rbcS mRNA was accumulated with diurnal variation but easily influenced by light and low temperature.

  12. Localization of eight additional genes in the human major histocompatibility complex, including the gene encoding the casein kinase II {beta} subunit (CSNK2B)

    Energy Technology Data Exchange (ETDEWEB)

    Albertella, M.R.; Jones, H.; Thomson, W. [Oxford Univ. (United Kingdom)] [and others

    1996-09-01

    A wide range of autoimmune and other diseases are known to be associated with the major histocompatibility complex. Many of these diseases are linked to the genes encoding the polymorphic histocompatibility complex. Many of these diseases are linked to the genes encoding the polymorphic histocompatibility antigens in the class I and class II regions, but some appear to be more strongly associated with genes in the central 1100-kb class III region, making it important to characterize this region fully for the presence of novel genes. An {approximately}220-kb segment of DNA in the class III region separating the Hsp70 (HSPA1L) and BAT1 (D6S8IE) genes, which was previously known to contain 14 genes. Genomic DNA fragments spanning the gaps between the known genes were used as probes to isolate cDNAs corresponding to five new genes within this region. Evidence from Northern blot analysis and exon trapping experiments that suggested the presence of at least two more new genes was also obtained. Partial cDNA and complete exonic genomic sequencing of one of the new genes has identified it as the casein kinase II{beta} subunit (CSNK2B). Two of the other novel genes lie within a region syntenic to that implicated in susceptibility to experimental allergic orchitis in the mouse, an autoimmune disease of the testis, and represent additional candidates for the Orch-1 locus associated with this disease. In addition, characterization of the 13-kb intergenic gap separating the RD (D6545) and G11 (D6S60E) genes has revealed the presence of a gene encoding a 1246-amino-acid polypeptide that shows significant sequence similarity to the yeast anti-viral Ski2p gene product. 49 refs., 8 figs.

  13. Targeted deletion of the mouse α2 nicotinic acetylcholine receptor subunit gene (Chrna2) potentiates nicotine-modulated behaviors.

    Science.gov (United States)

    Lotfipour, Shahrdad; Byun, Janet S; Leach, Prescott; Fowler, Christie D; Murphy, Niall P; Kenny, Paul J; Gould, Thomas J; Boulter, Jim

    2013-05-01

    Baseline and nicotine-modulated behaviors were assessed in mice harboring a null mutant allele of the nicotinic acetylcholine receptor (nAChR) subunit gene α2 (Chrna2). Homozygous Chrna2(-/-) mice are viable, show expected sex and Mendelian genotype ratios, and exhibit no gross neuroanatomical abnormalities. A broad range of behavioral tests designed to assess genotype-dependent effects on anxiety (elevated plus maze and light/dark box), motor coordination (narrow bean traverse and gait), and locomotor activity revealed no significant differences between mutant mice and age-matched wild-type littermates. Furthermore, a panel of tests measuring traits, such as body position, spontaneous activity, respiration, tremors, body tone, and startle response, revealed normal responses for Chrna2-null mutant mice. However, Chrna2(-/-) mice do exhibit a mild motor or coordination phenotype (a decreased latency to fall during the accelerating rotarod test) and possess an increased sensitivity to nicotine-induced analgesia in the hotplate assay. Relative to wild-type, Chrna2(-/-) mice show potentiated nicotine self-administration and withdrawal behaviors and exhibit a sex-dependent enhancement of nicotine-facilitated cued, but not trace or contextual, fear conditioning. Overall, our results suggest that loss of the mouse nAChR α2 subunit has very limited effects on baseline behavior but does lead to the potentiation of several nicotine-modulated behaviors.

  14. Preparation of Polyclonal Antibodies of Rubisco Large and Small Subunits and Their Application in the Functional Analysis of the Genes

    Institute of Scientific and Technical Information of China (English)

    Peng-Da MA; Tian-Cheng LU; Xiao-Fu ZHOU; Xiao-Juan ZHU; Xing-Zhi WANG

    2004-01-01

    Spinach Rubisco (ribulose-l,5-bisphosphate carboxylase/oxygenase) large (rbcL) and small (rbcS) subunits were separated by SDS-PAGE, and protein amount and purity were determined by Bradford assay. Polyclonal antibodies against rbcL and rbcS subunit were generated in female BALB/c mice and had no cross-reaction with each other. A total of 81 μg antigens were used and 0.3 ml anti-sera with titer of 1:5000were yielded. The antibodies were also applicable to study rbcL and rbcS in tobacco plant Nicotiana benthamiana. Potato virus X vector pGR107 induced silencing of rbcS gene by Agrobacterium in Nicotiana benthaniana was performed. The expression level ofrbcL and rbcS was lower in rbcS silenced plants than that in control plants as detected by the corresponding antibodies. This implied that the expression of rbcL was regulated by rbcS.

  15. Analysis of IL-12 p40 subunit gene and IFN-γ G5644A polymorphisms in Idiopathic Pulmonary Fibrosis

    Directory of Open Access Journals (Sweden)

    Welsh Kenneth I

    2003-06-01

    Full Text Available Abstract Background Genes encoding cytokine mediators are prime candidates for genetic analysis in conditions with T-helper (Th cell disease driven imbalance. Idiopathic Pulmonary Fibrosis (IPF is a predominantly Th2 mediated disease associated with a paucity of interferon-gamma (IFN-γ. The paucity of IFN-γ may favor the development of progressive fibrosis in IPF. Interleukin-12 (IL-12 plays a key role in inducing IFN-γ production. The aim of the current study was to assess whether the 1188 (A/C 3'UTR single nucleotide polymorphism (SNP in the IL-12 p40 subunit gene which was recently found to be functional and the 5644 (G/A 3' UTR SNP of the IFN-γ gene were associated with susceptibility to IPF. Methods We investigated the allelic distribution in these loci in UK white Caucasoid subjects comprising 73 patients with IPF and 157 healthy controls. The SNPs were determined using the polymerase chain reaction in association with sequence-specific primers incorporating mismatches at the 3'-end. Results Our results showed that these polymorphisms were distributed similarly in the IPF and control groups Conclusion We conclude that these two potentially important candidate gene single nucleotide polymorphisms are not associated with susceptibility to IPF.

  16. Phylogenetic relationships between Vorticella convallaria and other species inferred from small subunit rRNA gene sequences.

    Science.gov (United States)

    Itabashi, Takeshi; Mikami, Kazuyuki; Fang, Jie; Asai, Hiroshi

    2002-08-01

    Vorticellid ciliates generally dwell in freshwater. In nature, the species have up until now been identified by comparison with previous descriptions. It is difficult to identify between species of the genus Vorticella, because the morphological markers of vorticellid ciliates described in reports are limited and variable. Unfortunately, culturing them has only succeeded with certain species such as Vorticella convallaria, but many others have been impossible to culture. To find out whether the sequence of a small subunit rRNA gene was an appropriate marker to identify vorticellid ciliates, the gene was aligned and compared. Finding a new convenient method will contribute to research on vorticellid ciliates. In strains of V. convallaria, classified morphologically, some varieties of the SSrRNA gene sequences were recognized, but there were large variations within the same species. According to the phylogenetic tree, these strains are closely related. However, the difference was not as big as between Vorticella and Carchesium. In addition, Carchesium constructed a distinct clade from the genus Vorticella and Epistylis. These results show the possibility that the SSrRNA gene is one of the important markers to identify species of Vorticella. This study is first to approach and clarify the complicated taxa in the genus Vorticella.

  17. Molecular evolution and nucleotide sequences of the maize plastid genes for the alpha subunit of CF1 (atpA) and the proteolipid subunit of CF0 (atpH).

    Science.gov (United States)

    Rodermel, S R; Bogorad, L

    1987-05-01

    The nucleotide sequences of the maize plastid genes for the alpha subunit of CF1 (atpA) and the proteolipid subunit of CF0 (atpH) are presented. The evolution of these genes among higher plants is characterized by a transition mutation bias of about 2:1 and by rates of synonymous and nonsynonymous substitution which are much lower than similar rates for genes from other sources. This is consistent with the notion that the plastid genome is evolving conservatively in primary sequence. Yet, the mode and tempo of sequence evolution of these and other plastid-encoded coupling factor genes are not the same. In particular, higher rates of nonsynonymous substitution in atpE (the gene for the epsilon subunit of CF1) and higher rates of synonymous substitution in atpH in the dicot vs. monocot lineages of higher plants indicate that these sequences are likely subject to different evolutionary constraints in these two lineages. The 5'- and 3'-transcribed flanking regions of atpA and atpH from maize, wheat and tobacco are conserved in size, but contain few putative regulatory elements which are conserved either in their spatial arrangement or sequence complexity. However, these regions likely contain variable numbers of "species-specific" regulatory elements. The present studies thus suggest that the plastid genome is not a passive participant in an evolutionary process governed by a more rapidly changing, readily adaptive, nuclear compartment, but that novel strategies for the coordinate expression of genes in the plastid genome may arise through rapid evolution of the flanking sequences of these genes.

  18. The C. elegans nuclear receptor gene fax-1 and homeobox gene unc-42 coordinate interneuron identity by regulating the expression of glutamate receptor subunits and other neuron-specific genes.

    Science.gov (United States)

    Wightman, Bruce; Ebert, Bryan; Carmean, Nicole; Weber, Katherine; Clever, Sheila

    2005-11-01

    The fax-1 gene of the nematode C. elegans encodes a conserved nuclear receptor that is the ortholog of the human PNR gene and functions in the specification of neuron identities. Mutations in fax-1 result in locomotion defects. FAX-1 protein accumulates in the nuclei of 18 neurons, among them the AVA, AVB, and AVE interneuron pairs that coordinate body movements. The identities of AVA and AVE interneurons are defective in fax-1 mutants; neither neuron expresses the NMDA receptor subunits nmr-1 and nmr-2. Other ionotropic glutamate receptor subunits are expressed normally in the AVA and AVE neurons. The unc-42 homeobox gene also regulates AVA and AVE identity; however, unc-42 mutants display the complementary phenotype: NMDA receptor subunit expression is normal, but some non-NMDA glutamate receptor subunits are not expressed. These observations support a combinatorial role for fax-1 and unc-42 in specifying AVA and AVE identity. However, in four other neuron types, fax-1 is regulated by unc-42, and both transcriptional regulators function in the regulation of the opt-3 gene in the AVE neurons and the flp-1 and ncs-1 genes in the AVK neurons. Therefore, while fax-1 and unc-42 act in complementary parallel pathways in some cells, they function in overlapping or linear pathways in other cellular contexts, suggesting that combinatorial relationships among transcriptional regulators are complex and cannot be generalized from one neuron type to another.

  19. Mitochondrial bioenergetics and redox state are unaltered in Trypanosoma cruzi isolates with compromised mitochondrial complex I subunit genes.

    Science.gov (United States)

    Carranza, Julio César; Kowaltowski, Alicia J; Mendonça, Marco Aurélio G; de Oliveira, Thays C; Gadelha, Fernanda R; Zingales, Bianca

    2009-06-01

    In trypanosomatids the involvement of mitochondrial complex I in NADH oxidation has long been debated. Here, we took advantage of natural Trypanosoma cruzi mutants which present conspicuous deletions in ND4, ND5 and ND7 genes coding for complex I subunits to further investigate its functionality. Mitochondrial bioenergetics of wild type and complex I mutants showed no significant differences in oxygen consumption or respiratory control ratios in the presence of NADH-linked substrates or FADH(2)-generating succinate. No correlation could be established between mitochondrial membrane potentials and ND deletions. Since release of reactive oxygen species occurs at complex I, we measured mitochondrial H(2)O(2) formation induced by different substrates. Significant differences not associated to ND deletions were observed among the parasite isolates, demonstrating that these mutations are not important for the control of oxidant production. Our data support the notion that complex I has a limited function in T. cruzi.

  20. Molecular phylogeny of silk-producing insects based on 16S ribosomal RNA and cytochrome oxidase subunit I genes

    Indian Academy of Sciences (India)

    B. Mahendran; S. K. Ghosh; S. C. Kundu

    2006-04-01

    We have examined the molecular-phylogenetic relationships between nonmulberry and mulberry silkwormspecies that belong to the families Saturniidae, Bombycidae and Lasiocampidae using 16S ribosomal RNA (16S rRNA) and cytochrome oxidase subunit I (coxI) gene sequences. Aligned nucleotide sequences of 16S rRNA and coxI from 14 silk-producing species were used for construction of phylogenetic trees by maximum likelihood and maximum parsimony methods. The tree topology on the basis of 16S rRNA supports monophyly for members of Saturniidae and Bombycidae. Weighted parsimony analysis weighted towards transversions relative to transitions (ts, tv4) for coxI resulted in more robust bootstrap support over unweighted parsimony and favours the 16S rRNA tree topology. Combined analysis reflected clear biogeographic pattern, and agrees with morphological and cytological data.

  1. Functional characterization of the promoter for the mouse SPTLC2 gene, which encodes subunit 2 of serine palmitoyltransferase

    Science.gov (United States)

    Linn, Stephen C.; Andras, Lindsay M.; Kim, Hee-Sook; Wei, Jia; Nagiec, M. Marek; Dickson, Robert C.; Merrill, Alfred H.

    2006-01-01

    A series of luciferase reporter constructs was prepared from a 1035-bp fragment of mouse genomic DNA flanking the 5′ -coding sequence for the SPTLC2 subunit of serine palmitoyltransferase, the initial enzyme of de novo sphingolipid biosynthesis. The full-length DNA fragment promoted strong reporter gene expression in NIH3T3 cells while deletion and site-directed mutagenesis indicated that the proximal 335 bp contain initiator and downstream promoter elements, two proximal GC boxes that appear to stimulate transcription in a cooperative manner, and several additional elements whose activity cannot be accounted for by known factor binding sites. These findings provide insight into the control mechanisms for transcription of mammalian SPTLC2. PMID:17070807

  2. Phylogenetic position of Dysteria derouxi (Ciliophora:Phyllopharyngea: Dysteriida) inferred from the small subunit ribosomal RNA gene sequence

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    The complete small subunit rRNA (SSrRNA) gene sequence of a marine ciliate, Dysteria derouxi Gong and Song, 2004, was determined to be of 1 708 nucleotides. The phylogenetic position of this species within the class Phyllopharyngea was deduced using distance matrix, maximum parsimony and maximum likelihood methods. Dysteria derouxi, together with other available ciliates of the class Phyllopharyngea, forms a monophyletic clade with strong bootstrap support in the distance matrix, maximum parsimony and likelihood tree construction methods, while the dysterids are, as a monophyletic group, phylogenetically close to the clade of chlamydodontids [values of 100% LS(least-squares), 100% NJ(neighbor-joining)]. In addition, the trees indicate that dysteriids may be a higher or specialized group within the class, which corresponds well to the morphology and infraciliature.

  3. Phylogenetic position of three Condylostoma species(Protozoa,Ciliophora,Heterotricheal inferred from the small subunit rRNA gene sequence

    Institute of Scientific and Technical Information of China (English)

    Wenbo Guo; Shan Gao; Weibo Song; Khaled A.S.A1-Rasheid; Chen Shao; Miao Miao; Saleh A.A1-Farraj; Saleh A.A1-Qurishy; Zigui Chen; Zhenzhen Yi

    2008-01-01

    The systematically poorly known ciliate genus Conadylostoma was erected by Vincent in 1826.About 10 morphotypes have been reported,but any molecular investigations concerning this group SO far are lacking.In this work,the small subunit ribosomal RNA (SS rRNA)gene of three marine Conaylostoma species was sequenced,by which the phylogenetic trees were constructed by distance-matrix,maximum parsimony and Bayesian inference methods.The results show that(1)all the trees have similar topologies with high supports;(2)Condylostoma is mostly related to the genus Condylostentor;and(3)three Condylostoma species as well as Conadylostentor auriculatus cluster together and form a sister group with other heterotrichs.This is moderately consistent with the assessment of phylo-genetic relationships of Conaylostoma-related heterotrichs from the morphological information.The phylogenetic relationship of some other related heterotrichs,Peritromus,Fotlictllina,Stentor and Blepharisma,has been also discussed.

  4. Identification of a new human mtDNA polymorphism (A14290G in the NADH dehydrogenase subunit 6 gene

    Directory of Open Access Journals (Sweden)

    M. Houshmand

    2006-06-01

    Full Text Available Leber's hereditary optic neuropathy (LHON is a maternally inherited form of retinal ganglion cell degeneration leading to optic atrophy in young adults. Several mutations in different genes can cause LHON (heterogeneity. The ND6 gene is one of the mitochondrial genes that encodes subunit 6 of complex I of the respiratory chain. This gene is a hot spot gene. Fourteen Persian LHON patients were analyzed with single-strand conformational polymorphism and DNA sequencing techniques. None of these patients had four primary mutations, G3460A, G11788A, T14484C, and G14459A, related to this disease. We identified twelve nucleotide substitutions, G13702C, T13879C, T14110C, C14167T, G14199T, A14233G, G14272C, A14290G, G14365C, G14368C, T14766C, and T14798C. Eleven of twelve nucleotide substitutions had already been reported as polymorphism. One of the nucleotide substitutions (A14290G has not been reported. The A14290G nucleotide substitution does not change its amino acid (glutamic acid. We looked for base conservation using DNA star software (MEGALIGN program as a criterion for pathogenic or nonpathogenic nucleotide substitution in A14290G. The results of ND6 gene alignment in humans and in other species (mouse, cow, elegans worm, and Neurospora crassa mold revealed that the 14290th base was not conserved. Fifty normal controls were also investigated for this polymorphism in the Iranian population and two had A14290G polymorphism (4%. This study provides evidence that the mtDNA A14290G allele is a new nonpathogenic polymorphism. We suggest follow-up studies regarding this polymorphism in different populations.

  5. The RFC2 gene encoding a subunit of replication factor C of Saccharomyces cerevisiae.

    OpenAIRE

    Noskov, V; Maki, S.; Kawasaki, Y.; Leem, S H; Ono, B; Araki, H; Pavlov, Y; Sugino, A

    1994-01-01

    Replication Factor C (RF-C) of Saccharomyces cerevisiae is a complex that consists of several different polypeptides ranging from 120- to 37 kDa (Yoder and Burgers, 1991; Fien and Stillman, 1992), similar to human RF-C. We have isolated a gene, RFC2, that appears to be a component of the yeast RF-C. The RFC2 gene is located on chromosome X of S. cerevisiae and is essential for cell growth. Disruption of the RFC2 gene led to a dumbbell-shaped terminal morphology, common to mutants having a def...

  6. The Vacuolar ATPase from Entamoeba histolytica: Molecular cloning of the gene encoding for the B subunit and subcellular localization of the protein

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    Luna-Arias Juan

    2008-12-01

    Full Text Available Abstract Background Entamoeba histolytica is a professional phagocytic cell where the vacuolar ATPase plays a key role. This enzyme is a multisubunit complex that regulates pH in many subcellular compartments, even in those that are not measurably acidic. It participates in a wide variety of cellular processes such as endocytosis, intracellular transport and membrane fusion. The presence of a vacuolar type H+-ATPase in E. histolytica trophozoites has been inferred previously from inhibition assays of its activity, the isolation of the Ehvma1 and Ehvma3 genes, and by proteomic analysis of purified phagosomes. Results We report the isolation and characterization of the Ehvma2 gene, which encodes for the subunit B of the vacuolar ATPase. This polypeptide is a 55.3 kDa highly conserved protein with 34 to 80% identity to orthologous proteins from other species. Particularly, in silico studies showed that EhV-ATPase subunit B displays 78% identity and 90% similarity to its Dictyostelium ortholog. A 462 bp DNA fragment of the Ehvma2 gene was expressed in bacteria and recombinant polypeptide was used to raise mouse polyclonal antibodies. EhV-ATPase subunit B antibodies detected a 55 kDa band in whole cell extracts and in an enriched fraction of DNA-containing organelles named EhkOs. The V-ATPase subunit B was located by immunofluorescence and confocal microscopy in many vesicles, in phagosomes, plasma membrane and in EhkOs. We also identified the genes encoding for the majority of the V-ATPase subunits in the E. histolytica genome, and proposed a putative model for this proton pump. Conclusion We have isolated the Ehvma2 gene which encodes for the V-ATPase subunit B from the E. histolytica clone A. This gene has a 154 bp intron and encodes for a highly conserved polypeptide. Specific antibodies localized EhV-ATPase subunit B in many vesicles, phagosomes, plasma membrane and in EhkOs. Most of the orthologous genes encoding for the EhV-ATPase subunits

  7. Detection of the enzymatically-active polyhydroxyalkanoate synthase subunit gene, phaC, in cyanobacteria via colony PCR.

    Science.gov (United States)

    Lane, Courtney E; Benton, Michael G

    2015-12-01

    A colony PCR-based assay was developed to rapidly determine if a cyanobacterium of interest contains the requisite genetic material, the PHA synthase PhaC subunit, to produce polyhydroxyalkanoates (PHAs). The test is both high throughput and robust, owing to an extensive sequence analysis of cyanobacteria PHA synthases. The assay uses a single detection primer set and a single reaction condition across multiple cyanobacteria strains to produce an easily detectable positive result - amplification via PCR as evidenced by a band in electrophoresis. In order to demonstrate the potential of the presence of phaC as an indicator of a cyanobacteria's PHA accumulation capabilities, the ability to produce PHA was assessed for five cyanobacteria with a traditional in vivo PHA granule staining using an oxazine dye. The confirmed in vivo staining results were then compared to the PCR-based assay results and found to be in agreement. The colony PCR assay was capable of successfully detecting the phaC gene in all six of the diverse cyanobacteria tested which possessed the gene, while exhibiting no undesired product formation across the nine total cyanobacteria strains tested. The colony PCR quick prep provides sufficient usable DNA template such that this assay could be readily expanded to assess multiple genes of interest simultaneously.

  8. Inefficiency in GM2 ganglioside elimination by human lysosomal beta-hexosaminidase beta-subunit gene transfer to fibroblastic cell line derived from Sandhoff disease model mice.

    Science.gov (United States)

    Itakura, Tomohiro; Kuroki, Aya; Ishibashi, Yasuhiro; Tsuji, Daisuke; Kawashita, Eri; Higashine, Yukari; Sakuraba, Hitoshi; Yamanaka, Shoji; Itoh, Kohji

    2006-08-01

    Sandhoff disease (SD) is an autosomal recessive GM2 gangliosidosis caused by the defect of lysosomal beta-hexosaminidase (Hex) beta-subunit gene associated with neurosomatic manifestations. Therapeutic effects of Hex subunit gene transduction have been examined on Sandhoff disease model mice (SD mice) produced by the allelic disruption of Hexb gene encoding the murine beta-subunit. We demonstrate here that elimination of GM2 ganglioside (GM2) accumulated in the fibroblastic cell line derived from SD mice (FSD) did not occur when the HEXB gene only was transfected. In contrast, a significant increase in the HexB (betabeta homodimer) activity toward neutral substrates, including GA2 (asialo-GM2) and oligosaccharides carrying the terminal N-acetylglucosamine residues at their non-reducing ends (GlcNAc-oligosaccharides) was observed. Immunoblotting with anti-human HexA (alphabeta heterodimer) serum after native polyacrylamide gel electrophoresis (Native-PAGE) revealed that the human HEXB gene product could hardly form the chimeric HexA through associating with the murine alpha-subunit. However, co-introduction of the HEXA encoding the human alpha-subunit and HEXB genes caused significant corrective effect on the GM2 degradation by producing the human HexA. These results indicate that the recombinant human HexA could interspeciesly associate with the murine GM2 activator protein to degrade GM2 accumulated in the FSD cells. Thus, therapeutic effects of the recombinant human HexA isozyme but not human HEXB gene product could be evaluated by using the SD mice.

  9. Molecular characterization of Fasciola hepatica and phylogenetic analysis based on mitochondrial (nicotiamide adenine dinucleotide dehydrogenase subunit I and cytochrome oxidase subunit I) genes from the North-East of Iran

    Science.gov (United States)

    Reaghi, Saber; Haghighi, Ali; Harandi, Majid Fasihi; Spotin, Adel; Arzamani, Kourosh; Rouhani, Soheila

    2016-01-01

    Aim: Fascioliasis is one of the most zoonotic diseases with global extension. As the epidemiological distribution of Fasciola may lead to various genetic patterns of the parasite, the aim of this study is to identify Fasciola hepatica based on spermatogenesis, and phylogenetic analysis using mitochondrial (nicotiamide adenine dinucleotide dehydrogenase subunit I [ND1] and cytochrome oxidase subunit I) gene marker. Materials and Methods: In this study, 90 F. hepatica collected from 30 cattle at slaughterhouse located in three different geographical locations in the North-East of Iran were evaluated based on spermatogenetic ability and internal transcribed spacer 1 gene restriction fragment length polymorphism pattern. Genetic diversity and phylogenetic relationship using mtDNA gene marker for the isolates from the North-East of Iran, and other countries were then analyzed. Results: Partial sequences of mtDNA showed eight haplotypes in both genes. The phylogenic analysis using neighbor joining as well as maximum likelihood methods showed similar topologies of trees. Pairwise fixation index between different F. hepatica populations calculated from the nucleotide data set of ND1 gene are statistically significant and show the genetic difference. Conclusion: F. hepatica found in this region of Iran has different genetic structures through the other Fasciola populations in the world. PMID:27733809

  10. Design of a molecular method for subspecies specific identification of Klebsiella pneumoniae by using the 16S ribosomal subunit gene

    Directory of Open Access Journals (Sweden)

    Nelson Enrique Arenas

    2009-12-01

    Full Text Available Introduction: Rhinoscleroma is caused by Klebsiella pneumoniae subsp. rhinoscleromatis and the ozena infections caused by K. pneumoniae subsp. ozaenae, both infections affect the upper respiratory tract. In the first clinical phases the symptoms are unspecific, and the disease can be misdiagnosed as a common cold, therefore antimicrobial therapy cannot reach effective results and patients must be following up for several years since the infection became chronic. Objective: To identify Klebsiella subspecies using a specific assay based on amplicons restriction of a gene which encodes 16S subunit ribosomal (rDNA16S. Methodology: Specific restriction patterns were generated; using reported sequences from rDNA16S gene and bioinformatics programs MACAW, PFE, GENEDOC and GENE RUNNER. Amplification and restriction assays were standardized. Results: Predictions in silico allowed us to propose an algorithm for Klebsiella species and subspecies identification. Two reference strains were included and two clinical isolates which were biotyped and identified by the proposed method. rDNA16S gene restriction patterns showed differences regarding the initially identified species for conventional methods. Additionally two patterns of bands were observed for K. pneumoniae subsp. rhinoscleromatis, indicating the polymorphisms presence in the rDNA16S gene. Conclusions: We confirmed the difficulty to identify K. pneumoniae subspecies by conventional methods. Implementation of this technique could allow accurate and rapid differentiation among K. pneumoniae subsp. ozaenae and K. pneumoniae subsp. rhinoscleromatis the aetiological agents of two frequently misdiagnosed infections. Antimicrobial therapy usually could be ineffective, especially in chronic patients. Finally we consider very important to enlarge the study by using more clinical and reference strains.

  11. Design of a molecular method for subspecies specific identification of Klebsiella pneumoniae by using the 16S ribosomal subunit gene.

    Directory of Open Access Journals (Sweden)

    Nelson Enrique Arenas

    2009-12-01

    Full Text Available Introduction: Rhinoscleroma is caused by Klebsiella pneumoniae rhinoscleromatis and the ozena infections caused by K. pneumoniae ozaenae, both infections affect the upper respiratory tract. In the first clinical phases the symptoms are unspecific, and the disease can be misdiagnosed as a common cold, therefore antimicrobial therapy cannot reach effective results and patients must be following up for several years since the infection became chronic. Objective: To identify Klebsiella subspecies using a specific assay based on amplicons restriction of a gene which encodes 16S subunit ribosomal (rDNA16S.Methodology: Specific restriction patterns were generated; using reported sequences from rDNA16S gene and bioinformatics programs MACAW, PFE, GENEDOC and GENE RUNNER. Amplification and restriction assays were standardized. Results: Predictions in silico allowed to propose an algorithm for Klebsiella species and subspecies identification. Two reference strains were included and two clinical isolates which were biotyped and identified by the proposed method. rDNA16S gene restriction patterns showed differences regarding the initially identified species for conventional methods. Additionally two patterns of bands were observed for K. pneumoniae rhinoscleromatis, indicating the polymorphisms presence in the rDNA16S gene. Conclusions: It was confirmed the difficulty to identify K. pneumoniae subspecies by conventional methods. Implementation of this technique could allow an accurate and rapid differentiation among K. pneumoniae ozaenae and K. pneumoniae rhinoscleromatis aetiological agents of two frequently misdiagnosed infections. Antimicrobial therapy usually could be ineffective, especially in chronic patients. Finally it is considered very important to enlarge the study by using more clinical and reference strains.

  12. Cloning of a yeast gene coding for the glutamate synthase small subunit (GUS2) by complementation of Saccharomyces cerevisiae and Escherichia coli glutamate auxotrophs.

    Science.gov (United States)

    González, A; Membrillo-Hernández, J; Olivera, H; Aranda, C; Macino, G; Ballario, P

    1992-02-01

    A Saccharomyces cerevisiae glutamate auxotroph, lacking NADP-glutamate dehydrogenase (NADP-GDH) and glutamate synthase (GOGAT) activities, was complemented with a yeast genomic library. Clones were obtained which still lacked NADP-GDH but showed GOGAT activity. Northern analysis revealed that the DNA fragment present in the complementing plasmids coded for a 1.5kb mRNA. Since the only GOGAT enzyme so far purified from S. cerevisiae is made up of a small and a large subunit, the size of the mRNA suggested that the cloned DNA fragment could code for the GOGAT small subunit. Plasmids were purified and used to transform Escherichia coli glutamate auxotrophs. Transformants were only recovered when the recipient strain was an E. coli GDH-less mutant lacking the small GOGAT subunit. These data show that we have cloned the structural gene coding for the yeast small subunit (GUS2). Evidence is also presented indicating that the GOGAT enzyme which is synthesized in the E. coli transformants is a hybrid comprising the large E. coli subunit and the small S. cerevisiae subunit.

  13. Phenotypical Manifestations of Mutations in the Genes Encoding Subunits of the Cardiac Sodium Channel

    NARCIS (Netherlands)

    Wilde, Arthur A. M.; Brugada, Ramon

    2011-01-01

    Variations in the gene encoding for the major sodium channel (Na(v)1.5) in the heart, SCN5A, has been shown to cause a number of arrhythmia syndromes (with or without structural changes in the myocardium), including the long-QT syndrome (type 3), Brugada syndrome, (progressive) cardiac conduction di

  14. Genes encoding biotin carboxylase subunit of acetyl-CoA carboxylase from Brassica napus and parental species: cloning, expression patterns, and evolution

    Science.gov (United States)

    Comparative genomics is a useful tool to investigate gene and genome evolution. Biotin carboxylase (BC), an important subunit of heteromeric ACCase that is a rate-limiting enzyme in fatty acid biosynthesis in dicots, catalyzes ATP, biotin-carboxyl-carrier protein and CO2 to form carboxybiotin-carbo...

  15. Phylogeny, clinical associations, and diagnostic utility of the pilin subunit gene (sfpA) of sorbitol-fermenting, enterohemorrhagic Escherichia coli O157:H-

    NARCIS (Netherlands)

    Friedrich, Alexander W; Nierhoff, Katja V; Bielaszewska, Martina; Mellmann, Alexander; Karch, Helge

    2004-01-01

    The plasmid-borne sfpA gene encodes the pilin subunit in sorbitol-fermenting (SF) enterohemorrhagic Escherichia coli (EHEC) O157:H-. We investigated the distribution of sfpA among 600 E. coli isolates comprising the complete E. coli standard reference (ECOR) and diarrheagenic E. coli (DEC) strain co

  16. Cloning and expression of catalytic domain of Abl protein tyrosine kinase gene in E. coli

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Protein tyrosine kinases (PTKs) regulate cell proliferation, differentiation and are involved in signal transduction. Uncontrolled signaling from receptor tyrosine kinases to intracellular tyrosine kinases can lead to inflamma tory responses and diseases such as cancer and atherosclerosis. Thus, inhibitors that block the activity of tyrosine kinases or the signaling pathways of PTKs activation could be assumed as the potential candidate for drug development. On this assumption, we cloned and expressed the Abl PTK gene in E. coli, and purified the PTK, which was used to screen the PTK inhibitors from the extracts of Chinese herbs. The catalytic domain sequence of PTK gene was amplified by PCR us ing the cDNA of abl from Abelson murine leukemia virus as template. The amplified fragment was then cloned into the GST-tagged expression vector pGEX2T. The recombinant plasmid was transformed into host cell E. coli DH5α and was induced to express PTK protein. The expression of the protein was detected using SDS-PAGE. The result showed that a specific protein was induced to express after 12 min induction, and reached peak level about 40% of the host total pro tein after 4 h induction. The molecular weight of the fusion protein was about 58 kD. The purified GST-PTK fusion pro tein presented higher activity for tyrosine phosphorylation.

  17. Gene expression profiles and phosphorylation patterns of AMP-activated protein kinase subunits in various mesenchymal cell types

    Institute of Scientific and Technical Information of China (English)

    Wang Yugang; Fan Qiming; Ma Rui; Lin Wentao; Tang Tingting

    2014-01-01

    Background Recent studies on bone have shown an endocrine role of the skeleton,which could be impaired in various human diseases,including osteoporosis,obesity,and diabetes-associated bone diseases.As a sensor and regulator of energy metabolism,AMP-activated protein kinase (AMPK) may also play an important role in the regulation of bone metabolism.The current study aimed to establish the expression profiles and phosphorylation patterns of AMPK subunits in several mesenchymal cell types.Methods Reverse transcription-polymerase chain reaction (PCR) for relative quantification,real-time PCR for absolute quantification,and Western blotting were used to investigate the gene expression profiles and phosphorylation patterns of AMPK subunits in several mesenchymal cell types,including primary human mesenchymal stem cells (hMSCs) and hFOB,Saos-2,C3H/10T1/2,MC3T3-E1,3T3-L1,and C2C12 cells.Results AMPKα1 and AMPKβ1 mRNAs were abundantly expressed in all cell types.AMPKY1 mRNA was abundantly expressed in C3H/10T1/2,MC3T3-E1,3T3-L1,and C2C12 but not detected in human-derived cell types.AMPKY2 mRNA was mildly expressed in all cell types.AMPKα1 protein was highly expressed in all cell types and AMPKα2 protein was highly expressed only in hFOB and Saos-2 cells.AMPKβ1 protein was abundantly expressed in all cell types except for Saos-2,in which AMPKβ2 protein overwhelmed AMPKβ1 expression.AMPKy1 and AMPKY2 proteins were expressed in C3H/10T1/2,MC3T3-E1,3T3-L1,and C2C12 cells and only AMPKY2 protein was expressed in hMSCs,hFOB and Saos2 cells.AMPKα was phosphorylated at Thr172 and Ser485 and AMPKβ1 was phosphorylated at Ser108 and Ser182 in all cell types with a specific pattern in each cell type.Conclusion The combination of AMPK α,β,and Y subunits and phosphorylation of AMPKα (Thr172 and Ser485) and AMPKβ1 (Ser108 and Ser182) showed a specific pattern in each cell type.

  18. Role of the Rubisco small subunit. Final report for period May 1, 1997--April 30,2000

    Energy Technology Data Exchange (ETDEWEB)

    Spreitzer, Robert J.

    2000-10-04

    CO{sub 2} and O{sub 2} are mutually competitive at the active site of ribulose-1,5-biphosphate (RuBP) carboxylase/oxygenase (Rubisco). Rubisco contains two subunits, each present in eight copies. The 15-kD small subunit is coded by a family of nuclear RbcS genes. Until now, the role of the small subunit in Rubisco structure or catalytic efficiency is not known. Because of other work in eliminating the two RbcS genes in the green algo Chlamydomonas reinhardtii, it is now possible to address questions about the structure-function relationships of the eukaryotic small subunit. There are three specific aims in this project: (1) Alanine scanning mutagenesis is being used to dissect the importance of the {beta}A/{beta}B loop, a feature unique to the eukaryotic small subunit. (2) Random mutagenesis is being used to identify additional residues or regions of the small subunit that are important for holoenzyme assembly and function. (3) Attempts are being made to express foreign small subunits in Chlamydomonas to examine the contribution of small subunits to holoenzyme assembly, catalytic efficiency, and CO{sub 2}/O{sub 2} specificity.

  19. Sequence Variation of the Pertussis Toxin S1 Subunit Encoding Gene in the Clinical Isolates of Bordetella pertussis in Iran

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    Hosseinpour

    2015-08-01

    Full Text Available Background Whooping cough (pertussis is an acute respiratory disease caused by Bordetella pertussis (B. pertussis. Pertussis toxin is an important virulence factor of B. pertussis and plays a major role in the immune and inflammatory responses. Likewise, allelic variations in the genes of virulence factors have led to the non-responsiveness of the new strains to both whole-cell and acellular vaccines. Given the importance of pertussis vaccine, we sought to address the lack of fundamental studies on the polymorphisms of the virulence genes of B. pertussis in Iran. Objectives The aim of this study was to identify the polymorphisms of the pertussis toxin S1 subunit (ptxS1 gene in the circulating strains and compare them to the vaccine strain. Patients and Methods In this study, 50 strains of B. pertussis isolated from patients with pertussis were investigated in the pertussis reference laboratory of Pasteur institute of Iran. Cultivation, biochemical tests, and the specific antisera were used to confirm B. pertussis. The sequencing of the polymerase chain reaction products was performed to determine the ptxS1 alleles, and B. pertussis 134 was studied as the vaccine strain. Results The results showed that all the strains had the dominant allele ptxS1A. There were differences between the alleles of the clinical strains and the vaccine strain. Conclusions In recent years, a significant increase in the incidence of pertussis has been reported worldwide. Our findings regarding the allelic shift of the ptxS1 gene are similar to those reported in many European and American countries showing the difference of the dominant allele of ptxS1 between the circulating isolates and the vaccine strains.

  20. Establishment of a continuous culture system for Entamoeba muris and analysis of the small subunit rRNA gene

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    Kobayashi S.

    2009-06-01

    Full Text Available We established a culture system for Entamoeba muris (MG-EM-01 strain isolated from a Mongolian gerbil using a modified Balamuth’s egg yolk infusion medium supplemented with 4% adult bovine serum and Bacteroides fragilis cocultured with Escherichia coli. Further, encystation was observed in the culture medium. The morphological characteristics of E. muris are similar to those of Entamoeba coli (E. coli; moreover, the malic isoenzyme electrophoretic band, which shows species-specific electrophoretic mobility, of E. muris had almost the same mobility as that observed with the malic isoenzyme electrophorectic band of E. coli (UZG-EC-01 strain isolated from a gorilla. We determined the small subunit rRNA (SSU-rRNA gene sequence of the MG-EM-01 strain, and this sequence was observed to show 82.7% homology with that of the UZG-EC-01 strain. Further, the resultant phylogenetic tree for molecular taxonomy based on the SSU-rRNA genes of the 21 strains of the intestinal parasitic amoeba species indicated that the MG-EM-01 strain was most closely related to E. coli.

  1. Genetic diversity of Echinococcus granulosus in southwest China determined by the mitochondrial NADH dehydrogenase subunit 2 gene.

    Science.gov (United States)

    Wang, Jiahai; Wang, Ning; Hu, Dandan; Zhong, Xiuqin; Wang, Shuxian; Gu, Xiaobin; Peng, Xuerong; Yang, Guangyou

    2014-01-01

    We evaluated genetic diversity and structure of Echinococcus granulosus by analyzing the complete mitochondrial NADH dehydrogenase subunit 2 (ND2) gene in 51 isolates of E. granulosus sensu stricto metacestodes collected at three locations in this region. We detected 19 haplotypes, which formed a distinct clade with the standard sheep strain (G1). Hence, all 51 isolates were identified as E. granulosus sensu stricto (G1-G3). Genetic relationships among haplotypes were not associated with geographical divisions, and fixation indices (Fst) among sampling localities were low. Hence, regional populations of E. granulosus in the southwest China are not differentiated, as gene flow among them remains high. This information is important for formulating unified region-wide prevention and control measures. We found large negative Fu's Fs and Tajima's D values and a unimodal mismatch distribution, indicating that the population has undergone a demographic expansion. We observed high genetic diversity among the E. granulosus s. s. isolates, indicating that the parasite population in this important bioregion is genetically robust and likely to survive and spread. The data from this study will prove valuable for future studies focusing on improving diagnosis and prevention methods and developing robust control strategies.

  2. Apparent selection intensity for the cytochrome oxidase subunit I gene varies with mode of reproduction in echinoderms.

    Science.gov (United States)

    Foltz, David W; Hrincevich, Adam W; Rocha-Olivares, Axayácatl

    2004-10-01

    When most amino acid substitutions in protein-coding genes are slightly deleterious rather than selectively neutral, life history differences can potentially modify the effective population size or the selective regime, resulting in altered ratios of non-synonymous to synonymous substitutions among taxa. We studied substitution patterns for the mitochondrial cytochrome oxidase subunit I (COI) gene in a sea star genus (Leptasterias spp.) with an obligate brood-protecting mode of reproduction and small-scale population genetic subdivision, and compared the results to available COI sequences in nine other genera of echinoderms with pelagic larvae: three sea stars, five sea urchins and one brittle star. We predicted that this life history difference would be associated with differences in the ratio of non-synonymous (dN) to synonymous (dS) substitution rates. Leptasterias had a significantly greater dN/dS ratio (both between species and within species), a significantly smaller transition/transversion rate ratio, and a significantly lower average nucleotide diversity within species, than did the non-brooding genera. Other explanations for the results, such as altered mutation rates or selective sweeps, were not supported by the data analysis. These findings highlight the potential influence of reproductive traits and other life history factors on patterns of nucleotide substitution within and between species.

  3. A split and rearranged nuclear gene encoding the iron-sulfur subunit of mitochondrial succinate dehydrogenase in Euglenozoa

    Directory of Open Access Journals (Sweden)

    Gray Michael W

    2009-02-01

    Full Text Available Abstract Background Analyses based on phylogenetic and ultrastructural data have suggested that euglenids (such as Euglena gracilis, trypanosomatids and diplonemids are members of a monophyletic lineage termed Euglenozoa. However, many uncertainties are associated with phylogenetic reconstructions for ancient and rapidly evolving groups; thus, rare genomic characters become increasingly important in reinforcing inferred phylogenetic relationships. Findings We discovered that the iron-sulfur subunit (SdhB of mitochondrial succinate dehydrogenase is encoded by a split and rearranged nuclear gene in Euglena gracilis and trypanosomatids, an example of a rare genomic character. The two subgenic modules are transcribed independently and the resulting mRNAs appear to be independently translated, with the two protein products imported into mitochondria, based on the presence of predicted mitochondrial targeting peptides. Although the inferred protein sequences are in general very divergent from those of other organisms, all of the required iron-sulfur cluster-coordinating residues are present. Moreover, the discontinuity in the euglenozoan SdhB sequence occurs between the two domains of a typical, covalently continuous SdhB, consistent with the inference that the euglenozoan 'half' proteins are functional. Conclusion The discovery of this unique molecular marker provides evidence for the monophyly of Euglenozoa that is independent of evolutionary models. Our results pose questions about the origin and timing of this novel gene arrangement and the structure and function of euglenozoan SdhB.

  4. A Novel Approach to Functional Analysis of the Ribulose Bisphosphate Carboxylase Small Subunit Gene by Agrobacterium-Mediated Gene Silencing

    Institute of Scientific and Technical Information of China (English)

    Xiao-Fu Zhou; Peng-Da Ma; Ren-Hou Wang; Bo Liu; Xing-Zhi Wang

    2006-01-01

    A novel approach to virus-induced post-transcriptional gene silencing for studying the function of the ribulose bisphosphate carboxylase small subunlt (rbcS) gene was established and optimized using potato virus X vector and Nicotiana benthamiana as experimental material. The analysis of silencing phenomena,transcriptional level, protein expression, and pigment measurement showed that the expression of the rbcS endogenous gene was inactivated by the expression of a 500-bp homologous cDNA fragment carried in the virus vector.

  5. Initial analysis of the hemocyanin subunit type 1 (Hc1 gene) from Locusta migratoria manilensis.

    Science.gov (United States)

    Yin, Hong; Guan, Ni; Dong, Lijun; Yue, Qiaoyun; Yin, Xiangchu; Zhang, Daochuan

    2012-03-01

    Hemocyanins are copper-containing (Cu(+)) proteins that transport oxygen in many arthropods hemolymph. We characterized Hc1 gene from the grasshopper species Locusta migratoria manilensis. In particular, we cloned and sequenced the corresponding cDNAs and studied their expression at different developmental stages. The cDNA of Hc1 gene (GenBank accession no.:HQ213937) is 2271 bp in length and the open reading frame is 2016 bp, which encodes a 672 amino acids protein with a calculated molecular mass of 77.9 kD and the isoelectric point of 6.06. Sequence alignment analysis result showed that this gene shares 94.7% identity with Schistocerca americana EHP. In addition, analysis of quantitative RT-PCR indicated that, LmiHc1 was expressed in the embyro (24, 39, 62, 86, 144, and 193 h after hatch), nymphs (1st instar, 2nd instar, 3rd instar, 4th instar and 5th instar) and in adult. These results showed that Hc1 plays an important role in grasshopper, which may be related to an enhanced oxygen supply. Phylogenetic analysis of insecta based on Hc1 are basically consistent with the morphology.

  6. Identification and characterization of human neuronal voltage-gated calcium channel gamma 3 subunit gene

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    By homologous expressed sequence tag (EST) searching,one EST (GenBank: W29095) was obtained,which shows 75% identity in 435 bp overlap with the coding sequence of mouse Cacng2 gene. A 1 545 bp cDNA fragment was obtained from the nested polymerase chain reaction (PCR) and rapid applification of cDNA end (RACE) reaction in the human brain prefrontal cortex cDNA library and the human brain Ready cDNA with the primers designed on W29095. The fragment contained a 948-bp open reading frame (ORF) encoding 315 amino acids,and was named CACNG3. As it was identical to a BAC clone (GenBank: AC004125) from chromosome 16p12-p13.1,the CACNG3 gene was mapped to human chromosome 16p12-p13.1,and the coding region was composed of 4 exons. Reverse transcription PCR (RT-PCR) analysis showed that the CACNG3 gene expressed in human adult brain and fetal brain. Single strand comformation polymorphism (SSCP) analysis was performed in 3 pedigrees with autosomal recessive retinitis pigmentosa,8 pedigrees with autosomal recessive retinitis pigmentosa accompanied by deafness and 2 pedigrees with epilepsy,but no mutation was detected.

  7. New insight into the role of the β3 subunit of the GABAA-R in development, behavior, body weight regulation, and anesthesia revealed by conditional gene knockout

    Directory of Open Access Journals (Sweden)

    Hileman Stanley M

    2007-10-01

    Full Text Available Abstract Background The β3 subunit of the γ-aminobutyric acid type A receptor (GABAA-R has been reported to be important for palate formation, anesthetic action, and normal nervous system function. This subunit has also been implicated in the pathogenesis of Angelman syndrome and autism spectrum disorder. To further investigate involvement of this subunit, we previously produced mice with a global knockout of β3. However, developmental abnormalities, compensation, reduced viability, and numerous behavioral abnormalities limited the usefulness of that murine model. To overcome many of these limitations, a mouse line with a conditionally inactivated β3 gene was engineered. Results Gene targeting and embryonic stem cell technologies were used to create mice in which exon 3 of the β3 subunit was flanked by loxP sites (i.e., floxed. Crossing the floxed β3 mice to a cre general deleter mouse line reproduced the phenotype of the previously described global knockout. Pan-neuronal knockout of β3 was achieved by crossing floxed β3 mice to Synapsin I-cre transgenic mice. Palate development was normal in pan-neuronal β3 knockouts but ~61% died as neonates. Survivors were overtly normal, fertile, and were less sensitive to etomidate. Forebrain selective knockout of β3 was achieved using α CamKII-cre transgenic mice. Palate development was normal in forebrain selective β3 knockout mice. These knockouts survived the neonatal period, but ~30% died between 15–25 days of age. Survivors had reduced reproductive fitness, reduced sensitivity to etomidate, were hyperactive, and some became obese. Conclusion Conditional inactivation of the β3 gene revealed novel insight into the function of this GABAA-R subunit. The floxed β3 knockout mice described here will be very useful for conditional knockout studies to further investigate the role of the β3 subunit in development, ethanol and anesthetic action, normal physiology, and pathophysiologic processes.

  8. Nucleotide sequences of two fimbrial major subunit genes, pmpA and ucaA, from canine-uropathogenic Proteus mirabilis strains.

    Science.gov (United States)

    Bijlsma, I G; van Dijk, L; Kusters, J G; Gaastra, W

    1995-06-01

    Proteus mirabilis strains were isolated from dogs with urinary tract infection (UTI) and fimbriae were prepared from two strains. The N-terminal amino acid sequences of the major fimbrial subunits were determined and both sequences appeared identical to the N-terminal amino acid sequence of a urinary cell adhesin (UCA) (Wray, S. K., Hull, S. I., Cook, R. G., Barrish, J. & Hull, R. A., 1986, Infect Immun 54, 43-49). The genes of two different major fimbrial subunits were cloned using oligonucleotide probes that were designed on the basis of the N-terminal UCA sequence. Nucleotide sequencing revealed the complete ucaA gene of 540 bp (from strain IVB247) encoding a polypeptide of 180 amino acids, including a 22 amino acid signal sequence peptide, and the pmpA (P. mirabilis P-like pili) gene of 549 bp (from strain IVB219) encoding a polypeptide of 183 amino acids, including a 23 amino acid signal sequence. Hybridization experiments gave clear indications of the presence of both kinds of fimbriae in many UTI-related canine P. mirabilis isolates. However, the presence of these fimbriae could not be demonstrated in P. vulgaris or other Proteus-related species. Database analysis of amino acid sequences of major subunit proteins revealed that the UcaA protein shares about 56% amino acid identity with the F17A and F111A major fimbrial subunits from bovine enterotoxigenic Escherichia coli. In turn, the PmpA protein more closely resembled the pyelonephritis-associated pili (Pap)-like major subunit protein from UTI-related E. coli. The evolutionary relationship of UcaA, PmpA and various other fimbrial subunit proteins is presented in a phylogenetic tree.

  9. CK2(beta)tes gene encodes a testis-specific isoform of the regulatory subunit of casein kinase 2 in Drosophila melanogaster

    DEFF Research Database (Denmark)

    Kalmykova, Alla I; Shevelyov, Yuri Y; Polesskaya, Oksana O

    2002-01-01

    An earlier described CK2(beta)tes gene of Drosophila melanogaster is shown to encode a male germline specific isoform of regulatory beta subunit of casein kinase 2. Western-analysis using anti-CK2(beta)tes Ig revealed CK2(beta)tes protein in Drosophila testes extract. Expression of a CK2(beta...... and coimmunoprecipitation analysis of protein extract from Drosophila testes, we demonstrated an association between CK2(beta)tes and CK2alpha. Northern-analysis has shown that another regulatory (beta') subunit found recently in D. melanogaster genome is also testis-specific. Thus, we describe the first example of two...

  10. Cloning and characterization of genes encoding alpha and beta subunits of glutamate-gated chloride channel protein in Cylicocyclus nassatus.

    Science.gov (United States)

    Tandon, Ritesh; LePage, Keith T; Kaplan, Ray M

    2006-11-01

    The invertebrate glutamate-gated chloride channels (GluCls) are receptor molecules and targets for the avermectin-milbemycin (AM) group of anthelmintics. Mutations in GluCls are associated with ivermectin resistance in the soil dwelling nematode Caenorhabditis elegans and the parasitic nematode Cooperia oncophora. In this study, full-length cDNAs encoding alpha and beta subunits of GluCl were cloned and sequenced in Cylicocyclus nassatus, a common and important cyathostomin nematode parasite of horses. Both genes possess the sequence characteristics typical of GluCls, and phylogenetic analysis confirms that these genes are evolutionarily closely related to GluCls of other nematodes and flies. Complete coding sequences of C. nassatus GluCl-alpha and GluCl-beta were subcloned into pTL1 mammalian expression vector, and proteins were expressed in COS-7 cells. Ivermectin-binding characteristics were determined by incubating COS-7 cell membranes expressing C. nassatus GluCl-alpha and GluCl-beta proteins with [(3)H]ivermectin. In competitive binding experiments, fitting the data to a one site competition model, C. nassatus GluCl-alpha was found to bind [(3)H]ivermectin with a high amount of displaceable binding (IC(50)=208 pM). Compared to the mock-transfected COS-7 cells, the means of [(3)H]ivermectin binding were significantly different for C. nassatus GluCl-alpha and the Haemonchus contortus GluCl (HcGluCla) (p=0.018 and 0.023, respectively) but not for C. nassatus GluCl-beta (p=0.370). This is the first report of orthologs of GluCl genes and in vitro expression of an ivermectin-binding protein in a cyathostomin species. These data suggest the likelihood of a similar mechanism of action of AM drugs in these parasites, and suggest that mechanisms of resistance may also be similar.

  11. Cloning and expression analysis of GhDET3, a vacuolar H+-ATPase subunit C gene, from cotton

    Institute of Scientific and Technical Information of China (English)

    Zhongyi Xiao; Kunling Tan; Mingyu Hu; Peng Liao; Kuijun Chen; Ming Luo

    2008-01-01

    Vacuolar H+-ATPase was regarded as a key enzyme promoting the fiber cell elongation in cotton (Gossypium hirsuturm L.) through regulating turgot-driven pressure involved in polarity expansion of single cell fiber. The DET3, a V-ATPase subunit C, plays an impor-tant role in assembling subunits and regulating the enzyme activity, and is involved in Brassinosteroid-induced cell elongation. To ana-lyze the function of GhDET3 on the elongation of cotton fibers, seven candidates of ESTs were screened and contigged for a 5'-upstream sequence, and the 3'-RACE technique was used to clone the 3'-downstream sequence for the full length of GhDET3 gene. The full length of the target clone was 1,340 bp, including a 10 bp 5'-UTR, an ORF of 1,134 bp, and a 196 bp 3'-UTR. This cDNA sequence encoded a polypepide of 377 amino acid residues with a predicted molecular mass of 43 kDa and a basic isoelectric point of 5.58. Furthermore, a length of 3,410 bp sequence from genomic DNA of GhDET3 was also cloned by PCR. The deduced amino acid sequence had a high ho-mology with DET3 from Arabidopsis, rice, and maize. Quantitative real-time PCR (qRT-PCR) analysis showed that the GhDET3 expres-sion pattern was ubiquitous in all the tissues and organs detected. The result also revealed that the accumulation of GhDET3 mRNA reached the highest profile at the fiber elongation stage in 12 DPA (days post anthesis) fibers, compared with the lowest level at the fiber initiation stage in 0 DPA ovules (with fibers). The transcript accumulation in fibers and ovules shared the similar variation tendency. In addition, in vitro ovule culture experiment demonstrated that exogenous 24-EBL treatment to 4 DPA ovules (with fibers) was capable of increasing the expression level of GhDET3, and the mRNA accumulation of GhDET3 increased in transgenic FBP7::GhDET2 cotton fibers in vivo. These results indicate that GhDET3 gene plays a crucial role in cotton fiber elongation.

  12. The human mitochondrial NADH: Ubiquinone oxidoreductase 51-kDa subunit oxidoreductase 51-kDa subunit maps adjacent to the glutathione S-transferase P1-1 gene on chromosome 11q13

    Energy Technology Data Exchange (ETDEWEB)

    Spencer, S.R.; Taylor, J.B.; Cowell, I.G.; Xia, C.L.; Pemble, S.E.; Ketterer, B. (Univ. College and Middlesex School of Medicine, London (United Kingdom))

    1992-12-01

    The soluble glutathione transferases (GSTs) are a family of dimeric isoenymes catalyzing the conjugation of glutathione to hydrophobic electropiles. Their subunits can be grouped into four families, alpha, mu, pi, and theta, on the basis of their primary structures. In man, the pi class is represented by a single gene, GSTP1-1 (GST[pi]) localized to human chromosome 11, band q13. The oncogenes INT2, HSTF1, and PRAD1 are also localized at 11q13, and together with the GSTP1 locus and other gene loci mapped to 11q13, i.e., BCL1 and EMS1, they form a unit of DNA approximately 2000-2500 kb, known as the 11q13 amplicon, which is often amplified in a range of solid tumors. Any gene locus at 11q13 is of interest because it may influence tumorigenesis. 14 refs., 1 fig.

  13. Suggestive association between the C825T polymorphism of the G-protein beta3 subunit gene (GNB3) and clinical improvement with antipsychotics in schizophrenia.

    Science.gov (United States)

    Müller, Daniel J; De Luca, Vincenzo; Sicard, Tricia; King, Nicole; Hwang, Rudi; Volavka, Jan; Czobor, Pal; Sheitman, Brian B; Lindenmayer, Jean-Pierre; Citrome, Leslie; McEvoy, Joseph P; Lieberman, Jeffrey A; Meltzer, Herbert Y; Kennedy, James L

    2005-10-01

    G-proteins are composed of alpha, beta and gamma subunits. Once activated, these subunits play a major role in the conversion of external receptor activation into intracellular signals. The functional C825T polymorphism of the beta3 subunit gene (GNB3) has recently been shown to modulate antidepressant response, with the T-allele conferring an increased signaling and being associated with favorable antidepressant response. We hypothesized that this polymorphism may be associated with response to antipsychotics in a population of 145 chronic schizophrenic patients deriving from two study-samples and being mainly treated with clozapine for up to 6 months. Overall, the C/C genotype was significantly associated with relative clinical improvement as measured by Brief Psychiatric Rating Scale (BPRS) change scores after 6 and 12 weeks (ppoint to the role of intracellular mechanisms in antipsychotic response.

  14. Genetic factors affecting gene transcription and catalytic activity of UDP-glucuronosyltransferases in human liver.

    Science.gov (United States)

    Liu, Wanqing; Ramírez, Jacqueline; Gamazon, Eric R; Mirkov, Snezana; Chen, Peixian; Wu, Kehua; Sun, Chang; Cox, Nancy J; Cook, Edwin; Das, Soma; Ratain, Mark J

    2014-10-15

    The aim of this study was to discover cis- and trans-acting factors significantly affecting mRNA expression and catalytic activity of human hepatic UDP-glucuronosyltransferases (UGTs). Transcription levels of five major hepatic UGT1A (UGT1A1, UGT1A3, UGT1A4, UGT1A6 and UGT1A9) and five UGT2B (UGT2B4, UGT2B7, UGT2B10, UGT2B15 and UGT2B17) genes were quantified in human liver tissue samples (n = 125) using real-time PCR. Glucuronidation activities of 14 substrates were measured in 47 livers. We genotyped 167 tagSNPs (single-nucleotide polymorphisms) in UGT1A (n = 43) and UGT2B (n = 124), as well as the known functional UGT1A1*28 and UGT2B17 CNV (copy number variation) polymorphisms. Transcription levels of 15 transcription factors (TFs) known to regulate these UGTs were quantified. We found that UGT expression and activity were highly variable among the livers (median and range of coefficient of variations: 135%, 74-217% and 52%, 39-105%, respectively). CAR, PXR and ESR1 were found to be the most important trans-regulators of UGT transcription (median and range of correlation coefficients: 46%, 6-58%; 47%, 9-58%; and 52%, 24-75%, respectively). Hepatic UGT activities were mainly determined by UGT gene transcription levels. Twenty-one polymorphisms were significantly (FDR-adjusted P transcription and testosterone glucuronidation rate, in addition to that attributable to the UGT2B17 CNV. Our study discovered novel pharmacogenetic markers and provided detailed insight into the genetic network regulating hepatic UGTs.

  15. IDENTIFICATION OF 3 HUMAN PSEUDOGENES FOR SUBUNIT-VIB OF CYTOCHROME-C-OXIDASE - A MOLECULAR RECORD OF GENE EVOLUTION

    NARCIS (Netherlands)

    TAANMAN, JW; SCHRAGE, C; REUVEKAMP, P; BIJL, J; HARTOG, M; DEVRIES, H; AGSTERIBBE, E

    1991-01-01

    Three pseudogenes for the nuclear-encoded subunit VIb of cytochrome c oxidase (COX) were isolated by screening a human genomic library with cloned human cDNA coding for COX subunit VIb. The nucleotide sequences of the pseudogenes, designated PSI-COX6b-1, PSI-COX6b-2 and PSI-COX6b-3, were determined.

  16. Molecular characterization of Echinococcus granulosus from Peru by sequencing of the mitochondrial cytochrome C oxidase subunit 1 gene.

    Science.gov (United States)

    Sánchez, Elizabeth; Cáceres, Omar; Náquira, César; Garcia, David; Patiño, Gladys; Silvia, Herrera; Volotão, Aline C; Fernandes, Octavio

    2010-09-01

    Echinococcus granulosus, the etiologic agent of cystic echinococcosis (CE) in humans and other animal species, is distributed worldwide. Ten intra-specific variants, or genotypes (G1-G10), have been defined based on genetic diversity. To determine the genotypes present in endemic areas of Peru, samples were collected from cattle (44), sheep (41) and humans (14) from Junín, Puno Huancavelica, Cusco, Arequipa and Ayacucho. DNA was extracted from protoscolex and/or germinal layers derived from 99 E. granulosus isolates and used as templates to amplify the mitochondrial cytochrome C oxidase subunit 1 gene. The resulting polymerase chain reaction products were sequenced and further examined by sequence analysis. All isolates, independent of the host, exhibited the G1 genotype. Phylogenetic analysis showed that three isolates from Ayacucho shared the same cluster with microvariant G1(4). The G1 genotype is considered the most widespread and infectious form of E. granulosus worldwide and our results confirm that the same patterns apply to this country. Therefore, these findings should be taken into consideration in developing prevention strategies and control programs for CE in Peru.

  17. Mutations in Two Genes Encoding Different Subunits of a Receptor Signaling Complex Result in an Identical Disease Phenotype

    Science.gov (United States)

    Paloneva, Juha; Manninen, Tuula; Christman, Grant; Hovanes, Karine; Mandelin, Jami; Adolfsson, Rolf; Bianchin, Marino; Bird, Thomas; Miranda, Roxana; Salmaggi, Andrea; Tranebjærg, Lisbeth; Konttinen, Yrjö; Peltonen, Leena

    2002-01-01

    Polycystic lipomembranous osteodysplasia with sclerosing leukoencephalopathy (PLOSL), also known as “Nasu-Hakola disease,” is a globally distributed recessively inherited disease leading to death during the 5th decade of life and is characterized by early-onset progressive dementia and bone cysts. Elsewhere, we have identified PLOSL mutations in TYROBP (DAP12), which codes for a membrane receptor component in natural-killer and myeloid cells, and also have identified genetic heterogeneity in PLOSL, with some patients carrying no mutations in TYROBP. Here we complete the molecular pathology of PLOSL by identifying TREM2 as the second PLOSL gene. TREM2 forms a receptor signaling complex with TYROBP and triggers activation of the immune responses in macrophages and dendritic cells. Patients with PLOSL have no defects in cell-mediated immunity, suggesting a remarkable capacity of the human immune system to compensate for the inactive TYROBP-mediated activation pathway. Our data imply that the TYROBP-mediated signaling pathway plays a significant role in human brain and bone tissue and provide an interesting example of how mutations in two different subunits of a multisubunit receptor complex result in an identical human disease phenotype. PMID:12080485

  18. Strong inhibition of fimbrial 3 subunit gene transcription by a novel downstream repressive element in Bordetella pertussis.

    Science.gov (United States)

    Chen, Qing; Boulanger, Alice; Hinton, Deborah M; Stibitz, Scott

    2014-08-01

    The Bvg-regulated promoters for the fimbrial subunit genes fim2 and fim3 of Bordetella pertussis behave differently from each other both in vivo and in vitro. In vivo Pfim2 is significantly stronger than Pfim3 , even though predictions based on the DNA sequences of BvgA-binding motifs and core promoter elements would indicate the opposite. In vitro Pfim3 demonstrated robust BvgA∼P-dependent transcriptional activation, while none was seen with Pfim2 . This apparent contradiction was investigated further. By swapping sequence elements we created a number of hybrid promoters and assayed their strength in vivo. We found that, while Pfim3 promoter elements upstream of the +1 transcriptional start site do indeed direct Bvg-activated transcription more efficiently than those of Pfim2 , the overall promoter strength of Pfim3  in vivo is reduced due to sequences downstream of +1 that inhibit transcription more than 250-fold. This element, the DRE (downstream repressive element), was mapped to the 15 bp immediately downstream of the Pfim3 +1. Placing the DRE in different promoter contexts indicated that its activity was not specific to fim promoters, or even to Bvg-regulated promoters. However it does appear to be specific to Bordetella species in that it did not function in Escherichia coli.

  19. Molecular characterization of Echinococcus granulosusfrom Peru by sequencing of the mitochondrial cytochrome C oxidase subunit 1 gene

    Directory of Open Access Journals (Sweden)

    Elizabeth Sánchez

    2010-09-01

    Full Text Available Echinococcus granulosus, the etiologic agent of cystic echinococcosis (CE in humans and other animal species, is distributed worldwide. Ten intra-specific variants, or genotypes (G1-G10, have been defined based on genetic diversity. To determine the genotypes present in endemic areas of Peru, samples were collected from cattle (44, sheep (41 and humans (14 from Junín, Puno Huancavelica, Cusco, Arequipa and Ayacucho. DNA was extracted from protoscolex and/or germinal layers derived from 99 E. granulosus isolates and used as templates to amplify the mitochondrial cytochrome C oxidase subunit 1 gene. The resulting polymerase chain reaction products were sequenced and further examined by sequence analysis. All isolates, independent of the host, exhibited the G1 genotype. Phylogenetic analysis showed that three isolates from Ayacucho shared the same cluster with microvariant G1(4. The G1 genotype is considered the most widespread and infectious form of E. granulosusworldwide and our results confirm that the same patterns apply to this country. Therefore, these findings should be taken into consideration in developing prevention strategies and control programs for CE in Peru.

  20. Prevalent ciliate symbiosis on copepods: high genetic diversity and wide distribution detected using small subunit ribosomal RNA gene.

    Science.gov (United States)

    Guo, Zhiling; Liu, Sheng; Hu, Simin; Li, Tao; Huang, Yousong; Liu, Guangxing; Zhang, Huan; Lin, Senjie

    2012-01-01

    Toward understanding the genetic diversity and distribution of copepod-associated symbiotic ciliates and the evolutionary relationships with their hosts in the marine environment, we developed a small subunit ribosomal RNA gene (18S rDNA)-based molecular method and investigated the genetic diversity and genotype distribution of the symbiotic ciliates on copepods. Of the 10 copepod species representing six families collected from six locations of Pacific and Atlantic Oceans, 9 were found to harbor ciliate symbionts. Phylogenetic analysis of the 391 ciliate 18S rDNA sequences obtained revealed seven groups (ribogroups), six (containing 99% of all the sequences) belonging to subclass Apostomatida, the other clustered with peritrich ciliate Vorticella gracilis. Among the Apostomatida groups, Group III were essentially identical to Vampyrophrya pelagica, and the other five groups represented the undocumented ciliates that were close to Vampyrophrya/Gymnodinioides/Hyalophysa. Group VI ciliates were found in all copepod species but one (Calanus sinicus), and were most abundant among all ciliate sequences obtained, indicating that they are the dominant symbiotic ciliates universally associated with copepods. In contrast, some ciliate sequences were found only in some of the copepods examined, suggesting the host selectivity and geographic differentiation of ciliates, which requires further verification by more extensive sampling. Our results reveal the wide occurrence and high genetic diversity of symbiotic ciliates on marine copepods and highlight the need to systematically investigate the host- and geography-based genetic differentiation and ecological roles of these ciliates globally.

  1. Prevalent ciliate symbiosis on copepods: high genetic diversity and wide distribution detected using small subunit ribosomal RNA gene.

    Directory of Open Access Journals (Sweden)

    Zhiling Guo

    Full Text Available Toward understanding the genetic diversity and distribution of copepod-associated symbiotic ciliates and the evolutionary relationships with their hosts in the marine environment, we developed a small subunit ribosomal RNA gene (18S rDNA-based molecular method and investigated the genetic diversity and genotype distribution of the symbiotic ciliates on copepods. Of the 10 copepod species representing six families collected from six locations of Pacific and Atlantic Oceans, 9 were found to harbor ciliate symbionts. Phylogenetic analysis of the 391 ciliate 18S rDNA sequences obtained revealed seven groups (ribogroups, six (containing 99% of all the sequences belonging to subclass Apostomatida, the other clustered with peritrich ciliate Vorticella gracilis. Among the Apostomatida groups, Group III were essentially identical to Vampyrophrya pelagica, and the other five groups represented the undocumented ciliates that were close to Vampyrophrya/Gymnodinioides/Hyalophysa. Group VI ciliates were found in all copepod species but one (Calanus sinicus, and were most abundant among all ciliate sequences obtained, indicating that they are the dominant symbiotic ciliates universally associated with copepods. In contrast, some ciliate sequences were found only in some of the copepods examined, suggesting the host selectivity and geographic differentiation of ciliates, which requires further verification by more extensive sampling. Our results reveal the wide occurrence and high genetic diversity of symbiotic ciliates on marine copepods and highlight the need to systematically investigate the host- and geography-based genetic differentiation and ecological roles of these ciliates globally.

  2. Overexpression of nuclear protein kinase CK2 α catalytic subunit (CK2α as a poor prognosticator in human colorectal cancer.

    Directory of Open Access Journals (Sweden)

    Kai-Yuan Lin

    Full Text Available BACKGROUND: Colorectal cancer (CRC is one of the most common malignancies but the current therapeutic approaches for advanced CRC are less efficient. Thus, novel therapeutic approaches are badly needed. The purpose of this study is to investigate the involvement of nuclear protein kinase CK2 α subunit (CK2α in tumor progression, and in the prognosis of human CRC. METHODOLOGY/PRINCIPAL FINDINGS: Expression levels of nuclear CK2α were analyzed in 245 colorectal tissues from patients with CRC by immunohistochemistry, quantitative real-time PCR and Western blot. We correlated the expression levels with clinicopathologic parameters and prognosis in human CRC patients. Overexpression of nuclear CK2α was significantly correlated with depth of invasion, nodal status, American Joint Committee on Cancer (AJCC staging, degree of differentiation, and perineural invasion. Patients with high expression levels of nuclear CK2α had a significantly poorer overall survival rate compared with patients with low expression levels of nuclear CK2α. In multi-variate Cox regression analysis, overexpression of nuclear CK2α was proven to be an independent prognostic marker for CRC. In addition, DLD-1 human colon cancer cells were employed as a cellular model to study the role of CK2α on cell growth, and the expression of CK2α in DLD-1 cells was inhibited by using siRNA technology. The data indicated that CK2α-specific siRNA treatment resulted in growth inhibition. CONCLUSIONS/SIGNIFICANCE: Taken together, overexpression of nuclear CK2α can be a useful marker for predicting the outcome of patients with CRC.

  3. Comparison of sequences of hypervariable region (HVR subunit S-1 gene of field isolate I-37 infectious bronchitis virus with Connecticut serotype

    Directory of Open Access Journals (Sweden)

    N.L.P Indi Dharmayanti

    2003-06-01

    Full Text Available Infectious Bronchitis is a contagious and acute respiratory disease in chickens caused by infectious bronchitis virus (IBV.Antigenic differences in IBV are associated with changes in the sequence of the spike glycoprotein (S. The subunit S1 which demonstrates more sequence variability than S-2 have been identified as hypervariable region (HVR-1 and 2. There were several IB virus field isolates included I-37 have been identified in Indonesia by serum neutralization method. However, gene sequence variation in HVR subunit S-1 had not yet been identified. Isolate I-37 was close to the serotype Connecticut 46 (Conn 46. The aim of this study is to identify sequence variation of HVR subunit S-1 gene of isolate I-37 produced by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR and sequencing. Several procedures were carried out in the study including virus titration, propagation and was concentrated from the allantoic fluid infected with IBV. Then, RNA was extracted for RTPCR. urther the product was sequnced and its homology with IBV references from GenBank was compared by GenMac version 8.0. Result showed that isolate I-37 produced 515 bp of amplification product. Isolate I-37 and Conn 46 are same serotype, yet their HVR subunit S-1 nucleotides and amino acids (protein differ by 6.9% and 15.6% respectively. It might be concluded that isolate I-37 was variant of Conn 46.

  4. DNMT3B isoforms without catalytic activity stimulate gene body methylation as accessory proteins in somatic cells.

    Science.gov (United States)

    Duymich, Christopher E; Charlet, Jessica; Yang, Xiaojing; Jones, Peter A; Liang, Gangning

    2016-04-28

    Promoter DNA methylation is a key epigenetic mechanism for stable gene silencing, but is correlated with expression when located in gene bodies. Maintenance and de novo DNA methylation by catalytically active DNA methyltransferases (DNMT1 and DNMT3A/B) require accessory proteins such as UHRF1 and DNMT3L. DNMT3B isoforms are widely expressed, although some do not have active catalytic domains and their expression can be altered during cell development and tumourigenesis, questioning their biological roles. Here, we show that DNMT3B isoforms stimulate gene body methylation and re-methylation after methylation-inhibitor treatment. This occurs independently of the isoforms' catalytic activity, demonstrating a similar functional role to the accessory protein DNMT3L, which is only expressed in undifferentiated cells and recruits DNMT3A to initiate DNA methylation. This unexpected role for DNMT3B suggests that it might substitute for the absent accessory protein DNMT3L to recruit DNMT3A in somatic cells.

  5. The subunit gene Ldα1 of nicotinic acetylcholine receptors plays important roles in the toxicity of imidacloprid and thiamethoxam against Leptinotarsa decemlineata.

    Science.gov (United States)

    Qu, Yang; Chen, Jinhua; Li, Chenge; Wang, Qiang; Guo, Wenchao; Han, Zhaojun; Jiang, Weihua

    2016-02-01

    Nicotinic acetylcholine receptors (nAChRs) are pentameric ACh-gated ion channels. It is believed that nAChRs composed of different subunits may vary in their function and toxicological characteristics. Neonicotinoids are activators of nAChRs and important insecticides that are extensively used for crop protection and resistance has been developed by some pests. They are also major insecticides for the control of Leptinotarsa decemlineata, which is a destructive defoliator pest that invaded the Xinjiang region of China in the 1990s. However, little is known about the constitution or subunits of the target in this pest. In this study, the full-length cDNAs encoding four new nAChR subunits (named Ldα3, Ldα6, Ldα10, and Ldβ1) were cloned from L. decemlineata. These genes encode 822-, 753-, 672-, and 759-amino acid proteins, respectively, which share typical features of insect nAChRs subunits and closely resemble the corresponding subunits of the nAChRs from Tribolium castaneum. Temporal and spatial expression analyses showed that these genes, as well as the previously identified Ldα1, Ldα2, and Ldα8 genes, are widely expressed in all developmental stages, including eggs, larvae of various instars, pupae, and adults. All genes monitored were expressed at higher levels in the head than in the thorax and abdomen, except for Ldα10. Dietary ingestion of double-stranded RNA bacterially expressed for Ldα1 (dsLdα1) significantly reduced the mRNA level of Ldα1 in treated larvae and adults by 48.0% and 78.6%, respectively. Among the non-target genes, Ldα3, Ldα9, and Ldβ1 were significantly up-regulated in larvae. A toxicity bioassay showed that dsLdα1 treatment greatly decreased the sensitivity to imidacloprid and thiamethoxam in adults. The larval susceptibility to thiamethoxam but not to imidacloprid was also reduced because of the lower down-regulation of Ldα1. Thus, our results suggest that Ldα1 encodes a subunit of a functional nAChR that mediates the

  6. EFFECTS OF OUABAIN AND DIGOXIN ON THE GENE EXPRESSION OF SODIUM PUMP α-SUBUNIT ISOFORM IN AORTIC SMOOTH MUSCLE OF RATS

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Objective To compare the effects of ouabain and digoxin on both the systolic blood pressure and sodium pump α-subunit isoforms gene expression in the aortic smooth muscle of rats. Methods Normal SpragueDawley rats were injected with ouabain (20μg·kg-1 ·d-1 ,i. p),digoxin (32 μg·kg-1 ·d-1,i. p)and normal saline once a day, respectively, and indirect systolic blood pressure was recorded once a week. Six weeks later,all the rats were killed and sodium pump α1-,α2-,and α3-subunit mRNA levels were detected in the aortic smooth muscle with reverse transcription polymerase chain reaction(RT-PCR) method. Results The systolic blood pressure of rats infused with ouabain increased significantly at the end of week 6 [132. 6± 9. 0 mmHg (1 mmHg = 0. 133 kPa)vs 115. 7±8.2mmHg, P <0. 01] ,while no difference of blood pressure was found between digoxin group and NS group (P>0.05).The expression of sodium pump α-subunit isoforms in aortic smooth muscle was regulated by either ouabain or digoxin:both ouabain and digoxin increased α1- and α3-subunit expression, α2-subunit decreased in digoxin group but unchanged in ouabain group. Conclusion These results suggest that both ouabain and digoxin could regulate sodium pump α-subunit isoform expression, which might be related to the physiological roles of endogenous ouabain and might be responsible for the difference between the pharmacological and toxicological effects of ouabain and digoxin,including their effects on blood pressure.

  7. Absence of the A4 peptide in the G4 glycinin subunit of soybean cultivar Enrei is caused by a point mutation in the Gy4 gene

    Directory of Open Access Journals (Sweden)

    Yu Kangfu

    2005-01-01

    Full Text Available Functional properties of soy proteins for food are closely related to the composition of their storage protein subunits. Using base excision sequence scanning (BESS, we show that the absence of the A4 peptide in the G4 glycinin subunit of the soybean (Glycine max L. cultivar Enrei was caused by the same point mutation in the Gy4 gene as previously reported in the soybean cultivar Raiden. Although the genetic relationship between Raiden and Enrei is not known, the same point mutation in their Gy4 genes may indicate that they probably share a related origin. The application of BESS to identify single nucleotide polymorphisms (SNPs as co-dominant markers for marker-assisted selection (MAS of a recessive null allele is also discussed.

  8. Characterization of low-molecular-weight-glutenin subunit genes from the D-genome of Triticum aestivum, Aegilops crassa, Ae. cylindrica and Ae. tauschii

    NARCIS (Netherlands)

    Naghavi, M.R.; Ahmadi, S.; Shanejat-Boushehri, A.A.; Komaei, G.; Struik, P.C.

    2013-01-01

    Twenty low-molecular-weight-glutenin subunit (LMW-GS) gene sequences from the D-genome from Aegilops crassa (2n ¼ 4x ¼ 28), Ae. cylindrica (2n ¼ 4x ¼ 28), Ae. tauschii (2n ¼ 2x ¼ 14) and Triticum aestivum (2n ¼ 6x ¼ 42) were obtained using five sets of specific allele primer pairs. Only the sequence

  9. Cloning and sequence analysis of the gene encoding 19-kD subunit of Complex I from Dunaliella salina.

    Science.gov (United States)

    Liu, Yi; Qiao, Dai Rong; Zheng, Hong Bo; Dai, Xu Lan; Bai, Lin Han; Zeng, Jing; Cao, Yi

    2008-09-01

    NADH:ubiquinone oxidoreductase (complex I ) of the mitochondrial respiratory chain catalyzes the transfer of electrons from NADH to ubiquinone coupled to proton translocation across the membrane. The cDNA sequence of Dunaliella salina mitochondrial NADH: ubiquinone oxidoreductase 19-kD subunit contains a 682-bp ORF encoding a protein with an apparent molecular mass of 19 kD. The sequence has been submitted to the GenBank database under Accession No. EF566890 (cDNA sequences) and EF566891 (genomic sequence). The deduced amino-acid sequence is 74% identical to Chlamydomonas reinhardtii mitochondrial NADH:ubiquinone oxidoreductase 18-kD subunit. The 19-kD subunit mRNA expression was observed in oxygen deficiency, salt treatment, and rotenone treatment with lower levels. It demonstrate that the 19-kD subunit of Complex I from Dunaliella salina is regulated by these stresses.

  10. The NapF protein of the Escherichia coli periplasmic nitrate reductase system: demonstration of a cytoplasmic location and interaction with the catalytic subunit, NapA.

    Science.gov (United States)

    Nilavongse, Arjaree; Brondijk, T Harma C; Overton, Tim W; Richardson, David J; Leach, Emily R; Cole, Jeffrey A

    2006-11-01

    The periplasmic nitrate reductase of Escherichia coli is important during anaerobic growth in low-nitrate environments. The nap operon encoding this nitrate reductase comprises seven genes including a gene, napF, that encodes a putative cytoplasmic iron-sulphur protein of uncertain subcellular location and function. In this study, N-terminal sequence analysis, cell fractionation coupled with immunoblotting and construction of LacZ and PhoA fusion proteins were used together to establish that NapF is located in the E. coli cytoplasm. A bacterial two-hybrid protein-protein interaction system was used to demonstrate that NapF interacted in the cytoplasm with the terminal oxidoreductase NapA, but that it did not self-associate or interact with other electron-transport components of the Nap system, NapC, NapG or NapH, or with another cytoplasmic component, NapD. NapF, purified as a His(6)-tagged protein, exhibited spectral properties characteristic of an iron-sulphur protein. This protein was able to pull down NapA from soluble extracts of E. coli. A growth-based assay for NapF function in intact cell cultures was developed and applied to assess the effect of mutation of a number of conserved amino acids. It emerged that neither a highly conserved N-terminal double-arginine motif, nor a conserved proline motif, is essential for NapF-dependent growth. The combined data indicate that NapF plays one or more currently unidentified roles in the post-translational modification of NapA prior to the export of folded NapA via the twin-arginine translocation pathway into the periplasm.

  11. Chromosomal location of genes for novel glutenin subunits and gliadins in wild emmer wheat (Triticum turgidum L. var. dicoccoides).

    Science.gov (United States)

    Xu, S S; Khan, K; Klindworth, D L; Faris, J D; Nygard, G

    2004-05-01

    The glutenin and gliadin proteins of wild emmer wheat, Triticum turgidum L. var. dicoccoides, have potential for improvement of durum wheat ( T. turgidum L. var. durum) quality. The objective of this study was to determine the chromosomes controlling the high molecular weight (HMW) glutenin subunits and gliadin proteins present in three T. turgidum var. dicoccoides accessions (Israel-A, PI-481521, and PI-478742), which were used as chromosome donors in Langdon durum- T. turgidum var. dicoccoides (LDN-DIC) chromosome substitution lines. The three T. turgidum var. dicoccoides accessions, their respective LDN-DIC substitution lines, and a number of controls with known HMW glutenin subunits were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), urea/SDS-PAGE, and acid polyacrylamide gel electrophoresis (A-PAGE). The results revealed that all three T. turgidum var. dicoccoides accessions possess Glu-A1 alleles that are the same as or similar to those reported previously. However, each T. turgidum var. dicoccoides accession had a unique Glu-B1 allele. PI-478742 had an unusual 1Bx subunit, which had mobility slightly slower than the 1Ax subunit in 12% SDS-PAGE gels. The subunits controlled by chromosome 1B of PI-481521 were slightly faster in mobility than the subunits of the Glu-B1n allele, and the 1By subunit was identified as band 8. The 1B subunits of Israel-A had similar mobility to subunits 14 and 16. The new Glu-B1 alleles were designated as Glu-B1be in Israel-A, Glu-B1bf in PI-481521, and Glu-B1bg in PI-478742. Results from A-PAGE revealed that PI-481521, PI-478742, and Israel-A had eight, 12, and nine unique gliadin bands, respectively, that were assigned to specific chromosomes. The identified glutenin subunits and gliadin proteins in the LDN-DIC substitution lines provide the basis for evaluating their effects on end-use quality, and they are also useful biochemical markers for identifying specific chromosomes or chromosome

  12. Phylogenetic positions of two marine ciliates, Metanophrys similis and Pseudocohnilembus hargisi (Protozoa, Ciliophora, Scuticociliatia), inferred from complete small subunit rRNA gene sequences

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    The small subunit rRNA (SSrRNA) gene was sequenced for two marine scuticociliates Metanophrys similis and Pseudocohnilembus hargisi. The results show that this gene comprises 1763 and 1753 nucleotides in the two marine ciliates respectively.Metanophrys similis is phylogenetically closely related to the clade containing Mesanophrys carcini and Anophyroides haemophila, which branches basally to other species within the order Philasterida. Pseudocohnilembus hargisi groups with its congener, P. marinus, with strong bootstrap support. Paranophrys magna groups with the clade including Cohnilembus and Uronema, representing a sister clade to that containing the two Pseudocohnilembus species.

  13. The acquired radioresistance in HeLa cells under conditions mimicking hypoxia was attenuated by a decreased expression of HIF subunit genes induced by RNA interference

    Energy Technology Data Exchange (ETDEWEB)

    Doi, Nobutaka [Department of Radiological Sciences, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama 930-0194 (Japan); New Products Research & Development, Gene Engineering Division, NIPPON GENE Co., Ltd. (Japan); Ogawa, Ryohei, E-mail: ogawa@med.u-toyama.ac.jp [Department of Radiological Sciences, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama 930-0194 (Japan); Cui, Zheng-Guo [Department of Public Health, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama (Japan); Morii, Akihiro; Watanabe, Akihiko [Department of Urology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama (Japan); Kanayama, Shinji; Yoneda, Yuko [New Products Research & Development, Gene Engineering Division, NIPPON GENE Co., Ltd. (Japan); Kondo, Takashi [Department of Radiological Sciences, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama 930-0194 (Japan)

    2015-05-01

    The cancer cells residing in the hypoxic layer are resistant to radiation and these are ones responsible for cancer recurrence after radiation therapy. One of the reasons why hypoxic cancer cells acquire radioresistance may be attributable to changes in the gene expression profile by the activation of hypoxia inducible factors (HIFs). However, the details underlying this process remain unknown. In this study, we investigated the effects of knockdown of HIF subunit genes to elucidate how HIF subunit genes may be involved in the radioresistance acquired by HeLa cells following exposure to a hypoxia mimic. Interestingly, HIF-1α and HIF-2α seemed mutually complementary for each other when either of them was suppressed. We thus suppressed the expression of both genes simultaneously. To do this, we developed a short hairpin RNA (shRNA) targeting a high homology region between HIF-1α and HIF-2α. It was shown that the expression of the shRNA effectively suppressed the acquisition of radioresistance following the hypoxia mimic. Moreover, it was confirmed that suppression of both subunits resulted in the downregulation of stem cell markers and the suppression of spheroid formation during the hypoxia mimicking-conditions. This shRNA-mediated knockdown method targeting a common region shared by a family of genes may offer a new candidate cancer treatment. - Highlights: • Incubation with CoCl{sub 2} confers radioresistance to HeLa cells. • Both HIF-1α and HIF-2α are involved in the acquisition of radioresistance. • An shRNA to a homology region of HIF-1α and HIF-2α suppressed the radioresistance. • The shRNA decreased cells with stem cell markers and a stem cell phenotype.

  14. Neuron-specific specificity protein 4 bigenomically regulates the transcription of all mitochondria- and nucleus-encoded cytochrome c oxidase subunit genes in neurons.

    Science.gov (United States)

    Johar, Kaid; Priya, Anusha; Dhar, Shilpa; Liu, Qiuli; Wong-Riley, Margaret T T

    2013-11-01

    Neurons are highly dependent on oxidative metabolism for their energy supply, and cytochrome c oxidase (COX) is a key energy-generating enzyme in the mitochondria. A unique feature of COX is that it is one of only four proteins in mammalian cells that are bigenomically regulated. Of its thirteen subunits, three are encoded in the mitochondrial genome and ten are nuclear-encoded on nine different chromosomes. The mechanism of regulating this multisubunit, bigenomic enzyme poses a distinct challenge. In recent years, we found that nuclear respiratory factors 1 and 2 (NRF-1 and NRF-2) mediate such bigenomic coordination. The latest candidate is the specificity factor (Sp) family of proteins. In N2a cells, we found that Sp1 regulates all 13 COX subunits. However, we discovered recently that in primary neurons, it is Sp4 and not Sp1 that regulates some of the key glutamatergic receptor subunit genes. The question naturally arises as to the role of Sp4 in regulating COX in primary neurons. The present study utilized multiple approaches, including chromatin immunoprecipitation, promoter mutational analysis, knockdown and over-expression of Sp4, as well as functional assays to document that Sp4 indeed functionally regulate all 13 subunits of COX as well as mitochondrial transcription factors A and B. The present study discovered that among the specificity family of transcription factors, it is the less known neuron-specific Sp4 that regulates the expression of all 13 subunits of mitochondrial cytochrome c oxidase (COX) enzyme in primary neurons. Sp4 also regulates the three mitochondrial transcription factors (TFAM, TFB1M, and TFB2M) and a COX assembly protein SURF-1 in primary neurons.

  15. Cystatins as calpain inhibitors: engineered chicken cystatin- and stefin B-kininogen domain 2 hybrids support a cystatin-like mode of interaction with the catalytic subunit of mu-calpain.

    Science.gov (United States)

    Díaz, B G; Gross, S; Assfalg-Machleidt, I; Pfeiler, D; Gollmitzer, N; Gabrijelcic-Geiger, D; Stubbs, M T; Fritz, H; Auerswald, E A; Machleidt, W

    2001-01-01

    Within the cystatin superfamily, only kininogen domain 2 (KD2) is able to inhibit mu- and m-calpain. In an attempt to elucidate the structural requirements of cystatins for calpain inhibition, we constructed recombinant hybrids of human stefin B (an intracellular family 1 cystatin) with KD2 and deltaL110 deletion mutants of chicken cystatin-KD2 hybrids. Substitution of the N-terminal contact region of stefin B by the corresponding KD2 sequence resulted in a calpain inhibitor of Ki = 188 nM. Deletion of L110, which forms a beta-bulge in family 1 and 2 cystatins but is lacking in KD2, improved inhibition of mu-calpain 4- to 8-fold. All engineered cystatins were temporary inhibitors of calpain due to slow substrate-like cleavage of a single peptide bond corresponding to Gly9-Ala10 in chicken cystatin. Biomolecular interaction analysis revealed that, unlike calpastatin, the cystatin-type inhibitors do not bind to the calmodulin-like domain of the small subunit of calpain, and their interaction with the mu-calpain heterodimer is completely prevented by a synthetic peptide comprising subdomain B of calpastatin domain 1. Based on these results we propose that (i) cystatin-type calpain inhibitors interact with the active site of the catalytic domain of calpain in a similar cystatin-like mode as with papain and (ii) the potential for calpain inhibition is due to specific subsites within the papain-binding regions of the general cystatin fold.

  16. Novel insights into the composition, variation, organization, and expression of the low-molecular-weight glutenin subunit gene family in common wheat.

    Science.gov (United States)

    Zhang, Xiaofei; Liu, Dongcheng; Zhang, Jianghua; Jiang, Wei; Luo, Guangbin; Yang, Wenlong; Sun, Jiazhu; Tong, Yiping; Cui, Dangqun; Zhang, Aimin

    2013-04-01

    Low-molecular-weight glutenin subunits (LMW-GS), encoded by a complex multigene family, play an important role in the processing quality of wheat flour. Although members of this gene family have been identified in several wheat varieties, the allelic variation and composition of LMW-GS genes in common wheat are not well understood. In the present study, using the LMW-GS gene molecular marker system and the full-length gene cloning method, a comprehensive molecular analysis of LMW-GS genes was conducted in a representative population, the micro-core collections (MCC) of Chinese wheat germplasm. Generally, >15 LMW-GS genes were identified from individual MCC accessions, of which 4-6 were located at the Glu-A3 locus, 3-5 at the Glu-B3 locus, and eight at the Glu-D3 locus. LMW-GS genes at the Glu-A3 locus showed the highest allelic diversity, followed by the Glu-B3 genes, while the Glu-D3 genes were extremely conserved among MCC accessions. Expression and sequence analysis showed that 9-13 active LMW-GS genes were present in each accession. Sequence identity analysis showed that all i-type genes present at the Glu-A3 locus formed a single group, the s-type genes located at Glu-B3 and Glu-D3 loci comprised a unique group, while high-diversity m-type genes were classified into four groups and detected in all Glu-3 loci. These results contribute to the functional analysis of LMW-GS genes and facilitate improvement of bread-making quality by wheat molecular breeding programmes.

  17. Determination of the relative expression levels of rubisco small subunit genes in Arabidopsis by rapid amplification of cDNA ends.

    Science.gov (United States)

    Yoon, M; Putterill, J J; Ross, G S; Laing, W A

    2001-04-15

    Multigene families are common in higher organisms. However, due to the close similarities between members, it is often difficult to assess the individual contribution of each gene to the overall expression of the family. In Arabidopsis thaliana, there are four genes encoding the small subunits (SSU) of ribulose-1.5-bisphosphate carboxylase oxygenase (rubisco) whose nucleotide sequences are up to 98.4% identical. In order to overcome the technical limitations associated with gene-specific probes (or primers) commonly used in existing methods, we developed a new gene expression assay based on the RACE (rapid amplification of cDNA ends) technique with a single pair of primers. With this RACE gene expression assay, we were able to determine the relative transcript levels between four Arabidopsis SSU genes. We found that the relative SSU gene expression differed significantly between plants grown at different temperatures. Our observation raises the possibility that an adaptation of rubisco to the environment may be achieved through the specific synthesis of the SSU proteins, which is determined by the relative expression levels between the SSU genes.

  18. Identification of Botrytis cinerea genes up-regulated during infection and controlled by the Galpha subunit BCG1 using suppression subtractive hybridization (SSH).

    Science.gov (United States)

    Schulze Gronover, Christian; Schorn, Corinna; Tudzynski, Bettina

    2004-05-01

    The Galpha subunit BCG1 plays an important role during the infection of host plants by Botrytis cinerea. Delta bcg1 mutants are able to conidiate, penetrate host leaves, and produce small primary lesions. However, in contrast to the wild type, the mutants completely stop invasion of plant tissue at this stage; secondary lesions have never been observed. Suppression subtractive hybridization (SSH) was used to identify fungal genes whose expression on the host plant is specifically affected in bcg1 mutants. Among the 22 differentially expressed genes, we found those which were predicted to encode proteases, enzymes involved in secondary metabolism, and others encoding cell wall-degrading enzymes. All these genes are highly expressed during infection in the wild type but not in the mutant. However, the genes are expressed in both the wild type and the mutant under certain conditions in vitro. Most of the BCG1-controlled genes are still expressed in adenylate cyclase (bac) mutants in planta, suggesting that BCG1 is involved in at least one additional signaling cascade in addition to the cAMP-depending pathway. In a second SSH approach, 1,500 clones were screened for those that are specifically induced by the wild type during the infection of bean leaves. Of the 22 BCG1-controlled genes, 11 also were found in the in planta SSH library. Therefore, SSH technology can be successfully applied to identify target genes of signaling pathways and differentially expressed genes in planta.

  19. Nuclear-cytoplasmic conflict in pea (Pisum sativum L.) is associated with nuclear and plastidic candidate genes encoding acetyl-CoA carboxylase subunits.

    Science.gov (United States)

    Bogdanova, Vera S; Zaytseva, Olga O; Mglinets, Anatoliy V; Shatskaya, Natalia V; Kosterin, Oleg E; Vasiliev, Gennadiy V

    2015-01-01

    In crosses of wild and cultivated peas (Pisum sativum L.), nuclear-cytoplasmic incompatibility frequently occurs manifested as decreased pollen fertility, male gametophyte lethality, sporophyte lethality. High-throughput sequencing of plastid genomes of one cultivated and four wild pea accessions differing in cross-compatibility was performed. Candidate genes for involvement in the nuclear-plastid conflict were searched in the reconstructed plastid genomes. In the annotated Medicago truncatula genome, nuclear candidate genes were searched in the portion syntenic to the pea chromosome region known to harbor a locus involved in the conflict. In the plastid genomes, a substantial variability of the accD locus represented by nucleotide substitutions and indels was found to correspond to the pattern of cross-compatibility among the accessions analyzed. Amino acid substitutions in the polypeptides encoded by the alleles of a nuclear locus, designated as Bccp3, with a complementary function to accD, fitted the compatibility pattern. The accD locus in the plastid genome encoding beta subunit of the carboxyltransferase of acetyl-coA carboxylase and the nuclear locus Bccp3 encoding biotin carboxyl carrier protein of the same multi-subunit enzyme were nominated as candidate genes for main contribution to nuclear-cytoplasmic incompatibility in peas. Existence of another nuclear locus involved in the accD-mediated conflict is hypothesized.

  20. Switch in glutamate receptor subunit gene expression in CA1 subfield of hippocampus following global ischemia in rats.

    OpenAIRE

    Pellegrini-Giampietro, D E; Zukin, R.S.; Bennett, M V; Cho, S; Pulsinelli, W. A.

    1992-01-01

    Severe, transient global ischemia of the brain induces delayed damage to specific neuronal populations. Sustained Ca2+ influx through glutamate receptor channels is thought to play a critical role in postischemic cell death. Although most kainate-type glutamate receptors are Ca(2+)-impermeable, Ca(2+)-permeable kainate receptors have been reported in specific kinds of neurons and glia. Recombinant receptors assembled from GluR1 and/or GluR3 subunits in exogenous expression systems are permeab...

  1. SWR1 Chromatin-Remodeling Complex Subunits and H2A.Z Have Non-overlapping Functions in Immunity and Gene Regulation in Arabidopsis.

    Science.gov (United States)

    Berriri, Souha; Gangappa, Sreeramaiah N; Kumar, S Vinod

    2016-07-06

    Incorporation of the histone variant H2A.Z into nucleosomes by the SWR1 chromatin remodeling complex is a critical step in eukaryotic gene regulation. In Arabidopsis, SWR1c and H2A.Z have been shown to control gene expression underlying development and environmental responses. Although they have been implicated in defense, the specific roles of the complex subunits and H2A.Z in immunity are not well understood. In this study, we analyzed the roles of the SWR1c subunits, PHOTOPERIOD-INDEPENDENT EARLY FLOWERING1 (PIE1), ACTIN-RELATED PROTEIN6 (ARP6), and SWR1 COMPLEX 6 (SWC6), as well as H2A.Z, in defense and gene regulation. We found that SWR1c components play different roles in resistance to different pathogens. Loss of PIE1 and SWC6 function as well as depletion of H2A.Z led to reduced basal resistance, while loss of ARP6 fucntion resulted in enhanced resistance. We found that mutations in PIE1 and SWC6 resulted in impaired effector-triggered immunity. Mutation in SWR1c components and H2A.Z also resulted in compromised jasmonic acid/ethylene-mediated immunity. Genome-wide expression analyses similarly reveal distinct roles for H2A.Z and SWR1c components in gene regulation, and suggest a potential role for PIE1 in the regulation of the cross talk between defense signaling pathways. Our data show that although they are part of the same complex, Arabidopsis SWR1c components could have non-redundant functions in plant immunity and gene regulation.

  2. Relationship between reduced nicotinamide adenine dinucleotide phosphate oxidase subunit p22phox gene polymorphism and obstructive sleep apnea-hypopnea syndrome in the Chinese Han population

    Institute of Scientific and Technical Information of China (English)

    LIU Hui-guo; LIU Kui; ZHOU Yan-ning; XU Yong-jian

    2009-01-01

    Background Increased production of reactive oxygen species (ROS) is thought to play a major role in the pathogenesis of obstructive sleep apnea-hypopnea syndrome (OSAHS). The reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex is an important source of ROS. The p22phox subunit is polymorphic with a C242T variant that changes histidine-72 for a tyrosine in the potential heme binding site. This study aimed to investigate the relationship between NADPH oxidase subunit p22phox gene polymorphism and OSAHS. Methods The genotypes of p22phox polymorphism were determined by polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP) assay in 176 unrelated subjects of the Han population in southern region of China (including 107 OSAHS subjects and 69 non-OSAHS subjects), while the plasma concentration of superoxide dismutase (SOD) was detected in the two groups, and p22phox mRNA expression in peripheral blood mononuclear cell (PBMC) was determined with reverse transcription polymerase chain reaction (RT-PCR).Results The phagocyte NADPH oxidase subunit p22phox mRNA expression was significantly increased in the OSAHS group than that in the non-OSAHS group (P<0.01). Compared with the non-OSAHS control group ((85.31±9.23) U/ml), the levels of SOD were lower in patients with OSAHS ((59.65±11.61) U/ml (P<0.01). There were significant differences in genotypes distribution in p22phox polymorphism between the two groups (P=0.02). Compared with the non-OSAHS control group, the OSAHS group had a significantly higher T allele frequency in p22phox polymorphism (P=0.03). There were independent effects of p22phox polymorphism on body mass index (BMI), neck circumference (NC), waist-to-hip ratio (WHR) in the OSAHS group, and the carriers of the T allele of p22phox polymorphism had greater NC, WHR, systolic blood pressure (SBP), diastolic blood pressure (DBP) and apnea-hypopnea index (AHI) (P <0.05), but the carriers of the T allele had lower SOD

  3. Isolation and characterization of BetaM protein encoded by ATP1B4 - a unique member of the Na,K-ATPase {beta}-subunit gene family

    Energy Technology Data Exchange (ETDEWEB)

    Pestov, Nikolay B. [Department of Physiology and Pharmacology, University of Toledo College of Medicine, 3000 Arlington Ave., Toledo, OH 43614 (United States); Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow 117997 (Russian Federation); Zhao, Hao [Department of Physiology and Pharmacology, University of Toledo College of Medicine, 3000 Arlington Ave., Toledo, OH 43614 (United States); Basrur, Venkatesha [Department of Pathology, University of Michigan Medical School, Ann Arbor, MI 48109 (United States); Modyanov, Nikolai N., E-mail: nikolai.modyanov@utoledo.edu [Department of Physiology and Pharmacology, University of Toledo College of Medicine, 3000 Arlington Ave., Toledo, OH 43614 (United States)

    2011-09-09

    Highlights: {yields} Structural properties of BetaM and Na,K-ATPase {beta}-subunits are sharply different. {yields} BetaM protein is concentrated in nuclear membrane of skeletal myocytes. {yields} BetaM does not associate with a Na,K-ATPase {alpha}-subunit in skeletal muscle. {yields} Polypeptide chain of the native BetaM is highly sensitive to endogenous proteases. {yields} BetaM in neonatal muscle is a product of alternative splice mRNA variant B. -- Abstract: ATP1B4 genes represent a rare instance of the orthologous gene co-option that radically changed functions of encoded BetaM proteins during vertebrate evolution. In lower vertebrates, this protein is a {beta}-subunit of Na,K-ATPase located in the cell membrane. In placental mammals, BetaM completely lost its ancestral role and through acquisition of two extended Glu-rich clusters into the N-terminal domain gained entirely new properties as a muscle-specific protein of the inner nuclear membrane possessing the ability to regulate gene expression. Strict temporal regulation of BetaM expression, which is the highest in late fetal and early postnatal myocytes, indicates that it plays an essential role in perinatal development. Here we report the first structural characterization of the native eutherian BetaM protein. It should be noted that, in contrast to structurally related Na,K-ATPase {beta}-subunits, the polypeptide chain of BetaM is highly sensitive to endogenous proteases that greatly complicated its isolation. Nevertheless, using a complex of protease inhibitors, a sample of authentic BetaM was isolated from pig neonatal skeletal muscle by a combination of ion-exchange and lectin-affinity chromatography followed by SDS-PAGE. Results of the analysis of the BetaM tryptic digest using MALDI-TOF and ESI-MS/MS mass spectrometry have demonstrated that native BetaM in neonatal skeletal muscle is a product of alternative splice mRNA variant B and comprised of 351 amino acid residues. Isolated BetaM protein was

  4. A genetic suppressor of two dominant temperature-sensitive lethal proteasome mutants of Drosophila melanogaster is itself a mutated proteasome subunit gene.

    Science.gov (United States)

    Neuburger, Peter J; Saville, Kenneth J; Zeng, Jue; Smyth, Kerrie-Ann; Belote, John M

    2006-07-01

    Two dominant temperature-sensitive (DTS) lethal mutants of Drosophila melanogaster are Pros26(1) and Prosbeta2(1), previously known as DTS5 and DTS7. Heterozygotes for either mutant die as pupae when raised at 29 degrees , but are normally viable and fertile at 25 degrees . Previous studies have identified these as missense mutations in the genes encoding the beta6 and beta2 subunits of the 20S proteasome, respectively. In an effort to isolate additional proteasome-related mutants a screen for dominant suppressors of Pros26(1) was carried out, resulting in the identification of Pros25(SuDTS) [originally called Su(DTS)], a missense mutation in the gene encoding the 20S proteasome alpha2 subunit. Pros25(SuDTS) acts in a dominant manner to rescue both Pros26(1) and Prosbeta2(1) from their DTS lethal phenotypes. Using an in vivo protein degradation assay it was shown that this suppression occurs by counteracting the dominant-negative effect of the DTS mutant on proteasome activity. Pros25(SuDTS) is a recessive polyphasic lethal at ambient temperatures. The effects of these mutants on larval neuroblast mitosis were also examined. While Prosbeta2(1) shows a modest increase in the number of defective mitotic figures, there were no defects seen with the other two mutants, other than slightly reduced mitotic indexes.

  5. Disease-associated mutations in the HSPD1 gene encoding the large subunit of the mitochondrial HSP60/HSP10 chaperonin complex

    Directory of Open Access Journals (Sweden)

    Peter Bross

    2016-08-01

    Full Text Available Heat shock protein 60 (HSP60 forms together with heat shock protein 10 (HSP10 double-barrel chaperonin complexes that are essential for folding to the native state of proteins in the mitochondrial matrix space. Two extremely rare monogenic disorders have been described that are caused by missense mutations in the HSPD1 gene that encodes the HSP60 subunit of the HSP60/HSP10 chaperonin complex. Investigations of the molecular mechanisms underlying these disorders have revealed that different degrees of reduced HSP60 function produce distinct neurological phenotypes. While mutations with deleterious or strong dominant negative effects are not compatible with life, HSPD1 gene variations found in the human population impair HSP60 function and depending on the mechanism and degree of HSP60 dys- and malfunction cause different phenotypes. We here summarize the knowledge on the effects of disturbances of the function of the HSP60/HSP10 chaperonin complex by disease-associated mutations.

  6. Characterization and variation of a human inwardly-rectifying-K-channel gene (KCNJ6): a putative ATP-sensitive K-channel subunit.

    Science.gov (United States)

    Sakura, H; Bond, C; Warren-Perry, M; Horsley, S; Kearney, L; Tucker, S; Adelman, J; Turner, R; Ashcroft, F M

    1995-06-26

    The ATP-sensitive K-channel plays a central role in insulin release from pancreatic beta-cells. We report here the cloning of the gene (KCNJ6) encoding a putative subunit of a human ATP-sensitive K-channel expressed in brain and beta-cells, and characterisation of its exon-intron structure. Screening of a somatic cell mapping panel and fluorescent in situ hybridization place the gene on chromosome 21 (21q22.1-22.2). Analysis of single-stranded conformational polymorphisms revealed the presence of two silent polymorphisms (Pro-149: CCG-CCA and Asp-328: GAC-GAT) with similar frequencies in normal and non-insulin-dependent diabetic patients.

  7. Loss-of-function mutations of retromer large subunit genes suppress the phenotype of an Arabidopsis zig mutant that lacks Qb-SNARE VTI11.

    Science.gov (United States)

    Hashiguchi, Yasuko; Niihama, Mitsuru; Takahashi, Tetsuya; Saito, Chieko; Nakano, Akihiko; Tasaka, Masao; Morita, Miyo Terao

    2010-01-01

    Arabidopsis thaliana zigzag (zig) is a loss-of-function mutant of Qb-SNARE VTI11, which is involved in membrane trafficking between the trans-Golgi network and the vacuole. zig-1 exhibits abnormalities in shoot gravitropism and morphology. Here, we report that loss-of-function mutants of the retromer large subunit partially suppress the zig-1 phenotype. Moreover, we demonstrate that three paralogous VPS35 genes of Arabidopsis have partially overlapping but distinct genetic functions with respect to zig-1 suppression. Tissue-specific complementation experiments using an endodermis-specific SCR promoter show that expression of VPS35B or VPS35C cannot complement the function of VPS35A. The data suggest the existence of functionally specialized paralogous VPS35 genes that nevertheless share common functions.

  8. Loss-of-Function Mutations of Retromer Large Subunit Genes Suppress the Phenotype of an Arabidopsis zig Mutant That Lacks Qb-SNARE VTI11[C][W

    Science.gov (United States)

    Hashiguchi, Yasuko; Niihama, Mitsuru; Takahashi, Tetsuya; Saito, Chieko; Nakano, Akihiko; Tasaka, Masao; Morita, Miyo Terao

    2010-01-01

    Arabidopsis thaliana zigzag (zig) is a loss-of-function mutant of Qb-SNARE VTI11, which is involved in membrane trafficking between the trans-Golgi network and the vacuole. zig-1 exhibits abnormalities in shoot gravitropism and morphology. Here, we report that loss-of-function mutants of the retromer large subunit partially suppress the zig-1 phenotype. Moreover, we demonstrate that three paralogous VPS35 genes of Arabidopsis have partially overlapping but distinct genetic functions with respect to zig-1 suppression. Tissue-specific complementation experiments using an endodermis-specific SCR promoter show that expression of VPS35B or VPS35C cannot complement the function of VPS35A. The data suggest the existence of functionally specialized paralogous VPS35 genes that nevertheless share common functions. PMID:20086190

  9. Association of polymorphisms in calpain 1, (mu/I) large subunit, calpastatin, and cathepsin D genes with meat quality traits in double-muscled Piemontese cattle.

    Science.gov (United States)

    Ribeca, Cinzia; Bonfatti, Valentina; Cecchinato, Alessio; Albera, Andrea; Maretto, Fabio; Gallo, Luigi; Carnier, Paolo

    2013-04-01

    Five single-nucleotide polymorphisms (SNPs) located in the calpain 1, (mu/I) large subunit (CAPN1), calpastatin (CAST), and cathepsin D (CTSD) genes were analyzed in a large sample of Piemontese cattle. The aim of this study was to evaluate allele and genotype frequencies of these SNPs and to investigate associations of CAPN1, CAST, and CTSD gene variants with meat quality traits. Minor allele frequencies ranged from 30 to 48%. The presence of the A allele at CAPN530 increased yellowness and drip loss. The CAST282 G allele was associated with an increased drip loss compared to the C allele, and the CAST2959 A allele decreased redness compared to the G allele.

  10. Neurotransmitter release: vacuolar ATPase V0 sector c-subunits in possible gene or cell therapies for Parkinson's, Alzheimer's, and psychiatric diseases.

    Science.gov (United States)

    Higashida, Haruhiro; Yokoyama, Shigeru; Tsuji, Chiharu; Muramatsu, Shin-Ichi

    2017-01-01

    We overview the 16-kDa proteolipid mediatophore, the transmembrane c-subunit of the V0 sector of the vacuolar proton ATPase (ATP6V0C) that was shown to mediate the secretion of acetylcholine. Acetylcholine, serotonin, and dopamine (DA) are released from cell soma and/or dendrites if ATP6V0C is expressed in cultured cells. Adeno-associated viral vector-mediated gene transfer of ATP6V0C into the caudate putamen enhanced the depolarization-induced overflow of endogenous DA in Parkinson-model mice. Motor impairment was ameliorated in hemiparkinsonian model mice when ATP6V0C was expressed with DA-synthesizing enzymes. The review discusses application in the future as a potential tool for gene therapy, cell transplantation therapy, and inducible pluripotent stem cell therapy in neurological diseases, from the view point of recent findings regarding vacuolar ATPase.

  11. Cloning and sequencing of the genes coding for the A and B subunits of vacuolar-type Na(+)-ATPase from Enterococcus hirae. Coexistence of vacuolar- and F0F1-type ATPases in one bacterial cell.

    Science.gov (United States)

    Takase, K; Yamato, I; Kakinuma, Y

    1993-06-05

    The eubacterium Enterococcus hirae ATCC 9790 possesses a H(+)-translocating ATPase, and the deduced amino acid sequences of the genes coding for this enzyme have indicated that it is a typical F0F1-type ATPase (Shibata, C., Ehara, T., Tomura, K., Igarashi, K., and Kobayashi, H. (1992) J. Bacteriol. 174, 6117-6124). We cloned the ntpA and ntpB genes coding for the A and B subunits, respectively, of Na(+)-translocating ATPase from the same bacterium, and the full amino acid sequences of the two subunits were deduced from the nucleotide sequence. The A (593 amino acid residues) and B (458 amino acid residues) subunits were highly homologous (48-60% identical) to the A (large or alpha) and the B (small or beta) subunits, respectively, of vacuolar-type H(+)-ATPases which have been found in eukaryotic endomembrane systems (Neurospora crassa, Saccharomyces cerevisiae, Arabidopsis thaliana, and carrot) and archaebacterial cell membranes (Sulfolobus acidocaldarius and Methanosarcina barkeri). The A and B subunits of Na(+)-ATPase showed about 23-28% identities with the beta and alpha subunits of E. hirae F1-ATPase and of Escherichia coli F1-ATPase, respectively. These results indicate that E. hirae Na(+)-ATPase belongs to the vacuolar-type ATPase. This is the first demonstration that both genes for V- and F-type ATPases are functionally expressed in one bacterial cell.

  12. Analysis and validation of genome-specific DNA variations in 5' flanking conserved sequences of wheat low-molecular-weight glutenin subunit genes

    Institute of Scientific and Technical Information of China (English)

    LONG; Hai; WEI; Yuming

    2006-01-01

    The thirty-three 5' flanking conserved sequences of the known low-molecular-weight subunit (LMW-GS) genes have been divided into eight clusters, which was in agreement with the classification based on the deduced N-terminal protein sequences. The DNA polymorphism between the eight clusters was obtained by sequence alignment, and a total of 34 polymorphic positions were observed in the approximately 200 bp regions, among which 18 polymorphic positions were candidate SNPs. Seven cluster-specific primer sets were designed for seven out of eight clusters containing cluster-specific bases, with which the genomic DNA of the ditelosomic lines of group 1 chromosomes of a wheat variety 'Chinese Spring' was employed to carry out chromosome assignment. The subsequent cloning and DNA sequencing of PCR fragments validated the sequences specificity of the 5' flanking conserved sequences between LMW-GS gene groups in different genomes. These results suggested that the coding and 5' flanking regions of LMW-GS genes are likely to have evolved in a concerted fashion. The seven primer sets developed in this study could be used to isolate the complete ORFs of seven groups of LMW-GS genes, respectively, and therefore possess great value for further research in the contributions of a single LMW-GS gene to wheat quality in the complex genetic background and the efficient selections of quality-related components in breeding programs.

  13. Phylogenetic diversity of ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit genes of bacterioplankton in the East China Sea

    Institute of Scientific and Technical Information of China (English)

    ZENG Yonghui; JIAO Nianzhi; CAI Haiyuan; CHEN Xihan; WEI Chaoling

    2004-01-01

    Phylogenetic diversity of Form I and Form Ⅱ ribulose-1, 5-bisphosphate carboxylase/oxygenase (RubisCO) large subunit (rbcL) genes in the inshore and offshore areas of the East China Sea were investigated. Two new primer sets were designed for amplifying partial sequences of rbcL genes from Proteobacteria. Four rbcL gene clone libraries were constructed by amplification and cloning of approximately 640~800 bp sequences of bacterioplankton populations.The method of screening library by denaturing gradient gel electrophoresis (DGGE) was introduced. The results show that the diversity of Form I is higher in offshore waters with higher salinity and lower productivity, while that of Form Ⅱ is higher at the inshore station where salinity is lower and productivity is higher. Several clusters of sequences obtained are deeply rooted and show low similarity (60%~78%) to the known rbcL in existing databases.The degree of diversity of rbcL genes is directly related to environmental variables, including temperature, salinity,pH, dissolved oxygen, etc. These results indicate that rbcL gene can be used as an effective indicator for genetic diversity and population variability of bacterioplankton with the ability of carbon dioxide fixation in the sea.

  14. Association of polymorphisms in nicotinic acetylcholine receptor alpha 4 subunit gene (CHRNA4), mu-opioid receptor gene (OPRM1), and ethanol-metabolizing enzyme genes with alcoholism in Korean patients.

    Science.gov (United States)

    Kim, Soon Ae; Kim, Jong-Woo; Song, Ji-Young; Park, Sunny; Lee, Hee Jae; Chung, Joo-Ho

    2004-01-01

    Findings obtained from several studies indicate that ethanol enhances the activity of alpha4beta2 neuronal nicotinic acetylcholine receptor and support the possibility that a polymorphism of the nicotinic acetylcholine receptor alpha4 subunit gene (CHRNA4) modulates enhancement of nicotinic receptor function by ethanol. To identify the association between the CfoI polymorphism of the CHRNA4 and alcoholism, we examined distribution of genotypes and allele frequencies in Korean patients diagnosed with alcoholism (n = 127) and Korean control subjects without alcoholism (n = 185) with polymerase chain reaction-restriction fragment length polymorphism methods. We were able to detect the association between the CfoI polymorphism of the CHRNA4 and alcoholism in Korean patients (genotype P = .023; allele frequency P = .047). The genotypes and allele frequencies of known polymorphisms in other alcoholism candidate genes, such as alcohol metabolism-related genes [alcohol dehydrogenase 2 (ADH2), aldehyde dehydrogenase 2 (ALDH2), alcohol dehydrogenase 3 (ADH3), and cytochrome P450 2E1 (CYP2E1)] and mu-opioid receptor gene (OPRM1), were studied. The polymorphisms of ADH2, ALDH2, and CYP2E1 were significantly different in Korean patients with alcoholism and Korean control subjects without alcoholism, but ADH3 and OPRM1 did not differ between the two groups.

  15. Analysis of the type IV fimbrial-subunit gene fimA of Xanthomonas hyacinthi: application in PCR-mediated detection of yellow disease in Hyacinths.

    Science.gov (United States)

    van Doorn, J; Hollinger, T C; Oudega, B

    2001-02-01

    A sensitive and specific detection method was developed for Xanthomonas hyacinthi; this method was based on amplification of a subsequence of the type IV fimbrial-subunit gene fimA from strain S148. The fimA gene was amplified by PCR with degenerate DNA primers designed by using the N-terminal and C-terminal amino acid sequences of trypsin fragments of FimA. The nucleotide sequence of fimA was determined and compared with the nucleotide sequences coding for the fimbrial subunits in other type IV fimbria-producing bacteria, such as Xanthomonas campestris pv. vesicatoria, Neisseria gonorrhoeae, and Moraxella bovis. In a PCR internal primers JAAN and JARA, designed by using the nucleotide sequences of the variable central and C-terminal region of fimA, amplified a 226-bp DNA fragment in all X. hyacinthi isolates. This PCR was shown to be pathovar specific, as assessed by testing 71 Xanthomonas pathovars and bacterial isolates belonging to other genera, such as Erwinia and Pseudomonas. Southern hybridization experiments performed with the labelled 226-bp DNA amplicon as a probe suggested that there is only one structural type IV fimbrial-gene cluster in X. hyacinthi. Only two Xanthomonas translucens pathovars cross-reacted weakly in PCR. Primers amplifying a subsequence of the fimA gene of X. campestris pv. vesicatoria (T. Ojanen-Reuhs, N. Kalkkinen, B. Westerlund-Wikström, J. van Doorn, K. Haahtela, E.-L. Nurmiaho-Lassila, K. Wengelink, U. Bonas, and T. K. Korhonen, J. Bacteriol. 179: 1280-1290, 1997) were shown to be pathovar specific, indicating that the fimbrial-subunit sequences are more generally applicable in xanthomonads for detection purposes. Under laboratory conditions, approximately 1,000 CFU of X. hyacinthi per ml could be detected. In inoculated leaves of hyacinths the threshold was 5,000 CFU/ml. The results indicated that infected hyacinths with early symptoms could be successfully screened for X. hyacinthi with PCR.

  16. Dynamic expression of genes encoding subunits of inward rectifier potassium (Kir) channels in the yellow fever mosquito Aedes aegypti.

    Science.gov (United States)

    Yang, Zhongxia; Statler, Bethanie-Michelle; Calkins, Travis L; Alfaro, Edna; Esquivel, Carlos J; Rouhier, Matthew F; Denton, Jerod S; Piermarini, Peter M

    2017-02-01

    Inward rectifier potassium (Kir) channels play fundamental roles in neuromuscular, epithelial, and endocrine function in mammals. Recent research in insects suggests that Kir channels play critical roles in the development, immune function, and excretory physiology of fruit flies and/or mosquitoes. Moreover, our group has demonstrated that mosquito Kir channels may serve as valuable targets for the development of novel insecticides. Here we characterize the molecular expression of 5 mRNAs encoding Kir channel subunits in the yellow fever mosquito, Aedes aegypti: Kir1, Kir2A-c, Kir2B, Kir2B', and Kir3. We demonstrate that 1) Kir mRNA expression is dynamic in whole mosquitoes, Malpighian tubules, and the midgut during development from 4th instar larvae to adult females, 2) Kir2B and Kir3 mRNA levels are reduced in 4th instar larvae when reared in water containing an elevated concentration (50mM) of KCl, but not NaCl, and 3) Kir mRNAs are differentially expressed in the Malpighian tubules, midgut, and ovaries within 24h after blood feeding. Furthermore, we provide the first characterization of Kir mRNA expression in the anal papillae of 4th instar larval mosquitoes, which indicates that Kir2A-c is the most abundant. Altogether, the data provide the first comprehensive characterization of Kir mRNA expression in Ae. aegypti and offer insights into the putative physiological roles of Kir subunits in this important disease vector.

  17. Cloning, characterization, and expression of a novel member of proteasomal subunits gene in turbot,Scophthalmus maximus

    Institute of Scientific and Technical Information of China (English)

    ZHANG Bo; WANG Xianli; SONG Wenping; ZHENG Debin; MA Chao; XIAO Guangxia

    2015-01-01

    The proteasome is a large, polymeric protease complex responsible for the degradation of intracellular pro-teins and generation of peptides that bind to class I major histocompatibility complex (MHC) molecules. This study identified a new member of proteasomal subunits in turbots (Scophthalmus maximus). The full-length cDNA sequence of turbot proteasomal subunit was obtained. Sequence analysis indicated that its primary structure is highly similar to that ofLMP7 from other vertebrates. The relationship between the turbotLMP7 expression and immune responses to pathogen infection was reported. Quantitative reverse transcriptase polymerase chain reaction showed thatLMP7 was expressed differently in various tissues, with higher expression in the spleen, liver, muscle, and skin. TheLMP7 expression was the highest at 96 h after challenge with lymphocyctis disease virus (LCDV) and at 12 h after challenge withVibrio anguillarum in the turbot liver, kidney, and spleen. Furthermore, theLMP7 expression distinctly increased in turbot kidney cells at 24 h after challenge withV. anguillarumand at 96 h after challenge with LCDV. These results indicate that the turbot LMP7 protein participates in immune responses and may play a significant role in the immune process.

  18. M89V Sialic acid Acetyl Esterase (SIAE) and all other non-synonymous common variants of this gene are catalytically normal.

    Science.gov (United States)

    Chellappa, Vasant; Taylor, Kendra N; Pedrick, Kathryn; Donado, Carlos; Netravali, Ilka Arun; Haider, Khaleda; Cariappa, Annaiah; Dalomba, Natasha F; Pillai, Shiv

    2013-01-01

    Catalytically defective rare variants of Sialic acid Acetyl Esterase (SIAE) have previously been linked to autoimmunity. Studies presented here confirm that the M89V SIAE protein and all other products of common variant alleles of SIAE are catalytically normal. Although overexpressing transfected non-lymphoid cells secrete small amounts of SIAE that can associate with the cell surface, normal human lymphocytes do not exhibit cell surface SIAE, supporting genetic evidence in mice that indicates that this protein functions in a lymphocyte intrinsic manner. Analyses of the plasma proteome also indicate that SIAE is not secreted in vivo. A re-analysis exclusively of catalytically defective rare variant alleles of SIAE in subjects in which this gene was completely sequenced confirmed an association of SIAE with autoimmunity. A subset of catalytically defective rare variant SIAE alleles has previously been typed in a large genotyping study comparing a diverse group of disease subjects and controls; our re-analysis of this data shows that catalytically defective alleles are enriched in disease subjects. These data suggest that SIAE may be associated with autoimmunity and that further study of catalytically defective rare variant SIAE alleles in terms of autoimmune disease susceptibility is strongly warranted.

  19. M89V Sialic acid Acetyl Esterase (SIAE and all other non-synonymous common variants of this gene are catalytically normal.

    Directory of Open Access Journals (Sweden)

    Vasant Chellappa

    Full Text Available Catalytically defective rare variants of Sialic acid Acetyl Esterase (SIAE have previously been linked to autoimmunity. Studies presented here confirm that the M89V SIAE protein and all other products of common variant alleles of SIAE are catalytically normal. Although overexpressing transfected non-lymphoid cells secrete small amounts of SIAE that can associate with the cell surface, normal human lymphocytes do not exhibit cell surface SIAE, supporting genetic evidence in mice that indicates that this protein functions in a lymphocyte intrinsic manner. Analyses of the plasma proteome also indicate that SIAE is not secreted in vivo. A re-analysis exclusively of catalytically defective rare variant alleles of SIAE in subjects in which this gene was completely sequenced confirmed an association of SIAE with autoimmunity. A subset of catalytically defective rare variant SIAE alleles has previously been typed in a large genotyping study comparing a diverse group of disease subjects and controls; our re-analysis of this data shows that catalytically defective alleles are enriched in disease subjects. These data suggest that SIAE may be associated with autoimmunity and that further study of catalytically defective rare variant SIAE alleles in terms of autoimmune disease susceptibility is strongly warranted.

  20. Genetic Differences of Mitten Crabs Based on RFLP Analysis on Mitochondrial Cytochrome Oxidase Subunit I (COI) Gene

    Institute of Scientific and Technical Information of China (English)

    HU Pengfei; WANG Qian; DAI Wei; WANG Xiaomei

    2008-01-01

    The genetic differences of 15 mitten crab populations from 6 river systems in mainland China and 1 population from Russia were studied based on RFLP analysis of mitochondrial cytochrome oxidase subunit I (COI).The results showed that Tas I-RFLP pattern could be used as a genetic marker to distinguish Eriocheir hepuensis from Eriocheir sinensis, Eriocheirjaponica and Eriocheir leptognathus;genetic distances among 13 populations ofEriocheir sinensis range from 0 to 0.015, indicating that they were different geographic strains;the subspecies status ofEriocheir sinensis and Eriocheir hepuensis (population from Nanliujiang) were considered owning to their genetic distances of 0.02-0.044,indicating that genetic divergence between them was low; Eriocheir leptognathus (population from Nanpaihe, Tianjin) was the most distant taxon with genetic distances value of 0.147-0.195,which could be defined as genetic distances between species in genus Eriocheir.

  1. Understanding the mechanisms of ATPase beta family genes for cellular thermotolerance in crossbred bulls

    Science.gov (United States)

    Deb, Rajib; Sajjanar, Basavaraj; Singh, Umesh; Alex, Rani; Raja, T. V.; Alyethodi, Rafeeque R.; Kumar, Sushil; Sengar, Gyanendra; Sharma, Sheetal; Singh, Rani; Prakash, B.

    2015-12-01

    Na+/K+-ATPase is an integral membrane protein composed of a large catalytic subunit (alpha), a smaller glycoprotein subunit (beta), and gamma subunit. The beta subunit is essential for ion recognition as well as maintenance of the membrane integrity. Present study was aimed to analyze the expression pattern of ATPase beta subunit genes (ATPase B1, ATPase B2, and ATPase B3) among the crossbred bulls under different ambient temperatures (20-44 °C). The present study was also aimed to look into the relationship of HSP70 with the ATPase beta family genes. Our results demonstrated that among beta family genes, transcript abundance of ATPase B1 and ATPase B2 is significantly ( P P < 0.01) with HSP70, representing that the change in the expression pattern of these genes is positive and synergistic. These may provide a foundation for understanding the mechanisms of ATPase beta family genes for cellular thermotolerance in cattle.

  2. The carB gene encoding the large subunit of carbamoylphosphate synthetase from Lactococcus lactis is transcribed monocistronically

    DEFF Research Database (Denmark)

    Martinussen, Jan; Hammer, Karin

    1998-01-01

    the requirement for carbamoylphosphate in pyrimidine biosynthesis through degradation by the arginine deiminase pathway. The expression of the carB gene is subject to regulation at the level of transcription by pyrimidines most probably by an attenuator mechanism. Upstream of the carB gene, an open reading frame...... to be an isolated transcriptional unit. Carbamoylphosphate is a precursor in the biosynthesis of both pyrimidine nucleotides and arginine. By mutant analysis L. lactis is shown to possess only one carB gene; the same gene product is thus required for both biosynthetic pathways. Furthermore, arginine may satisfy...

  3. The Characteristics of Cytochrome C Oxidase Gene Subunit I in Wild Silkmoth Cricula trifenestrata Helfer and Its Evaluation for Species Marker

    Directory of Open Access Journals (Sweden)

    Suriana

    2012-08-01

    Full Text Available The study was conducted to assess the characteristics of partial gene of cytochrome C oxidase subunit I (COI of wild silkmoth Cricula trifenestrata, and to detect the diagnostic sites from these gene for evaluation as species marker. A total of fifteen larvae of C. tifenestrata were collected from Bogor, Purwakarta, and Bantul Regencies. Genomic DNA was extracted from silk gland of individual larvae, then amplified by PCR method and sequenced. DNA sequencing was done to characterize their nucleotide and amino acid contents. The results showed that 595 nucleotides at the 5 ‘end of COI gene of C. tifenestrata was conserved at the species level, but varies at the family level. Nucleotide dominated by thymine and adenine bases (± 70%. There were 25 diagnostic sites for C. tifenestrata, and four diagnostic sites for genus level. One hundred eigthty nine (189 amino acids were alignment, and only one percent of the genes was varied among species. The 107th amino acid (valine and 138th (threonine were diagnostics amino acid for C. tifenestrata. Based on nucleotides and amino acids sequences, the phylogeny showed that C. tifenestrata lied on the same nodes with Antheraea, so the Saturniidae family is monophyletic.

  4. An intact F1ATPase alpha-subunit gene and a pseudogene with differing genomic organization are detected in both male-fertile and CMS petunia mitochondria.

    Science.gov (United States)

    Yesodi, V; Hauschner, H; Tabib, Y; Firon, N

    1997-11-01

    The gene copies for the alpha-subunit of the mitochondrial F1ATPase (atpA) were isolated and characterized in both male-fertile and cytoplasmic male sterile (CMS) petunia. Two copies, an intact gene and a truncated gene, were detected in both cytoplasms. The accumulated data, based upon a comparison of the sequences (the open reading frames as well as the 5' and 3' flanking regions) of the two atpA copies, both in male-fertile and CMS Petunia, indicate that: (1) they differ in their genomic organization and (2) a common progenitor cytoplasm, containing two copies of an intact atpA sequence, served as the origin for the atpA copies of the fertility and CMS-inducing cytoplasms. Homologous recombination through the progenitor intact atpA sequences is assumed to have caused the rearrangement in the 3' portion of the atpA open reading frame and the generation of the truncated atpA gene. It is thus suggested that the atpA pseudogenes, in both male-fertile and CMS cytoplasms, originated from a common progenitor atpA pseudogene sequence.

  5. Increase of AMPA receptor glutamate receptor 1 subunit and B-cell receptor-associated protein 31 gene expression in hippocampus of fatigued mice.

    Science.gov (United States)

    Kamakura, Masaki; Tamaki, Keisuke; Sakaki, Toshiyuki; Yoneda, Yukio

    2005-10-14

    Central fatigue is an indispensable biosignal for maintaining life, but the neuronal and molecular mechanisms involved remain unclear. In this study, we searched for genes differentially expressed in the hippocampus of fatigued mice to elucidate the mechanisms underlying fatigue. Mice were forced to swim in an adjustable-current water pool, and the maximum swimming time (endurance) until fatigue was measured thrice. Fatigued and nonfatigued mice with equal swimming capacity and body weight were compared. We found that the genes of GluR1 and B-cell receptor-associated protein 31 (Bap31), which acts as a transport molecule in the secretory pathway or as a mediator of apoptosis, were upregulated in the hippocampus of fatigued mice, and increases of GluR1 and Bap31 were confirmed by Northern blotting and real-time PCR. No change of gene expression of AMPA receptor subunits other than GluR1 was observed. These results suggest that a compositional change of AMPA receptor (increase of GluR1) and upregulation of the Bap31 gene may be implicated in fatigue in mice.

  6. SNP detection in Na/K ATP-ase gene α1 subunit of bisexual and parthenogenetic Artemia strains by RFLP screening.

    Science.gov (United States)

    Manaffar, R; Zare, S; Agh, N; Abdolahzadeh, N; Soltanian, S; Sorgeloos, P; Bossier, P; Van Stappen, G

    2011-01-01

    In order to find a marker for differentiating between a bisexual and a parthenogenetic Artemia strain, Exon-7 of the Na/K ATPase α(1) subunit gene was screened by RFLP technique. The results revealed a constant synonymous SNP (single nucleotide polymorphism) in digestion by the Tru1I enzyme that was consistent with these two types of Artemia. This SNP was identified as an accurate molecular marker for discrimination between bisexual and parthenogenetic Artemia. According to the Nei's genetic distance (1973), the lowest genetic distance was found between individuals from Artemia urmiana Günther 1890 and parthenogenetic populations, making the described marker the first marker to easily distinguish between these two cooccurring species.

  7. Population structure of the Monocelis lineata (Proseriata, Monocelididae species complex assessed by phylogenetic analysis of the mitochondrial Cytochrome c Oxidase subunit I (COI gene

    Directory of Open Access Journals (Sweden)

    Daria Sanna

    2009-01-01

    Full Text Available Monocelis lineata consists of a complex of sibling species, widespread in the Mediterranean and Atlantic Ocean. Previous genetic analysis placed in evidence at least four sibling species. Nevertheless, this research was not conclusive enough to fully resolve the complex or to infer the phylogeny/phylogeography of the group. We designed specific primers aiming at obtaining partial sequences of the mtDNA gene Cytochrome c Oxidase subunit I (COI of M. lineata, and have identified 25 different haplotypes in 32 analyzed individuals. The dendrogram generated by Neighbor-Joining analysis confirmed the differentiation between Atlantic and Mediterranean siblings, as well as the occurrence of at least two Mediterranean sibling species. Thus validated, the method here presented appears as a valuable tool in population genetics and biodiversity surveys on the Monocelis lineata complex.

  8. Differentiation of expression proifles of two calcineurin subunit genes in chicken skeletal muscles during early postnatal growth depending on anatomical location of muscles and breed

    Institute of Scientific and Technical Information of China (English)

    SHAN Yan-ju; XU Wen-juan; SHU Jing-ting; ZHANG Ming; SONG Wei-tao; TAO Zhi-yun; ZHU Chun-hong; LI Hui-fang

    2016-01-01

    Calcineurin (Cn or CaN) is implicated in the control of skeletal muscle ifber phenotype and hypertrophy. However, little information is available concerning the expression of Cn in chickens. In the present study, the expression of two Cn subunit genes (CnAα andCnB1) was quantiifed by qPCR in the lateral gastrocnemius (LG, mainly composing of red fast-twitch myoifbers), the soleus (mainly composing of red slow-twitch myoifbers) and the extensor digitorum longus (EDL, mainly composing of white fast-twitch myoifbers) from Qingyuan partridge chickens (QY, slow-growing chicken breed) and Recessive White chickens (RW, fast-growing chicken breed) on different days (1, 8, 22, 36, 50 and 64 days post-hatching). Although CnAα andCnB1 gene expressions were variable with different trends in different skeletal muscles in the two chicken breeds during postnatal growth, it is highly muscle phenotype and breed speciifc. In general, the levels ofCnAαandCnB1gene expressions of the soleus were lower than those of EDL and LG in both chicken breeds at the same stages. Compared be-tween the two chicken breeds, the levels ofCnAα gene expression of the three skeletal muscles in QY chickens were higher than those in RW chickens on days 1 and 22. However, on day 64, the levels of bothCnAα andCnB1 gene expressions of the three skeletal muscles were lower in QY chickens than those in RW chickens. Correlation analysis of the levels of CnAα andCnB1 gene expressions of the same skeletal muscle showed that there were positive correlations for al three skeletal muscle tissues in two chicken breeds. These results provide some valuable clues to understand the role of Cn in the development of chicken skeletal muscles, with a function that may be related to meat quality.

  9. An upstream initiator caspase 10 of snakehead murrel Channa striatus, containing DED, p20 and p10 subunits: molecular cloning, gene expression and proteolytic activity.

    Science.gov (United States)

    Arockiaraj, Jesu; Gnanam, Annie J; Muthukrishnan, Dhanaraj; Pasupuleti, Mukesh; Milton, James; Singh, Arun

    2013-02-01

    Caspase 10 (CsCasp10) was identified from a constructed cDNA library of freshwater murrel (otherwise called snakehead) Channa striatus. The CsCasp10 is 1838 base pairs (bp) in length and it is encoding 549 amino acid (aa) residues. CsCasp10 amino acid contains two death effector domains (DED) in the N-terminal at 2-77 and 87-154 and it contains caspase family p20 domain (large subunit) and caspase family p10 domain (small subunit) in the C-terminal at 299-425 and 449-536 respectively. Pairwise analysis of CsCasp10 showed the highest sequence similarity (79%) with caspase 10 of Paralichthys olivaceus. Moreover, the phylogenetic analysis showed that CsCasp10 is clustered together with other fish caspase 10, formed a sister group with caspase 10 from other lower vertebrates including amphibian, reptile and birds and finally clustered together with higher vertebrates such as mammals. Significantly (P < 0.05) highest CsCasp10 gene expression was noticed in gills and lowest in intestine. Furthermore, the CsCasp10 gene expression in C. striatus was up-regulated in gills by fungus Aphanomyces invadans and bacteria Aeromonas hydrophila induction. The proteolytic activity was analyzed using the purified recombinant CsCasp10 protein. The results showed the proteolytic activity of CsCasp10 for caspase 10 substrate was 2.5 units per μg protein. Moreover, the proteolytic activities of CsCasp10 in kidney and spleen induced by A. invadans and A. hydrophila stimulation were analyzed by caspase 10 activity assay kit. All these results showed that CsCasp10 are participated in immunity of C. striatus against A. invadans and A. hydrophila infection.

  10. Association of Common Polymorphisms in the Nicotinic Acetylcholine Receptor Alpha4 Subunit Gene with an Electrophysiological Endophenotype in a Large Population-Based Sample.

    Directory of Open Access Journals (Sweden)

    A Mobascher

    Full Text Available Variation in genes coding for nicotinic acetylcholine receptor (nAChR subunits affect cognitive processes and may contribute to the genetic architecture of neuropsychiatric disorders. Single nucleotide polymorphisms (SNPs in the CHRNA4 gene that codes for the alpha4 subunit of alpha4/beta2-containing receptors have previously been implicated in aspects of (mostly visual attention and smoking-related behavioral measures. Here we investigated the effects of six synonymous but functional CHRNA4 exon 5 SNPs on the N100 event-related potential (ERP, an electrophysiological endophenotype elicited by a standard auditory oddball. A total of N = 1,705 subjects randomly selected from the general population were studied with electroencephalography (EEG as part of the German Multicenter Study on nicotine addiction. Two of the six variants, rs1044396 and neighboring rs1044397, were significantly associated with N100 amplitude. This effect was pronounced in females where we also observed an effect on reaction time. Sequencing of the complete exon 5 region in the population sample excluded the existence of additional/functional variants that may be responsible for the observed effects. This is the first large-scale population-based study investigation the effects of CHRNA4 SNPs on brain activity measures related to stimulus processing and attention. Our results provide further evidence that common synonymous CHRNA4 exon 5 SNPs affect cognitive processes and suggest that they also play a role in the auditory system. As N100 amplitude reduction is considered a schizophrenia-related endophenotype the SNPs studied here may also be associated with schizophrenia outcome measures.

  11. The archipelago ubiquitin ligase subunit acts in target tissue to restrict tracheal terminal cell branching and hypoxic-induced gene expression.

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    Nathan T Mortimer

    Full Text Available The Drosophila melanogaster gene archipelago (ago encodes the F-box/WD-repeat protein substrate specificity factor for an SCF (Skp/Cullin/F-box-type polyubiquitin ligase that inhibits tumor-like growth by targeting proteins for degradation by the proteasome. The Ago protein is expressed widely in the fly embryo and larva and promotes degradation of pro-proliferative proteins in mitotically active cells. However the requirement for Ago in post-mitotic developmental processes remains largely unexplored. Here we show that Ago is an antagonist of the physiologic response to low oxygen (hypoxia. Reducing Ago activity in larval muscle cells elicits enhanced branching of nearby tracheal terminal cells in normoxia. This tracheogenic phenotype shows a genetic dependence on sima, which encodes the HIF-1α subunit of the hypoxia-inducible transcription factor dHIF and its target the FGF ligand branchless (bnl, and is enhanced by depletion of the Drosophila Von Hippel Lindau (dVHL factor, which is a subunit of an oxygen-dependent ubiquitin ligase that degrades Sima/HIF-1α protein in metazoan cells. Genetic reduction of ago results in constitutive expression of some hypoxia-inducible genes in normoxia, increases the sensitivity of others to mild hypoxic stimulus, and enhances the ability of adult flies to recover from hypoxic stupor. As a molecular correlate to these genetic data, we find that Ago physically associates with Sima and restricts Sima levels in vivo. Collectively, these findings identify Ago as a required element of a circuit that suppresses the tracheogenic activity of larval muscle cells by antagonizing the Sima-mediated transcriptional response to hypoxia.

  12. PKA-chromatin association at stress responsive target genes from Saccharomyces cerevisiae.

    Science.gov (United States)

    Baccarini, Leticia; Martínez-Montañés, Fernando; Rossi, Silvia; Proft, Markus; Portela, Paula

    2015-11-01

    Gene expression regulation by intracellular stimulus-activated protein kinases is essential for cell adaptation to environmental changes. There are three PKA catalytic subunits in Saccharomyces cerevisiae: Tpk1, Tpk2, and Tpk3 and one regulatory subunit: Bcy1. Previously, it has been demonstrated that Tpk1 and Tpk2 are associated with coding regions and promoters of target genes in a carbon source and oxidative stress dependent manner. Here we studied five genes, ALD6, SED1, HSP42, RPS29B, and RPL1B whose expression is regulated by saline stress. We found that PKA catalytic and regulatory subunits are associated with both coding regions and promoters of the analyzed genes in a stress dependent manner. Tpk1 and Tpk2 recruitment was completely abolished in catalytic inactive mutants. BCY1 deletion changed the binding kinetic to chromatin of each Tpk isoform and this strain displayed a deregulated gene expression in response to osmotic stress. In addition, yeast mutants with high PKA activity exhibit sustained association to target genes of chromatin-remodeling complexes such as Snf2-catalytic subunit of the SWI/SNF complex and Arp8-component of INO80 complex, leading to upregulation of gene expression during osmotic stress. Tpk1 accumulation in the nucleus was stimulated upon osmotic stress, while the nuclear localization of Tpk2 and Bcy1 showed no change. We found that each PKA subunit is transported into the nucleus by a different β-karyopherin pathway. Moreover, β-karyopherin mutant strains abolished the chromatin association of Tpk1 or Tpk2, suggesting that nuclear localization of PKA catalytic subunits is required for its association to target genes and properly gene expression.

  13. Regulation of the nuclear gene that encodes the alpha-subunit of the mitochondrial F0F1-ATP synthase complex. Activation by upstream stimulatory factor 2.

    Science.gov (United States)

    Breen, G A; Jordan, E M

    1997-04-18

    We have previously identified several positive cis-acting regulatory regions in the promoters of the bovine and human nuclear-encoded mitochondrial F0F1-ATP synthase alpha-subunit genes (ATPA). One of these cis-acting regions contains the sequence 5'-CACGTG-3' (an E-box), to which a number of transcription factors containing a basic helix-loop-helix motif can bind. This E-box element is required for maximum activity of the ATPA promoter in HeLa cells. The present study identifies the human transcription factor, upstream stimulatory factor 2 (USF2), as a nuclear factor that binds to the ATPA E-box and demonstrates that USF2 plays a critical role in the activation of the ATPA gene in vivo. Evidence includes the following. Antiserum directed against USF2 recognized factors present in HeLa nuclear extracts that interact with the ATPA promoter in mobility shift assays. Wild-type USF2 proteins synthesized from expression vectors trans-activated the ATPA promoter through the E-box, whereas truncated USF2 proteins devoid of the amino-terminal activation domains did not. Importantly, expression of a dominant-negative mutant of USF2 lacking the basic DNA binding domain but able to dimerize with endogenous USF proteins significantly reduced the level of activation of the ATPA promoter caused by ectopically coexpressed USF2, demonstrating the importance of endogenous USF2 in activation of the ATPA gene.

  14. The largest subunit of RNA polymerase II as a new marker gene to study assemblages of arbuscular mycorrhizal fungi in the field.

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    Herbert Stockinger

    Full Text Available Due to the potential of arbuscular mycorrhizal fungi (AMF, Glomeromycota to improve plant growth and soil quality, the influence of agricultural practice on their diversity continues to be an important research question. Up to now studies of community diversity in AMF have exclusively been based on nuclear ribosomal gene regions, which in AMF show high intra-organism polymorphism, seriously complicating interpretation of these data. We designed specific PCR primers for 454 sequencing of a region of the largest subunit of RNA polymerase II gene, and established a new reference dataset comprising all major AMF lineages. This gene is known to be monomorphic within fungal isolates but shows an excellent barcode gap between species. We designed a primer set to amplify all known lineages of AMF and demonstrated its applicability in combination with high-throughput sequencing in a long-term tillage experiment. The PCR primers showed a specificity of 99.94% for glomeromycotan sequences. We found evidence of significant shifts of the AMF communities caused by soil management and showed that tillage effects on different AMF taxa are clearly more complex than previously thought. The high resolving power of high-throughput sequencing highlights the need for quantitative measurements to efficiently detect these effects.

  15. Phylogenetic relationships within Taenia taeniaeformis variants and other taeniid cestodes inferred from the nucleotide sequence of the cytochrome c oxidase subunit I gene.

    Science.gov (United States)

    Okamoto, M; Bessho, Y; Kamiya, M; Kurosawa, T; Horii, T

    1995-01-01

    Nucleotide sequence variations in a region of the mitochondrial cytochrome c oxidase subunit I (COI) gene (391 bp) were examined within seven species of the genus Taenia and two species of the genus Echinococcus, including ten isolates of T. taeniaeformis and six isolates of E. multilocularis. More than a 12% rate of nucleotide differences between taeniid species was found, allowing the species to be distinguished. In E. multilocularis, no sequence variation was observed among isolates, regardless of the host (gray red-backed vole, tundra vole, pig, Norway rat) or area (Japan, Alaska) from which each metacestode had been isolated. In contrast, six distinct sequences were detected among the ten T. taeniaeformis isolates examined. The level of nucleotide variation in the COI gene within T. taeniaeformis isolates except for one isolate from the gray red-backed vole (TtACR), which has been proposed as a distinct strain or a different species, was about 0.3%-4.1%, whereas the COI gene sequence for TtACR differed from those of the other isolates, with levels being 9.0%-9.5%. Phylogenetic trees were then inferred from these sequence data using two different algorithms.

  16. Characterization of a gene from chromosome 1B encoding the large subunit of ADPglucose pyrophosphorylase from wheat: evolutionary divergence and differential expression of Agp2 genes between leaves and developing endosperm.

    Science.gov (United States)

    Thorneycroft, David; Hosein, Felicia; Thangavelu, Madan; Clark, Joanna; Vizir, Igor; Burrell, Michael M; Ainsworth, Charles

    2003-07-01

    A full-length genomic clone containing the gene encoding the large subunit of the ADPglucose pyrophosphorylase (Agp2), was isolated from a genomic library prepared from etiolated shoots of hexaploid wheat (Triticum aestivum L., cv, Chinese Spring). The coding region of this gene is identical to one of the cDNA clones previously isolated from a developing wheat grain cDNA library and is therefore an actively transcribed gene. The sequence represented by the cDNA spans 4.8 kb of the genomic clone and contains 15 introns. 2852 bp of DNA flanking the transcription start site of the gene was cloned upstream of the GUS (beta-glucuronidase) reporter gene. This Agp2::GUS construct and promoter deletions were used to study the pattern of reporter gene expression in both transgenic tobacco and wheat plants. Histochemical analysis of GUS expression in transgenic tobacco demonstrated that the reporter gene was expressed in guard cells of leaves and throughout the seed. In transgenic wheat, reporter gene expression was confined to the endosperm and aleurone with no expression in leaves. The cloned Agp2 gene was located to chromosome 1B by gene-specific PCR with nullisomic-tetrasomic lines. Northern analysis demonstrated that the Agp2 genes are differentially expressed in leaves and developing endosperm; while all three classes of Agp2 genes are transcribed in developing wheat grain endosperm, only one is transcribed in leaves. The differences between the Agp2 genes are discussed in relation to the evolution of hexaploid wheat.

  17. A Cyclin Dependent Kinase Regulatory Subunit (CKS) Gene of Pigeonpea Imparts Abiotic Stress Tolerance and Regulates Plant Growth and Development in Arabidopsis.

    Science.gov (United States)

    Tamirisa, Srinath; Vudem, Dashavantha R; Khareedu, Venkateswara R

    2017-01-01

    Frequent climatic changes in conjunction with other extreme environmental factors are known to affect growth, development and productivity of diverse crop plants. Pigeonpea, a major grain legume of the semiarid tropics, endowed with an excellent deep-root system, is known as one of the important drought tolerant crop plants. Cyclin dependent kinases (CDKs) are core cell cycle regulators and play important role in different aspects of plant growth and development. The cyclin-dependent kinase regulatory subunit gene (CKS) was isolated from the cDNA library of pigeonpea plants subjected to drought stress. Pigeonpea CKS (CcCKS) gene expression was detected in both the root and leaf tissues of pigeonpea and was upregulated by polyethylene glycol (PEG), mannitol, NaCl and abscisic acid (ABA) treatments. The overexpression of CcCKS gene in Arabidopsis significantly enhanced tolerance of transgenics to drought and salt stresses as evidenced by different physiological parameters. Under stress conditions, transgenics showed higher biomass, decreased rate of water loss, decreased MDA levels, higher free proline contents, and glutathione levels. Moreover, under stress conditions transgenics exhibited lower stomatal conductance, lower transpiration, and higher photosynthetic rates. However, under normal conditions, CcCKS-transgenics displayed decreased plant growth rate, increased cell size and decreased stomatal number compared to those of wild-type plants. Real-time polymerase chain reaction revealed that CcCKS could regulate the expression of both ABA-dependent and ABA-independent genes associated with abiotic stress tolerance as well as plant growth and development. As such, the CcCKS seems promising and might serve as a potential candidate gene for enhancing the abiotic stress tolerance of crop plants.

  18. Knockdown of the Rhipicephalus microplus cytochrome c oxidase subunit III gene is associated with a failure of Anaplasma marginale transmission.

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    Thais D Bifano

    Full Text Available Rhipicephalus microplus is an obligate hematophagous ectoparasite of cattle and an important biological vector of Anaplasma marginale in tropical and subtropical regions. The primary determinants for A. marginale transmission are infection of the tick gut, followed by infection of salivary glands. Transmission of A. marginale to cattle occurs via infected saliva delivered during tick feeding. Interference in colonization of either the tick gut or salivary glands can affect transmission of A. marginale to naïve animals. In this study, we used the tick embryonic cell line BME26 to identify genes that are modulated in response to A. marginale infection. Suppression-subtractive hybridization libraries (SSH were constructed, and five up-regulated genes {glutathione S-transferase (GST, cytochrome c oxidase sub III (COXIII, dynein (DYN, synaptobrevin (SYN and phosphatidylinositol-3,4,5-triphosphate 3-phosphatase (PHOS} were selected as targets for functional in vivo genomic analysis. RNA interference (RNAi was used to determine the effect of tick gene knockdown on A. marginale acquisition and transmission. Although RNAi consistently knocked down all individually examined tick genes in infected tick guts and salivary glands, only the group of ticks injected with dsCOXIII failed to transmit A. marginale to naïve calves. To our knowledge, this is the first report demonstrating that RNAi of a tick gene is associated with a failure of A. marginale transmission.

  19. Analysis of the cytochrome c oxidase subunit 1 (COX1) gene reveals the unique evolution of the giant panda.

    Science.gov (United States)

    Hu, Yao-Dong; Pang, Hui-Zhong; Li, De-Sheng; Ling, Shan-Shan; Lan, Dan; Wang, Ye; Zhu, Yun; Li, Di-Yan; Wei, Rong-Ping; Zhang, He-Min; Wang, Cheng-Dong

    2016-11-05

    As the rate-limiting enzyme of the mitochondrial respiratory chain, cytochrome c oxidase (COX) plays a crucial role in biological metabolism. "Living fossil" giant panda (Ailuropoda melanoleuca) is well-known for its special bamboo diet. In an effort to explore functional variation of COX1 in the energy metabolism behind giant panda's low-energy bamboo diet, we looked at genetic variation of COX1 gene in giant panda, and tested for its selection effect. In 1545 base pairs of the gene from 15 samples, 9 positions were variable and 1 mutation leaded to an amino acid sequence change. COX1 gene produces six haplotypes, nucleotide (pi), haplotype diversity (Hd). In addition, the average number of nucleotide differences (k) is 0.001629±0.001036, 0.8083±0.0694 and 2.517, respectively. Also, dN/dS ratio is significantly below 1. These results indicated that giant panda had a low population genetic diversity, and an obvious purifying selection of the COX1 gene which reduces synthesis of ATP determines giant panda's low-energy bamboo diet. Phylogenetic trees based on the COX1 gene were constructed to demonstrate that giant panda is the sister group of other Ursidae.

  20. Role of positron emission tomography and bone scintigraphy in the evaluation of bone involvement in metastatic pheochromocytoma and paraganglioma: specific implications for succinate dehydrogenase enzyme subunit B gene mutations.

    NARCIS (Netherlands)

    Zelinka, T.; Timmers, H.J.L.M.; Kozupa, A.; Chen, C.C.; Carrasquillo, J.A.; Reynolds, J.C.; Ling, A.; Eisenhofer, G.; Lazurova, I.; Adams, K.T.; Whatley, M.A.; Widimsky, J.Jr.; Pacak, K.

    2008-01-01

    We performed a retrospective analysis of 71 subjects with metastatic pheochromocytoma and paraganglioma (30 subjects with mutation of succinate dehydrogenase enzyme subunit B (SDHB) gene and 41 subjects without SDHB mutation). Sixty-nine percent presented with bone metastases (SDHB +/-: 77% vs 63%),

  1. Molecular cloning and functional expression of the Equine K+ channel KV11.1 (Ether à Go-Go-related/KCNH2 gene) and the regulatory subunit KCNE2 from equine myocardium

    DEFF Research Database (Denmark)

    Pedersen, Philip Juul; Thomsen, Kirsten Brolin; Olander, Emma Rie;

    2015-01-01

    The KCNH2 and KCNE2 genes encode the cardiac voltage-gated K+ channel KV11.1 and its auxiliary β subunit KCNE2. KV11.1 is critical for repolarization of the cardiac action potential. In humans, mutations or drug therapy affecting the KV11.1 channel are associated with prolongation of the QT inter...

  2. Gene expression profile of sodium channel subunits in the anterior cingulate cortex during experimental paclitaxel-induced neuropathic pain in mice

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    Willias Masocha

    2016-11-01

    Full Text Available Paclitaxel, a chemotherapeutic agent, causes neuropathic pain whose supraspinal pathophysiology is not fully understood. Dysregulation of sodium channel expression, studied mainly in the periphery and spinal cord level, contributes to the pathogenesis of neuropathic pain. We examined gene expression of sodium channel (Nav subunits by real time polymerase chain reaction (PCR in the anterior cingulate cortex (ACC at day 7 post first administration of paclitaxel, when mice had developed paclitaxel-induced thermal hyperalgesia. The ACC was chosen because increased activity in the ACC has been observed during neuropathic pain. In the ACC of vehicle-treated animals the threshold cycle (Ct values for Nav1.4, Nav1.5, Nav1.7, Nav1.8 and Nav1.9 were above 30 and/or not detectable in some samples. Thus, comparison in mRNA expression between untreated control, vehicle-treated and paclitaxel treated animals was done for Nav1.1, Nav1.2, Nav1.3, Nav1.6, Nax as well as Navβ1–Navβ4. There were no differences in the transcript levels of Nav1.1–Nav1.3, Nav1.6, Nax, Navβ1–Navβ3 between untreated and vehicle-treated mice, however, vehicle treatment increased Navβ4 expression. Paclitaxel treatment significantly increased the mRNA expression of Nav1.1, Nav1.2, Nav1.6 and Nax, but not Nav1.3, sodium channel alpha subunits compared to vehicle-treated animals. Treatment with paclitaxel significantly increased the expression of Navβ1 and Navβ3, but not Navβ2 and Navβ4, sodium channel beta subunits compared to vehicle-treated animals. These findings suggest that during paclitaxel-induced neuropathic pain (PINP there is differential upregulation of sodium channels in the ACC, which might contribute to the increased neuronal activity observed in the area during neuropathic pain.

  3. Gene expression profile of sodium channel subunits in the anterior cingulate cortex during experimental paclitaxel-induced neuropathic pain in mice

    Science.gov (United States)

    2016-01-01

    Paclitaxel, a chemotherapeutic agent, causes neuropathic pain whose supraspinal pathophysiology is not fully understood. Dysregulation of sodium channel expression, studied mainly in the periphery and spinal cord level, contributes to the pathogenesis of neuropathic pain. We examined gene expression of sodium channel (Nav) subunits by real time polymerase chain reaction (PCR) in the anterior cingulate cortex (ACC) at day 7 post first administration of paclitaxel, when mice had developed paclitaxel-induced thermal hyperalgesia. The ACC was chosen because increased activity in the ACC has been observed during neuropathic pain. In the ACC of vehicle-treated animals the threshold cycle (Ct) values for Nav1.4, Nav1.5, Nav1.7, Nav1.8 and Nav1.9 were above 30 and/or not detectable in some samples. Thus, comparison in mRNA expression between untreated control, vehicle-treated and paclitaxel treated animals was done for Nav1.1, Nav1.2, Nav1.3, Nav1.6, Nax as well as Navβ1–Navβ4. There were no differences in the transcript levels of Nav1.1–Nav1.3, Nav1.6, Nax, Navβ1–Navβ3 between untreated and vehicle-treated mice, however, vehicle treatment increased Navβ4 expression. Paclitaxel treatment significantly increased the mRNA expression of Nav1.1, Nav1.2, Nav1.6 and Nax, but not Nav1.3, sodium channel alpha subunits compared to vehicle-treated animals. Treatment with paclitaxel significantly increased the expression of Navβ1 and Navβ3, but not Navβ2 and Navβ4, sodium channel beta subunits compared to vehicle-treated animals. These findings suggest that during paclitaxel-induced neuropathic pain (PINP) there is differential upregulation of sodium channels in the ACC, which might contribute to the increased neuronal activity observed in the area during neuropathic pain. PMID:27896032

  4. Extracellular signal-regulated kinase mediates gonadotropin subunit gene expression and LH release responses to endogenous gonadotropin-releasing hormones in goldfish.

    Science.gov (United States)

    Klausen, Christian; Booth, Morgan; Habibi, Hamid R; Chang, John P

    2008-08-01

    The possible involvement of extracellular signal-regulated kinase (ERK) in mediating the stimulatory actions of two endogenous goldfish gonadotropin-releasing hormones (salmon (s)GnRH and chicken (c)GnRH-II) on gonadotropin synthesis and secretion was examined. Western blot analysis revealed the presence of ERK and phosphorylated (p)ERK in goldfish brain, pituitary, liver, ovary, testis and muscle tissue extracts, as well as extracts of dispersed goldfish pituitary cells and HeLa cells. Interestingly, a third ERK-like immunoreactive band of higher molecular mass was detected in goldfish tissue and pituitary cell extracts in addition to the ERK1-p44- and ERK2-p42-like immunoreactive bands. Incubation of primary cultures of goldfish pituitary cells with either a PKC-activating 4beta-phorbol ester (TPA) or a synthetic diacylglycerol, but not a 4alpha-phorbol ester, elevated the ratio of pERK/total (t)ERK for all three ERK isoforms. The stimulatory effects of TPA were attenuated by the PKC inhibitor GF109203X and the MEK inhibitor PD98059. sGnRH and cGnRH-II also elevated the ratio of pERK/tERK for all three ERK isoforms, in a time-, dose- and PD98059-dependent manner. In addition, treatment with PD98059 reduced the sGnRH-, cGnRH-II- and TPA-induced increases in gonadotropin subunit mRNA levels in Northern blot studies and sGnRH- and cGnRH-II-elicited LH release in cell column perifusion studies with goldfish pituitary cells. These results indicate that GnRH and PKC can activate ERK through MEK in goldfish pituitary cells. More importantly, the present study suggests that GnRH-induced gonadotropin subunit gene expression and LH release involve MEK/ERK signaling in goldfish.

  5. Alternative-splicing in the exon-10 region of GABA(A receptor beta(2 subunit gene: relationships between novel isoforms and psychotic disorders.

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    Cunyou Zhao

    Full Text Available BACKGROUND: Non-coding single nucleotide polymorphisms (SNPs in GABRB2, the gene for beta(2-subunit of gamma-aminobutyric acid type A (GABA(A receptor, have been associated with schizophrenia (SCZ and quantitatively correlated to mRNA expression and alternative splicing. METHODS AND FINDINGS: Expression of the Exon 10 region of GABRB2 from minigene constructs revealed this region to be an "alternative splicing hotspot" that readily gave rise to differently spliced isoforms depending on intron sequences. This led to a search in human brain cDNA libraries, and the discovery of two novel isoforms, beta(2S1 and beta(2S2, bearing variations in the neighborhood of Exon-10. Quantitative real-time PCR analysis of postmortem brain samples showed increased beta(2S1 expression and decreased beta(2S2 expression in both SCZ and bipolar disorder (BPD compared to controls. Disease-control differences were significantly correlated with SNP rs187269 in BPD males for both beta(2S1 and beta(2S2 expressions, and significantly correlated with SNPs rs2546620 and rs187269 in SCZ males for beta(2S2 expression. Moreover, site-directed mutagenesis indicated that Thr(365, a potential phosphorylation site in Exon-10, played a key role in determining the time profile of the ATP-dependent electrophysiological current run-down. CONCLUSION: This study therefore provided experimental evidence for the importance of non-coding sequences in the Exon-10 region in GABRB2 with respect to beta(2-subunit splicing diversity and the etiologies of SCZ and BPD.

  6. New genes encoding subunits of a cytochrome bc1-analogous complex in the respiratory chain of the hyperthermoacidophilic crenarchaeon Sulfolobus acidocaldarius.

    Science.gov (United States)

    Hiller, A; Henninger, T; Schäfer, G; Schmidt, C L

    2003-04-01

    The soxL gene from Sulfolobus acidocaldarius (DSM 639) encodes a Rieske iron-sulfur protein. In this study we report the identification of two open reading frames in its downstream region. The first one, named soxN, codes for a membrane protein bearing a resemblance to the b-type cytochromes of the cytochrome bc1 and b6f complexes. The protein is predicted to contain at least 10 transmembrane helices and features the two conserved histidine pairs coordinating the heme groups of these cytochromes. The second open reading frame, named odsN, encodes a soluble protein of unknown function. The genomic region displays a complex transcription pattern. Northern blot and RT-PCR analyses revealed the presence of mono- and bi-cistronic transcripts as well as a tri-cistronic transcript of soxL and cbsAB, encoding the mono-heme cytochrome b558/566. Phylogenetic analyses of the genes of the soxLN pair and of other archaeal gene pairs encoding Rieske iron-sulfur proteins and b-type cytochromes revealed an identical branching patterns for both protein families, suggesting an evolutionary link of these genes provided by the functional interaction of the proteins. On the basis of the findings of this study and the previously studied properties of the soxL and cbsA proteins, we propose the occurrence of a novel cytochrome bc1-analogous complex in the membranes of Sulfolobus, consisting of the cytochrome b homolog soxN, the Rieske protein soxL, the high potential cytochrome cbsA, as well as the non-redox-active subunits cbsB and odsN.

  7. Gene splicing of an invertebrate beta subunit (LCavβ in the N-terminal and HOOK domains and its regulation of LCav1 and LCav2 calcium channels.

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    Taylor F Dawson

    Full Text Available The accessory beta subunit (Ca(vβ of calcium channels first appear in the same genome as Ca(v1 L-type calcium channels in single-celled coanoflagellates. The complexity of this relationship expanded in vertebrates to include four different possible Ca(vβ subunits (β1, β2, β3, β4 which associate with four Ca(v1 channel isoforms (Ca(v1.1 to Ca(v1.4 and three Ca(v2 channel isoforms (Ca(v2.1 to Ca(v2.3. Here we assess the fundamentally-shared features of the Ca(vβ subunit in an invertebrate model (pond snail Lymnaea stagnalis that bears only three homologous genes: (LCa(v1, LCa(v2, and LCa(vβ. Invertebrate Ca(vβ subunits (in flatworms, snails, squid and honeybees slow the inactivation kinetics of Ca(v2 channels, and they do so with variable N-termini and lacking the canonical palmitoylation residues of the vertebrate β2a subunit. Alternative splicing of exon 7 of the HOOK domain is a primary determinant of a slow inactivation kinetics imparted by the invertebrate LCa(vβ subunit. LCa(vβ will also slow the inactivation kinetics of LCa(v3 T-type channels, but this is likely not physiologically relevant in vivo. Variable N-termini have little influence on the voltage-dependent inactivation kinetics of differing invertebrate Ca(vβ subunits, but the expression pattern of N-terminal splice isoforms appears to be highly tissue specific. Molluscan LCa(vβ subunits have an N-terminal "A" isoform (coded by exons: 1a and 1b that structurally resembles the muscle specific variant of vertebrate β1a subunit, and has a broad mRNA expression profile in brain, heart, muscle and glands. A more variable "B" N-terminus (exon 2 in the exon position of mammalian β3 and has a more brain-centric mRNA expression pattern. Lastly, we suggest that the facilitation of closed-state inactivation (e.g. observed in Ca(v2.2 and Ca(vβ3 subunit combinations is a specialization in vertebrates, because neither snail subunit (LCa(v2 nor LCa(vβ appears to be compatible

  8. Effects of Catalytic Subunitαof Protein Phosphatase 2A on Cell Migration of Mouse Embryo Fibroblasts%蛋白磷酸酶2A 催化亚基α在小鼠胚胎成纤维细胞迁移中的作用研究

    Institute of Scientific and Technical Information of China (English)

    王庆华; 王生存; 李斌; 刘春; 吴刘成; 王旭; 邵义祥

    2016-01-01

    利用体外基因敲除技术检测小鼠胚胎成纤维细胞(mouse embryonic fibroblasts,MEFs)周期和迁移能力的变化,探究蛋白磷酸酶2A 的 Cα亚基在 MEFs 细胞迁移过程中的作用。用 Cαfl/fl的纯合子雄鼠与雌鼠1∶2配对,取 E12.5 d 的胚胎制作小鼠胚胎成纤维细胞。利用表达 Cre 重组酶(Ad-Cre-EGFP)以及GFP 荧光蛋白(Ad-EGFP)的腺病毒载体,对 P3代 MEFs 进行感染,分别用 PCR,RT-PCR 和 Western blot方法进行基因型鉴定。同时利用流式分选技术检测感染后 MEFs 的细胞周期的变化,利用细胞划痕试验检测了其细胞迁移能力的变化。结果显示,胰酶消化的方法成功获得了小鼠胚胎成纤维细胞,DNA,RNA 和蛋白水平的鉴定获得了3对3的野生型和基因敲除 MEFs。流式分选发现 Ad-Cre 处理的 MEFs 与 Ad-EG-FP 处理的 MEFs 相比,处于 G2期的细胞比例为30.8%±2.57%,高于 Ad-EGFP MEFs 的23.9%±2.46%,并且细胞碎片的比例也远高于后者。划痕试验表明,Cα亚基缺失后细胞迁移能力下降,12 h 就显著低于对照组。结果表明,腺病毒携带的 Cre 重组酶能够在体外有效地敲除掉 MEFs 中的蛋白磷酸酶2A 的Cα亚基,PP2A Cα亚基的缺失会导致 MEFs 细胞周期倾向于阻滞在 G2期,并会降低细胞迁移的能力。%To investigate the effects of catalytic subunit α of protein phosphatase 2A (PP2A)on cell cycle and cell migration in mouse embryo fibroblasts(MEFs),the mouse embryos were got at E12.5 by mating the heterozygotes of Cαsubunit conditional knockout male and female homozygous mice at the ratio of 1∶2.MEFs were prepared by trypsin digestion and genotyped by PCR,RT-PCR and Western blot to identify the genetic basis of each embryo.Adenovirus associated Cre recombinase and GFP immunofluorescence protein were constructed and delivered to the P3 MEFs to knockout the Cαgene,GFP was used as control

  9. Role of the Rubisco Small Subunit

    Energy Technology Data Exchange (ETDEWEB)

    Spreitzer, Robert Joseph [Univ. of Nebraska, Lincoln, NE (United States)

    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

  10. 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.

    Science.gov (United States)

    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.

  11. Impairment of the tRNA-splicing endonuclease subunit 54 (tsen54) gene causes neurological abnormalities and larval death in zebrafish models of pontocerebellar hypoplasia.

    Science.gov (United States)

    Kasher, Paul R; Namavar, Yasmin; van Tijn, Paula; Fluiter, Kees; Sizarov, Aleksander; Kamermans, Maarten; Grierson, Andrew J; Zivkovic, Danica; Baas, Frank

    2011-04-15

    Pontocerebellar hypoplasia (PCH) represents a group (PCH1-6) of neurodegenerative autosomal recessive disorders characterized by hypoplasia and/or atrophy of the cerebellum, hypoplasia of the ventral pons, progressive microcephaly and variable neocortical atrophy. The majority of PCH2 and PCH4 cases are caused by mutations in the TSEN54 gene; one of the four subunits comprising the tRNA-splicing endonuclease (TSEN) complex. We hypothesized that TSEN54 mutations act through a loss of function mechanism. At 8 weeks of gestation, human TSEN54 is expressed ubiquitously in the brain, yet strong expression is seen within the telencephalon and metencephalon. Comparable expression patterns for tsen54 are observed in zebrafish embryos. Morpholino (MO) knockdown of tsen54 in zebrafish embryos results in loss of structural definition in the brain. This phenotype was partially rescued by co-injecting the MO with human TSEN54 mRNA. A developmental patterning defect was not associated with tsen54 knockdown; however, an increase in cell death within the brain was observed, thus bearing resemblance to PCH pathophysiology. Additionally, N-methyl-N-nitrosourea mutant zebrafish homozygous for a tsen54 premature stop-codon mutation die within 9 days post-fertilization. To determine whether a common disease pathway exists between TSEN54 and other PCH-related genes, we also monitored the effects of mitochondrial arginyl-tRNA synthetase (rars2; PCH1 and PCH6) knockdown in zebrafish. Comparable brain phenotypes were observed following the inhibition of both genes. These data strongly support the hypothesis that TSEN54 mutations cause PCH through a loss of function mechanism. Also we suggest that a common disease pathway may exist between TSEN54- and RARS2-related PCH, which may involve a tRNA processing-related mechanism.

  12. Fifteen novel mutations in the mitochondrial NADH dehydrogenase subunit 1, 2, 3, 4, 4L, 5 and 6 genes from Iranian patients with Leber's hereditary optic neuropathy (LHON).

    Science.gov (United States)

    Rezvani, Zahra; Didari, Elmira; Arastehkani, Ahoura; Ghodsinejad, Vadieh; Aryani, Omid; Kamalidehghan, Behnam; Houshmand, Massoud

    2013-12-01

    Leber's hereditary optic neuropathy (LHON) is an optic nerve dysfunction resulting from mutations in mitochondrial DNA (mtDNA), which is transmitted in a maternal pattern of inheritance. It is caused by three primary point mutations: G11778A, G3460A and T14484C; in the mitochondrial genome. These mutations are sufficient to induce the disease, accounting for the majority of LHON cases, and affect genes that encode for the different subunits of mitochondrial complexes I and III of the mitochondrial respiratory chain. Other mutations are secondary mutations associated with the primary mutations. The purpose of this study was to determine MT-ND variations in Iranian patients with LHON. In order to determine the prevalence and distribution of mitochondrial mutations in the LHON patients, their DNA was studied using PCR and DNA sequencing analysis. Sequencing of MT-ND genes from 35 LHON patients revealed a total of 44 nucleotide variations, in which fifteen novel variations-A14020G, A13663G, C10399T, C4932A, C3893G, C10557A, C12012A, C13934T, G4596A, T12851A, T4539A, T4941A, T13255A, T14353C and del A 4513-were observed in 27 LHON patients. However, eight patients showed no variation in the ND genes. These mutations contribute to the current database of mtDNA polymorphisms in LHON patients and may facilitate the definition of disease-related mutations in human mtDNA. This research may help to understand the disease mechanism and open up new diagnostic opportunities for LHON.

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

    Science.gov (United States)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Beckers, M.C.; Ernst, E.; Gros, P. [McGill Univ., Montreal (Canada)

    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. 17 refs., 1 fig., 3 tabs.

  15. A multilevel prediction of physiological response to challenge: Interactions among child maltreatment, neighborhood crime, endothelial nitric oxide synthase gene (eNOS), and GABA(A) receptor subunit alpha-6 gene (GABRA6).

    Science.gov (United States)

    Lynch, Michael; Manly, Jody Todd; Cicchetti, Dante

    2015-11-01

    Physiological response to stress has been linked to a variety of healthy and pathological conditions. The current study conducted a multilevel examination of interactions among environmental toxins (i.e., neighborhood crime and child maltreatment) and specific genetic polymorphisms of the endothelial nitric oxide synthase gene (eNOS) and GABA(A) receptor subunit alpha-6 gene (GABRA6). One hundred eighty-six children were recruited at age 4. The presence or absence of child maltreatment as well as the amount of crime that occurred in their neighborhood during the previous year were determined at that time. At age 9, the children were brought to the lab, where their physiological response to a cognitive challenge (i.e., change in the amplitude of the respiratory sinus arrhythmia) was assessed and DNA samples were collected for subsequent genotyping. The results confirmed that complex Gene × Gene, Environment × Environment, and Gene × Environment interactions were associated with different patterns of respiratory sinus arrhythmia reactivity. The implications for future research and evidence-based intervention are discussed.

  16. Nuclear respiratory factor 2 regulates the expression of the same NMDA receptor subunit genes as NRF-1: both factors act by a concurrent and parallel mechanism to couple energy metabolism and synaptic transmission.

    Science.gov (United States)

    Priya, Anusha; Johar, Kaid; Wong-Riley, Margaret T T

    2013-01-01

    Neuronal activity and energy metabolism are tightly coupled processes. Previously, we found that nuclear respiratory factor 1 (NRF-1) transcriptionally co-regulates energy metabolism and neuronal activity by regulating all 13 subunits of the critical energy generating enzyme, cytochrome c oxidase (COX), as well as N-methyl-d-aspartate (NMDA) receptor subunits 1 and 2B, GluN1 (Grin1) and GluN2B (Grin2b). We also found that another transcription factor, nuclear respiratory factor 2 (NRF-2 or GA-binding protein) regulates all subunits of COX as well. The goal of the present study was to test our hypothesis that NRF-2 also regulates specific subunits of NMDA receptors, and that it functions with NRF-1 via one of three mechanisms: complementary, concurrent and parallel, or a combination of complementary and concurrent/parallel. By means of multiple approaches, including in silico analysis, electrophoretic mobility shift and supershift assays, in vivo chromatin immunoprecipitation of mouse neuroblastoma cells and rat visual cortical tissue, promoter mutations, real-time quantitative PCR, and western blot analysis, NRF-2 was found to functionally regulate Grin1 and Grin2b genes, but not any other NMDA subunit genes. Grin1 and Grin2b transcripts were up-regulated by depolarizing KCl, but silencing of NRF-2 prevented this up-regulation. On the other hand, over-expression of NRF-2 rescued the down-regulation of these subunits by the impulse blocker TTX. NRF-2 binding sites on Grin1 and Grin2b are conserved among species. Our data indicate that NRF-2 and NRF-1 operate in a concurrent and parallel manner in mediating the tight coupling between energy metabolism and neuronal activity at the molecular level.

  17. Distribution of genotypes C825T polymorphism G-protein β3-subunit gene in patients with hypertension depending on body mass index

    Directory of Open Access Journals (Sweden)

    Prystupa L.N.

    2015-09-01

    Full Text Available The aim of the study was to investigate the frequency of genotypes of C825T polymorphism G-protein β3-subunit gene (GNB3 in patients with arterial hypertension (AH, depending on body mass index (BMI. The study involved 155 patients with verified diagnosis of AH (study group and 50 healthy individuals (control group. The patients of the main group were divided into 3 groups according to BMI: I - 35 patients with normal body weight, II - 38 patients with overweight, III - 82 patients with obesity. We used general clinical, anthropometric, instrumental, molecular-genetic and statistical methods. Probability of differences in the frequency of alleles and genotypes was determined using χ² criteria. Pairwise comparison of groups was made using nonparametric Mann-Whitney test. The difference was considered statistically significant at p <0,05. Investigation of the distribution of genotypes C825T polymorphism GNB3 in patients with AH according to BMI showed statistically significant increase in the frequency of genotypes C / T and T / T and T allele in patients with overweight and obesity as compared with patients with normal body weight (χ² = 26 8; p <0.001. The risk of weight increase in AH patients with T allele carriers is 2,2 times higher than in C allele carriers. Association of C825T polymorphism of GNB3 with a tendency to obesity and overweight in patients with AH was proved.

  18. Genetic structure of the snakehead murrel, Channa striata (channidae) based on the cytochrome c oxidase subunit I gene: Influence of historical and geomorphological factors.

    Science.gov (United States)

    Jamsari, Amirul Firdaus Jamaluddin; Jamaluddin, Jamsari Amirul Firdaus; Pau, Tan Min; Siti-Azizah, Mohd Nor

    2011-01-01

    Nucleotide sequences of a partial cytochrome c oxidase subunit I gene were used to assess the manner in which historical processes and geomorphological effects may have influenced genetic structuring and phylogeographic patterns in Channa striata. Assaying was based on individuals from twelve populations in four river systems, which were separated into two regions, the eastern and western, of the biodiversely rich state of Perak in central Peninsular Malaysia. In 238 specimens, a total of 368-bp sequences with ten polymorphic sites and eleven unique haplotypes were detected. Data on all the twelve populations revealed incomplete divergence due to past historical coalescence and the short period of separation. Nevertheless, SAMOVA and F(ST) revealed geographical structuring existed to a certain extent in both regions. For the eastern region, the data also showed that the upstream populations were genetically significantly different compared to the mid- and downstream ones. It is inferred that physical barriers and historical processes played a dominant role in structuring the genetic dispersal of the species. A further inference is that the Grik, Tanjung Rambutan and Sungkai are potential candidates for conservation and aquaculture programmes since they contained most of the total diversity in this area.

  19. Genetic structure of the snakehead murrel, Channa striata (channidae based on the cytochrome c oxidase subunit I gene: influence of historical and geomorphological factors

    Directory of Open Access Journals (Sweden)

    Jamsari Amirul Firdaus Jamaluddin

    2011-01-01

    Full Text Available Nucleotide sequences of a partial cytochrome c oxidase subunit I gene were used to assess the manner in which historical processes and geomorphological effects may have influenced genetic structuring and phylogeographic patterns in Channa striata. Assaying was based on individuals from twelve populations in four river systems, which were separated into two regions, the eastern and western, of the biodiversely rich state of Perak in central Peninsular Malaysia. In 238 specimens, a total of 368-bp sequences with ten polymorphic sites and eleven unique haplotypes were detected. Data on all the twelve populations revealed incomplete divergence due to past historical coalescence and the short period of separation. Nevertheless, SAMOVA and F ST revealed geographical structuring existed to a certain extent in both regions. For the eastern region, the data also showed that the upstream populations were genetically significantly different compared to the mid- and downstream ones. It is inferred that physical barriers and historical processes played a dominant role in structuring the genetic dispersal of the species. A further inference is that the Grik, Tanjung Rambutan and Sungkai are potential candidates for conservation and aquaculture programmes since they contained most of the total diversity in this area.

  20. Correlation of oxygen consumption, cytochrome c oxidase, and cytochrome c oxidase subunit I gene expression in the termination of larval diapause in the bamboo borer, Omphisa fuscidentalis.

    Science.gov (United States)

    Singtripop, Tippawan; Saeangsakda, Manasawan; Tatun, Nujira; Kaneko, Yu; Sakurai, Sho

    2007-09-01

    The moth Omphisa fuscidentalis (Lepidoptera, Pyralidae) is a univoltine insect with a larval diapause period lasting up to 9 months. We studied changes in O(2) consumption in conjunction with cytochrome c oxidase activity and cytochrome c oxidase subunit I (cox1) gene expression. O(2) consumption changed within a day, showing a supradian rhythm with a ca.12-h cycle at 25 degrees C. During the first two-thirds of the diapause period, from October to March, O(2) consumption was constant until January and then increased by March. Topical application of methoprene, a juvenile hormone analog (JHA), to diapausing larvae terminated the diapause and was associated with an increase in O(2) consumption rate at diapause termination. In JHA-treated larvae, cytochrome c oxidase activity in fat bodies was high at the beginning of the prepupal period and highest at pupation. cox1 expression in fat bodies displayed a transient peak 8 days after JHA application and peaked in the prepupal period. Taken together, our results show that the break of diapause by JHA is associated with the activation of cox1, bringing about an increase in cytochrome c oxidase activity, followed by an increase in O(2) consumption rate.

  1. The effect of high glucose levels on the hypermethylation of protein phosphatase 1 regulatory subunit 3C (PPP1R3C) gene in colorectal cancer

    Indian Academy of Sciences (India)

    Soo Kyung Lee; Ji Wook Moon; Yong Woo Lee; Jung Ok Lee; Su Jin Kim; Nami Kim; Jin Kim; Hyeon Soo Kim; Sun-Hwa Park

    2015-03-01

    DNA methylation is an epigenetic event that occurs frequently in colorectal cancer (CRC). Increased glucose level is a strong risk factor for CRC. Protein phosphatase 1 regulatory subunit 3C (PPP1R3C) modulates glycogen metabolism, particularly glycogen synthesis. The aim of this study was to investigate the effect of high glucose levels on DNA methylation of PPP1R3C in CRC. PPP1R3C was significantly hypermethylated in CRC tissues (76/105, 72.38%, < 0.05) and colon cancer cell lines ( < 0.05). CRC tissues obtained from patients with high glucose levels showed that the methylation of PPP1R3C was lower than in patients who had normal levels of glucose. When DLD-1 cells were cultured under conditions of high glucose, the methylation of PPP1R3C was repressed. The expression of PPP1R3C was inversely related to methylation status. In addition, a promoter luciferase assay showed that the transcriptional activity of PPP1R3C was increased in high glucose culture conditions. The number of cells decreased when PPP1R3C was silenced in DLD-1 cells. These results suggest that PPP1R3C, a novel hypermethylated gene in CRC, may play a critical role in cancer cell growth in association with glucose levels.

  2. Expression of the gene for large subunit of m-calpain is elevated in skeletal muscle from Duchenne muscular dystrophy patients

    Indian Academy of Sciences (India)

    Tajamul Hussain; Harleen Mangath; C. Sundaram; M. P. J. S. Anandaraj

    2000-08-01

    Calpain is an intracellular nonlysosomal protease involved in essential regulatory or processing functions of the cell, mediated by physiological concentrations of Ca2+. However, in an environment of abnormal intracellular calcium, such as that seen in Duchenne muscular dystrophy (DMD), calpain is suggested to cause degeneration of muscle owing to enhanced activity. To test whether the reported increase in calpain activity in DMD results from de novo synthesis of the protease, we have assessed the quantitative changes in mRNA specific for m-calpain. mRNA isolated from DMD and control muscle was analysed by dot blot hybridization using a cDNA probe for the large subunit of m-calpain. Compared to control a four-fold increase in specific mRNAwas observed in dystrophic muscle. This enhanced expression of the m-calpain gene in dystrophic condition suggests that the reported increase in m-calpain activity results from de novo synthesis of protease and underlines the important role of m-calpain in DMD.

  3. Reconsideration of the phylogenetic positions of five peritrich genera, Vorticella, Pseudovorticella, Zoothamnopsis, Zoothamnium, and Epicarchesium (Ciliophora, Peritrichia, Sessilida), based on small subunit rRNA gene sequences.

    Science.gov (United States)

    Li, Lifang; Song, Weibo; Warren, Alan; Shin, Mann Kyoon; Chen, Zigui; Ji, Daode; Sun, Ping

    2008-01-01

    In order to re-evaluate the systematics of sessilid peritrich ciliates, small subunit (SSU) rRNA gene sequences were determined for 12 species belonging to five genera: Vorticella, Pseudovorticella, Epicarchesium, Zoothamnium, and Zoothamnopsis. Phylogenetic trees were deduced using Bayesian inference, maximum parsimony, and maximum likelihood methods. The phylogenetic analyses suggest that (1) sessilids which have stalks with continuous myonemes that contract in a zig-zag fashion form a separate clade from those which have stalks that contract independently and in a spiral fashion, supporting the separation of the family Zoothamniidae from the family Vorticellidae and (2) Epicarchesium and Pseudovorticella, both of which have reticulate silverline systems, are more closely related to each other than to other vorticellids, suggesting that differences in the silverline system (i.e. transverse vs. reticulate) may be the result of genuine evolutionary divergence among sessilid peritrichs. However, the newly sequenced Zoothamnopsis sinica, which has a reticulate silverline pattern, nests within the unresolved Zoothamnium species that have transverse silverline patterns. Thus, there were at least two evolutions of the reticulate silverline pattern character state from a plesiomorphic transverse state in the peritrichid ciliates. The molecular work demonstrates the genus Zoothamnium to be paraphyletic in relation to morphological studies, and suggests that Astylozoon, Opisthonecta, and Vorticella microstoma possibly share a SSU rRNA secondary structure in the helix E10-1 region.

  4. [DISTRIBUTION OF GENOTYPES OF C825T POLYMORPHISM β3-SUBUNIT G-PROTEIN GENE IN PATIENTS WITH ARTERIAL HYPERTENSION ACCORDING THE DEGREE OF OBESITY].

    Science.gov (United States)

    Moiseyenko, I; Prystupa, L; Garbuzova, V; Pogorielova, O; Opolonskaya, N

    2015-01-01

    Arterial hypertension (AH) and obesity - risk factors for cardiovascular diseases and their complications, leading to high morbidity and mortality. These nosologies notedly linked, because have common etiological factors, pathophysiological mechanisms and genetic determination. The aim this research was to analyze the distribution of genotypes of the C825T polymorphism of β3-subunit G-protein gene (GNB3) according the degree of obesity and to assess the risk of obesity in patients with AH. Patients were divided into three groups according the degree of obesity. We used clinical, anthropometric, instrumental, molecular-genetic and statistical methods. The significance of differences of alleles and genotypes frequency was determined by test χ². For comparing the groups used nonparametric Mann-Whitney and Kruskal-Wallis tests. A value of phypertension and obesity (χ² = 27,976, p obesity. The risk of obesity in T allele carriers was in 2.2 times higher than in C allele carriers in patients with AH. In summary, our study showed association of C825T polymorphism of the GNB3 with obesity, but did not prove the association this with the degree of obesity i patients with AH.

  5. The roles of Na⁺/K⁺-ATPase α-subunit gene from the ridgetail white prawn Exopalaemon carinicauda in response to salinity stresses.

    Science.gov (United States)

    Li, Jitao; Ma, Peng; Liu, Ping; Chen, Ping; Li, Jian

    2015-02-01

    Na(+)/K(+)-ATPase (NAK) is one important transporter protein and plays a key role in maintaining osmotic homeostasis in low and high salinity acclimation in variety of crustacean species. The ridgetail white prawn Exopalaemon carinicauda is an euryhaline and economic shrimp species in China, but it remains unclear about its mechanism of salinity adaption. In this study, a full-length of Na(+)/K(+)-ATPase α-subunit (α-NAK) cDNA was cloned from E. carinicauda by using rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of α-NAK was of 3680 bp, containing an open reading frame (ORF) of 3030 bp encoding a polypeptide of 1009 amino acids with the predicted molecular weight of 112.27 kDa. Eight transmembrane domains and two sites of phosphorylation and ATP binding were identified in E. carinicauda α-NAK. BLAST analysis revealed that the sequence of α-NAK amino acids of E. carinicauda shared more than 75% homologies with those of other crustacean. Real time quantitative RT-PCR analysis indicated that E. carinicauda α-NAK gene could be detected in all the tested tissues with highest expression level in gill. The expression profiles of E. carinicauda α-NAK transcripts were analyzed in gill and hepatopancreas tissues after salinity stresses. The results showed that the expression level of E. carinicauda α-NAK gene in both gill and hepatopancreas reached peak at different time after low and high salinity stresses, and showed different expression profiles. The expression profiles of proPO transcripts in gills after salinity stresses also indicated α-NAK and proPO played synergistic actions for salinity responses in E. carinicauda. These results indicated that E. carinicauda α-NAK involved in stress responses against salinity.

  6. Cloning and Sequence Analysis of Lateolabrax japonicus Glucose-6-phosphatase Catalytic Subunit (G6PC) cDNA and Its 5'-flanking Region%鲈鱼6-磷酸葡萄糖酶催化亚基(G6PC)cDNA和5’侧翼序列的克隆及分析

    Institute of Scientific and Technical Information of China (English)

    钱云霞; 郑伟贤; 宋娟娟

    2011-01-01

    6-磷酸葡萄糖酶(glucose-6-phosphatase,G6Pase,EC 3.1.3.9)催化6-磷酸葡萄糖水解产生磷酸和葡萄糖,即糖异生途径和糖原分解途径的最后一个限速反应.我们用SMART RACE技术从鲈鱼(Lateolabraxjaponicus)肝脏中克隆了6-磷酸葡萄糖酶催化亚基(glucose-6-phosphatase catalytic subunit,G6PG)基因cDNA序列.该序列全长1651 bp,5’端非翻译区75 bp,3’端非翻译区496 bp,开放阅读框1080bp,可编码一个由359个氨基酸组成的蛋白,该蛋白理论分子量40.45kD、等电点9.30(GenBank登录号:HQ317736.1).氨基酸序列分析表明,鲈鱼G6PC与胡瓜鱼(Osmerus mordax)、翘嘴红鲌(Erythroculter ilishaeformis)、慈鲷(Haplochromis nub ilus)、斑马鱼(Danio rerio)、黑青斑河鲀(Tetraodon nigroviridis)、金头鲷(Sparus aurata)、非洲爪蟾(Xenopus laevis、牛(Bos taurus)、人(Homo sapiens)、小鼠(Mus musculus)及大鼠(Rattus norvegicus)11个物种的同源性达58%以上,其中与胡瓜鱼同源性最高,为85%.RT-PCR分析G6PC基因在鲈鱼肌、心、眼、脑、鳃、肝、肠、肾、脂和脾10个组织中的表达,在肝、肠和肾中有较高的表达,在脑和鳃只有微弱的表达.用基因组步移技术克隆了G6PC基因的5’侧翼区的序列1 243bp,表明该序列含有2个TATA box位点,2个胰岛素作用序列(insulin response sequence,IRS)及C/EBPb、CRE-BP、HNF-3b等潜在的转录因子结合位点.本研究结果表明,鲈鱼G6PC基因在肝、肠和肾中有高表达,其5’侧翼区存在多个转录因子结合位点,该结果为研究G6PC基因的表达调控机制提供了基础资料.%Glucose-6-phosphatase (G6Pase, EC3.1.3.9) catalyzes the hydrolysis of glucose-6-phosphate into glucose, the final step in the gluconeogenic and glycogenolytic pathways. A full-length cDNA of glucose-6-phosphatase catalytic subunit (G6PC) from Lateolabrax japordcus was amplified by SMART RACE method. The cDNA was 1 651 bp in size, with a 75 bp 5'-UTR, 496 bp 3'-UTR and

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

    Science.gov (United States)

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

    1999-01-01

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

  8. [Molecular cloning of the DNA sequence of activin beta A subunit gene mature peptides from panda and related species and its application in the research of phylogeny and taxonomy].

    Science.gov (United States)

    Wang, Xiao-Jing; Wang, Xiao-Xing; Wang, Ya-Jun; Wang, Xi-Zhong; He, Guang-Xin; Chen, Hong-Wei; Fei, Li-Song

    2002-09-01

    Activin, which is included in the transforming growth factor-beta (TGF beta) superfamily of proteins and receptors, is known to have broad-ranging effects in the creatures. The mature peptide of beta A subunit of this gene, one of the most highly conserved sequence, can elevate the basal secretion of follicle-stimulating hormone (FSH) in the pituitary and FSH is pivotal to organism's reproduction. Reproduction block is one of the main reasons which cause giant panda to extinct. The sequence of Activin beta A subunit gene mature peptides has been successfully amplified from giant panda, red panda and malayan sun bear's genomic DNA by using polymerase chain reaction (PCR) with a pair of degenerate primers. The PCR products were cloned into the vector pBlueScript+ of Esherichia coli. Sequence analysis of Activin beta A subunit gene mature peptides shows that the length of this gene segment is the same (359 bp) and there is no intron in all three species. The sequence encodes a peptide of 119 amino acid residues. The homology comparison demonstrates 93.9% DNA homology and 99% homology in amino acid among these three species. Both GenBank blast search result and restriction enzyme map reveal that the sequences of Activin beta A subunit gene mature peptides of different species are highly conserved during the evolution process. Phylogeny analysis is performed with PHYLIP software package. A consistent phylogeny tree has been drawn with three different methods. The software analysis outcome accords with the academic view that giant panda has a closer relationship to the malayan sun bear than the red panda. Giant panda should be grouped into the bear family (Uersidae) with the malayan sun bear. As to the red panda, it would be better that this animal be grouped into the unique family (red panda family) because of great difference between the red panda and the bears (Uersidae).

  9. Interactions between beta D372 and gamma subunit N-terminus residues gamma K9 and gamma S12 are important to catalytic activity catalyzed by Escherichia coli F1F0-ATP synthase.

    Science.gov (United States)

    Lowry, David S; Frasch, Wayne D

    2005-05-17

    Substitution of Escherichia coli F(1)F(0) ATP synthase residues betaD372 or gammaS12 with groups that are unable to form a hydrogen bond at this location decreased ATP synthase-dependent cell growth by 2 orders of magnitude, eliminated the ability of F(1)F(0) to catalyze ATPase-dependent proton pumping in inverted E. coli membranes, caused a 15-20% decrease in the coupling efficiency of the membranes as measured by the extent of succinate-dependent acridine orange fluorescence quenching, but increased soluble F(1)-ATPase activity by about 10%. Substitution of gammaK9 to eliminate the ability to form a salt bridge with betaD372 decreased soluble F(1)-ATPase activity and ATPase-driven proton pumping by 2-fold but had no effect on the proton gradient induced by addition of succinate. Mutations to eliminate the potential to form intersubunit hydrogen bonds and salt bridges between other less highly conserved residues on the gamma subunit N-terminus and the beta subunits had little effect on ATPase or ATP synthase activities. These results suggest that the betaD372-gammaK9 salt bridge contributes significantly to the rate-limiting step in ATP hydrolysis of soluble F(1) while the betaD372-gammaS12 hydrogen bond may serve as a component of an escapement mechanism for ATP synthesis in which alphabetagamma intersubunit interactions provide a means to make substrate binding a prerequisite of proton gradient-driven gamma subunit rotation.

  10. The intein of the Thermoplasma A-ATPase A subunit: Structure, evolution and expression in E. coli

    Directory of Open Access Journals (Sweden)

    Gogarten J Peter

    2001-11-01

    Full Text Available Abstract Background Inteins are selfish genetic elements that excise themselves from the host protein during post translational processing, and religate the host protein with a peptide bond. In addition to this splicing activity, most reported inteins also contain an endonuclease domain that is important in intein propagation. Results The gene encoding the Thermoplasma acidophilum A-ATPase catalytic subunit A is the only one in the entire T. acidophilum genome that has been identified to contain an intein. This intein is inserted in the same position as the inteins found in the ATPase A-subunits encoding gene in Pyrococcus abyssi, P. furiosus and P. horikoshii and is found 20 amino acids upstream of the intein in the homologous vma-1 gene in Saccharomyces cerevisiae. In contrast to the other inteins in catalytic ATPase subunits, the T. acidophilum intein does not contain an endonuclease domain. T. acidophilum has different codon usage frequencies as compared to Escherichia coli. Initially, the low abundance of rare tRNAs prevented expression of the T. acidophilum A-ATPase A subunit in E. coli. Using a strain of E. coli that expresses additional tRNAs for rare codons, the T. acidophilum A-ATPase A subunit was successfully expressed in E. coli. Conclusions Despite differences in pH and temperature between the E. coli and the T. acidophilum cytoplasms, the T. acidophilum intein retains efficient self-splicing activity when expressed in E. coli. The small intein in the Thermoplasma A-ATPase is closely related to the endonuclease containing intein in the Pyrococcus A-ATPase. Phylogenetic analyses suggest that this intein was horizontally transferred between Pyrococcus and Thermoplasma, and that the small intein has persisted in Thermoplasma apparently without homing.

  11. Phylogenetic position of Linguatula arctica and Linguatula serrata (Pentastomida) as inferred from the nuclear 18S rRNA gene and the mitochondrial cytochrome c oxidase subunit I gene.

    Science.gov (United States)

    Gjerde, Bjørn

    2013-10-01

    Genomic DNA was isolated from a Linguatula serrata female expelled from a dog imported to Norway from Romania and from four Linguatula arctica females collected from semi-domesticated reindeer from northern Norway and subjected to PCR amplification of the complete nuclear 18S rRNA gene and a 1,045-bp portion of the mitochondrial cytochrome c oxidase subunit I gene (cox1). The two species differed at two of 1,830 nucleotide positions (99.9% identity) of the complete 18S rRNA gene sequences and at 102 of 1,045 nucleotide positions (90.2% identity) of the partial cox1 sequences. The four isolates of L. arctica showed no genetic variation in either gene. The new cox1 primers may facilitate the diagnosis of various developmental stages of L. arctica and L. serrata in their hosts. In separate phylogenetic analyses using the maximum likelihood method on sequence data from either gene, L. arctica and L. serrata clustered with members of the order Cephalobaenida rather than with members of the order Porocephalida, in which the genus Linguatula is currently placed based on morphological characters. The phylogenetic relationship of L. arctica, L. serrata and other pentastomids to other metazoan groups could not be clearly resolved, but the pentastomids did not seem to have a sister relationship to crustaceans of the subclass Branchiura as found in other studies. A more extensive taxon sampling, including molecular characterisation of more pentastomid taxa across different genera, seems to be necessary in order to estimate the true relationship of the Pentastomida to other metazoan groups.

  12. G-protein beta3 subunit gene variant is unlikely to have a significant influence on serum uric acid level in Japanese workers.

    Science.gov (United States)

    Suwazono, Yasushi; Kobayashi, Etsuko; Uetani, Mirei; Miura, Katsuyuki; Morikawa, Yuko; Ishizaki, Masao; Kido, Teruhiko; Nakagawa, Hideaki; Nogawa, Koji

    2006-06-01

    The C825T variant of the G-protein beta3 subunit (GNB3) gene has attracted renewed attention as a candidate gene for obesity, hypertension and hyperuricemia. The main role of G-protein is to translate signals from the cell surface into a cellular response. The 825T allele is associated with a splice variant of GNB3 protein and enhanced G-protein activation. We examined the relationship between this variant and the risk of hyperuricemia in Japanese workers. The study subjects were 1,452 men and 1,169 women selected from 3,834 men and 2,591 women in 1997. On the basis of common clinical criteria, hyperuricemia I was defined as serum uric acid >or= 7.0 mg/dl in men and 6.0 mg/dl in women or taking antihyperuricemic medication. The hyperuricemia I group consisted of 186 men and 20 women and its control of 1,266 men and 1,149 women. Hyperuricemia II was defined as serum uric acid > 5.7 mg/dl (median) in men and 3.9 mg/dl (median) in women or taking antihyperuricemic medication. The hyperuricemic II group consisted of 684 men and 570 women and its control of 768 men and 599 women. To replicate previous significant results in young Caucasian men, we selected these criteria because the authors of the study in young Caucasian men adopted the median in their subjects as a cut-off. The statistical power was estimated as 99% based on the significant results in Caucasians. Genotype and allele distributions in men and women with hyperuricemia I and II were not significantly different from those in the corresponding control groups. Logistic regression analysis on hyperuricemia I and II, and multiple regression on serum uric acid level demonstrated no significant effect of the C825T genotype. Despite the sufficient statistical power, this study could not demonstrate the significant influence of C825T on hyperuricemia or serum uric acid. The targeting of this polymorphism is unlikely to be beneficial in the prevention of hyperuricemia in the general Japanese population.

  13. 5'-AMP-activated protein kinase activity and subunit expression in exercise-trained human skeletal muscle

    DEFF Research Database (Denmark)

    Nielsen, Jakob Nis; Mustard, Kirsty J.W.; Graham, Drew A.

    2002-01-01

    5'-AMP-activated protein kinase (AMPK) has been proposed to be a pivotal factor in cellular responses to both acute exercise and exercise training. To investigate whether protein levels and gene expression of catalytic (alpha(1), alpha(2)) and regulatory (beta(1), beta(2), gamma(1), gamma(2), gam...... muscle has increased alpha(1)-AMPK protein levels and blunted AMPK activation during exercise.......5'-AMP-activated protein kinase (AMPK) has been proposed to be a pivotal factor in cellular responses to both acute exercise and exercise training. To investigate whether protein levels and gene expression of catalytic (alpha(1), alpha(2)) and regulatory (beta(1), beta(2), gamma(1), gamma(2), gamma......(3)) AMPK subunits and exercise-induced AMPK activity are influenced by exercise training status, muscle biopsies were obtained from seven endurance exercise-trained and seven sedentary young healthy men. The alpha(1)- and alpha(2)-AMPK mRNA contents in trained subjects were both 117 +/- 2...

  14. The NDUFB6 subunit of the mitochondrial respiratory chain complex I is required for electron transfer activity: A proof of principle study on stable and controlled RNA interference in human cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Loublier, Sandrine; Bayot, Aurelien; Rak, Malgorzata; El-Khoury, Riyad; Benit, Paule [Inserm U676, Hopital Robert Debre, F-75019 Paris (France); Universite Paris 7, Faculte de medecine Denis Diderot, IFR02 Paris (France); Rustin, Pierre, E-mail: pierre.rustin@inserm.fr [Inserm U676, Hopital Robert Debre, F-75019 Paris (France); Universite Paris 7, Faculte de medecine Denis Diderot, IFR02 Paris (France)

    2011-10-22

    Highlights: {yields} NDUFB6 is required for activity of mitochondrial complex I in human cell lines. {yields} Lentivirus based RNA interference results in frequent off target insertions. {yields} Flp-In recombinase mediated miRNA insertion allows gene-specific extinction. -- Abstract: Molecular bases of inherited deficiencies of mitochondrial respiratory chain complex I are still unknown in a high proportion of patients. Among 45 subunits making up this large complex, more than half has unknown function(s). Understanding the function of these subunits would contribute to our knowledge on mitochondrial physiology but might also reveal that some of these subunits are not required for the catalytic activity of the complex. A direct consequence of this finding would be the reduction of the number of candidate genes to be sequenced in patients with decreased complex I activity. In this study, we tested two different methods to stably extinct complex I subunits in cultured cells. We first found that lentivirus-mediated shRNA expression frequently resulted in the unpredicted extinction of additional gene(s) beside targeted ones. This can be ascribed to uncontrolled genetic material insertions in the genome of the host cell. This approach thus appeared inappropriate to study unknown functions of a gene. Next, we found it possible to specifically extinct a CI subunit gene by direct insertion of a miR targeting CI subunits in a Flp site (HEK293 Flp-In cells). By using this strategy we unambiguously demonstrated that the NDUFB6 subunit is required for complex I activity, and defined conditions suitable to undertake a systematic and stable extinction of the different supernumerary subunits in human cells.

  15. Fusion of the Tumor-Suppressor Gene CHEK2 and the Gene for the Regulatory Subunit B of Protein Phosphatase 2 PPP2R2A in Childhood Teratoma

    Directory of Open Access Journals (Sweden)

    Yuesheng Jin

    2006-05-01

    Full Text Available We characterized the molecular genetic consequences of a balanced chromosome translocation t(8;22(p21; q12, which occurred as the sole cytogenetic aberration in short-term cultured cells from an intrathoracic mature teratoma in a 15-year-old girl. Fluorescence in situ hybridization and reverse transcription- polymerase chain reaction disclosed that t(8;22 resulted in the fusion of the genes PPP2R2A and CHEK2, with an inserted fragment belonging to class I endogenous retrovirus-related sequences at the junction. Sequencing of the two genes did not reveal any additional mutation. None of the three detected PPP2R2A/CHEK2 fusion transcripts resulted in an in-frame PPP2R2A/CHEK2 chimerical open reading frame; however, in all of them, the known open reading frame of CHEK2 was preserved. Thus, promoter swapping leading to deregulated CHEK2 expression would be the most likely oncogenic mechanism. Whereas inactivating mutations of CHEK2 previously have been described in a variety of sporadic tumors and in inherited cancer-predisposing syndromes, PPP2R2A, encoding a regulatory subunit of the multimeric enzyme phosphatase 2, has not been directly implicated in tumorigenesis. Our findings suggest that deregulation of CHEK2 and/or PPP2R2A is of pathogenetic importance in at least a subset of germ cell tumors.

  16. Two thyroid hormone regulated genes, the beta-subunits of nerve growth factor (NGFB) and thyroid stimulating hormone (TSHB), are located less than 310 kb apart in both human and mouse genomes.

    Science.gov (United States)

    Dracopoli, N C; Rose, E; Whitfield, G K; Guidon, P T; Bale, S J; Chance, P A; Kourides, I A; Housman, D E

    1988-08-01

    Two thyroid hormone regulated genes, the beta-subunits of nerve growth factor (NGFB) and thyroid stimulating hormone (TSHB), have been assigned to mouse chromosome 3 and human chromosome 1p22. We have used the techniques of linkage analysis and pulsed field gel electrophoresis to determine the proximity of these two antithetically regulated genes in this conserved linkage group. Four novel restriction fragment length polymorphisms were identified at the human TSHB gene. Two-point linkage analysis between TSHB and NGFB in 46 families, including the Centre d'Etude du Polymorphisme Humain (CEPH) reference panel, demonstrated no recombination (theta = 0.00, Z = 42.8). Analysis of this region by pulsed field gel electrophoresis showed that the genes for TSHB and NGFB are located less than 310 kb apart in man and 220 kb in the mouse.

  17. Genetic predisposition to essential hypertension in a Mongolian population Detecting the C825T polymorphism of the G-protein beta 3 subunit gene

    Institute of Scientific and Technical Information of China (English)

    Chunyu Zhang; Shigang Zhao; Guangming Niu; Rile Hu; Zhiguang Wang; Mingfang Jiang; Rile Hu

    2007-01-01

    BACKGROUND: The prevalences of hypertension, cerebrovascular diseases, etc. are higher in Mongolian population because of the influence of various factors including genetics, geography, diet, etc. Therefore, it is helpful for prevention to develop researches on the genetics of various diseases including hypertension in Mongolian population.OBJECTIVE: To analyze the association between C825T polymorphisms of G-protein beta 3 subunit gene (GNB3), the important candidate gene of various disease of cardiovascular system, and Mongolian patients with essential hypertension.DESIGN: A comparative observation.SETTINGS: Department of Neurology, the First Affiliated Hospital of Inner Mongolia Medical College;Wulate Houqi Red Cross Society.PARTICIPANTS: Totally 267 Mongolian residents, whose blood relations of 3 generations were all Mongolians, were selected from Wulate Houqi, Inner Mongolia. The patients were screened based on the diagnostic standard of hypertension set by WHO in 1999, and the enrolled subjects were divided into two groups according to the level of blood pressure: ① Normal blood pressure group (n =124): 64 males and 60 females, systolic blood pressure (SBP) < 140 mm Hg (1 mm Hg=0.133 kPa), diastolic blood pressure (DBP) <90 mm Hg; ② Essential hypertension group (n =143): 71 males and 72 females, including 60 patients with simple high SBP (SBP ranged 145 to 195 mm Hg, whereas DBP < 90 mm Hg).METHODS: Peripheral venous blood (5 mL) was drawn from all the subjects, the genome DNA was extracted, and the polymorphisms of the GNB3 C825T genotype were detected with the Sequenom system.Polymerase chain reaction (PCR) experiment and SNP detection were performed in Beijing Huada gene laboratory. Then the univariate analysis of variance was applied in the sample comparison among groups, and the chi-square test was used to compare the genotypes and allele frequencies. The odd ratio (OR) and 95% confidence interval (CI)were calculated.MAIN OUTCOME MEASURES: The

  18. Cloning, Characterization, and Distribution of an mRNA Encoding a H+-ATPase α Subunit in the Mantle of Pearl Oyster, Pinctada fucata

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Mitochondrial ATP synthase is responsible for the production of the majority of the cellular ATP,which is composed of two major units: F0 and F1. Although much is known about the active complex (5 subunits (αβγδε)), the role of the α subunit in the catalytic mechanism remains unclear, particularly in bivalve animals. This study first cloned and identified the full-length sequence of the mitochondrial H+-ATP synthase α subunit cDNA gene in Pinctada fucata using the reverse transcriptase polymerase chain reaction (RT-PCR)technique. The Pinctada fucata mitochondrial H+-ATP synthase α subunit contains 1991 nucleotides, with the translation start site at nt 48 (ATG) and the stop codon at nt 1660 (TAA), encoding a polypeptide 553 amino acids in length, which shares high similarity to that of other animals (81% identity to fruit fly, 82% to carp, and 83% to humans). Alignment analysis of the well-conserved amino acid domains in the ATPase α subunit, the α/β signal transduction domain, showed that two residues (Asp358 and Asn359) differ from any other ATP synthase α subunit. In situ hybridization analysis was used to reveal the wide-spread distribution of mitochondrial H+-ATP synthase in various tissues in Pinctada fucata. This work will help further research on pearl energy metabolism to increase the output and quality of pearls to more efficiently utilize our rich pearl oyster resources.

  19. The Cleavage and Polyadenylation Specificity Factor 6 (CPSF6) Subunit of the Capsid-recruited Pre-messenger RNA Cleavage Factor I (CFIm) Complex Mediates HIV-1 Integration into Genes.

    Science.gov (United States)

    Rasheedi, Sheeba; Shun, Ming-Chieh; Serrao, Erik; Sowd, Gregory A; Qian, Juan; Hao, Caili; Dasgupta, Twishasri; Engelman, Alan N; Skowronski, Jacek

    2016-05-27

    HIV-1 favors integration into active genes and gene-enriched regions of host cell chromosomes, thus maximizing the probability of provirus expression immediately after integration. This requires cleavage and polyadenylation specificity factor 6 (CPSF6), a cellular protein involved in pre-mRNA 3' end processing that binds HIV-1 capsid and connects HIV-1 preintegration complexes to intranuclear trafficking pathways that link integration to transcriptionally active chromatin. CPSF6 together with CPSF5 and CPSF7 are known subunits of the cleavage factor I (CFIm) 3' end processing complex; however, CPSF6 could participate in additional protein complexes. The molecular mechanisms underpinning the role of CPSF6 in HIV-1 infection remain to be defined. Here, we show that a majority of cellular CPSF6 is incorporated into the CFIm complex. HIV-1 capsid recruits CFIm in a CPSF6-dependent manner, which suggests that the CFIm complex mediates the known effects of CPSF6 in HIV-1 infection. To dissect the roles of CPSF6 and other CFIm complex subunits in HIV-1 infection, we analyzed virologic and integration site targeting properties of a CPSF6 variant with mutations that prevent its incorporation into CFIm We show, somewhat surprisingly, that CPSF6 incorporation into CFIm is not required for its ability to direct preferential HIV-1 integration into genes. The CPSF5 and CPSF7 subunits appear to have only a minor, if any, role in this process even though they appear to facilitate CPSF6 binding to capsid. Thus, CPSF6 alone controls the key molecular interactions that specify HIV-1 preintegration complex trafficking to active chromatin.

  20. Inverted repeat of Olisthodiscus luteus chloroplast DNA contains genes for both subunits of ribulose-1,5-bisphosphate carboxylase and the 32,000-dalton QB protein: Phylogenetic implications

    Science.gov (United States)

    Reith, Michael; Cattolico, Rose Ann

    1986-01-01

    The chloroplast DNA of the chromophytic alga Olisthodiscus luteus has been physically mapped with four restriction enzymes. An inverted repeat of 22 kilobase pairs is present in this 150-kilobase-pair plastid genome. The inverted repeat contains the genes for the large and small subunit polypeptides of ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) and also codes for the 32,000-dalton QB protein. These observations demonstrate that significant differences exist in chloroplast genome structure and organization among major plant taxa. Images PMID:16578794

  1. Bioinformatic Analysis on Catalytic Subunits of Calcium-activated Neutral Protease-1 and-2%人钙激活中性蛋白酶-1及-2催化亚基的生物信息学分析

    Institute of Scientific and Technical Information of China (English)

    徐国强; 胡晓霞; 潘娅; 董宇华; 张金娟; 张敏; 张祥令; 臧贵勇; 熊英

    2011-01-01

    Objective: To compare the function of human calpain-1 and calpain-2, two classic subfamily proteins of calcium-activated neutral protease. Methods: The RefSeq and amino acid sequences of human calpain-1 and -2 were acquired from the protein database of American national center for biotechnology (NCBI). The general protein annotation and domain information of calpain-1 and -2 were obtained from UniProt database. DNAMAN software was used to compare the primary structure of domains of the two calpains, and the distribution of modified sites were detected with protein data bank (PBD) and protein modification database(PhosphoSit Plus) jointly. String 9.0 was used in the analysis of protein-protein interaction. Results: As analysis of domain showing, calpain catalytic domain of the two calpains were highly conserved, while the variation in EF-hand domain was relatively large. The post-translational modification (PTM) in calpain-1 was only on lys ( K) 84, while in calpain-2 was more complex which could be found on the surface, in the cleft and inside the molecular in different forms. The protein-protein interaction analysis indicated that there were only few proteins commonly in both interaction maps, while most proteins were specific in individual maps of the two calpains, which associated with different cell activities. Conclusion; The catalytic domains of human calpain-1 and calpain-2 are highly conserved. The functional differences between them are mainly caused by the alteration of regulating sites (EF-hand domain and PTM sites).%目的:对人钙激活中性蛋白酶( calpain)的2个亚家族成员calpain-1和calpain-2进行功能的比较分析。方法:以美国生物技术国家中心( NCBI)的蛋白质数据库获取人calpain-1和calpain-2的参考序列号(RefSeq)和氨基酸序列,通过UniProt数据库检索2个蛋白的一般蛋白质功能注释,获取相关结构域信息;利用DNA-MAN软件对结构域进行序列比对,用蛋白质结构数

  2. Analysis of iron- and sulfur-oxidizing bacteria in a treatment plant of acid rock drainage from a Japanese pyrite mine by use of ribulose-1, 5-bisphosphate carboxylase/oxygenase large-subunit gene.

    Science.gov (United States)

    Kamimura, Kazuo; Okabayashi, Ai; Kikumoto, Mei; Manchur, Mohammed Abul; Wakai, Satoshi; Kanao, Tadayoshi

    2010-03-01

    Iron- and sulfur-oxidizing bacteria in a treatment plant of acid rock drainage (ARD) from a pyrite mine in Yanahara, Okayama prefecture, Japan, were analyzed using the gene (cbbL) encoding the large subunit of ribulose-1, 5-bisphosphate carboxylase/oxygenase (RubisCO). Analyses of partial sequences of cbbL genes from Acidithiobacillus ferrooxidans, Acidithiobacillus thiooxidans and Acidithiobacillus caldus strains revealed the diversity in their cbbL gene sequences. In contrast to the presence of two copies of form I cbbL genes (cbbL1 and cbbL2) in A. ferrooxidans genome, A. thiooxidans and A. caldus had a single copy of form I cbbL gene in their genomes. A phylogenetic analysis based on deduced amino acid sequences from cbbL genes detected in the ARD treatment plant and their close relatives revealed that 89% of the total clones were affiliated with A. ferrooxidans. Clones loosely affiliated with the cbbL from A. thiooxidans NB1-3 or Thiobacillus denitrificans was also detected in the treatment plant. cbbL gene sequences of iron- or sulfur-oxidizing bacteria isolated from the ARD and the ARD treatment plant were not detected in the cbbL libraries from the treatment plant, suggesting the low frequencies of isolates in the samples.

  3. Protein Kinase A Subunit Balance Regulates Lipid Metabolism in Caenorhabditis elegans and Mammalian Adipocytes.

    Science.gov (United States)

    Lee, Jung Hyun; Han, Ji Seul; Kong, Jinuk; Ji, Yul; Lv, Xuchao; Lee, Junho; Li, Peng; Kim, Jae Bum

    2016-09-23

    Protein kinase A (PKA) is a cyclic AMP (cAMP)-dependent protein kinase composed of catalytic and regulatory subunits and involved in various physiological phenomena, including lipid metabolism. Here we demonstrated that the stoichiometric balance between catalytic and regulatory subunits is crucial for maintaining basal PKA activity and lipid homeostasis. To uncover the potential roles of each PKA subunit, Caenorhabditis elegans was used to investigate the effects of PKA subunit deficiency. In worms, suppression of PKA via RNAi resulted in severe phenotypes, including shortened life span, decreased egg laying, reduced locomotion, and altered lipid distribution. Similarly, in mammalian adipocytes, suppression of PKA regulatory subunits RIα and RIIβ via siRNAs potently stimulated PKA activity, leading to potentiated lipolysis without increasing cAMP levels. Nevertheless, insulin exerted anti-lipolytic effects and restored lipid droplet integrity by antagonizing PKA action. Together, these data implicate the importance of subunit stoichiometry as another regulatory mechanism of PKA activity and lipid metabolism.

  4. Specific Inhibition of Herpes Simplex Virus DNA Polymerase by Helical Peptides Corresponding to the Subunit Interface

    Science.gov (United States)

    Digard, Paul; Williams, Kevin P.; Hensley, Preston; Brooks, Ian S.; Dahl, Charles E.; Coen, Donald M.

    1995-02-01

    The herpes simplex virus DNA polymerase consists of two subunits-a catalytic subunit and an accessory subunit, UL42, that increases processivity. Mutations affecting the extreme C terminus of the catalytic subunit specifically disrupt subunit interactions and ablate virus replication, suggesting that new antiviral drugs could be rationally designed to interfere with polymerase heterodimerization. To aid design, we performed circular dichroism (CD) spectroscopy and analytical ultracentrifugation studies, which revealed that a 36-residue peptide corresponding to the C terminus of the catalytic subunit folds into a monomeric structure with partial α-helical character. CD studies of shorter peptides were consistent with a model where two separate regions of α-helix interact to form a hairpin-like structure. The 36-residue peptide and a shorter peptide corresponding to the C-terminal 18 residues blocked UL42-dependent long-chain DNA synthesis at concentrations that had no effect on synthesis by the catalytic subunit alone or by calf thymus DNA polymerase δ and its processivity factor. These peptides, therefore, represent a class of specific inhibitors of herpes simplex virus DNA polymerase that act by blocking accessory-subunit-dependent synthesis. These peptides or their structures may form the basis for the synthesis of clinically effective drugs.

  5. Structural Basis for the Catalytic Activity of Human Serine/Threonine Protein Phosphatase type 5 (PP5)

    Science.gov (United States)

    Swingle, Mark R.; Ciszak, Ewa M.; Honkanen, Richard E.

    2004-01-01

    Serine/threonine protein phosphatase-5 (PP5) is a member of the PPP-gene family of protein phosphatases that is widely expressed in mammalian tissues and is highly conserved among eukaryotes. PP5 associates with several proteins that affect signal transduction networks, including the glucocorticoid receptor (GR)-heat shock protein-90 (Hsp90)-heterocomplex, the CDC16 and CDC27 subunits of the anaphase-promoting complex, elF2alpha kinase, the A subunit of PP2A, the G12-alpha / G13-alpha subunits of heterotrimeric G proteins and DNA-PK. The catalytic domain of PP5 (PP5c) shares 35-45% sequence identity with the catalytic domains of other PPP-phosphatases, including protein phosphatase-1 (PP1), -2A (PP2A), -2B / calcineurin (PP2B), -4 (PP4), -6 (PP6), and -7 (PP7). Like PP1, PP2A and PP4, PP5 is also sensitive to inhibition by okadaic acid, microcystin, cantharidin, tautomycin, and calyculin A. Here we report the crystal structure of the PP5 catalytic domain (PP5c) at a resolution of 1.6 angstroms. From this structure we propose a mechanism for PP5-mediated hydrolysis of phosphoprotein substrates, which requires the precise positioning of two metal ions within a conserved Asp(sup 271)-M(sub 1):M(sub 2)-W(sup 1)-His(sup 304)-Asp(sup 274) catalytic motif. The structure of PP5c provides a possible structural basis for explaining the exceptional catalytic proficiency of protein phosphatases, which are among the most powerful known catalysts. Resolution of the entire C-terminus revealed a novel subdomain, and the structure of the PP5c should also aid development of type-specific inhibitors.

  6. Structure of protein kinase CK2: dimerization of the human beta-subunit

    DEFF Research Database (Denmark)

    Boldyreff, B; Mietens, U; Issinger, O G

    1996-01-01

    Protein kinase CK2 has been shown to be elevated in all so far investigated solid tumors and its catalytic subunit has been shown to serve as an oncogene product. CK2 is a heterotetrameric serine-threonine kinase composed of two catalytic (alpha and/or alpha') and two regulatory beta...

  7. Molecular cloning of Schistosoma mansoni calcineurin subunits and immunolocalization to the excretory system.

    Science.gov (United States)

    Mecozzi, B; Rossi, A; Lazzaretti, P; Kady, M; Kaiser, S; Valle, C; Cioli, D; Klinkert, M Q

    2000-10-01

    In order to explain the schistosomicidal effect of cyclosporin A, the hypothesis was advanced that the drug, complexed with cyclophilin, inhibits the phosphatase activity of parasite calcineurin (CN), with mechanisms similar to those operating in its immunosuppressive action. As a preparatory step to the testing of this hypothesis, we report the molecular cloning of both CN subunits in Schistosoma mansoni. The catalytic (A) subunit has a predicted sequence of 607 amino acids and shows substantial similarity to other cloned CNs, except for the carboxy-terminal end that is highly divergent. The regulatory (B) subunit consists of 169 amino acids that are 86% identical to those of the human counterpart and, from its anomalous electrophoretic mobility, it appears to be myristoylated. The results of Southern blotting experiments are compatible with the existence of multiple genes for CNA and a single gene for CNB. Western blots showed that both subunits are present at all stages of the parasite life cycle and can be detected both in the soluble and in the membrane fraction. Immunofluorescence confocal microscopy revealed a striking concentration of the anti-CNA reactivity in 6-8 discrete spots in the schistosomula and in distinct spots along the body of the adult parasite, corresponding to the expected localization of flame cells. Both patterns were confirmed by a perfect co-localization of the anti-CNA signal with that of a previously characterized anti-flame cell monoclonal antibody. The preferential confinement of schistosome CN to the protonephridial system suggests that the enzyme in the parasite may fulfil similar functions to those performed in mammalian kidneys.

  8. Na+ channel β subunits: Overachievers of the ion channel family

    Directory of Open Access Journals (Sweden)

    William J Brackenbury

    2011-09-01

    Full Text Available Voltage gated Na+ channels (VGSCs in mammals contain a pore-forming α subunit and one or more β subunits. There are five mammalian β subunits in total: β1, β1B, β2, β3, and β4, encoded by four genes: SCN1B-SCN4B. With the exception of the SCN1B splice variant, β1B, the β subunits are type I topology transmembrane proteins. In contrast, β1B lacks a transmembrane domain and is a secreted protein. A growing body of work shows that VGSC β subunits are multifunctional. While they do not form the ion channel pore, β subunits alter gating, voltage-dependence, and kinetics of VGSC α subunits and thus regulate cellular excitability in vivo. In addition to their roles in channel modulation, β subunits are members of the immunoglobulin (Ig superfamily of cell adhesion molecules (CAMs and regulate cell adhesion and migration. β subunits are also substrates for sequential proteolytic cleavage by secretases. An example of the multifunctional nature of β subunits is β1, encoded by SCN1B, that plays a critical role in neuronal migration and pathfinding during brain development, and whose function is dependent on Na+ current and γ-secretase activity. Functional deletion of SCN1B results in Dravet Syndrome, a severe and intractable pediatric epileptic encephalopathy. β subunits are emerging as key players in a wide variety of pathophysiologies, including epilepsy, cardiac arrhythmia, multiple sclerosis, Huntington’s disease, neuropsychiatric disorders, neuropathic and inflammatory pain, and cancer. β subunits mediate multiple signaling pathways on different timescales, regulating electrical excitability, adhesion, migration, pathfinding, and transcription. Importantly, some β subunit functions may operate independent of α subunits. Thus, β subunits perform critical roles during development and disease. As such, they may prove useful in disease diagnosis and therapy.

  9. Cloning and activity analysis of in vitro expression of plant NAD-IDH genes

    Institute of Scientific and Technical Information of China (English)

    CHEN Defu; CHEN Xiwen

    2004-01-01

    The plant NAD+-dependent isocitrate dehydrogenase (NAD-IDH) is an important multifunctional enzyme. The cDNAs encoding three different subunits of the NAD-IDH were cloned successfully from leaves of Arabidopsis thaliana ecotype Columbia gl1 and three lines (Shaan 2A, Shaan 2B and Ken C1) of Brassica napus by RT-PCR method. By searching the sequences in GenBank Database, it is shown that these sequences from B. napus were novel. Their encoding regions of a functional protein were then inserted into the multiple cloning sites of pMID1 vector for the expression of recombinant protein. All the recombinants were successfully expressed in the in vitro expression system. When the transcripts of subunits 0, 1 and 2 were added together to the in vitro system with 36 μg/mL of protein disulfide isomerase, the expressed products had NAD-IDH enzymatic activity. Comparison of different kinds of gene and molecular ratio of the transcripts showed that the NAD-IDH enzyme is composed of three subunits designated subunit 0, subunit 1 and subunit 2. All the three subunits are essential to catalytic activity. Missing subunit 0 could abolish activity. Missing either subunit 1 or subunit 2 would cause severe impact on activity. Deletions, which would cause frameshift mutation or nonsense mutation, were also found in some transcripts of subunit 1 gene from Shaan 2A and Shaan 2B of B. napus. The mutated subunit 1 lost its proper function. This may explain why there is difference of the NAD-IDH activity among three lines of B. napus.

  10. The gene sml0013 of Synechocystis species strain PCC 6803 encodes for a novel subunit of the NAD(P)H oxidoreductase or complex I that is ubiquitously distributed among Cyanobacteria.

    Science.gov (United States)

    Schwarz, Doreen; Schubert, Hendrik; Georg, Jens; Hess, Wolfgang R; Hagemann, Martin

    2013-11-01

    The NAD(P)H oxidoreductase or complex I (NDH1) complex participates in many processes such as respiration, cyclic electron flow, and inorganic carbon concentration in the cyanobacterial cell. Despite immense progress in our understanding of the structure-function relation of the cyanobacterial NDH1 complex, the subunits catalyzing NAD(P)H docking and oxidation are still missing. The gene sml0013 of Synechocystis 6803 encodes for a small protein of unknown function for which homologs exist in all completely known cyanobacterial genomes. The protein exhibits weak similarities to the NDH-dependent flow6 (NDF6) protein, which was reported from Arabidopsis (Arabidopsis thaliana) chloroplasts as a NDH subunit. An sml0013 inactivation mutant of Synechocystis 6803 was generated and characterized. It showed only weak differences regarding growth and pigmentation in various culture conditions; most remarkably, it exhibited a glucose-sensitive phenotype in the light. The genome-wide expression pattern of the Δsml0013::Km mutant was almost identical to the wild type when grown under high CO2 conditions as well as after shifts to low CO2 conditions. However, measurements of the photosystem I redox kinetic in cells of the Δsml0013::Km mutant revealed differences, such as a decreased capability of cyclic electron flow as well as electron flow into respiration in comparison with the wild type. These results suggest that the Sml0013 protein (named NdhP) represents a novel subunit of the cyanobacterial NDH1 complex, mediating its coupling either to the respiratory or the photosynthetic electron flow.

  11. Genotypic to expression profiling of bovine calcium channel, voltage-dependent, alpha-2/delta subunit 1 gene, and their association with bovine mastitis among Frieswal (HFX Sahiwal) crossbred cattle of Indian origin.

    Science.gov (United States)

    Deb, Rajib; Singh, Umesh; Kumar, Sushil; Kumar, Arun; Singh, Rani; Sengar, Gyanendra; Mann, Sandeep; Sharma, Arjava

    2014-04-01

    Calcium channel, voltage-dependent, alpha-2/delta subunit 1 (CACNA2D1) gene is considered to be an important noncytokine candidate gene influencing mastitis. Scanty of reports are available until today regarding the role play of CACNA2D1 gene on the susceptibility of bovine mastitis. We interrogated the CACNA2D1 G519663A [A>G] SNP by PCR-RFLP among two hundreds Frieswal (HF X Sahiwal) crossbred cattle of Indian origin. Genotypic frequency of AA (51.5, n=101) was comparatively higher than AG (35, n=70) and GG (14.5, n=29). Association of Somatic cell score (SCS) with genotypes revealed that, GG genotypes showing lesser count (less susceptible to mastitis) compare to AA and AG. Relative expression of CACNA2D1 transcript (in milk samples) was significantly higher among GG than AG and AA. Further we have also isolated blood sample from the all groups and PBMCs were cultured from each blood sample as per the standard protocol. They were treated with Calcium channel blocker and the expression level of the CACNA2D1 gene was evaluated by Real Time PCR. Results show that expression level decline in each genotypic group after treatment and expression level of GG are again significantly higher than AA and AG. Thus, it may be concluded that GG genotypic animals are favorable for selecting disease resistant breeds.

  12. 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

    Energy Technology Data Exchange (ETDEWEB)

    Lalioti, M.D.; Rossier, C.; Antonarakis, S.E. [Univ. of Geneva Medical School (Switzerland)] [and others

    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.

  13. Rotation of subunits during catalysis by Escherichia coli F1-ATPase.

    Science.gov (United States)

    Duncan, T M; Bulygin, V V; Zhou, Y; Hutcheon, M L; Cross, R L

    1995-11-21

    During oxidative and photo-phosphorylation, F0F1-ATP synthases couple the movement of protons down an electrochemical gradient to the synthesis of ATP. One proposed mechanistic feature that has remained speculative is that this coupling process requires the rotation of subunits within F0F1. Guided by a recent, high-resolution structure for bovine F1 [Abrahams, J. P., Leslie, A. G., Lutter, R. & Walker, J. E. (1994) Nature (London) 370, 621-628], we have developed a critical test for rotation of the central gamma subunit relative to the three catalytic beta subunits in soluble F1 from Escherichia coli. In the bovine F1 structure, a specific point of contact between the gamma subunit and one of the three catalytic beta subunits includes positioning of the homolog of E. coli gamma-subunit C87 (gamma C87) close to the beta-subunit 380DELSEED386 sequence. A beta D380C mutation allowed us to induce formation of a specific disulfide bond between beta and gamma C87 in soluble E. coli F1. Formation of the crosslink inactivated beta D380C-F1, and reduction restored full activity. Using a dissociation/reassembly approach with crosslinked beta D380C-F1, we incorporated radiolabeled beta subunits into the two noncrosslinked beta-subunit positions of F1. After reduction of the initial nonradioactive beta-gamma crosslink, only exposure to conditions for catalytic turnover results in similar reactivities of unlabeled and radiolabeled beta subunits with gamma C87 upon reoxidation. The results demonstrate that gamma subunit rotates relative to the beta subunits during catalysis.

  14. Comparative Analysis of Eubacterial DNA Polymerase Ⅲ Alpha Subunits

    Institute of Scientific and Technical Information of China (English)

    Xiao-Qian Zhao; Jian-Fei Hu; Jun Yu

    2006-01-01

    DNA polymerase Ⅲ is one of the five eubacterial DNA polymerases that is responsible for the replication of DNA duplex. Among the ten subunits of the DNA polymerase Ⅲ core enzyme, the alpha subunit catalyzes the reaction for polymerizing both DNA strands. In this study, we extracted genomic sequences of the alpha subunit from 159 sequenced eubacterial genomes, and carried out sequencebased phylogenetic and structural analyses. We found that all eubacterial genomes have one or more alpha subunits, which form either homodimers or heterodimers.Phylogenetic and domain structural analyses as well as copy number variations of the alpha subunit in each bacterium indicate the classification of alpha subunit into four basic groups: polC, dnaE1, dnaE2, and dnaE3. This classification is of essence in genome composition analysis. We also consolidated the naming convention to avoid further confusion in gene annotations.

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

    Science.gov (United States)

    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.

  16. Identification of Bovine, Pig and Duck Meat Species in Mixtures and in Meat Products on the Basis of the mtDNA Cytochrome Oxidase Subunit I (COI Gene Sequence

    Directory of Open Access Journals (Sweden)

    Spychaj Anita

    2016-03-01

    Full Text Available The aim of this study was to develop a method using PCR and self-designed primers on the basis of the mtDNA cytochrome oxidase subunit I (COI gene sequence to enable direct identification of the meat of three species of animals, i.e. bovines, pigs and ducks, in the single type sample, in meat mixtures and meat products. The mixtures comprised up to six meat species including apart from beef, pork and duck also chicken, turkey and goose meat. The obtained results indicate the possibility of qualitative identification of the aforementioned meat species in all types of investigated food products. The maximum length of PCR products did not exceed 300 bp, which was supposed to favour the amplification of DNA from meat products which are usually thermally processed and/or exposed to high pressure. PCR primers hybridised selectively with bovine, pig and duck DNA, showing total species specificity.

  17. Functional analysis of the glycogen binding subunit CG9238/Gbs-70E of protein phosphatase 1 in Drosophila melanogaster.

    Science.gov (United States)

    Kerekes, Éva; Kókai, Endre; Páldy, Ferenc Sándor; Dombrádi, Viktor

    2014-06-01

    The product of the CG9238 gene that we termed glycogen binding subunit 70E (Gbs-70E) was characterized by biochemical and molecular genetics methods. The interaction between Gbs-70E and all catalytic subunits of protein phosphatase 1 (Pp1-87B, Pp1-9C, Pp1-96A and Pp1-13C) of Drosophila melanogaster was confirmed by pairwise yeast two-hybrid tests, co-immunoprecipitation and pull down experiments. The binding of Gbs-70E to glycogen was demonstrated by sedimentation analysis. With RT-PCR we found that the mRNAs coding for the longer Gbs-70E PB/PC protein were expressed in all developmental stages of the fruit flies while the mRNA for the shorter Gbs-70E PA was restricted to the eggs and the ovaries of the adult females. The development specific expression of the shorter splice variant was not conserved in different Drosophila species. The expression level of the gene was manipulated by P-element insertions and gene deletion to analyze the functions of the gene product. A small or moderate reduction in the gene expression resulted in no significant changes, however, a deletion mutant expressing very low level of the transcript lived shorter and exhibited reduced glycogen content in the imagos. In addition, the gene deletion decreased the fertility of the fruit flies. Our results prove that Gbs-70E functions as the glycogen binding subunit of protein phosphatase 1 that regulates glycogen content and plays a role in the development of eggs in D. melanogaster.

  18. Mitochondrial cytochrome c oxidase subunit II variations predict adverse prognosis in cytogenetically normal acute myeloid leukaemia.

    Science.gov (United States)

    Silkjaer, Trine; Nyvold, Charlotte Guldborg; Juhl-Christensen, Caroline; Hokland, Peter; Nørgaard, Jan Maxwell

    2013-10-01

    Alterations in the two catalytic genes cytochrome c oxidase subunits I and II (COI and COII) have recently been suggested to have an adverse impact on prognosis in patients with acute myeloid leukaemia (AML). In order to explore this in further detail, we sequenced these two mitochondrial genes in diagnostic bone marrow or blood samples in 235 patients with AML. In 37 (16%) patients, a non-synonymous variation in either COI or COII could be demonstrated. No patients harboured both COI and COII non-synonymous variations. Twenty-four (10%) patients had non-synonymous variations in COI, whereas 13 (6%) patients had non-synonymous variations in COII. The COI and COII are essential subunits of cytochrome c oxidase that is the terminal enzyme in the oxidative phosphorylation complexes. In terms of disease course, we observed that in patients with a normal cytogenetic analysis at disease presentation (CN-AML) treated with curative intent, the presence of a non-synonymous variation in the COII was an adverse prognostic marker for both overall survival and disease-free survival (DFS) in both univariate (DFS; hazard ratio (HR) 4.4, P = 0.006) and multivariate analyses (DFS; HR 7.2, P = 0.001). This is the first demonstration of a mitochondrial aberration playing an adverse prognostic role in adult AML, and we argue that its role as a potentially novel adverse prognostic marker in the subset of CN-AML should be explored further.

  19. Dissecting the sterol C-4 demethylation process in higher plants. From structures and genes to catalytic mechanism.

    Science.gov (United States)

    Rahier, Alain

    2011-03-01

    Sterols become functional only after removal of the two methyl groups at C-4. This review focuses on the sterol C-4 demethylation process in higher plants. An intriguing aspect in the removal of the two C-4 methyl groups of sterol precursors in plants is that it does not occur consecutively as it does in yeast and animals, but is interrupted by several enzymatic steps. Each C-4 demethylation step involves the sequential participation of three individual enzymatic reactions including a sterol methyl oxidase (SMO), a 3β-hydroxysteroid-dehydrogenase/C4-decarboxylase (3βHSD/D) and a 3-ketosteroid reductase (SR). The distant location of the two C-4 demethylations in the sterol pathway requires distinct SMOs with respective substrate specificity. Combination of genetic and molecular enzymological approaches allowed a thorough identification and functional characterization of two distinct families of SMOs genes and two 3βHSD/D genes. For the latter, these studies provided the first molecularly and functionally characterized HSDs from a short chain dehydrogenase/reductase family in plants, and the first data on 3-D molecular interactions of an enzyme of the postoxidosqualene cyclase sterol biosynthetic pathway with its substrate in animals, yeast and higher plants. Characterization of these three new components involved in C-4 demethylation participates to the completion of the molecular inventory of sterol synthesis in higher plants.

  20. 大豆7S蛋白β亚基基因RNAi表达载体构建%Construction of the RNAi Expression Vector of Soybean 7S Protein β-subunit Gene

    Institute of Scientific and Technical Information of China (English)

    戴嘉乐; 马建; 付永平; 曲静; 王丕武

    2012-01-01

    In this study we used β-subunit gene of 7S protein as the target genes(Gene number: AB030840) ,tken obtained The current version does not support copying Cyrillic text to the Clipboard. pression vector of 7S protein (β-subunil was successfully constructed. This study provided foundation for the application of RNA interference technology to reduce soybean allergens and improve quality of soybean proteins.%以大豆7S蛋白β亚基基因为靶基因(基因编号:AB030840),利用RT-PCR克隆得到大豆7S蛋白β亚基基因核心序列398 bp,构建了以抗除草剂基因BAR为筛选标记的安全植物RNAi表达载体BAR-7αp-β.分子生物学检测表明7S蛋白β亚基基因的表达载体构建成功.研究结果为应用RNA干扰技术降低大豆过敏原,提高大豆蛋白品质奠定了基础.

  1. Targeted deletion of one or two copies of the G protein β subunit Gβ5 gene has distinct effects on body weight and behavior in mice.

    Science.gov (United States)

    Wang, Qiang; Levay, Konstantin; Chanturiya, Tatyana; Dvoriantchikova, Galina; Anderson, Karen L; Bianco, Suzy D C; Ueta, Cintia B; Molano, R Damaris; Pileggi, Antonello; Gurevich, Eugenia V; Gavrilova, Oksana; Slepak, Vladlen Z

    2011-11-01

    We investigated the physiological role of Gβ5, a unique G protein β subunit that dimerizes with regulators of G protein signaling (RGS) proteins of the R7 family instead of Gγ. Gβ5 is essential for stability of these complexes, so that its knockout (KO)causes degradation of the entire Gβ5-R7 family. We report that the Gβ5-KO mice remain leaner than the wild type (WT) throughout their lifetime and are resistant to a high-fat diet. They have a 5-fold increase in locomotor activity, increased thermogenesis, and lower serum insulin, all of which correlate with a higher level of secreted epinephrine. Heterozygous (HET) mice are 2-fold more active than WT mice. Surprisingly, with respect to body weight, the HET mice display a phenotype opposite to that of the KO mice: by the age of 6 mo, they are ≥ 15% heavier than the WT and have increased adiposity, insulin resistance, and liver steatosis. These changes occur in HET mice fed a normal diet and without apparent hyperphagia, mimicking basic characteristics of human metabolic syndrome. We conclude that even a partial reduction in Gβ5-R7 level can perturb normal animal metabolism and behavior. Our data on Gβ5 haploinsufficient mice may explain earlier observations of genetic linkage between R7 family mutations and obesity in humans.

  2. Diversity of low-molecular-weight-glutenin subunit genes associated with D-genome in Triticum aestivum, Aegilops crassa, A. cylindrica and A. tauschii

    Directory of Open Access Journals (Sweden)

    Fatemeh Vafadar Shamasbi

    2016-09-01

    Full Text Available The 40% of endosperm protein of common wheat is composed of Low Molecular Weight (LMW Glutenin Subunits. To examine variation in the D genome of wheat, 98 accessions from different areas of Iran were studied using the five Glu-D3-specific pair primers. The amplification percentages of all primer pair sets were 80.61%, 92.86%, 79.59%, 90.82% and 67.35%, respectively. In comparison of the four species, the most observed bands of the first primer pair were found in Ae. tauschii samples. For the second primer pair, the most frequency of the amplified product was found in the T. aestivum samples. For the third primer pair, the Ae. cylindrical samples had the most amplified band. For the fourth primer pair, the most amplified band was found in the T. aestivum samples. The Ae. cylindrica samples had the most frequency band for the fifth primer pair. Based on dendrogram analysis, the accessions were divided in to 18 categories; and also 42 accessions had a bond for any PCR reaction. It is hoped that the result will be effective in molecular analysis and breeding of native landrace plants.

  3. Protein-disulfide Isomerase Displaces the Cholera Toxin A1 Subunit from the Holotoxin without Unfolding the A1 Subunit*

    OpenAIRE

    Taylor, Michael; Banerjee, Tuhina; Ray, Supriyo; Tatulian, Suren A.; Teter, Ken

    2011-01-01

    Protein-disulfide isomerase (PDI) has been proposed to exhibit an “unfoldase” activity against the catalytic A1 subunit of cholera toxin (CT). Unfolding of the CTA1 subunit is thought to displace it from the CT holotoxin and to prepare it for translocation to the cytosol. To date, the unfoldase activity of PDI has not been demonstrated for any substrate other than CTA1. An alternative explanation for the putative unfoldase activity of PDI has been suggested by recent structural studies demons...

  4. Microprocessor complex subunit DiGeorge syndrome critical region gene 8 (Dgcr8) is required for schwann cell myelination and myelin maintenance.

    Science.gov (United States)

    Lin, Hsin-Pin; Oksuz, Idil; Hurley, Edward; Wrabetz, Lawrence; Awatramani, Rajeshwar

    2015-10-02

    We investigated the role of a key component of the Microprocessor complex, DGCR8, in the regulation of myelin formation and maintenance. We found that conditionally ablating Dgcr8 in Schwann cells (SCs) during development results in an arrest of SC differentiation. Dgcr8 conditional knock-out (cKO) SCs fail to form 1:1 relationships with axons or, having achieved this, fail to form myelin sheaths. The expression of genes normally found in immature SCs, such as sex-determining region Y-box 2 (Sox2), is increased in Dgcr8 cKO SCs, whereas the expression of myelin-related genes, including the master regulatory transcription factor early growth response 2 (Egr2), is decreased. Additionally, expression of a novel gene expression program involving sonic hedgehog (Shh), activated de novo in injured nerves, is elevated in Dgcr8 cKOs but not in Egr2 null mice, a model of SC differentiation arrest, suggesting that the injury-related gene expression program in Dgcr8 cKOs cannot be attributed to differentiation arrest. Inducible ablation of Dgcr8 in adult SCs results in gene expression changes similar to those found in cKOs, including an increase in the expression of Sox2 and Shh. Analyses of these nerves mainly reveal normal myelin thickness and axon size distribution but some dedifferentiated SCs and increased macrophage infiltration. Together our data suggest that Dgcr8 is responsible for modulation of gene expression programs underlying myelin formation and maintenance as well as suppression of an injury-related gene expression program.

  5. Induction of T helper 1 response by immunization of BALB/c mice with the gene encoding the second subunit of Echinococcus granulosus antigen B (EgAgB8/2

    Directory of Open Access Journals (Sweden)

    Boutennoune H.

    2012-05-01

    Full Text Available A pre-designed plasmid containing the gene encoding the second subunit of Echinococcus granulosus AgB8 (EgAgB8/2 was used to study the effect of the immunization route on the immune response in BALB/c mice. Mice were immunized with pDRIVEEgAgB8/ 2 or pDRIVE empty cassette using the intramuscular (i.m., intranasal (i.n. or the epidermal gene gun (g.g. routes. Analysis of the antibody response and cytokine data revealed that gene immunization by the i.m. route induced a marked bias towards a T helper type 1 (Th1 immune response as characterized by high IFN-γ gene expression and a low IgG1/IgG2a reactivity index (R.I. ratio of 0.04. The i.n. route showed a moderate IFN-γ expression but a higher IgG1/IgG2a R.I. ratio of 0.25 indicating a moderate Th1 response. In contrast, epidermal g.g. immunization induced a Th2 response characterized by high IL-4 expression and the highest IgG1/IgG2a R.I. ratio of 0.58. In conclusion, this study showed the advantage of genetic immunization using the i.m. route and i.n. over the epidermal g.g. routes in the induction of Th1 immunity in response to E. granulosus AgB gene immunization.

  6. Transcriptional regulation of the nuclear gene encoding the alpha-subunit of the mammalian mitochondrial F1F0 ATP synthase complex: role for the orphan nuclear receptor, COUP-TFII/ARP-1.

    Science.gov (United States)

    Jordan, Elzora M; Worley, Teri; Breen, Gail A M

    2003-03-11

    Our laboratory has been studying the transcriptional regulation of the nuclear gene (ATPA) that encodes the alpha-subunit of the mammalian mitochondrial F1F0 ATP synthase complex. We have previously determined that the regulatory factor, upstream stimulatory factor 2 (USF2), can stimulate transcription of the ATPA gene through the cis-acting regulatory element 1 in the upstream promoter of this gene. In this study, we used the yeast one-hybrid screening method to identify another factor, COUP-TFII/ARP-1, which also binds to the ATPA cis-acting regulatory element 1. Binding of the orphan nuclear receptor, COUP-TFII/ARP-1, to the ATPA regulatory element 1 was confirmed using electrophoretic mobility shift experiments, and COUP-TFII/ARP-1-containing complexes were detected in HeLa cell nuclear extracts. A mutational analysis indicated that the binding site for COUP-TFII/ARP-1 in the ATPA regulatory element 1 is an imperfect direct repeat of a nuclear receptor response element (A/GGGTCA) with a spacer of three nucleotides. Functional assays in HeLa cells showed that COUP-TFII/ARP-1 represses the ATPA promoter activity in a dose- and sequence-dependent manner. Furthermore, cotransfection assays demonstrated that COUP-TFII/ARP-1 inhibits the USF2-mediated activation of the wild-type ATPA gene promoter but not a mutant promoter that is defective in COUP-TFII/ARP-1-binding. Overexpression of USF2 reversed the COUP-TFII/ARP-1-mediated repression of the ATPA promoter. Mobility shift assays revealed that COUP-TFII/ARP-1 and USF2 compete for binding to the ATPA regulatory element 1. Thus, the ATPA gene is regulated by a multifunctional binding site through which the transcription factors, COUP-TFII/ARP-1 and USF2, bind and exert their antagonistic effects.

  7. First molecular cloning and gene expression analysis of teleost CD42 (glycoprotein Ib beta chain) GPIb-IX-V subunit from rock bream, Oplegnathus fasciatus.

    Science.gov (United States)

    Jeong, Ji-Min; Kim, Ju-Won; Kim, Do-Hyung; Park, Chan-Il

    2015-04-01

    CD42 is a platelet membrane glycoprotein Ib that plays a key role in haemostasis and thrombin-induced platelet activation. Here, we report the molecular cloning and sequence analysis of the CD42c gene from rock bream (Oplegnathus fasciatus). Rock bream CD42 (RbCD42c) gene expression profiles were determined after infection with Streptococcus iniae, Edwardsiella tarda and red seabream iridovirus (RSIV). The full-length RbCD42c cDNA contained an open reading frame of 624 bp encoding 207 amino acids. The deduced amino acid sequences of the leucine-rich repeat (LRR)-N terminal and LRR-C terminal were conserved between fish and mammals. RbCD42c was highly expressed in red blood cells, spleen, gill, liver and kidney of healthy rock bream. The RbCD42c gene was not significantly up- or downregulated after E. tarda exposure. However, RbCD42c gene expression was upregulated in kidney, spleen and gill after S. iniae infection. RbCD42c was upregulated in spleen, liver and gill, but downregulated in kidney 24 and 48 h after RSIV infection. These results suggest that RbCD42c has different expression patterns after infection with bacterial or viral pathogens. This gene may be directly involved in haemostasis.

  8. 霍乱毒素B亚单位与志贺毒素2B亚单位融合表达及抗原性检测%Clone and express ctb-stx2 b fusion gene in Enterohemrrhagic escherichia coli O157:H7 Shigeai toxin 2B subunit and V cholera toxin B subunit and the detection of their immunogenicity

    Institute of Scientific and Technical Information of China (English)

    李振军; 孙强正; 景怀琦; 徐建国

    2008-01-01

    Objective To clone and express the fusion gene encoding Enterohemrrhagic escherichia coli O157:H7(EHEC O157:H7)Shigela toxin 2B subunit(Stx2B)and vibrio cholera toxin B subunit (CTB)as well as to detect the immunogenicity and GM1-binding ability of fusion protein.Methods To design a primer to amplify stx2b gene and ctb-stx2b fusion gene encoding Stx2B and CTB-Stx2B respectively and to clone the genes into express plasmid pET30a(+)C in order to construct pET30a-ctb-stx2b after T-A sequencing was varified,then to transform constructed plasmid into E.coliBL21(DE3)induced by IPTG and purified by a purify kit and to detect molecular weight and immunogenicity by SDSPAGE and Western-blot.Results The amplified ctb-stx2b fragments appeared to he 750 bp and gene sequence was identical to designed sequence.The prokaryotic expression system pET30a-ctb-stx2b/BL21 could express protein weight about Mr20×103and the expressed protein could react to CTB monoclone anti-body.The fusion protein CTB-Stx2B could bind GM1.Conclusion CTB-Stx2B had successfully been expressed in prokaryotic while the expressed protein had good immunogenicity and GM1-Binding ability.This study provided information on further EHEC O157:H7 vaccine research.%目的 克隆表达出血性大肠埃希菌(EHEC)O157:H7志贺毒素2B亚单位(Stx2B)与霍乱毒素B亚单位(CTB)的融合蛋白(CTB-Stx2B),并检测其抗原性和与神经节苷脂(GM1)结合能力.方法 设计引物扩增融合蛋白CTB-Stx2B的编码基因ctb-stx2b,T-A克隆测序验证后克隆入原核表达质粒pET30a(+)C,构建表达质粒pET30a(+)-ctb-stx2b,转化E.coliBL21(DE3),IPTG诱导表达、纯化,获得目的蛋白CTB-Stx2B,SDS-PAGE和Western-blot检测其抗原性和形成五聚体的能力;GM1-ELISA法检测其与GM1结合能力.结果 扩增出约750 bp的目的片段,测序鉴定与设计序列一致;原核表达质粒pET30a(+)-ctb-stx2b转化E.coliBL21(DE3)后,经酶切和PCR扩增鉴定正确;IPTG

  9. 微孢子虫核糖体小亚单位RNA(ssUrRNA)基因%Small Subunit Ribosomal RNA Genes of Microsporidia

    Institute of Scientific and Technical Information of China (English)

    王见杨; 黄可威; 毛西成; 赵 昀; 陆长德

    2001-01-01

    微孢子虫是广泛分布于自然界的细胞内原虫类寄生物。它们可寄生于整个生物界。微孢子虫是真核生物,但其核糖体及核糖体RNA(rRNA)为原核生物型。为探讨9种家蚕病原性微孢子虫的种属地位及亲缘关系,对已广泛用于生物进化分类的核糖体小亚单位RNA(asurRNA)基因进行了研究。由微孢子虫ssurRNA基因序列同源性分析所构建的系统进化发育树及Southam杂交分析表明,这9种微孢子虫同为Nosema属,为同属不同种。%Microsporidia are ubiquitous intracellular parasitic protozoa infecting all types of animals. Their ribosomes and rRNAs are of prokaryotic size. In order to better understand their phylogenetic relationship and identify the uncertain species, the sequences of the small subunit ribosomal RNA (ssurRNA, 16 S rRNA) genesof nine microsporidia infectious to the silkworm, Bombyx mori, were determined. The results of phylogenetic trees and Southern blotting suggest all the nine strains of icrosporidia are various species of the genus Nosema.

  10. The search for mutations in the gene for the beta subunit of the cGMP phosphodiesterase (PDEB) in patients with autosomal recessive retinitis pigmentosa

    DEFF Research Database (Denmark)

    Riess, O; Noerremoelle, A; Weber, B

    1992-01-01

    including 196 bp of the 5' region of the PDEB gene have been assessed for mutations by using single-strand conformational polymorphism analysis in 14 patients from 13 unrelated families with autosomal recessive retinitis pigmentosa (ARRP). No disease-causing mutations were found in this group of affected...... individuals of seven different ancestries. However, a frequent intronic and two exonic polymorphisms (Leu489----Gln and Gly842----Gly) were identified. Segregation analysis using these polymorphic sites excludes linkage of ARRP to the PDEB gene in a family with two affected children....

  11. Expression of Kir3 gene and its subunits in human esophageal smooth muscle cells%人食管平滑肌细胞Kir3亚型表达的实验观察

    Institute of Scientific and Technical Information of China (English)

    卢强; 黄立军; 张志培; 刘同刚; 李小飞; 韩勇

    2012-01-01

    OBJECTIVE:To investigate the expression of Kir3 gene and its subunits in esophageal smooth muscles,a series of experiments was designed in this study. The difference of the expression between human esophageal longitudinal muscle(LM) and circular muscle(CM) cells were detected. METHODS:Normal esophageal smooth muscle was selected according to the 24 h esophageal pH testing,esophageal endoscopy.and HE staining. All primers were designed based on human gene sequences. The expression of Kir3. 1 - 3. 4 subunit mRNAs and total proteins were examined in human e-sophageal smooth muscle cells(SMCs) with the methods of reverse transcription polymerase chain reaction(RT-PCR) and western blot. RESULTS:The mRNA expressions of Kir3. 2,Kir3. 3,Kir3. 4 subunits were 0. 121 ±0. 015 and 0. 124± 0. 017,0. 255±0. 018 and 0. 295 ± 0. 028,0. 685 ± 0. 040 and 0. 693 ± 0. 037 respectively. The protein expressions of Kir3. 2.Kir3. 3,Kir3. 4 subunits were 0. 053±0. 010 and 0.068 ± 0.009,0. 160 ± 0.021 and 0. 192 ± 0.032,0.488 + 0.040 and 0. 504 + 0. 033 respectively. The most abundant expression was the Kir3. 4 subunit. The Kir3. 2 and Kir3. 4,Kir3. 3 and Kir3. 4 expression were both significantly differenUP0. 05). CONCLUSIONS:Expression of Kir 3. 2 - 3. 4 subunits was found in human esophageal SMCs except Kir3.1. The Kir3. 4 subunit was the most abundant expression. The expression of Kir3, 2 - 3. 4 was not significantly different in human esophageal LM and CM.%目的:探讨食管下段平滑肌中是否表达Kir3及其各亚型之间表达的差异性,同时明确食管环形平滑肌(CM)与纵行平滑肌(LM)之间表达的区别.方法:根据24 h食管pH值检测、食管镜检查及HE染色结果,分辨、筛选正常人食管平滑肌,按人Kir3.1~3.4序列设计并合成各自的高效引物,利用RT-PCR、蛋白质印迹法检测人食管平滑肌细胞中Kir3的表达及其亚型Kir3.1~3.4表达的差异.结果:在人食管LM、CM层平滑肌细

  12. Transcriptional regulators of Na, K-ATPase subunits

    Directory of Open Access Journals (Sweden)

    Zhiqin eLi

    2015-10-01

    Full Text Available 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 alpha-subunit, the beta-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 have been observed in many carcinoma, we will also discuss transcription factors that are associated with epithelial-to-mesenchymal transition, a crucial step in the progression of many tumors to malignant disease.

  13. Mutations in Nu1, the gene encoding the small subunit of bacteriophage lambda terminase, suppress the postcleavage DNA packaging defect of cosB mutations.

    Science.gov (United States)

    Cai, Z H; Hwang, Y; Cue, D; Catalano, C; Feiss, M

    1997-04-01

    The linear double-stranded DNA molecules in lambda virions are generated by nicking of concatemeric intracellular DNA by terminase, the lambda DNA packaging enzyme. Staggered nicks are introduced at cosN to generate the cohesive ends of virion DNA. After nicking, the cohesive ends are separated by terminase; terminase bound to the left end of the DNA to be packaged then binds the empty protein shell, i.e., the prohead, and translocation of DNA into the prohead occurs. cosB, a site adjacent to cosN, is a terminase binding site. cosB facilitates the rate and fidelity of the cosN cleavage reaction by serving as an anchoring point for gpNu1, the small subunit of terminase. cosB is also crucial for the formation of a stable terminase-DNA complex, called complex I, formed after cosN cleavage. The role of complex I is to bind the prohead. Mutations in cosB affect both cosB functions, causing mild defects in cosN cleavage and severe packaging defects. The lethal cosB R3- R2- R1- mutation contains a transition mutation in each of the three gpNu1 binding sites of cosB. Pseudorevertants of lambda cosB R3- R2- R1- DNA contain suppressor mutations affecting gpNu1. Results of experiments that show that two such suppressors, Nu1ms1 and Nu1ms3, do not suppress the mild cosN cleavage defect caused by the cosB R3- R2- R1- mutation but strongly suppress the DNA packaging defect are presented. It is proposed that the suppressing terminases, unlike the wild-type enzyme, are able to assemble a stable complex I with cosB R3- R2- R1- DNA. Observations on the adenosine triphosphatase activities and protease susceptibilities of gpNu1 of the Nu1ms1 and Nu1ms3 terminases indicate that the conformation of gpNu1 is altered in the suppressing terminases.

  14. Vorticella Linnaeus, 1767 (Ciliophora, Oligohymenophora, Peritrichia) is a grade not a clade: redefinition of Vorticella and the families Vorticellidae and Astylozoidae using molecular characters derived from the gene coding for small subunit ribosomal RNA.

    Science.gov (United States)

    Sun, Ping; Clamp, John; Xu, Dapeng; Kusuoka, Yasushi; Miao, Wei

    2012-01-01

    Recent phylogenetic analyses of the peritrich genus Vorticella have suggested that it might be paraphyletic, with one Vorticella species - Vorticella microstoma grouping with the swimming peritrichs Astylozoon and Opisthonecta in a distant clade. These results were based on very limited taxon sampling and thus could not be accepted as conclusive evidence for revising the generic classification. We tested paraphyly of the genus Vorticella by making a new analysis with a broad range of samples from three continents that yielded 52 new sequences of the gene coding for small subunit rRNA. Our results, together with the available sequences in Genbank, form a comprehensive set of data for the genus Vorticella. Analyses of these data showed that Vorticella microstoma morphotypes, Astylozoon, and Opisthonecta form a well-supported, monophyletic clade, that is distinct from and basal to the family Vorticellidae containing other species of Vorticella. Paraphyly of the genus Vorticella and family Vorticellidae was strongly confirmed by these results. Furthermore, the two clades of Vorticella identified by the SSU rRNA gene are so genetically diverse whereas the genetic distances within the one containing Vorticella microstoma morphotypes, Astylozoon, and Opisthonecta were so slight, which marked it as a separate family that must be defined by molecular characters in the absence of unifying morphological and morphogenetic characters. An emended characterization and status of the genus Vorticella, the families Vorticellidae and Astylozoidae are presented and discussed.

  15. Evidence for multiple promoters of the human IL-5 receptor alpha subunit gene: a novel 6-base pair element determines cell-specific promoter function.

    Science.gov (United States)

    Zhang, J; Kuvelkar, R; Cheewatrakoolpong, B; Williams, S; Egan, R W; Billah, M M

    1997-12-01

    In addition to a previously characterized promoter (P1), we now show the existence of a second promoter for the human IL-5Ralpha gene. Initially, a genomic region (P2) 5' upstream of human IL-5Ralpha exon 2 was cloned by an inverted PCR. The transcriptional start site was then mapped to a deoxycytidine (C) residue within P2 by analyzing cellular mRNA with both the 5' rapid amplification of cDNA end-PCR and S1 nuclease protection assays. Transfection of eosinophilic HL-60 cells with reporter gene constructs in which either P1 or P2 was linked to the bacterial chloramphenicol acetyltransferase (CAT) gene resulted in CAT expression; little or no CAT expression occurred in other myeloid and nonmyeloid cell lines. Deletion studies showed that a 66-bp region, ranging from -31 to +35, was sufficient to promote CAT expression in eosinophilic HL-60 cells. Analysis of linker-scanning mutants identified a novel 6-bp element (5' CTAATT 3') spanning -19 to -14 that was essential for P2 promoter activity. In electrophoretic mobility shift assays, a P2 region from -31 to +1 containing the unique 6-bp element, when used as a probe, formed a complex with a protein(s) that was found only in the eosinophilic cell line. This binding activity was lost upon replacement of the 6-bp element with a 6-bp linker, suggesting that this element likely serves as the binding site for an eosinophilic HL-60 cell-specific transcription factor(s). Together, these data suggest an important role for P2 promoter in the regulation of eosinophil-specific expression of the human IL-5 receptor alpha gene.

  16. P. berghei telomerase subunit TERT is essential for parasite survival.

    Science.gov (United States)

    Religa, Agnieszka A; Ramesar, Jai; Janse, Chris J; Scherf, Artur; Waters, Andrew P

    2014-01-01

    Telomeres define the ends of chromosomes protecting eukaryotic cells from chromosome instability and eventual cell death. The complex regulation of telomeres involves various proteins including telomerase, which is a specialized ribonucleoprotein responsible for telomere maintenance. Telomeres of chromosomes of malaria parasites are kept at a constant length during blood stage proliferation. The 7-bp telomere repeat sequence is universal across different Plasmodium species (GGGTTT/CA), though the average telomere length varies. The catalytic subunit of telomerase, telomerase reverse transcriptase (TERT), is present in all sequenced Plasmodium species and is approximately three times larger than other eukaryotic TERTs. The Plasmodium RNA component of TERT has recently been identified in silico. A strategy to delete the gene encoding TERT via double cross-over (DXO) homologous recombination was undertaken to study the telomerase function in P. berghei. Expression of both TERT and the RNA component (TR) in P. berghei blood stages was analysed by Western blotting and Northern analysis. Average telomere length was measured in several Plasmodium species using Telomere Restriction Fragment (TRF) analysis. TERT and TR were detected in blood stages and an average telomere length of ∼ 950 bp established. Deletion of the tert gene was performed using standard transfection methodologies and we show the presence of tert- mutants in the transfected parasite populations. Cloning of tert- mutants has been attempted multiple times without success. Thorough analysis of the transfected parasite populations and the parasite obtained from extensive parasite cloning from these populations provide evidence for a so called delayed death phenotype as observed in different organisms lacking TERT. The findings indicate that TERT is essential for P. berghei cell survival. The study extends our current knowledge on telomere biology in malaria parasites and validates further investigations to

  17. P. berghei telomerase subunit TERT is essential for parasite survival.

    Directory of Open Access Journals (Sweden)

    Agnieszka A Religa

    Full Text Available Telomeres define the ends of chromosomes protecting eukaryotic cells from chromosome instability and eventual cell death. The complex regulation of telomeres involves various proteins including telomerase, which is a specialized ribonucleoprotein responsible for telomere maintenance. Telomeres of chromosomes of malaria parasites are kept at a constant length during blood stage proliferation. The 7-bp telomere repeat sequence is universal across different Plasmodium species (GGGTTT/CA, though the average telomere length varies. The catalytic subunit of telomerase, telomerase reverse transcriptase (TERT, is present in all sequenced Plasmodium species and is approximately three times larger than other eukaryotic TERTs. The Plasmodium RNA component of TERT has recently been identified in silico. A strategy to delete the gene encoding TERT via double cross-over (DXO homologous recombination was undertaken to study the telomerase function in P. berghei. Expression of both TERT and the RNA component (TR in P. berghei blood stages was analysed by Western blotting and Northern analysis. Average telomere length was measured in several Plasmodium species using Telomere Restriction Fragment (TRF analysis. TERT and TR were detected in blood stages and an average telomere length of ∼ 950 bp established. Deletion of the tert gene was performed using standard transfection methodologies and we show the presence of tert- mutants in the transfected parasite populations. Cloning of tert- mutants has been attempted multiple times without success. Thorough analysis of the transfected parasite populations and the parasite obtained from extensive parasite cloning from these populations provide evidence for a so called delayed death phenotype as observed in different organisms lacking TERT. The findings indicate that TERT is essential for P. berghei cell survival. The study extends our current knowledge on telomere biology in malaria parasites and validates further

  18. Mutation in cpsf6/CFIm68 (Cleavage and Polyadenylation Specificity Factor Subunit 6) causes short 3'UTRs and disturbs gene expression in developing embryos, as revealed by an analysis of primordial germ cell migration using the medaka mutant naruto.

    Science.gov (United States)

    Sasado, Takao; Kondoh, Hisato; Furutani-Seiki, Makoto; Naruse, Kiyoshi

    2017-01-01

    Our previous studies analyzing medaka mutants defective in primordial germ cell (PGC) migration identified cxcr4b and cxcr7, which are both receptors of the chemokine sdf1/cxcl12, as key regulators of PGC migration. Among PGC migration mutants, naruto (nar) is unique in that the mutant phenotype includes gross morphological abnormalities of embryos, suggesting that the mutation affects a broader range of processes. A fine genetic linkage mapping and genome sequencing showed the nar gene encodes Cleavage and Polyadenylation Specificity Factor subunit 6 (CPSF6/CFIm68). CPSF6 is a component of the Cleavage Factor Im complex (CFIm) which plays a key role in pre-mRNA 3'-cleavage and polyadenylation. 3'RACE of sdf1a/b and cxcr7 transcripts in the mutant embryos indicated shorter 3'UTRs with poly A additions occurring at more upstream positions than wild-type embryos, suggesting CPSF6 functions to prevent premature 3'UTR cleavage. In addition, expression of the coding region sequences of sdf1a/b in nar mutants was more anteriorly extended in somites than wild-type embryos, accounting for the abnormally extended distribution of PGCs in nar mutants. An expected consequence of shortening 3'UTR is the escape from the degradation mechanism mediated by microRNAs interacting with distal 3'UTR sequence. The abnormal expression pattern of sdf1a coding sequence may be at least partially accounted for by this mechanism. Given the pleiotropic effects of nar mutation, further analysis using the nar mutant will reveal processes in which CPSF6 plays essential regulatory roles in poly A site selection and involvement of 3'UTRs in posttranscriptional gene regulation in various genes in vivo.

  19. Heterogeneity in hand veins responses to acetylcholine is not associated with polymorphisms in the G-protein beta3-subunit (C825T) and endothelial nitric oxide synthase (G894T) genes but with serum low density lipoprotein cholesterol.

    Science.gov (United States)

    Grossmann, M; Dobrev, D; Siffert, W; Kirch, W

    2001-06-01

    Vascular responses to acetylcholine (ACh) are notoriously variable, the reason for this phenomenon is unknown. We tested the hypothesis that the variability in venous response to acetylcholine may be associated with two recently identified genetic polymorphisms for proteins involved in the signal transduction pathway, i.e. the G-protein beta3-subunit (GNB3) and endothelial nitric oxide synthase (eNOS). The dorsal hand vein technique was used in 37 healthy subjects. Hand veins were preconstricted with the alpha1-adrenoceptor agonist phenylephrine and the venodilator response to local ACh infusion was measured with and without comedication of acetylsalicylic acid or co-infusion of N(G)-monomethyl-L-arginine (L-NMMA). In addition, all subjects received routine laboratory tests and 26 of them were genotyped for the C825T polymorphism of the GNB3 gene and for the G894T polymorphism of the eNOS gene. A striking variability in venous response to ACh was found with dilation observed in the low ACh concentration range and reduced dilation or even constriction at high concentrations. ACh-induced venodilation was mediated by muscarinic receptors and abolished in the presence of both acetylsalicylic acid and L-NMMA suggesting dependence on endothelium. We did not find any association of the variability in ACh response with GNB3 or eNOS allele status. On the other hand, a significant positive correlation between ACh responsiveness and low density lipoprotein-cholesterol status was detected. Two recently discovered gene polymorphisms are not responsible for the profound heterogeneity in venodilator response to ACh. Surprisingly, this variability appears to relate to the lipid status of the subjects. The exact nature of this new finding requires further study.

  20. Mutation in cpsf6/CFIm68 (Cleavage and Polyadenylation Specificity Factor Subunit 6) causes short 3'UTRs and disturbs gene expression in developing embryos, as revealed by an analysis of primordial germ cell migration using the medaka mutant naruto

    Science.gov (United States)

    Kondoh, Hisato; Furutani-Seiki, Makoto; Naruse, Kiyoshi

    2017-01-01

    Our previous studies analyzing medaka mutants defective in primordial germ cell (PGC) migration identified cxcr4b and cxcr7, which are both receptors of the chemokine sdf1/cxcl12, as key regulators of PGC migration. Among PGC migration mutants, naruto (nar) is unique in that the mutant phenotype includes gross morphological abnormalities of embryos, suggesting that the mutation affects a broader range of processes. A fine genetic linkage mapping and genome sequencing showed the nar gene encodes Cleavage and Polyadenylation Specificity Factor subunit 6 (CPSF6/CFIm68). CPSF6 is a component of the Cleavage Factor Im complex (CFIm) which plays a key role in pre-mRNA 3'-cleavage and polyadenylation. 3'RACE of sdf1a/b and cxcr7 transcripts in the mutant embryos indicated shorter 3’UTRs with poly A additions occurring at more upstream positions than wild-type embryos, suggesting CPSF6 functions to prevent premature 3’UTR cleavage. In addition, expression of the coding region sequences of sdf1a/b in nar mutants was more anteriorly extended in somites than wild-type embryos, accounting for the abnormally extended distribution of PGCs in nar mutants. An expected consequence of shortening 3'UTR is the escape from the degradation mechanism mediated by microRNAs interacting with distal 3’UTR sequence. The abnormal expression pattern of sdf1a coding sequence may be at least partially accounted for by this mechanism. Given the pleiotropic effects of nar mutation, further analysis using the nar mutant will reveal processes in which CPSF6 plays essential regulatory roles in poly A site selection and involvement of 3'UTRs in posttranscriptional gene regulation in various genes in vivo. PMID:28253363

  1. Genetic Construction of Truncated and Chimeric Metalloproteins Derived from the Alpha Subunit of Acetyl-CoA Synthase from Clostridium thermoaceticum

    Energy Technology Data Exchange (ETDEWEB)

    Huay-Keng Loke; Xiangshi Tan; Paul A. Lindahl

    2002-06-28

    In this study, a genetics-based method is used to truncate acetyl-coenzyme A synthase from Clostridium thermoaceticum (ACS), an alpha2beta2 tetrameric 310 kda bifunctional enzyme. ACS catalyzes the reversible reduction of CO2 to CO and the synthesis of acetyl-CoA from CO (or CO2 in the presence of low-potential reductants), CoA, and a methyl group bound to a corrinoid-iron sulfur protein (CoFeSP). ACS contains 7 metal-sulfur clusters of 4 different types called A, B, C, and D. The B, C, and D clusters are located in the 72 kda beta subunit while the A-cluster, a Ni-X-Fe4S4 cluster that serves as the active site for acetyl-CoA synthase activity, is located in the 82 kda alpha subunit. The extent to which the essential properties of the cluster, including catalytic, redox, spectroscopic, and substrate-binding properties, were retained as ACS was progressively truncated was determined. Acetyl-CoA synthase catalytic activity remained when the entire alpha subunit was removed, as long as CO, rather than CO2 and a low-potential reductant, was used as a substrate. Truncating an {approx} 30 kda region from the N-terminus of the alpha subunit yielded a 49 kda protein that lacked catalytic activity but exhibited A-cluster-like spectroscopic, redox, and CO binding properties. Further truncation afforded a 23 kda protein that lacked recognizable A-cluster properties except for UV-vis spectra typical of [Fe4S4]2+ clusters. Two chimeric proteins were constructed by fusing the gene encoding a ferredoxin from Chromatium vinosum to genes encoding the 49 kda and 82 kda fragments of the alpha subunit. The chimeric proteins exhibited EPR signals that were not the simple sum of the signals from the separate proteins, suggesting magnetic interactions between clusters. This study highlights the potential for using genetics to simplify the study of complex multi-centered metalloenzymes and to generate new complex metalloenzymes with interesting properties.

  2. The crystal structure of the complex of Zea mays alpha subunit with a fragment of human beta subunit provides the clue to the architecture of protein kinase CK2 holoenzyme

    DEFF Research Database (Denmark)

    Battistutta, R; Sarno, S; De Moliner, E

    2000-01-01

    The crystal structure of a complex between the catalytic alpha subunit of Zea mays CK2 and a 23-mer peptide corresponding the C-terminal sequence 181-203 of the human CK2 regulatory beta subunit has been determined at 3.16-A resolution. The complex, composed of two alpha chains and two peptides......, presents a molecular twofold axis, with each peptide interacting with both alpha chains. In the derived model of the holoenzyme, the regulatory subunits are positioned on the opposite side with respect to the opening of the catalytic sites, that remain accessible to substrates and cosubstrates. The beta...... subunit can influence the catalytic activity both directly and by promoting the formation of the alpha2 dimer, in which each alpha chain interacts with the active site of the other. Furthermore, the two active sites are so close in space that they can simultaneously bind and phosphorylate two...

  3. Genetic analysis of the cytoplasmic dynein subunit families.

    Directory of Open Access Journals (Sweden)

    K Kevin Pfister

    2006-01-01

    Full Text Available 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.

  4. 端粒酶催化亚单位基因启动子调控Hsv-tk/GCV系统对肺癌细胞A549选择性杀伤作用的研究%A study on selective killing effect of Hsv-tk/GCV driven by human telomerase catalytic subunit promoter on human lung cancer cell A549

    Institute of Scientific and Technical Information of China (English)

    唐小军; 王艳萍; 周清华; 车国卫; 陈小禾; 朱大兴

    2007-01-01

    目的 研究人端粒酶催化亚单位(human telomerase catalytic subunit,hTERT)基因启动子调控单纯疱疹胸苷激酶/更昔洛韦(herpes simplex virus-thymidine kinase/ganciclovir, Hsv-tk/GCV)治疗系统对人肺癌细胞株A549的选择体外杀伤作用.方法 (1)用脂质体法将hTERT启动子和sv40启动子调控的tk基因表达质粒(pGL3-hTp-tk和pGL3-sv40-tk)转染端粒酶阳性的人肺腺癌细胞株A549及端粒酶阴性的人胚肺成纤维细胞株MRC-5,用逆转录-PCR方法检测转染细胞中tk基因的表达情况; (2)用MTT法检测GCV对上述转染细胞体外增殖的抑制作用; (3) 用流式细胞仪检测GCV对上述转染细胞凋亡和细胞周期的影响. 结果 (1)转染pGL3-sv40-tk的细胞A549、MRC-5和转染pGL3-hTp-tk的A549均有tk mRNA表达,转染pGL3-hTp-tk的MRC-5无tk mRNA表达; (2)GCV对转染pGL3-sv40-tk的细胞A549、MRC-5和转染pGL3-hTp-tk的A549体外增殖均有明显抑制作用,对转染pGL3-hTp-tk的MRC-5无明显抑制作用; (3) 转染pGL3-sv40-tk的A549、MRC-5和转染pGL3-hTp-tk的A549细胞,GCV处理后细胞凋亡指数(21.58%、9.35%和23.19%)均显著高于转染pGL3-hTp的A549和MRC-5细胞(0.78%和0.55%)及空白对照A549和MRC-5细胞(2.17%和0.60%),转染pGL3-hTp-tk的MRC-5细胞凋亡指数(0.88%)无明显升高.结论 hTERT启动子调控Hsv-tk基因可以在肺癌细胞中选择性表达,hTERT调控的Hsv-tk/GCV治疗系统对肺癌细胞体外增殖具有靶向性抑制作用.

  5. Identification of a novel HMW glutenin subunit and comparison of its amino acid sequence with those of homologous subunits

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Aegilops tauschii is the donor of the D genome of common wheat (Triticum aestivum). Genetic variation of HMW glutenin subunits encoded by the Glu-1Dt locus of Ae. tauschii has been found to be higher than that specified by the Glu-1D locus in common wheat. In the present note, we report the identification of a novel HMW glutenin subunit, Dy13t, from Ae. tauschii. The newly identified subunit possessed an electrophoretic mobility that was faster than that of the Dy12 subunit of common wheat. The complete ORF of encoding the Dy13t subunit contained 624 codons (excluding the stop codons). The amino acid sequence deduced from the Dy13t gene ORF was the shortest among those of the previously reported subunits derived by the D genome. A further comparison of Dy13t amino acid sequence with those of the subunits characterized from the A, B, D, R genomes of Triticeae showed that the smaller size of the Dy13t subunit was associated with a reduction in the size of its repetitive domain.

  6. Study of genetic diversity of eukaryotic picoplankton in different oceanic regions by small-subunit rRNA gene cloning and sequencing.

    Science.gov (United States)

    Díez, B; Pedrós-Alió, C; Massana, R

    2001-07-01

    Very small eukaryotic organisms (picoeukaryotes) are fundamental components of marine planktonic systems, often accounting for a significant fraction of the biomass and activity in a system. Their identity, however, has remained elusive, since the small cells lack morphological features for identification. We determined the diversity of marine picoeukaryotes by sequencing cloned 18S rRNA genes in five genetic libraries from North Atlantic, Southern Ocean, and Mediterranean Sea surface waters. Picoplankton were obtained by filter size fractionation, a step that excluded most large eukaryotes and recovered most picoeukaryotes. Genetic libraries of eukaryotic ribosomal DNA were screened by restriction fragment length polymorphism analysis, and at least one clone of each operational taxonomic unit (OTU) was partially sequenced. In general, the phylogenetic diversity in each library was rather great, and each library included many different OTUs and members of very distantly related phylogenetic groups. Of 225 eukaryotic clones, 126 were affiliated with algal classes, especially the Prasinophyceae, the Prymnesiophyceae, the Bacillariophyceae, and the Dinophyceae. A minor fraction (27 clones) was affiliated with clearly heterotrophic organisms, such as ciliates, the chrysomonad Paraphysomonas, cercomonads, and fungi. There were two relatively abundant novel lineages, novel stramenopiles (53 clones) and novel alveolates (19 clones). These lineages are very different from any organism that has been isolated, suggesting that there are previously unknown picoeukaryotes. Prasinophytes and novel stramenopile clones were very abundant in all of the libraries analyzed. These findings underscore the importance of attempts to grow the small eukaryotic plankton in pure culture.

  7. 5-HT7 receptor is coupled to G alpha subunits of heterotrimeric G12-protein to regulate gene transcription and neuronal morphology.

    Science.gov (United States)

    Kvachnina, Elena; Liu, Guoquan; Dityatev, Alexander; Renner, Ute; Dumuis, Aline; Richter, Diethelm W; Dityateva, Galina; Schachner, Melitta; Voyno-Yasenetskaya, Tatyana A; Ponimaskin, Evgeni G

    2005-08-24

    The neurotransmitter serotonin (5-HT) plays an important role in the regulation of multiple events in the CNS. We demonstrated recently a coupling between the 5-HT4 receptor and the heterotrimeric G13-protein resulting in RhoA-dependent neurite retraction and cell rounding (Ponimaskin et al., 2002). In the present study, we identified G12 as an additional G-protein that can be activated by another member of serotonin receptors, the 5-HT7 receptor. Expression of 5-HT7 receptor induced constitutive and agonist-dependent activation of a serum response element-mediated gene transcription through G12-mediated activation of small GTPases. In NIH3T3 cells, activation of the 5-HT7 receptor induced filopodia formation via a Cdc42-mediated pathway correlating with RhoA-dependent cell rounding. In mouse hippocampal neurons, activation of the endogenous 5-HT7 receptors significantly increased neurite length, whereas stimulation of 5-HT4 receptors led to a decrease in the length and number of neurites. These data demonstrate distinct roles for 5-HT7R/G12 and 5-HT4R/G13 signaling pathways in neurite outgrowth and retraction, suggesting that serotonin plays a prominent role in regulating the neuronal cytoarchitecture in addition to its classical role as neurotransmitter.

  8. Association study of polymorphisms in the alpha 7 nicotinic acetylcholine receptor subunit and catechol-o-methyl transferase genes with sensory gating in first-episode schizophrenia.

    Science.gov (United States)

    Liu, Xia; Hong, Xiaohong; Chan, Raymond C K; Kong, Fanzhi; Peng, Zhizhen; Wan, Xiaona; Wang, Changqing; Cheng, Lu

    2013-10-30

    The purpose of the current study was to explore the association of auditory P50 sensory gating (P50) and prepulse inhibition (PPI) of schizophrenia with polymorphisms in the CHRNA7 and COMT genes. One hundred and fourty patients with schizophrenia participated in this study. They were administered the tests P50 and PPI. Moreover, three single nucleotide polymorphisms (SNPs) (rs2337980, rs1909884 and rs883473) in CHRNA7 and three SNPs (rs4680, rs737865 and rs165599) in COMT were selected to be genotyped by polyacrylamide gel microarray techniques. P50 index showed significant reduction in S2 amplitude between wild-type and mutation groups in the COMT rs4680. S1 amplitude of mutation group in the COMT rs737865 was also lower compared to wild-type group. PPI index revealed a shorter pulse latency of mutation group in the rs4680. The suppression ratio of mutation group was lower in COMT rs165599. Negative findings were shown between comparisons in all the CHRNA7 SNPs. We find that P50 and PPI may be influenced by COMT rs4680 polymorphisms in schizophrenia; more excitingly, we find that P50 might be influenced by COMT rs737865 polymorphisms and PPI may be influenced by COMT rs165599 polymorphisms in schizophrenia, and their mutations are associated with the reduction of the risk of P50 or PPI defects in schizophrenia. Futher studies with a larger number of subjects are needed to verify the present findings.

  9. Morphology, morphogenesis and small subunit rRNA gene sequence of a soil hypotrichous ciliate, Perisincirra paucicirrata (Ciliophora, Kahliellidae), from the shoreline of the Yellow River, North China.

    Science.gov (United States)

    Li, Fengchao; Xing, Yi; Li, Jiamei; Al-Rasheid, Khaled A S; He, Songke; Shao, Chen

    2013-01-01

    The morphology, morphogenesis, and 18S rRNA gene sequence of a soil hypotrichous ciliate Perisincirra paucicirrata, isolated from north China, were investigated. Perisincirra paucicirrata differs from its congeners in: (1) having a body length to width ratio in vivo of 4:1, (2) its adoral zone occupying between 15% and 25% of the total body length, and (3) the presence of two parabuccal cirri, three left (with 10-16 cirri each) and two right marginal rows (with 14-24 cirri each), and three dorsal kineties. Our study offers a first attempt to begin to map the morphogenetic processes of the genus, which are mainly characterised by the following: the formation of four frontal ventral transverse anlagens for each daughter cell, with the proter's anlage I originating from the reorganised anterior part of the parental paroral; the paroral and endoral anlage developed from the reorganised old endoral and do not contribute the first frontal cirrus; the frontoventral transverse anlage I contributing the left frontal cirrus; anlage II generating the middle frontal and the buccal cirri; anlage III developing the right frontal cirrus and the anterior parabuccal cirrus; and anlage IV contributing the posterior parabuccal cirrus. As an additional contribution, we judge that the inner one or the two right rows of P. kahli and P. longicirrata are marginal rows. Phylogenetic analysis based on SSU rDNA sequences suggests that Perisincirra is related to sporadotrichids, but provides no credible evidence for its taxonomic position.

  10. Prevalence of microsporidiosis due to Enterocytozoon bieneusi and Encephalitozoon (Septata) intestinalis among patients with AIDS-related diarrhea: determination by polymerase chain reaction to the microsporidian small-subunit rRNA gene.

    Science.gov (United States)

    Coyle, C M; Wittner, M; Kotler, D P; Noyer, C; Orenstein, J M; Tanowitz, H B; Weiss, L M

    1996-11-01

    Microsporidia are emerging as opportunistic pathogens in patients with AIDS. Enterocytozoon bieneusi and Encephalitozoon (Septata) intestinalis have been implicated in enteric infections in AIDS patients with chronic diarrhea, a wasting syndrome, and malabsorption. We used the polymerase chain reaction (PCR) and primers that amplify the conserved regions of the small-subunit rRNA (SSU-rRNA) gene of E. bieneusi and E. intestinalis in tissue specimens from HIV-infected patients with and without diarrhea to examine the association between microsporidia and diarrhea in patients with AIDS. Tissue specimens were obtained from 68 patients with AIDS and diarrhea (mean CD4 lymphocyte count, 21/mm3) and 43 AIDS patients without diarrhea (mean CD4 lymphocyte count, 60/mm3). By means of PCR with use of the SSU-rRNA primers specific for E. bieneusi and E. intestinalis, we found that 44% of patients with diarrhea were infected with microsporidia, whereas only 2.3% of the patients without diarrhea were infected with microsporidia (P < .001). There was a clear association between the presence of microsporidia and diarrhea. In addition, the SSU-rRNA primers proved to be sensitive and specific when used in this clinical setting.

  11. The Arabidopsis gene DIG6 encodes a large 60S subunit nuclear export GTPase 1 that is involved in ribosome biogenesis and affects multiple auxin-regulated development processes

    KAUST Repository

    Zhao, Huayan

    2015-08-13

    The circularly permuted GTPase large subunit GTPase 1 (LSG1) is involved in the maturation step of the 60S ribosome and is essential for cell viability in yeast. Here, an Arabidopsis mutant dig6 (drought inhibited growth of lateral roots) was isolated. The mutant exhibited multiple auxin-related phenotypes, which included reduced lateral root number, altered leaf veins, and shorter roots. Genetic mapping combined with next-generation DNA sequencing identified that the mutation occurred in AtLSG1-2. This gene was highly expressed in regions of auxin accumulation. Ribosome profiling revealed that a loss of function of AtLSG1-2 led to decreased levels of monosomes, further demonstrating its role in ribosome biogenesis. Quantitative proteomics showed that the expression of certain proteins involved in ribosome biogenesis was differentially regulated, indicating that ribosome biogenesis processes were impaired in the mutant. Further investigations showed that an AtLSG1-2 deficiency caused the alteration of auxin distribution, response, and transport in plants. It is concluded that AtLSG1-2 is integral to ribosome biogenesis, consequently affecting auxin homeostasis and plant development.

  12. Quantitative analysis of interferon alpha receptor subunit 1 and suppressor of cytokine signaling 1 gene transcription in blood cells of patients with chronic hepatitis C

    Directory of Open Access Journals (Sweden)

    Sedeño-Monge Virginia

    2010-09-01

    Full Text Available Abstract Background Interferon (IFN-α receptor 1 (ifnar1 and suppressor of cytokine signaling 1 (socs1 transcription levels were quantified in peripheral blood mononuclear cells (PBMC of 59 patients infected with hepatitis C virus (HCV and 17 non-infected individuals. Samples were obtained from patients infected with HCV that were either untreated or treated with IFN-α2 plus ribavirin for 1 year and divided into responders and non-responders based on viral load reduction 6 months after treatment. Ifnar1 and socs1 transcription was quantified by real-time RT-PCR, and the fold difference (2-ΔΔCT with respect to hprt housekeeping gene was calculated. Results Ifnar1 transcription increased significantly in HCV-infected patients either untreated (3.26 ± 0.31, responders (3.1 ± 0.23 and non-responders (2.18 ± 0.23 with respect to non-infected individuals (1 ± 0.34; P = 0.005. Ifnar1 transcription increased significantly (P = 0.003 in patients infected with HCV genotypes 1a (4.74 ± 0.25 and 1b (2.81 ± 0.25 but not in 1a1b (1.58 ± 0.21. No association was found of Ifnar1 transcription with disease progress, initial viral load or other clinical factors. With respect to socs1 transcription, values were similar for non-infected individuals (1 ± 0.28 and untreated patients (0.99 ± 0.41 but increased in responders (2.81 ± 0.17 and non-responder patients (1.67 ± 0.41. Difference between responder and non-responder patients was not statistically significant. Socs1 transcription increased in patients infected with HCV genotypes 1a and 1b (2.87 ± 0.45 and 2.22 ± 0.17, respectively but not in 1a1b (1.28 ± 0.40. Socs1 transcript was absent in three patients infected with HCV genotype 1b. A weak correlation between ifnar1 and socs1 transcription was found, when Spearman's correlation coefficient was calculated. Conclusion Our results suggest that HCV infection may up-regulate ifnar1 transcription. HCV genotypes differ in their capacity to affect

  13. Regulation of BK channels by auxiliary γ subunits

    Directory of Open Access Journals (Sweden)

    Jiyuan eZhang

    2014-10-01

    Full Text Available The large-conductance, calcium- and voltage-activated potassium (BK channel has the largest single-channel conductance among potassium channels and can be activated by both membrane depolarization and increases in intracellular calcium concentration. BK channels consist of pore-forming, voltage- and calcium-sensing α subunits, either alone or in association with regulatory subunits. BK channels are widely expressed in various tissues and cells including both excitable and non-excitable cells and display diverse biophysical and pharmacological characteristics. This diversity can be explained in part by posttranslational modifications and alternative splicing of the α subunit, which is encoded by a single gene, KCNMA1, as well as by tissue-specific β subunit modulation. Recently, a leucine-rich repeat-containing membrane protein, LRRC26, was found to interact with BK channels and cause an unprecedented large negative shift (~-140 mV in the voltage dependence of the BK channel activation. LRRC26 allows BK channels to open even at near-physiological calcium concentration and membrane voltage in non-excitable cells. Three LRRC26-related proteins, LRRC52, LRRC55, and LRRC38, were subsequently identified as BK channel modulators. These LRRC proteins are structurally and functionally distinct from the BK channel β subunits and were designated as γ subunits. The discovery of the γ subunits 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. Unlike BK channel β subunits, which have been intensively investigated both mechanistically and physiologically, our understanding of the γ subunits is very limited at this stage. This article reviews the structure, modulatory mechanisms, physiological relevance, and potential therapeutic implications of γ subunits as they are currently understood.

  14. Rubisco catalytic properties of wild and domesticated relatives provide scope for improving wheat photosynthesis.

    Science.gov (United States)

    Prins, Anneke; Orr, Douglas J; Andralojc, P John; Reynolds, Matthew P; Carmo-Silva, Elizabete; Parry, Martin A J

    2016-03-01

    Rubisco is a major target for improving crop photosynthesis and yield, yet natural diversity in catalytic properties of this enzyme is poorly understood. Rubisco from 25 genotypes of the Triticeae tribe, including wild relatives of bread wheat (Triticum aestivum), were surveyed to identify superior enzymes for improving photosynthesis in this crop. In vitro Rubisco carboxylation velocity (V c), Michaelis-Menten constants for CO2 (K c) and O2 (K o) and specificity factor (S c/o) were measured at 25 and 35 °C. V c and K c correlated positively, while V c and S c/o were inversely related. Rubisco large subunit genes (rbcL) were sequenced, and predicted corresponding amino acid differences analysed in relation to the corresponding catalytic properties. The effect of replacing native wheat Rubisco with counterparts from closely related species was analysed by modelling the response of photosynthesis to varying CO2 concentrations. The model predicted that two Rubisco enzymes would increase photosynthetic performance at 25 °C while only one of these also increased photosynthesis at 35 °C. Thus, under otherwise identical conditions, catalytic variation in the Rubiscos analysed is predicted to improve photosynthetic rates at physiological CO2 concentrations. Naturally occurring Rubiscos with superior properties amongst the Triticeae tribe can be exploited to improve wheat photosynthesis and crop productivity.

  15. The NDUFS4 nuclear gene of complex I of mitochondria and the cAMP cascade.

    Science.gov (United States)

    Papa, Sergio

    2002-09-10

    Results of studies on the role of the 18 kDa (IP) polypeptide subunit of complex I, encoded by the nuclear NDUFS4 gene, in isolated bovine heart mitochondria and human and murine cell cultures are presented.The mammalian 18 kDa subunit has in the carboxy-terminal sequence a conserved consensus site (RVS), which in isolated mitochondria is phosphorylated by cAMP-dependent protein kinase (PKA). The catalytic and regulatory subunits of PKA have been directly immunodetected in the inner membrane/matrix fraction of mammalian mitochondria. In the mitochondrial inner membrane a PP2Cgamma-type phosphatase has also been immunodetected, which dephosphorylates the 18 kDa subunit, phosphorylated by PKA. This phosphatase is Mg(2+)-dependent and inhibited by Ca(2+). In human and murine fibroblast and myoblast cultures "in vivo", elevation of intracellular cAMP level promotes phosphorylation of the 18 kDa subunit and stimulates the activity of complex I and NAD-linked mitochondrial respiration. Four families have been found with different mutations in the cDNA of the NDUFS4 gene. These mutations, transmitted by autosomal recessive inheritance, were associated in homozygous children with fatal neurological syndrome. All these mutations destroyed the phosphorylation consensus site in the C terminus of the 18 kDa subunit, abolished cAMP activation of complex I and impaired its normal assembly.

  16. 夏枯草RuBPCase大亚基基因的克隆与表达%Molecular Cloning and Expression Analysis of A Large Subunit Gene of RuBPCase ( PvrbcL ) from Prunella vulgaris

    Institute of Scientific and Technical Information of China (English)

    许锋; 蒋丽阳; 曹腾; 宁迎晶; 程水源

    2012-01-01

    In order to clone the large subunit gene (rbcL) of Rubisco-1,5-bisphosphate carboxylase/ oxylase (RuBPCase), one of the important enzyme involved in photosynthesis and photorespiration, from P. vulgaris, and so as to provide basic data for studying on the structure and function of RuBPCase and discussing the photosynthesis mechanism on the molecular level of P. vulgaris, a pair of specific primers was designed. An rbcL gene fragment, named PvrbcL., was cloned from P. vulgaris using genomic DNA as template. The results showed that the length of PvrbcL sequence was 837 bp, encoding a 278-amino-acid protein. The GenBank accession No. of PvrbcL gene was JN692563. The BLAST results showed that the homology of the PvrbcL nucleotide sequence with rbcL genes from Prunella grandiflora , Salvia pachyphylla , Nicotiana tabacum , Glycine max , Oryza sativa , Phaseolus vulgaris , Vitis vinifer, and Brassica napus was 86%~100%, and the homology of amino acid sequences was 92//o~ 100%. Northern blot analysis revealed that PvrbcL gene expressed in leaves and stems, and the highest level was detected in leaves. Developmental expression patter indicated that the expression level of PvrbcL gene was increased along with the growth of the leaves. The isolation and expression of PvrbcL gene could provide the basis for further studying the structure and function of RuBPCase in P. vulgaris.%为从夏枯草中克隆参与光合作用和光呼吸代谢的关键酶RuBPcase大亚基基因rbcL,为今后研究夏枯草RuBPcase的结构和功能以及从分子水平探讨夏枯草的光合作用机理提供必要的基础数据,以夏枯草基因组DNA为模板,采用PCR法对夏枯草RuBPCase大亚基基因(PvrbcL)的克隆与表达进行研究.结果表明:PCR法扩增出夏枯草RuBPcase大亚基基因片段PvrbcL,基因长837bp,编码278个氨基酸,GenBank登录号为JN692563;经BLAST序列比较,夏枯草PvrbcL基因核苷酸序列与大花夏枯草、鼠尾草、烟草、大豆、水稻

  17. Rubisco oligomers composed of linked small and large subunits assemble in tobacco plastids and have higher affinities for CO2 and O2.

    Science.gov (United States)

    Whitney, Spencer Michael; Kane, Heather Jean; Houtz, Robert L; Sharwood, Robert Edward

    2009-04-01

    Manipulation of Rubisco within higher plants is complicated by the different genomic locations of the large (L; rbcL) and small (S; RbcS) subunit genes. Although rbcL can be accurately modified by plastome transformation, directed genetic manipulation of the multiple nuclear-encoded RbcS genes is more challenging. Here we demonstrate the viability of linking the S and L subunits of tobacco (Nicotiana tabacum) Rubisco using a flexible 40-amino acid tether. By replacing the rbcL in tobacco plastids with an artificial gene coding for a S40L fusion peptide, we found that the fusions readily assemble into catalytic (S40L)8 and (S40L)16 oligomers that are devoid of unlinked S subunits. While there was little or no change in CO2/O2 specificity or carboxylation rate of the Rubisco oligomers, their Kms for CO2 and O2 were reduced 10% to 20% and 45%, respectively. In young maturing leaves of the plastome transformants (called ANtS40L), the S40L-Rubisco levels were approximately 20% that of wild-type controls despite turnover of the S40L-Rubisco oligomers being only slightly enhanced relative to wild type. The reduced Rubisco content in ANtS40L leaves is partly attributed to problems with folding and assembly of the S40L peptides in tobacco plastids that relegate approximately 30% to 50% of the S40L pool to the insoluble protein fraction. Leaf CO2-assimilation rates in ANtS40L at varying pCO2 corresponded with the kinetics and reduced content of the Rubisco oligomers. This fusion strategy provides a novel platform to begin simultaneously engineering Rubisco L and S subunits in tobacco plastids.

  18. Early expression of GABA(A) receptor delta subunit in the neonatal rat hippocampus.

    Science.gov (United States)

    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.

  19. Cloning, Expression and Sequence Analysis of the Subunit Gene Atp9 Unit in Trametes Gallica%粗毛栓菌atp9基因的克隆、表达及序列分析

    Institute of Scientific and Technical Information of China (English)

    雍彬; 司文杰; 陶宗娅; 严伟

    2011-01-01

    ATP合酶是生物体内能量代谢的关键酶,参与多种氧化磷酸化和光合磷酸化反应.atp9基因是ATP合酶的重要组成部分,其编码了ATP合酶A亚基上第9亚单位,与呼吸作用和光合作用密切相关.本研究利用atP9基因在进化过程中高度保守的特点,据已知近缘真菌基因序列,设计并合成了一对引物,以粗毛栓菌mRNA反转录得到的cDNA第一链为模板,PCR扩增得到atp9基因完整序列,并连接于原核表达载体pET32a(+)上.测序与序列分析表明:该克隆片段全长222 bp,共编码73个氨基酸,翻译的蛋白质分子量是7.35 kDa.转化大肠杆菌后经IPTG诱导,可高效表达外源融合蛋白,分子量大小与预测结果一致.经过同源比对和进化树分析,该克隆基因编码的氨基酸序列与可可丛枝病菌(Crinipellis perniciosa)和瓣环栓菌(Trametes cingulata strain ATCC 26747)中相对应的氨基酸序列相似度最高.本实验为未来进一步研究粗毛栓菌atp9基因和其蛋白功能,阐明其调控和作用机制奠定了基础.%ATP synthase is a key enzyme of energy metabolism, which was involved in a variety of oxidative phosphorylation and photophosphorylation in vivo. The atp9 gene encoding the ninth part of ATP synthase A sub-unit is an important component of ATP synthase. It is closely related with respiration and photosynthesis. For its highly conserved sequence in evolution, one pair of primers were designed and synthesized according to the known gene sequences from closely related fungi. The first strand of Trametes gallica Cdna was amplified by reverse transcription and then the complete atp9 gene sequences were obtained by PCR. And then the product of PCR was ligated to the prokaryotic expression vector pET32a ( + ) and transformed into E. Coll for the expression of fusion protein. The sequenced and bioinformatics analysis showed that: the complete length of atp9 gene was 222 bp, and the peptide it encoded had 73 amino acids with

  20. Phylogenetic relationships in the mushroom genus Coprinus and dark-spored allies based on sequence data from the nuclear gene coding for the large ribosomal subunit RNA: divergent domains, outgroups, and monophyly.

    Science.gov (United States)

    Hopple, J S; Vilgalys, R

    1999-10-01

    Phylogenetic relationships were investigated in the mushroom genus Coprinus based on sequence data from the nuclear encoded large-subunit rDNA gene. Forty-seven species of Coprinus and 19 additional species from the families Coprinaceae, Strophariaceae, Bolbitiaceae, Agaricaceae, Podaxaceae, and Montagneaceae were studied. A total of 1360 sites was sequenced across seven divergent domains and intervening sequences. A total of 302 phylogenetically informative characters was found. Ninety-eight percent of the average divergence between taxa was located within the divergent domains, with domains D2 and D8 being most divergent and domains D7 and D10 the least divergent. An empirical test of phylogenetic signal among divergent domains also showed that domains D2 and D3 had the lowest levels of homoplasy. Two equally most parsimonious trees were resolved using Wagner parsimony. A character-state weighted analysis produced 12 equally most parsimonious trees similar to those generated by Wagner parsimony. Phylogenetic analyses employing topological constraints suggest that none of the major taxonomic systems proposed for subgeneric classification is able to completely reflect phylogenetic relationships in Coprinus. A strict consensus integration of the two Wagner trees demonstrates the problematic nature of choosing outgroups within dark-spored mushrooms. The genus Coprinus is found to be polyphyletic and is separated into three distinct clades. Most Coprinus taxa belong to the first two clades, which together form a larger monophyletic group with Lacrymaria and Psathyrella in basal positions. A third clade contains members of Coprinus section Comati as well as the genus Leucocoprinus, Podaxis pistillaris, Montagnea arenaria, and Agaricus pocillator. This third clade is separated from the other species of Coprinus by members of the families Strophariaceae and Bolbitiaceae and the genus Panaeolus.

  1. Molecular identification of sibling species of Sclerodermus (Hymenoptera: Bethylidae that parasitize buprestid and cerambycid beetles by using partial sequences of mitochondrial DNA cytochrome oxidase subunit 1 and 28S ribosomal RNA gene.

    Directory of Open Access Journals (Sweden)

    Yuan Jiang

    Full Text Available The species belonging to Sclerodermus (Hymenoptera: Bethylidae are currently the most important insect natural enemies of wood borer pests, mainly buprestid and cerambycid beetles, in China. However, some sibling species of this genus are very difficult to distinguish because of their similar morphological features. To address this issue, we conducted phylogenetic and genetic analyses of cytochrome oxidase subunit I (COI and 28S RNA gene sequences from eight species of Sclerodermus reared from different wood borer pests. The eight sibling species were as follows: S. guani Xiao et Wu, S. sichuanensis Xiao, S. pupariae Yang et Yao, and Sclerodermus spp. (Nos. 1-5. A 594-bp fragment of COI and 750-bp fragment of 28S were subsequently sequenced. For COI, the G-C content was found to be low in all the species, averaging to about 30.0%. Sequence divergences (Kimura-2-parameter distances between congeneric species averaged to 4.5%, and intraspecific divergences averaged to about 0.09%. Further, the maximum sequence divergences between congeneric species and Sclerodermus sp. (No. 5 averaged to about 16.5%. All 136 samples analyzed were included in six reciprocally monophyletic clades in the COI neighbor-joining (NJ tree. The NJ tree inferred from the 28S rRNA sequence yielded almost identical results, but the samples from S. guani, S. sichuanensis, S. pupariae, and Sclerodermus spp. (Nos. 1-4 clustered together and only Sclerodermus sp. (No. 5 clustered separately. Our findings indicate that the standard barcode region of COI can be efficiently used to distinguish morphologically similar Sclerodermus species. Further, we speculate that Sclerodermus sp. (No. 5 might be a new species of Sclerodermus.

  2. Role of positron emission tomography and bone scintigraphy in the evaluation of bone involvement in metastatic pheochromocytoma and paraganglioma: specific implications for succinate dehydrogenase enzyme subunit B gene mutations.

    Science.gov (United States)

    Zelinka, Tomás; Timmers, Henri J L M; Kozupa, Anna; Chen, Clara C; Carrasquillo, Jorge A; Reynolds, James C; Ling, Alexander; Eisenhofer, Graeme; Lazúrová, Ivica; Adams, Karen T; Whatley, Millie A; Widimsky, Jirí; Pacak, Karel

    2008-03-01

    We performed a retrospective analysis of 71 subjects with metastatic pheochromocytoma and paraganglioma (30 subjects with mutation of succinate dehydrogenase enzyme subunit B (SDHB) gene and 41 subjects without SDHB mutation). Sixty-nine percent presented with bone metastases (SDHB +/-: 77% vs 63%), 39% with liver metastases (SDHB +/-: 27% vs 47%), and 32% with lung metastases (SDHB +/-: 37% vs 29%). The most common sites of bone involvement were thoracic spine (80%; SDHB+/-: 83% vs 77%), lumbar spine (78%; SDHB +/-: 78% vs 75%), and pelvic and sacral bones (78%; SDHB +/-: 91% vs 65%, P=0.04). Subjects with SDHB mutation also showed significantly higher involvement of long bones (SDHB +/-: 78% vs 30%, P=0.007) than those without the mutation. The best overall sensitivity in detecting bone metastases demonstrated positron emission tomography (PET) with 6-[(18)F]-fluorodopamine ([(18)F]-FDA; 90%), followed by bone scintigraphy (82%), computed tomography or magnetic resonance imaging (CT/MRI; 78%), 2-[(18)F]-fluoro-2-deoxy-d-glucose ([(18)F]-FDG) PET (76%), and scintigraphy with [(123/131)I]-metaiodobenzylguanidine (71%). In subjects with SDHB mutation, imaging modalities with best sensitivities for detecting bone metastases were CT/MRI (96%), bone scintigraphy (95%), and [(18)F]-FDG PET (92%). In subjects without SDHB mutations, the modality with the best sensitivity for bone metastases was [(18)F]-FDA PET (100%). In conclusion, bone scintigraphy should be used in the staging of patients with malignant pheochromocytoma and paraganglioma, particularly in patients with SDHB mutations. As for PET imaging, [(18)F]-FDG PET is highly recommended in SDHB mutation patients, whereas [(18)F]-FDA PET is recommended in patients without the mutation.

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

    Science.gov (United States)

    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.

  4. Putative pyrophosphate phosphofructose 1-kinase genes identified in sugar cane may be getting energy from pyrophosphate.

    Science.gov (United States)

    Suzuki, J; Mutton, M A; Ferro, M I T; Lemos, M V F; Pizauro, J M; Mutton, M J R; Di Mauro, S M Z

    2003-12-30

    Pyrophosphate-dependent phosphofructokinase (PPi-PFK) has been detected in several types of plant cells, but the gene has not been reported in sugar cane. Using Citrus paradisi PPi-PFK gene (AF095520 and AF095521) sequences to search the sugar cane EST database, we have identified both the alpha and beta subunits of this enzyme. The deduced amino acid sequences showed 76 and 80% similarity with the corresponding alpha and beta subunits of C. paradisi. A high degree of similarity was also observed among the PFK b subunits when the alignment of the sugar cane sequences was compared to those of Ricinus communis and Solanum tuberosum. It appears that alpha and beta are two distinct subunits; they were found at different concentrations in several sugar cane tissues. It remains to be determined if the different gene expression levels have some physiological importance and how they affect sucrose synthesis, export, and storage in vacuoles. A comparison between the amino acid sequences of b PFKs from a variety of organisms allowed us to identify the two critical Asp residues typical of this enzyme's activity site and the other binding sites; these residues are tightly conserved in all members of this protein family. Apparently, there are catalytic residues on the b subunit of the pyrophosphate-dependent enzyme.

  5. The identification of the nitrate assimilation related genes in the novel Bacillus megaterium NCT-2 accounts for its ability to use nitrate as its only source of nitrogen.

    Science.gov (United States)

    Shi, Weiwei; Lu, Wei; Liu, Qunlu; Zhi, Yuee; Zhou, Pei

    2014-03-01

    Bacillus megaterium NCT-2 is a novel bacterium that can utilize nitrate as its only nitrogen source for growth.The nitrate assimilation related genes that are involved in this process would be expected to be crucial. However, little is known about the genomic background of this bacterium,let alone the sequences of the nitrate assimilation related genes. In order to further investigate the nitrate assimilation function of the NCT-2, genome sequencing was performed.After obtaining the fine map of the NCT-2 genome, which was submitted to the NCBI GenBank (AHTF00000000), the sequences of the nitrate assimilation related genes (the nitrate reductase electron transfer subunit nasB and the nitrate reductase catalytic subunit nasC, the nitrite reductase [NAD(P)H]large subunit nasD and the nitrite reductase [NAD(P)H] small subunit nasE, and the glutamine synthetase glnA) were identified.Multiple alignments were performed to find out the sequence identities of the nitrate assimilation related genes to that of their similar species. Through KEGG signaling mapping search, the nitrate assimilation related genes were revealed to be located in the nitrogen metabolism signaling pathway. The putative 3D protein structures of these genes were modeled by SWISS MODEL, and shown to be highly similar to the nitrate assimilation related genes in the PDB database. Finally, the sequence validity of the nitrate assimilation related genes was verified by PCR with specifically designed primers.

  6. Further examination of seventeen mutations in Escherichia coli F1-ATPase beta-subunit.

    Science.gov (United States)

    Senior, A E; al-Shawi, M K

    1992-10-25

    Seventeen mutations in beta-subunit of Escherichia coli F1-ATPase which had previously been characterized in strain AN1272 (Mu-induced mutant) were expressed in strain JP17 (beta-subunit gene deletion). Six showed unchanged behavior, namely: C137Y; G142D; G146S; G207D; Y297F; and Y354F. Five failed to assemble F1F0 correctly, namely: G149I; G154I; G149I,G154I; G223D; and P403S,G415D. Six assembled F1F0 correctly, but with membrane ATPase lower than in AN1272, namely: K155Q; K155E; E181Q; E192Q; D242N; and D242V. AN1272 was shown to unexpectedly produce a small amount of wild-type beta-subunit; F1-ATPase activities reported previously in AN1272 were referable to hybrid enzymes containing both mutant and wild-type beta-subunits. Purified F1 was obtained from K155Q; K155E; E181Q; E192Q; and D242N mutants in JP17. Vmax ATPase values were lower, and unisite catalysis rate and equilibrium constants were perturbed to greater extent, than in AN1272. However, general patterns of perturbation revealed by difference energy diagrams were similar to those seen previously, and the new data correlated well in linear free energy relationships for reaction steps of unisite catalysis. Correlation between multisite and unisite ATPase activity was seen in the new enzymes. Overall, the data give strong support to previously proposed mechanisms of unisite catalysis, steady-state catalysis, and energy coupling in F1-ATPases (Al-Shawi, M. K., Parsonage, D. and Senior, A. E. (1990) J. Biol. Chem. 265, 4402-4410). The K155Q, K155E, D242N, and E181Q mutations caused 5000-fold, 4000-fold, 1800-fold, and 700-fold decrease, respectively, in Vmax ATPase, implying possibly direct roles for these residues in catalysis. Experiments with the D242N mutant suggested a role for residue beta D242 in catalytic site Mg2+ binding.

  7. Molecular pathways: targeting ETS gene fusions in cancer.

    Science.gov (United States)

    Feng, Felix Y; Brenner, J Chad; Hussain, Maha; Chinnaiyan, Arul M

    2014-09-01

    Rearrangements, or gene fusions, involving the ETS family of transcription factors are common driving events in both prostate cancer and Ewing sarcoma. These rearrangements result in pathogenic expression of the ETS genes and trigger activation of transcriptional programs enriched for invasion and other oncogenic features. Although ETS gene fusions represent intriguing therapeutic targets, transcription factors, such as those comprising the ETS family, have been notoriously difficult to target. Recently, preclinical studies have demonstrated an association between ETS gene fusions and components of the DNA damage response pathway, such as PARP1, the catalytic subunit of DNA protein kinase (DNAPK), and histone deactylase 1 (HDAC1), and have suggested that ETS fusions may confer sensitivity to inhibitors of these DNA repair proteins. In this review, we discuss the role of ETS fusions in cancer, the preclinical rationale for targeting ETS fusions with inhibitors of PARP1, DNAPK, and HDAC1, as well as ongoing clinical trials targeting ETS gene fusions.

  8. Expression of accessory colonization factor subunit A (ACFA) of Vibrio cholerae and ACFA fused to cholera toxin B subunit in transgenic tomato (Solanum lycopersicum).

    Science.gov (United States)

    Sharma, Manoj Kumar; Jani, Dewal; Thungapathra, M; Gautam, J K; Meena, L S; Singh, Yogendra; Ghosh, Amit; Tyagi, Akhilesh Kumar; Sharma, Arun Kumar

    2008-05-20

    In earlier study from our group, cholera toxin B subunit had been expressed in tomato for developing a plant-based vaccine against cholera. In the present investigation, gene for accessory colonization factor (acf) subunit A, earlier reported to be essential for efficient colonization in the intestine, has been expressed in Escherichia coli as well as tomato plants. Gene encoding for a chimeric protein having a fusion of cholera toxin B subunit and accessory colonization factor A was also expressed in tomato to generate more potent combinatorial antigen. CaMV35S promoter with a duplicated enhancer sequence was used for expression of these genes in tomato. Integration of transgenes into tomato genome was confirmed by PCR and Southern hybridization. Expression of the genes was confirmed at transcript and protein levels. Accessory colonization factor A and cholera toxin B subunit fused to this protein accumulated up to 0.25% and 0.08% of total soluble protein, respectively, in the fruits of transgenic plants. Whereas protein purified from E. coli, in combination with cholera toxin B subunit can be used for development of conventional subunit vaccine, tomato fruits expressing these proteins can be used together with tomato plants expressing cholera toxin B subunit for development of oral vaccine against cholera.

  9. 编码酶复合体Ⅰ亚单位的线粒体基因新突变导致的MELAS综合征%Novel mutations in the mitochondrial DNA encoded complex Ⅰ subunit genes associated with MELAS

    Institute of Scientific and Technical Information of China (English)

    赵丹华; 王朝霞; 李务荣; 洪道俊; 郑日亮; 孙永安; 张巍; 袁云

    2011-01-01

    目的:报道4例由编码酶复合体Ⅰ中NADH脱氢酶(ND)亚单位的线粒体基因(mtDNA)突变所导致的线粒体脑肌病患者,分析其临床及骨骼肌病理改变特点.方法:4例患者的发病年龄在6 ~21岁之间,病程在7~ 20年.其中1例为MELAS、3例为MELAS/Leigh叠加综合征.对4例患者进行肌肉活检和mtDNA全长测序检查.结果:骨骼肌病理检查发现1例同时存在破碎红纤维(RRFs)及SDH深染的血管(SSVs),2例仅有SSVs,另1例未见异常.4例患者均携带mtDNA编码的ND基因突变,分别为位于ND3编码区的T10191C(p.S45P)、ND4编码区的A11470C(p.K237N)、ND5编码区的T13046C(p.M237T)点突变以及累及ND5和ND6编码区的单一大片段缺失( 13025-13033:14417-14425),后3种突变均为新发现的致病性突变.结论:ND基因突变是导致部分MELAS或MELAS/Leigh叠加综合征患者的分子病理学基础,这些患者的骨骼肌病理检查常缺乏典型的线粒体脑肌病的病理改变,如RRFs.%Objective To report the clinical and myopathological features of 4 patients with mitochondrial encepha-lomyopathy associated with mutations in mitochondrial DNA (mtDNA) encoded NADH dehydrogenase (ND) subunit genes of complex I. Methods The onset age of 4 patients ranged from 6 to 21 years,with a clinical course from 7 to 20 years. A-mong them, 1 case was consistent with MELAS and 3 cases with MELAS/Leigh overlap syndrome. Muscle biopsy and whole sequencing of mtDNA were performed on these patients. Results Skeletal muscle biopsy disclosed both ragged-red fibers ( RRFs) and strongly succinate dehydrogenase-reactive vessels (SSVs) in one case,only SSVs without RRFs in two cases, and no abnormality in one. Whole sequencing of mtDNA revealed T10191C( p. S45P) in ND3 , A11470C( p. K237N) in ND4,T13046C( p. M237T) in ND5 and a single large-scale deletion ranging from 13025-13033 to 14417-14425 encompassing ND5 and ND6 in these patients respectively. Among them,Al 1470C,T13046C and the

  10. Sequence-specific cleavage of BM2 gene transcript of influenza B virus by 10-23 catalytic motif containing DNA enzymes significantly inhibits viral RNA translation and replication.

    Science.gov (United States)

    Kumar, Binod; Kumar, Prashant; Rajput, Roopali; Saxena, Latika; Daga, Mradul K; Khanna, Madhu

    2013-10-01

    One of the hallmarks of progression of influenza virus replication is the step involving the virus uncoating that occurs in the host cytoplasm. The BM2 ion channel protein of influenza B virus is highly conserved and is essentially required during the uncoating processes of virus, thus an attractive target for designing antiviral drugs. We screened several DNA enzymes (Dzs) containing the 10-23 catalytic motif against the influenza B virus BM2 RNA. Dzs directed against the predicted single-stranded bulge regions showed sequence-specific cleavage activities. The Dz209 not only showed significant intracellular reduction of BM2 gene expression in transient-expression system but also provided considerable protection against influenza B virus challenge in MDCK cells. Our findings suggest that the Dz molecule can be used as selective and effective inhibitor of viral RNA replication, and can be explored further for development of a potent therapeutic agent against influenza B virus infection.

  11. Catalytic combustor for hydrogen

    Energy Technology Data Exchange (ETDEWEB)

    Mercea, J.; Grecu, E.; Fodor, T.; Kreibik, S.

    1982-01-01

    The performance of catalytic combustors for hydrogen using platinum-supported catalysts is described. Catalytic plates of different sizes were constructed using fibrous and ceramic supports. The temperature distribution as well as the reaction efficiency as a function of the fuel input rate was determined, and a comparison between the performances of different plates is discussed.

  12. Identification of Na(+)-K(+)-ATPase beta-subunit in alveolar epithelial cells.

    Science.gov (United States)

    Zhang, X L; Danto, S I; Borok, Z; Eber, J T; Martín-Vasallo, P; Lubman, R L

    1997-01-01

    The Na(+)-K(+)-ATPase is a heterodimeric plasma membrane protein that consists of a catalytic alpha-subunit and a smaller glycosylated beta-subunit that has not been fully characterized in alveolar epithelial cells (AEC) to date. In this study, we identified the Na(+)-K(+)-ATPase beta-subunit protein in rat AEC and lung membranes using immunochemical techniques. Rat AEC grown in primary culture and rat lung, brain, and kidney membranes were solubilized in either 2% sodium dodecyl sulfate (SDS) sample buffer for SDS-polyacrylamide gel electrophoresis or in 1% Nonidet P-40 lysis buffer for immunoprecipitation studies. Na(+)-K(+)-ATPase beta-subunit was not detected in either AEC or lung membranes on Western blots when probed with a panel of antibodies (Ab) against beta-subunit isoforms, whereas brain and kidney beta-subunit were recognized as broad approximately 50-kDa bands. AEC, lung, and kidney membranes were immunoprecipitated with anti-beta Ab IEC 1/48, a monoclonal Ab that recognizes beta-subunit protein only in its undenatured state. The beta-subunit was detected in the immunoprecipitate (IP) from kidney membranes by several different anti-beta-subunit Ab. The beta-subunit was faintly detectable from AEC and lung IP as a broad approximately 50-kDa band when blotted with the polyclonal anti-beta 1-subunit Ab SpET but could not be detected by blotting with other anti-beta Ab. Treatment of the IP from kidney, lung, and AEC with N-glycosidase F for 2 h at 37 degrees C resulted in immunodetection of identical approximately 35 kDa bands when probed with all anti-beta 1 Ab on Western blots. From these results, we conclude that rat lung and AEC possess immunoreactive beta-subunit protein that is only readily detectable after deglycosylation. Because anti-beta Ab fail to detect the Na(+)-K(+)-ATPase beta-subunit in rat lung or AEC by standard Western blotting techniques under the conditions of these experiments, our results suggest that lung beta-subunit may be

  13. Dravet综合征的临床特点分析及SCN1A基因新突变%Clinical Analysis of Dravet Syndrome and Novel Gene Mutation of Voltage-Gated Sodium Channel α1-Su-bunit

    Institute of Scientific and Technical Information of China (English)

    王新华; 周水珍

    2011-01-01

    Objective To study the clinical characteristics of Dravet syndrome and to screen the voltage -gated sodium channel αl -subunit( SCNI A ) of a newly diagnosed child, hoping to find the gene mutation. Methods The clinical information of 3 Dravet syndrome children were collected,the blood sample of a new diagnostic child was provided. DNA was extracted from peripheral blood leukocytes using relax gene blood DNA system. The total 26 exons of SCN1A were amplified by polymerase chain reaction( PCR), and the PCR products were screened by Denaturing high performance liquid chromatography, then the abnormal fragments were sequenced by Sanger method in order to find the mutations of SCN 1A gene. Results 1. The common manifestations of 3 Dravet syndrome cases: onset during the first year of life; in all children, the seizures were associated with febrile seizures and they changed to afebrile seizures after 1 year; the forms of seizures included clonus,myoclonus and atypical absence;the seizures were difficult to control with anti -epileptic drugs; all children presented some degree of psychomotor development delay; there were sharp - slow waves, spike - slow waves and multi spike - slow waves in EEG of diapause. 2. A missense mutation of SCNI A gene (c. 2867T > G, M956R) was found in the Dravet syndrome child, which had not been reported up to Nov.2010. Conclusions Dravet syndrome is an epileptic encephalopathy with a bad prognosis,and it needs to be differentiate it frome febrile seizures. The missense mutation of SCNIA gene supports the relationship of SCNIA mutation and Dravet syndrom.%目的 分析Dravet综合征的临床特点,并对新诊断患儿进行SCN1A基因筛查,寻找基因突变.方法收集3例Dravet综合征患儿临床资料,留取例1患儿血样标本,提取外周血白细胞基因组DNA,对SCN1A全部外显子进行PCR扩增,通过变性高效液相色谱法对PCR产物进行突变片段筛查,对于变性高效液相色谱法筛查有异常的片段

  14. Structure of the ATP Synthase Catalytic Complex (F1) from Escherichia coli in an Autoinhibited conformation

    Energy Technology Data Exchange (ETDEWEB)

    G Cingolani; T Duncan

    2011-12-31

    ATP synthase is a membrane-bound rotary motor enzyme that is critical for cellular energy metabolism in all kingdoms of life. Despite conservation of its basic structure and function, autoinhibition by one of its rotary stalk subunits occurs in bacteria and chloroplasts but not in mitochondria. The crystal structure of the ATP synthase catalytic complex (F{sub 1}) from Escherichia coli described here reveals the structural basis for this inhibition. The C-terminal domain of subunit {var_epsilon} adopts a heretofore unknown, highly extended conformation that inserts deeply into the central cavity of the enzyme and engages both rotor and stator subunits in extensive contacts that are incompatible with functional rotation. As a result, the three catalytic subunits are stabilized in a set of conformations and rotational positions distinct from previous F{sub 1} structures.

  15. Maternal High Fat Diet Alters Skeletal Muscle Mitochondrial Catalytic Activity in Adult Male Rat Offspring

    Science.gov (United States)

    Pileggi, Chantal A.; Hedges, Christopher P.; Segovia, Stephanie A.; Markworth, James F.; Durainayagam, Brenan R.; Gray, Clint; Zhang, Xiaoyuan D.; Barnett, Matthew P. G.; Vickers, Mark H.; Hickey, Anthony J. R.; Reynolds, Clare M.; Cameron-Smith, David

    2016-01-01

    A maternal high-fat (HF) diet during pregnancy can lead to metabolic compromise, such as insulin resistance in adult offspring. Skeletal muscle mitochondrial dysfunction is one mechanism contributing to metabolic impairments in insulin resistant states. Therefore, the present study aimed to investigate whether mitochondrial dysfunction is evident in metabolically compromised offspring born to HF-fed dams. Sprague-Dawley dams were randomly assigned to receive a purified control diet (CD; 10% kcal from fat) or a high fat diet (HFD; 45% kcal from fat) for 10 days prior to mating, throughout pregnancy and during lactation. From weaning, all male offspring received a standard chow diet and soleus muscle was collected at day 150. Expression of the mitochondrial transcription factors nuclear respiratory factor-1 (NRF1) and mitochondrial transcription factor A (mtTFA) were downregulated in HF offspring. Furthermore, genes encoding the mitochondrial electron transport system (ETS) respiratory complex subunits were suppressed in HF offspring. Moreover, protein expression of the complex I subunit, NDUFB8, was downregulated in HF offspring (36%), which was paralleled by decreased maximal catalytic linked activity of complex I and III (40%). Together, these results indicate that exposure to a maternal HF diet during development may elicit lifelong mitochondrial alterations in offspring skeletal muscle. PMID:27917127

  16. Cytosolic phospholipase A2: a member of the signalling pathway of a new G protein α subunit in Sporothrix schenckii

    Directory of Open Access Journals (Sweden)

    González-Méndez Ricardo

    2009-05-01

    Full Text Available Abstract Background Sporothrix schenckii is a pathogenic dimorphic fungus, the etiological agent of sporotrichosis, a lymphocutaneous disease that can remain localized or can disseminate, involving joints, lungs, and the central nervous system. Pathogenic fungi use signal transduction pathways to rapidly adapt to changing environmental conditions and S. schenckii is no exception. S. schenckii yeast cells, either proliferate (yeast cell cycle or engage in a developmental program that includes proliferation accompanied by morphogenesis (yeast to mycelium transition depending on the environmental conditions. The principal intracellular receptors of environmental signals are the heterotrimeric G proteins, suggesting their involvement in fungal dimorphism and pathogenicity. Identifying these G proteins in fungi and their involvement in protein-protein interactions will help determine their role in signal transduction pathways. Results In this work we describe a new G protein α subunit gene in S. schenckii, ssg-2. The cDNA sequence of ssg-2 revealed a predicted open reading frame of 1,065 nucleotides encoding a 355 amino acids protein with a molecular weight of 40.9 kDa. When used as bait in a yeast two-hybrid assay, a cytoplasmic phospholipase A2 catalytic subunit was identified as interacting with SSG-2. The sspla2 gene, revealed an open reading frame of 2538 bp and encoded an 846 amino acid protein with a calculated molecular weight of 92.62 kDa. The principal features that characterize cPLA2 were identified in this enzyme such as a phospholipase catalytic domain and the characteristic invariable arginine and serine residues. A role for SSPLA2 in the control of dimorphism in S. schenckii is suggested by observing the effects of inhibitors of the enzyme on the yeast cell cycle and the yeast to mycelium transition in this fungus. Phospholipase A2 inhibitors such as AACOCF3 (an analogue of archidonic acid and isotetrandrine (an inhibitor of G protein

  17. The POLD3 subunit of DNA polymerase δ can promote translesion synthesis independently of DNA polymerase ζ

    OpenAIRE

    Hirota, Kouji; Yoshikiyo, Kazunori; Guilbaud, Guillaume; Tsurimoto, Toshiki; Murai, Junko; Tsuda, Masataka; Phillips, Lara G.; Narita, Takeo; Nishihara, Kana; Kobayashi, Kaori; Yamada, Kouich; Nakamura, Jun; Pommier, Yves; Lehmann, Alan; Sale, Julian E.

    2015-01-01

    The replicative DNA polymerase Polδ consists of a catalytic subunit POLD1/p125 and three regulatory subunits POLD2/p50, POLD3/p66 and POLD4/p12. The ortholog of POLD3 in Saccharomyces cerevisiae, Pol32, is required for a significant proportion of spontaneous and UV-induced mutagenesis through its additional role in translesion synthesis (TLS) as a subunit of DNA polymerase ζ. Remarkably, chicken DT40 B lymphocytes deficient in POLD3 are viable and able to replicate undamaged genomic DNA with ...

  18. Cloning and Expression of Pyruvate Dehydrogenase E 1-α Subunit Gene (pdha) in Mycoplasma ovipneumoniae and Its Immunologic Activity Evaluation%绵羊肺炎支原体(Mycoplasma ovipneumoniae)丙酮酸脱氢酶E1-α亚单位基因(pdha)的克隆、表达及其免疫学活性测定

    Institute of Scientific and Technical Information of China (English)

    许健; 储岳峰; 高鹏程; 赵萍; 贺英; 剡根强; 逯忠新

    2012-01-01

    丙酮酸脱氢酶α-亚单位(PDHA)在病原体丙酮酸脱氢酶的催化过程中发挥着重要作用.为表达绵羊肺炎支原体(Mycoplasma ovipneumoniae)PDHA蛋白并测定其免疫学活性,应用PCR方法扩增出绵羊肺炎支原体pdha基因并对其序列进行分析,将pdha基因中色氨酸密码子TGA优化为TGG后进行全基因合成,插入到pET32-a(+)载体上,构建了pET3 2-a(+ )-pdha重组质粒,将重组质粒转化到大肠杆菌(Escherichia coli)BL21中诱导表达PDHA蛋白,并通过免疫印迹及小鼠(Mus musculus)免疫试验对其免疫学活性进行测定.结果pdha基因全长1 125 bp,编码375 aa,(G+C)%为34.76%,第304~306位、379~381位、586~588位、592~594位、625~627位、811~813位、889~891位及964~966位TGA在支原体中编码色氨酸而不是作为终止密码子;基因序列比对及进化树分析显示,绵羊肺炎支原体pdha基因与10种支原体的pdha基因序列同源性为32.6%~85.3%,氨基酸序列同源性为39.3%~90.6%,基因序列和氨基酸序列均与猪肺炎支原体(M.hyopneumoniae)有同源性,分别为85.3%和90.6%;绵羊肺炎支原体pdha基因在33℃、IPTG 0.25 mmol/L诱导6h的表达条件下,表达量最高;重组的PDHA蛋白可与绵羊肺炎支原体高免血清具有免疫印迹条带,在免疫小鼠后血清抗体效价与对照组相比,均显著升高(P<0.05).本实验首次成功克隆表达了绵羊肺炎支原体pdha基因,并证明其重组PDHA蛋白具有较好的免疫学活性.为绵羊支原体肺炎基因工程疫苗及诊断研究提供候选靶标.%Pyruvate dehydrogenase El-a subunit (PDHA) plays an important role in the catalytic activity of pyruvate dehydrogenase of pathogens. In order to characterize the immunologic activity of the PDHA of Mycoplasma ovipneumoniae, we amplified and sequenced the pdha gene of M. Ovipneumoniae. After optimized with TGG instead of TGA for coding the amino acid of tryptophane, the pdha gene

  19. Influence of Conformation of M. tuberculosis RNase P Protein Subunit on Its Function.

    Directory of Open Access Journals (Sweden)

    Alla Singh

    Full Text Available RNase P is an essential enzyme that processes 5' end leader sequence of pre-tRNA to generate mature tRNA. The bacterial RNase Ps contain a RNA subunit and one protein subunit, where the RNA subunit contains the catalytic activity. The protein subunit which lacks any catalytic activity, relaxes the ionic requirements for holoenzyme reaction and is indispensable for pre-tRNA cleavage in vivo. In the current study, we reconstituted the M. tuberculosis RNase P holoenzyme in vitro. We prepared the RNase P protein through two different strategies that differ in the conditions under which the recombinant M. tuberculosis protein, expressed in E. coli was purified. The mycobacterial RNase P protein which was purified under native conditions subsequent to isolation from inclusion bodies and in vitro renaturation, was capable of cleaving pre-tRNA specifically without the requirement of RNase P RNA. However, the preparation that was purified under denaturing conditions and refolded subsequently lacked any inherent pre-tRNA processing activity and cleaved the substrate only as a component of the holoenzyme with the RNA subunit. We found that the two RNase P protein preparations attained alternative conformations and differed with respect to their stability as well.

  20. In Search of Small Molecule Inhibitors Targeting the Flexible CK2 Subunit Interface

    Directory of Open Access Journals (Sweden)

    Benoît Bestgen

    2017-02-01

    Full Text Available Protein kinase CK2 is a tetrameric holoenzyme composed of two catalytic (α and/or α’ subunits and two regulatory (β subunits. Crystallographic data paired with fluorescence imaging techniques have suggested that the formation of the CK2 holoenzyme complex within cells is a dynamic process. Although the monomeric CK2α subunit is endowed with a constitutive catalytic activity, many of the plethora of CK2 substrates are exclusively phosphorylated by the CK2 holoenzyme. This means that the spatial and high affinity interaction between CK2α and CK2β subunits is critically important and that its disruption may provide a powerful and selective way to block the phosphorylation of substrates requiring the presence of CK2β. In search of compounds inhibiting this critical protein–protein interaction, we previously designed an active cyclic peptide (Pc derived from the CK2β carboxy-terminal domain that can efficiently antagonize the CK2 subunit interaction. To understand the functional significance of this interaction, we generated cell-permeable versions of Pc, exploring its molecular mechanisms of action and the perturbations of the signaling pathways that it induces in intact cells. The identification of small molecules inhibitors of this critical interaction may represent the first-choice approach to manipulate CK2 in an unconventional way.

  1. Using yeast two-hybrid system to detect interactions of ATP synthase subunits from Spinacia oleracea

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Subunit interactions among the chloroplast ATP synthase subunits were studied using the yeast two-hybrid system. Various pairwise combinations of genes encoding a , b , g , d and e subunits of Spinach ATP synthase fused to the binding domain or activation domain of GAL4 DNA were introduced into yeast and then expression of a reporter gene encoding b -galactosidase was detected. Of all the combinations, that of g and e subunit genes showed the highest level of reporter gene expression, while those of a and b , a and e , b and e and b and d induced stable and significant reporter gene expression. The combination of d and e as well as that of d and g induced weak and unstable reporter gene expression. However, combinations of a and g , b and g and a and d did not induce reporter gene expression. These results suggested that specific and strong interactions between g and e , a and b , a and e , b and e and b and d subunits, and weak and transient interactions between d and e and d and g subunits occurred in the yeast cell in the two-hybrid system. These results give a new look into the structural change of ATP synthase during catalysis.

  2. Using yeast two-hybrid system to detect interactions of ATP synthase subunits from Spinacia oleracea

    Institute of Scientific and Technical Information of China (English)

    石晓冰; 魏家绵; 沈允钢

    2000-01-01

    Subunit interactions among the chloroplast ATP synthase subunits were studied using the yeast two-hybrid system. Various pairwise combinations of genes encoding a, p, y, 8 and e subunits of Spinach ATP synthase fused to the binding domain or activation domain of GAL4 DNA were introduced into yeast and then expression of a reporter gene encoding p-galactosidase was detected. Of all the combinations, that of y and e subunit genes showed the highest level of reporter gene expression, while those of a and p, a and e, p and e and p and 8 induced stable and significant reporter gene expression. The combination of 8 and e as well as that of 8 and y induced weak and unstable reporter gene expression. However, combinations of a and y, p and y and a and 8 did not induce reporter gene expression. These results suggested that specific and strong interactions between y and e, a and p, a and e, p and e and p and 8 subunits, and weak and transient interactions between 8 and e and 8 and y subunits occurred in the yeast

  3. AChR deficiency due to epsilon-subunit mutations : two common mutations in the Netherlands

    NARCIS (Netherlands)

    Faber, Catharina G.; Molenaar, Peter C.; Vles, Johannes S. H.; Bonifati, Domenic M.; Verschuuren, Jan J. G. M.; van Doorn, Pieter A.; Kuks, Jan B. M.; Wokke, John H. J.; Beeson, David; De Baets, Marc

    2009-01-01

    Congenital myasthenic syndromes are a clinically and genetically heterogeneous group of hereditary disorders affecting neuromuscular transmission. We have identified mutations within the acetylcholine receptor (AChR) epsilon-subunit gene underlying congenital myasthenic syndromes in nine patients (s

  4. Gene fusions with human carbonic anhydrase II for efficient expression and rapid single-step recovery of recombinant proteins: expression of the Escherichia coli F1-ATPase epsilon subunit.

    Science.gov (United States)

    Van Heeke, G; Shaw, R; Schnizer, R; Couton, J M; Schuster, S M; Wagner, F W

    1993-08-01

    A new expression vector was constructed which allows the overproduction in Escherichia coli of tripartite proteins consisting of human carbonic anhydrase isozyme II (hCAII), a peptide linker containing an enterokinase cleavage site, and a target protein of interest. Carbonic anhydrase is soluble and stable in E. coli and serves as a highly specific purification tag in the recovery of the fusion protein by a single affinity chromatography step. The enterokinase cleavage site was engineered into the construct to allow accurate and efficient release of the target protein. To demonstrate the practical value of this vector, the E. coli F1-ATPase epsilon subunit was expressed as a fusion with hCAII. After a single purification step, biologically active recombinant E. coli F1-ATPase epsilon subunit was recovered following proteolytic removal of the hCAII moiety.

  5. Characterization of the putative tryptophan synthase β-subunit from Mycobacterium tuberculosis

    Institute of Scientific and Technical Information of China (English)

    Hongbo Shen; Yanping Yang; Feifei Wang; Ying Zhang; Naihao Ye; Shengfeng Xu; Honghai Wang

    2009-01-01

    The increasing emergence of drug-resistant tuberculosis (TB)poses a serious threat to the control of this disease.It is in urgent need to develop new TB drugs.Tryptophan biosynthetic pathway plays an important role in the growth and replication of Mycobacterium tuberculosis(Mtb).The β-subunit of tryptophan synthase(TrpB)catalyzes the last step of the tryptophan biosynthetic pathway,and it might be a potential target for TB drug design.In this study,we overexpressed,purified,and characterized the putative TrpB-encoding gene Rv1612 in Mtb H37Rv.Results showed that Mtb His-TrpB optimal enzymatic activity is at pH 7.8 with 0.15 M Na+or 0.18 M Mg2+ at 37℃.Structure analysis indicated that Mtb TrpB exhibited a typical β/α barrel structure.The amino acid residues believed to interact with the enzyme cofactor pyridoxal-5'-phosphate were predicted by homology modeling and structure alignment.The role of these residues in catalytic activity of the Mtb His-TrpB was confirmed by site-directed mutagenesis.These results provided reassuring structural information for drug design based on TrpB.

  6. Cloning and analysis of the four genes coding for Bpu10I restriction-modification enzymes.

    Science.gov (United States)

    Stankevicius, K; Lubys, A; Timinskas, A; Vaitkevicius, D; Janulaitis, A

    1998-02-15

    The Bpu 10I R-M system from Bacillus pumilus 10, which recognizes the asymmetric 5'-CCTNAGC sequence, has been cloned, sequenced and expressed in Escherichia coli . The system comprises four adjacent, similarly oriented genes encoding two m5C MTases and two subunits of Bpu 10I ENase (34.5 and 34 kDa). Both bpu10IR genes either in cis or trans are needed for the manifestation of R. Bpu 10I activity. Subunits of R. Bpu 10I, purified to apparent homogeneity, are both required for cleavage activity. This heterosubunit structure distinguishes the Bpu 10I restriction endonuclease from all other type II restriction enzymes described previously. The subunits reveal 25% amino acid identity. Significant similarity was also identified between a 43 amino acid region of R. Dde I and one of the regions of higher identity shared between the Bpu 10I subunits, a region that could possibly include the catalytic/Mg2+binding center. The similarity between Bpu 10I and Dde I MTases is not limited to the conserved motifs (CM) typical for m5C MTases. It extends into the variable region that lies between CMs VIII and IX. Duplication of a progenitor gene, encoding an enzyme recognizing a symmetric nucleotide sequence, followed by concerted divergent evolution, may provide a possible scenario leading to the emergence of the Bpu 10I ENase, which recognizes an overall asymmetric sequence and cleaves within it symmetrically.

  7. Catalytic Functions of Standards

    NARCIS (Netherlands)

    K. Blind (Knut)

    2009-01-01

    textabstractThe three different areas and the examples have illustrated several catalytic functions of standards for innovation. First, the standardisation process reduces the time to market of inventions, research results and innovative technologies. Second, standards themselves promote the diffusi

  8. Catalytic Synthesis Lactobionic Acid

    Directory of Open Access Journals (Sweden)

    V.G. Borodina

    2014-07-01

    Full Text Available Gold nanoparticles are obtained, characterized and deposited on the carrier. Conducted catalytic synthesis of lactobionic acid from lactose. Received lactobionic acid identify on the IR spectrum.

  9. Catalytic distillation process

    Science.gov (United States)

    Smith, Jr., Lawrence A.

    1982-01-01

    A method for conducting chemical reactions and fractionation of the reaction mixture comprising feeding reactants to a distillation column reactor into a feed zone and concurrently contacting the reactants with a fixed bed catalytic packing to concurrently carry out the reaction and fractionate the reaction mixture. For example, a method for preparing methyl tertiary butyl ether in high purity from a mixed feed stream of isobutene and normal butene comprising feeding the mixed feed stream to a distillation column reactor into a feed zone at the lower end of a distillation reaction zone, and methanol into the upper end of said distillation reaction zone, which is packed with a properly supported cationic ion exchange resin, contacting the C.sub.4 feed and methanol with the catalytic distillation packing to react methanol and isobutene, and concurrently fractionating the ether from the column below the catalytic zone and removing normal butene overhead above the catalytic zone.

  10. Catalytic distillation process

    Science.gov (United States)

    Smith, L.A. Jr.

    1982-06-22

    A method is described for conducting chemical reactions and fractionation of the reaction mixture comprising feeding reactants to a distillation column reactor into a feed zone and concurrently contacting the reactants with a fixed bed catalytic packing to concurrently carry out the reaction and fractionate the reaction mixture. For example, a method for preparing methyl tertiary butyl ether in high purity from a mixed feed stream of isobutene and normal butene comprising feeding the mixed feed stream to a distillation column reactor into a feed zone at the lower end of a distillation reaction zone, and methanol into the upper end of said distillation reaction zone, which is packed with a properly supported cationic ion exchange resin, contacting the C[sub 4] feed and methanol with the catalytic distillation packing to react methanol and isobutene, and concurrently fractionating the ether from the column below the catalytic zone and removing normal butene overhead above the catalytic zone.

  11. Identification of the fifth subunit of Saccharomyces cerevisiae replication factor C.

    Science.gov (United States)

    Gary, S L; Burgers, M J

    1995-01-01

    Yeast replication factor C (RF-C) is a multipolypeptide complex required for chromosomal DNA replication. Previously this complex was known to consist of at least four subunits. We here report the identification of a fifth RF-C subunit from Saccharomyces cerevisiae, encoded by the RFC5 (YBR0810) gene. This subunit exhibits highest homology to the 38 kDa subunit (38%) of human RF-C (activator 1). Like the other four RFC genes, the RFC5 gene is essential for yeast viability, indicating an essential function for each subunit. RFC5 mRNA is expressed at steady-state levels throughout the mitotic cell cycle. Upon overexpression in Escherichia coli Rfc5p has an apparent molecular mass of 41 kDa. Overproduction of RF-C activity in yeast is dependent on overexpression of the RFC5 gene together with overexpression of the RFC1-4 genes, indicating that the RFC5 gene product forms an integral subunit of this replication factor. Images PMID:8559655

  12. Catalytic distillation structure

    Science.gov (United States)

    Smith, L.A. Jr.

    1984-04-17

    Catalytic distillation structure is described for use in reaction distillation columns, and provides reaction sites and distillation structure consisting of a catalyst component and a resilient component intimately associated therewith. The resilient component has at least about 70 volume % open space and is present with the catalyst component in an amount such that the catalytic distillation structure consists of at least 10 volume % open space. 10 figs.

  13. The phosphorylation pattern of bovine heart complex I subunits

    DEFF Research Database (Denmark)

    Palmisano, Giuseppe; Sardanelli, Anna Maria; Signorile, Anna;

    2007-01-01

    The phosphoproteome of bovine heart complex I of the respiratory chain has been analysed with a procedure based on nondenaturing gel electrophoretic separation of complex I from small quantities of mitochondria samples, in-gel digestion, in combination with phosphopeptide enrichment by titanium...... dioxide and MS. The results, complemented by analyses of purified samples of complex I, showed phosphorylation of five subunits of the complex, 42 kDa (human gene NDUFA10), ESSS, B14.5a (human gene NDUFA7), B14.5b (human gene NDUFC2) and B16.6 (GRIM-19). MS also revealed the presence of phosphorylated...

  14. Molecular architecture of the yeast Elongator complex reveals an unexpected asymmetric subunit arrangement.

    Science.gov (United States)

    Setiaputra, Dheva T; Cheng, Derrick Th; Lu, Shan; Hansen, Jesse M; Dalwadi, Udit; Lam, Cindy Hy; To, Jeffrey L; Dong, Meng-Qiu; Yip, Calvin K

    2017-02-01

    Elongator is a ~850 kDa protein complex involved in multiple processes from transcription to tRNA modification. Conserved from yeast to humans, Elongator is assembled from two copies of six unique subunits (Elp1 to Elp6). Despite the wealth of structural data on the individual subunits, the overall architecture and subunit organization of the full Elongator and the molecular mechanisms of how it exerts its multiple activities remain unclear. Using single-particle electron microscopy (EM), we revealed that yeast Elongator adopts a bilobal architecture and an unexpected asymmetric subunit arrangement resulting from the hexameric Elp456 subassembly anchored to one of the two Elp123 lobes that form the structural scaffold. By integrating the EM data with available subunit crystal structures and restraints generated from cross-linking coupled to mass spectrometry, we constructed a multiscale molecular model that showed the two Elp3, the main catalytic subunit, are located in two distinct environments. This work provides the first structural insights into Elongator and a framework to understand the molecular basis of its multifunctionality.

  15. Structural characterization of recombinant crustacyanin subunits from the lobster Homarus americanus.

    Science.gov (United States)

    Ferrari, Michele; Folli, Claudia; Pincolini, Elisa; McClintock, Timothy S; Rössle, Manfred; Berni, Rodolfo; Cianci, Michele

    2012-08-01

    Crustacean crustacyanin proteins are linked to the production and modification of carapace colour, with direct implications for fitness and survival. Here, the structural and functional properties of the two recombinant crustacyanin subunits H(1) and H(2) from the American lobster Homarus americanus are reported. The two subunits are structurally highly similar to the corresponding natural apo crustacyanin CRTC and CRTA subunits from the European lobster H. gammarus. Reconstitution studies of the recombinant crustacyanin proteins H(1) and H(2) with astaxanthin reproduced the bathochromic shift of 85-95 nm typical of the natural crustacyanin subunits from H. gammarus in complex with astaxanthin. Moreover, correlations between the presence of crustacyanin genes in crustacean species and the resulting carapace colours with the spectral properties of the subunits in complex with astaxanthin confirmed this genotype-phenotype linkage.

  16. Catalytic hydrotreating process

    Science.gov (United States)

    Karr, Jr., Clarence; McCaskill, Kenneth B.

    1978-01-01

    Carbonaceous liquids boiling above about 300.degree. C such as tars, petroleum residuals, shale oils and coal-derived liquids are catalytically hydrotreated by introducing the carbonaceous liquid into a reaction zone at a temperature in the range of 300.degree. to 450.degree. C and a pressure in the range of 300 to 4000 psig for effecting contact between the carbonaceous liquid and a catalytic transition metal sulfide in the reaction zone as a layer on a hydrogen permeable transition metal substrate and then introducing hydrogen into the reaction zone by diffusing the hydrogen through the substrate to effect the hydrogenation of the carbonaceous liquid in the presence of the catalytic sulfide layer.

  17. Gene therapy for hemophilia B mediated by recombinant adeno-associated viral vector with hFIXR338A, a high catalytic activity mutation of human coagulation factor IX

    Institute of Scientific and Technical Information of China (English)

    LU; Huazhong; (

    2001-01-01

    [1]Chang, J., Jin, J., Lollar, P. et al., Changing residue 338 in human factor IX from arginine to alanine causes an increase in catalytic activity, J. Bio. Chem., 1998, 273 (20): 12089-12094.[2]Lai, L., Chen, L., Zhou, H. et al., Clinical phenotype and genetic stability of factor IX gene knock out mice, J. Fudan Uni., 1999, 38 (4): 435-438.[3]Wu, Z. J., Wu, X. B., Hou, Y. D., Generation of a recombinant herps simplex virus which can provide packaging function for recombinant adeno-associated virus, Chinese Sci. Bull., 1999, 44 (8): 715-719.[4]Snyder, R. O., Miao, C. H., Patijn, G. A. et al., Persistent and therapeutic concentrations of human factor IX in mice after hepatic gene transfer of recombinant AAV vectors, Nat. Genet., 1997, 16 (3): 270-276.[5]Lai, L. H., Chen, L., Wang, J. M. et al., Skeletal muscle-specific expression of human blood coagulation factor IX rescues factor IX deficiency mouse by AAV-mediated gene transfer, Science in China, Ser. C, 1999, 42 (6): 628-634.[6]Snyder, R. O., Miao, C., Meuse, L. et al., Correction of hemophilia B in canine and murine models using recombinant adeno-associated viral vectors, Nat. Med., 1999, 5 (1): 64-70.[7]Kung, S. H., Hagstrom, J. N., Cass, D. et al., Human factor IX corrects the bleeding diathesis of mice with hemophilia B, Blood, 1998, 91(3): 784-790.[8]Hirt, B., Selective extraction of polyoma DNA from infected mouse cell culture, J. Mol. Biol., 1967, 26: 365-369.[9]Sambrook, J., Fritsch, E., Maniatis, T., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press, 1989, 6, 20-21.[10]Chao, H., Samulski, R. J., Bellinger, D. A. et al., Persistent expression of canine factor IX in hemophilia B canines, Gene Ther., 1999, 6: 1695-1704.[11]Kaufman, R. J., Advances toward gene therapy for hemophilia at the millennium, Hum. Gene Ther., 1999, 10 (13): 2091-2107.[12]Lu, D. R., Zhou, J. M., Zheng, B. et al., Stage I clinical trial of gene

  18. Structural and biochemical characterization of human PR70 in isolation and in complex with the scaffolding subunit of protein phosphatase 2A.

    Directory of Open Access Journals (Sweden)

    Rebecca Dovega

    Full Text Available Protein Phosphatase 2A (PP2A is a major Ser/Thr phosphatase involved in the regulation of various cellular processes. PP2A assembles into diverse trimeric holoenzymes, which consist of a scaffolding (A subunit, a catalytic (C subunit and various regulatory (B subunits. Here we report a 2.0 Å crystal structure of the free B''/PR70 subunit and a SAXS model of an A/PR70 complex. The crystal structure of B''/PR70 reveals a two domain elongated structure with two Ca2+ binding EF-hands. Furthermore, we have characterized the interaction of both binding partner and their calcium dependency using biophysical techniques. Ca2+ biophysical studies with Circular Dichroism showed that the two EF-hands display different affinities to Ca2+. In the absence of the catalytic C-subunit, the scaffolding A-subunit remains highly mobile and flexible even in the presence of the B''/PR70 subunit as judged by SAXS. Isothermal Titration Calorimetry studies and SAXS data support that PR70 and the A-subunit have high affinity to each other. This study provides additional knowledge about the structural basis for the function of B'' containing holoenzymes.

  19. 云南保山和普洱地区带绦虫线粒体DNA基因编码核糖体RNA小亚基基因序列分析%Analysis of the mitochondrial DNA-gene encoding ribosomal RNA small subunit gene sequence of Taenia cestode from Baoshan and Puer areas in Yunnan Province

    Institute of Scientific and Technical Information of China (English)

    刘爱波; 杨毅梅

    2011-01-01

    Objective To identify Taenia cestodes specimens collected from Baoshan and Puer regions of Yunnan Province by analyzing mitochondrial DNA gene encoding ribosomal RNA small subunit (mtDNA-12S rRNA) gene sequence. Methods The adult Taenia cestode samples were collected from Baoshan and Puer regions of Yunnan Province. The genomic DNA was extracted and mtDNA-12S rRAN gene was amplified by polymerase chain reaction (PCR), then sequenced.Combined with the known mtDNA-12S rRNA gene sequence of Taenia solium, Taenia saginata,Taenia asiatica in GenBank, homology tree and phylogenetic tree were constructed by DNA MAN software. Results Taenia cestode homology tree and phylogenetic tree showed that gene sequences of BS1, BS2, BS4 and BS5 were most close to YZ with identity of 99% and those of BS3, BS6, BST,PE1 and PE2 were most close to ND with identity of 99%. Conclusions Taenia saginata and Taenia asiatica can be found in Baoshan area, while Taenia saginata can be found in Puer area. The gene sequence of mtDNA-12S rRNA can be used for clarifying the three types of Taenia cestode.%目的 利用线粒体DNA基因编码核糖体RNA小亚基(mtDNA-12S rRNA)基因序列分析对采自云南保山、普洱地区的带绦虫标本进行鉴定.方法 选取保山(7条,BS1-BS7)、普洱(2条,PE1~PE2)带绦虫成虫节片,抽提基因组DNA,PCR扩增mtDNA-12S rRNA基因序列,并测序;结合GenBank中已知的猪带绦虫(ZD)、牛带绦虫(ND)、亚洲带绦虫(YZ)mtDNA-12S rRNA基因序列,经DNA MAN软件处理后构建同源树状图与系统发育树状图.结果 带绦虫同源树与系统发育树状图显示,BS1、BS2、BS4、BS5与YZ的同源性最近(99%).BS3、BS6、BS7、PE1、PE2与ND的同源性最近(99%).结论 云南保山存在牛带绦虫与亚洲带绦虫,普洱存在牛带绦虫,mtDNA-12S rRNA基因序列可用于三种带绦虫的分类研究.

  20. Isoform-specific Regulation of Sodium pump α-subunit Gene Expression i n Aortic Smooth Muscle Cell of 1-kidney-1-clip Hypertensive Rats%“一肾一夹”高血压大鼠动脉平滑肌细胞钠泵α亚单位的基因表达

    Institute of Scientific and Technical Information of China (English)

    原卫清; 王颢; 吕卓人

    2001-01-01

    Objective To investigate the gene expression of sodium pump α-subunit in aortic smooth muscle of 1-kidney-1-clip (1k1c) hypertensive rats.  Methods 1k1c hypertensive rats were prepared by partially ligating the left renal artery and removing the right kidney. 4 weeks later, all the rats were killed and sodium pump α1- , α2-, and α3-subunit in aortic smooth muscles were detected with reverse transcription p ol ymerase chain reaction (RT-PCR) method and immunohistochemical assay at both mR NA and protein levels, respectively.  Resulsts Changes in the expression of sodi um pump α-subunit gene were found in aortic smooth muscles of 1k1c hypertensiv e rats: α1-su bunit increased at both mRNA protein levels, while α2- and α3-subunits r emained without changes.  Conclusions There were great changes in the gene expression of so dium pump α-subunit in aor tic smooth muscles of 1k1c hypertensive rats, which might be related to the development of hypertension in this hypertensive model.%目的 探讨“一肾一夹(1k1c)”高血压大鼠动脉平滑肌细胞钠泵α亚单位各异构体基 因表达的改变,为高血压发病机制的深入研究提供理论及实验依据。方法 分别应用分子生物学 RT-PCR及免疫组化技术,探讨1k1c高血压大鼠动脉平滑肌细胞钠泵α1、α2及α3亚单位mR NA及蛋白 水平基因表达的改变。结果 1k1c高血压大鼠动脉平滑肌细胞钠泵α亚单位基因表达发生改变: 无论在mRNA或蛋白水平,α1亚单位表达增强,而α2与α3亚单位表达无改变。结论 1k1c高血压大鼠动脉平滑肌细胞钠泵α亚单位基因表达发生改变,这种 改变可能参与了该模型的血压升高 机制。

  1. Analysis of mucolipidosis II/III GNPTAB missense mutations identifies domains of UDP-GlcNAc:lysosomal enzyme GlcNAc-1-phosphotransferase involved in catalytic function and lysosomal enzyme recognition.

    Science.gov (United States)

    Qian, Yi; van Meel, Eline; Flanagan-Steet, Heather; Yox, Alex; Steet, Richard; Kornfeld, Stuart

    2015-01-30

    UDP-GlcNAc:lysosomal enzyme GlcNAc-1-phosphotransferase tags newly synthesized lysosomal enzymes with mannose 6-phosphate recognition markers, which are required for their targeting to the endolysosomal system. GNPTAB encodes the α and β subunits of GlcNAc-1-phosphotransferase, and mutations in this gene cause the lysosomal storage disorders mucolipidosis II and III αβ. Prior investigation of missense mutations in GNPTAB uncovered amino acids in the N-terminal region and within the DMAP domain involved in Golgi retention of GlcNAc-1-phosphotransferase and its ability to specifically recognize lysosomal hydrolases, respectively. Here, we undertook a comprehensive analysis of the remaining missense mutations in GNPTAB reported in mucolipidosis II and III αβ patients using cell- and zebrafish-based approaches. We show that the Stealth domain harbors the catalytic site, as some mutations in these regions greatly impaired the activity of the enzyme without affecting its Golgi localization and proteolytic processing. We also demonstrate a role for the Notch repeat 1 in lysosomal hydrolase recognition, as missense mutations in conserved cysteine residues in this domain do not affect the catalytic activity but impair mannose phosphorylation of certain lysosomal hydrolases. Rescue experiments using mRNA bearing Notch repeat 1 mutations in GNPTAB-deficient zebrafish revealed selective effects on hydrolase recognition that differ from the DMAP mutation. Finally, the mutant R587P, located in the spacer between Notch 2 and DMAP, was partially rescued by overexpression of the γ subunit, suggesting a role for this region in γ subunit binding. These studies provide new insight into the functions of the different domains of the α and β subunits.

  2. Phosphorylation of the regulatory beta-subunit of protein kinase CK2 by checkpoint kinase Chk1: identification of the in vitro CK2beta phosphorylation site

    DEFF Research Database (Denmark)

    Kristensen, Lars P; Larsen, Martin Røssel; Højrup, Peter;

    2004-01-01

    The regulatory beta-subunit of protein kinase CK2 mediates the formation of the CK2 tetrameric form and it has functions independent of CK2 catalytic subunit through interaction with several intracellular proteins. Recently, we have shown that CK2beta associates with the human checkpoint kinase Chk...... by the modification of Thr213 but it does require the presence of an active Chk1 kinase....

  3. Efficient reconstitution of functional Escherichia coli 30S ribosomal subunits from a complete set of recombinant small subunit ribosomal proteins.

    Science.gov (United States)

    Culver, G M; Noller, H F

    1999-06-01

    Previous studies have shown that the 30S ribosomal subunit of Escherichia coli can be reconstituted in vitro from individually purified ribosomal proteins and 16S ribosomal RNA, which were isolated from natural 30S subunits. We have developed a 30S subunit reconstitution system that uses only recombinant ribosomal protein components. The genes encoding E. coli ribosomal proteins S2-S21 were cloned, and all twenty of the individual proteins were overexpressed and purified. Reconstitution, following standard procedures, using the complete set of recombinant proteins and purified 16S ribosomal RNA is highly inefficient. Efficient reconstitution of 30S subunits using these components requires sequential addition of proteins, following either the 30S subunit assembly map (Mizushima & Nomura, 1970, Nature 226:1214-1218; Held et al., 1974, J Biol Chem 249:3103-3111) or following the order of protein assembly predicted from in vitro assembly kinetics (Powers et al., 1993, J MoI Biol 232:362-374). In the first procedure, the proteins were divided into three groups, Group I (S4, S7, S8, S15, S17, and S20), Group II (S5, S6, S9, Sll, S12, S13, S16, S18, and S19), and Group III (S2, S3, S10, S14, and S21), which were sequentially added to 16S rRNA with a 20 min incubation at 42 degrees C following the addition of each group. In the second procedure, the proteins were divided into Group I (S4, S6, S11, S15, S16, S17, S18, and S20), Group II (S7, S8, S9, S13, and S19), Group II' (S5 and S12) and Group III (S2, S3, S10, S14, and S21). Similarly efficient reconstitution is observed whether the proteins are grouped according to the assembly map or according to the results of in vitro 30S subunit assembly kinetics. Although reconstitution of 30S subunits using the recombinant proteins is slightly less efficient than reconstitution using a mixture of total proteins isolated from 30S subunits, it is much more efficient than reconstitution using proteins that were individually isolated

  4. Catalytic efficiency of designed catalytic proteins.

    Science.gov (United States)

    Korendovych, Ivan V; DeGrado, William F

    2014-08-01

    The de novo design of catalysts that mimic the affinity and specificity of natural enzymes remains one of the Holy Grails of chemistry. Despite decades of concerted effort we are still unable to design catalysts as efficient as enzymes. Here we critically evaluate approaches to (re)design of novel catalytic function in proteins using two test cases: Kemp elimination and ester hydrolysis. We show that the degree of success thus far has been modest when the rate enhancements seen for the designed proteins are compared with the rate enhancements by small molecule catalysts in solvents with properties similar to the active site. Nevertheless, there are reasons for optimism: the design methods are ever improving and the resulting catalyst can be efficiently improved using directed evolution.

  5. Catalytic Phosphination and Arsination

    Institute of Scientific and Technical Information of China (English)

    Kwong Fuk Yee; Chan Kin Shing

    2004-01-01

    The catalytic, user-friendly phosphination and arsination of aryl halides and triflates by triphenylphosphine and triphenylarsine using palladium catalysts have provided a facile synthesis of functionalized aryl phosphines and arsines in neutral media. Modification of the cynaoarisne yielded optically active N, As ligands which will be screened in various asymmetric catalysis.

  6. Construction of a hybrid β-hexosaminidase subunit capable of forming stable homodimers that hydrolyze GM2 ganglioside in vivo.

    Science.gov (United States)

    Tropak, Michael B; Yonekawa, Sayuri; Karumuthil-Melethil, Subha; Thompson, Patrick; Wakarchuk, Warren; Gray, Steven J; Walia, Jagdeep S; Mark, Brian L; Mahuran, Don

    2016-01-01

    Tay-Sachs or Sandhoff disease result from mutations in either the evolutionarily related HEXA or HEXB genes encoding respectively, the α- or β-subunits of β-hexosaminidase A (HexA). Of the three Hex isozymes, only HexA can interact with its cofactor, the GM2 activator protein (GM2AP), and hydrolyze GM2 ganglioside. A major impediment to establishing gene or enzyme replacement therapy based on HexA is the need to synthesize both subunits. Thus, we combined the critical features of both α- and β-subunits into a single hybrid µ-subunit that contains the α-subunit active site, the stable β-subunit interface and unique areas in each subunit needed to interact with GM2AP. To facilitate intracellular analysis and the purification of the µ-homodimer (HexM), CRISPR-based genome editing was used to disrupt the HEXA and HEXB genes in a Human Embryonic Kidney 293 cell line stably expressing the µ-subunit. In association with GM2AP, HexM was shown to hydrolyze a fluorescent GM2 ganglioside derivative both in cellulo and in vitro. Gene transfer studies in both Tay-Sachs and Sandhoff mouse models demonstrated that HexM expression reduced brain GM2 ganglioside levels.

  7. Recent advances in the production of recombinant subunit vaccines in Pichia pastoris.

    Science.gov (United States)

    Wang, Man; Jiang, Shuai; Wang, Yefu

    2016-04-01

    Recombinant protein subunit vaccines are formulated using defined protein antigens that can be produced in heterologous expression systems. The methylotrophic yeast Pichia pastoris has become an important host system for the production of recombinant subunit vaccines. Although many basic elements of P. pastoris expression system are now well developed, there is still room for further optimization of protein production. Codon bias, gene dosage, endoplasmic reticulum protein folding and culture condition are important considerations for improved production of recombinant vaccine antigens. Here we comment on current advances in the application of P. pastoris for the synthesis of recombinant subunit vaccines.

  8. Cereblon inhibits proteasome activity by binding to the 20S core proteasome subunit beta type 4.

    Science.gov (United States)

    Lee, Kwang Min; Lee, Jongwon; Park, Chul-Seung

    2012-10-26

    In humans, mutations in the gene encoding cereblon (CRBN) are associated with mental retardation. Although CRBN has been investigated in several cellular contexts, its function remains unclear. Here, we demonstrate that CRBN plays a role in regulating the ubiquitin-proteasome system (UPS). Heterologous expression of CRBN inhibited proteasome activity in a human neuroblastoma cell line. Furthermore, proteasome subunit beta type 4 (PSMB4), the β7 subunit of the 20S core complex, was identified as a direct binding partner of CRBN. These findings suggest that CRBN may modulate proteasome activity by directly interacting with the β7 subunit.

  9. Candidate genes in ocular dominance plasticity

    Directory of Open Access Journals (Sweden)

    M. Liset Rietman

    2012-02-01

    Full Text Available The objective of this study was to identify new candidate genes involved in experience-dependent plasticity. To this aim, we combined previously obtained data from recombinant inbred BXD strains on ocular dominance (OD plasticity and gene expression levels in the neocortex. We validated our approach using a list of genes which alter OD plasticity when inactivated. The expression levels of one fifth of these genes correlated with the amount of OD plasticity. Moreover, the two genes with the highest relative inter-strain differences were among the correlated genes. This suggests that correlation between gene expression levels and OD plasticity is indeed likely to point to genes with a causal role in modulating or generating plasticity in the visual cortex. After this validation on known plasticity genes, we identified new candidate genes by a multi-step approach. First, a list was compiled of all genes of which the expression level in BXD strains correlate with the amount of OD plasticity. To narrow this list to the more promising candidates, we took its cross-section with a list of genes co-regulated with the sensitive period for OD plasticity and a list of genes associated with pathways implicated in OD plasticity. This analysis resulted in a list of 32 candidate genes. The list contained unproven, but not surprising, candidates, such as the genes for IGF-1, NCAM1, NOGO-A, the gamma2 subunit of the GABA(A receptor, acetylcholine esterase and the catalytic subunit of cAMP-dependent protein kinase A. This was indicative of the viability of our approach, but more interesting were the novel candidate genes: Akap7, Akt1, Camk2d, Cckbr, Cd44, Crim1, Ctdsp2, Dnajc5, Gnai1, Itpka, Mapk8, Nbea, Nfatc3, Nlk, Npy5r, Phf21a, Phip, Ppm1l, Ppp1r1b, Rbbp4, Slc1a3, Slit2, Socs2, Spock3, St8sia1, Zfp207. The possible role of some of these candidates is discussed in the article.

  10. The elusive third subunit IIa of the bacterial B-type oxidases: the enzyme from the hyperthermophile Aquifex aeolicus.

    Directory of Open Access Journals (Sweden)

    Laurence Prunetti

    Full Text Available The reduction of molecular oxygen to water is catalyzed by complicated membrane-bound metallo-enzymes containing variable numbers of subunits, called cytochrome c oxidases or quinol oxidases. We previously described the cytochrome c oxidase II from the hyperthermophilic bacterium Aquifex aeolicus as a ba(3-type two-subunit (subunits I and II enzyme and showed that it is included in a supercomplex involved in the sulfide-oxygen respiration pathway. It belongs to the B-family of the heme-copper oxidases, enzymes that are far less studied than the ones from family A. Here, we describe the presence in this enzyme of an additional transmembrane helix "subunit IIa", which is composed of 41 amino acid residues with a measured molecular mass of 5105 Da. Moreover, we show that subunit II, as expected, is in fact longer than the originally annotated protein (from the genome and contains a transmembrane domain. Using Aquifex aeolicus genomic sequence analyses, N-terminal sequencing, peptide mass fingerprinting and mass spectrometry analysis on entire subunits, we conclude that the B-type enzyme from this bacterium is a three-subunit complex. It is composed of subunit I (encoded by coxA(2 of 59000 Da, subunit II (encoded by coxB(2 of 16700 Da and subunit IIa which contain 12, 1 and 1 transmembrane helices respectively. A structural model indicates that the structural organization of the complex strongly resembles that of the ba(3 cytochrome c oxidase from the bacterium Thermus thermophilus, the IIa helical subunit being structurally the lacking N-terminal transmembrane helix of subunit II present in the A-type oxidases. Analysis of the genomic context of genes encoding oxidases indicates that this third subunit is present in many of the bacterial oxidases from B-family, enzymes that have been described as two-subunit complexes.

  11. Structure of a thermophilic F1-ATPase inhibited by an ε-subunit: deeper insight into the ε-inhibition mechanism.

    Science.gov (United States)

    Shirakihara, Yasuo; Shiratori, Aya; Tanikawa, Hiromi; Nakasako, Masayoshi; Yoshida, Masasuke; Suzuki, Toshiharu

    2015-08-01

    F1-ATPase (F1) is the catalytic sector in F(o)F1-ATP synthase that is responsible for ATP production in living cells. In catalysis, its three catalytic β-subunits undergo nucleotide occupancy-dependent and concerted open-close conformational changes that are accompanied by rotation of the γ-subunit. Bacterial and chloroplast F1 are inhibited by their own ε-subunit. In the ε-inhibited Escherichia coli F1 structure, the ε-subunit stabilizes the overall conformation (half-closed, closed, open) of the β-subunits by inserting its C-terminal helix into the α3β3 cavity. The structure of ε-inhibited thermophilic F1 is similar to that of E. coli F1, showing a similar conformation of the ε-subunit, but the thermophilic ε-subunit stabilizes another unique overall conformation (open, closed, open) of the β-subunits. The ε-C-terminal helix 2 and hook are conserved between the two structures in interactions with target residues and in their positions. Rest of the ε-C-terminal domains are in quite different conformations and positions, and have different modes of interaction with targets. This region is thought to serve ε-inhibition differently. For inhibition, the ε-subunit contacts the second catches of some of the β- and α-subunits, the N- and C-terminal helices, and some of the Rossmann fold segments. Those contacts, as a whole, lead to positioning of those β- and α- second catches in ε-inhibition-specific positions, and prevent rotation of the γ-subunit. Some of the structural features are observed even in IF1 inhibition in mitochondrial F1.

  12. Mitsunobu Reactions Catalytic in Phosphine and a Fully Catalytic System.

    Science.gov (United States)

    Buonomo, Joseph A; Aldrich, Courtney C

    2015-10-26

    The Mitsunobu reaction is renowned for its mild reaction conditions and broad substrate tolerance, but has limited utility in process chemistry and industrial applications due to poor atom economy and the generation of stoichiometric phosphine oxide and hydrazine by-products that complicate purification. A catalytic Mitsunobu reaction using innocuous reagents to recycle these by-products would overcome both of these shortcomings. Herein we report a protocol that is catalytic in phosphine (1-phenylphospholane) employing phenylsilane to recycle the catalyst. Integration of this phosphine catalytic cycle with Taniguchi's azocarboxylate catalytic system provided the first fully catalytic Mitsunobu reaction.

  13. Characterisation of the tryptophan synthase alpha subunit in maize

    Directory of Open Access Journals (Sweden)

    Gierl Alfons

    2008-04-01

    Full Text Available Abstract Background In bacteria, such as Salmonella typhimurium, tryptophan is synthesized from indole-3-glycerole phosphate (IGP by a tryptophan synthase αββα heterotetramer. Plants have evolved multiple α (TSA and β (TSB homologs, which have probably diverged in biological function and their ability of subunit interaction. There is some evidence for a tryptophan synthase (TS complex in Arabidopsis. On the other hand maize (Zea mays expresses the TSA-homologs BX1 and IGL that efficiently cleave IGP, independent of interaction with TSB. Results In order to clarify, how tryptophan is synthesized in maize, two TSA homologs, hitherto uncharacterized ZmTSA and ZmTSAlike, were functionally analyzed. ZmTSA is localized in plastids, the major site of tryptophan biosynthesis in plants. It catalyzes the tryptophan synthase α-reaction (cleavage of IGP, and forms a tryptophan synthase complex with ZmTSB1 in vitro. The catalytic efficiency of the α-reaction is strongly enhanced upon complex formation. A 160 kD tryptophan synthase complex was partially purified from maize leaves and ZmTSA was identified as native α-subunit of this complex by mass spectrometry. ZmTSAlike, for which no in vitro activity was detected, is localized in the cytosol. ZmTSAlike, BX1, and IGL were not detectable in the native tryptophan synthase complex in leaves. Conclusion It was demonstrated in vivo and in vitro that maize forms a tryptophan synthase complex and ZmTSA functions as α-subunit in this complex.

  14. Abundant intergenic TAACTGA direct repeats and putative alternate RNA polymerase β´ subunits in marine Beggiatoaceae genomes: possible regulatory roles and origins

    Directory of Open Access Journals (Sweden)

    Barbara J. MacGregor

    2015-12-01

    Full Text Available The genome sequences of several giant marine sulfur-oxidizing bacteria present evidence of a possible post-transcriptional regulatory network that may have been transmitted to or from two distantly related bacteria lineages. The draft genome of a Cand. Maribeggiatoa filament from the Guaymas Basin (Gulf of California, Mexico seafloor contains 169 sets of TAACTGA direct repeats and one indirect repeat, with two to six copies per set. Related heptamers are rarely or never found as direct repeats. TAACTGA direct repeats are also found in some other Beggiatoaceae, Thiocystis violascens, a range of Cyanobacteria, and five Bacteroidetes. This phylogenetic distribution suggests they may have been transmitted horizontally, but no mechanism is evident. There is no correlation between total TAACTGA occurrences and repeats per genome. In most species the repeat units are relatively short, but longer arrays of up to 43 copies are found in several Bacteroidetes and Cyanobacteria. The majority of TAACTGA repeats in the Cand. Maribeggiatoa Orange Guaymas (BOGUAY genome are within several nucleotides upstream of a putative start codon, suggesting they may be binding sites for a post-transcriptional regulator. Candidates include members of the ribosomal protein S1, Csp (cold shock protein, and Csr (carbon storage regulator families. No pattern was evident in the predicted functions of the open reading frames (ORFs downstream of repeats, but some encode presumably essential products such as ribosomal proteins. Among these is an ORF encoding a possible alternate or modified RNA polymerase beta prime subunit, predicted to have the expected subunit interaction domains but lacking most catalytic residues. A similar ORF was found in the Thioploca ingrica draft genome, but in no others. In both species they are immediately upstream of putative sensor kinase genes with nearly identical domain structures. In the marine Beggiatoaceae, a role for the TAACTGA repeats in

  15. Effects of overexpression of PKAc genes on expressions of lignin-modifying enzymes by Pleurotus ostreatus.

    Science.gov (United States)

    Toyokawa, Chihana; Shobu, Misaki; Tsukamoto, Rie; Okamura, Saki; Honda, Yoichi; Kamitsuji, Hisatoshi; Izumitsu, Kousuke; Suzuki, Kazumi; Irie, Toshikazu

    2016-09-01

    We studied the role of genes encoding the cAMP-dependent protein kinase A catalytic subunit (PKAc) in the ligninolytic system in Pleurotus ostreatus. The wild-type P. ostreatus strain PC9 has two PKAc-encoding genes: PKAc1 and PKAc2 (protein ID 114122 and 85056). In the current study, PKAc1 and PKAc2 were fused with a β-tubulin promoter and introduced into strain PC9 to produce the overexpression strains PKAc1-97 and PKAc2-69. These strains showed significantly higher transcription levels of isozyme genes encoding lignin-modifying enzymes than strain PC9, but the specific gene expression patterns differed between the two recombinant strains. Both recombinants showed 2.05-2.10-fold faster degradation of beechwood lignin than strain PC9. These results indicate that PKAc plays an important role in inducing the wood degradation system in P. ostreatus.

  16. Recombinant cholera toxin B subunit in Escherichia coli: high-level secretion, purification, and characterization

    NARCIS (Netherlands)

    Slos, P.; Speck, D.; Accart, N.; Kolbe, H.V.; Schubnel, D.; Bouchon, B.; Bischoff, Rainer; Kieny, M.P.

    1994-01-01

    The gene coding for cholera toxin subunit B (CT-B) was fused to a modified ompA signal sequence and subsequently cloned into a high expression vector based on the regulatory signals of the arabinose operon of Salmonella typhimurium. Upon induction of gene expression in Escherichia coli, a product of

  17. Identification of differentially expressed radiation-induced genes in cervix carcinoma cells using suppression subtractive hybridization

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Jun Sang; Lee, Young Sook; Lee, Jeung Hoon; Lee, Woong Hee; Seo, Eun Young; Cho, Moon June [Chungnam National University, Daejeon (Korea, Republic of)

    2005-03-15

    A number of genes and their products are induced early or late following exposure of cells to ionizing radiation. These radiation-induced genes have various effects of irradiated cells and tissues. Suppression subtractive hybridization (SSH) based on PCR was used to identify the differentially expressed genes by radiation in cervix carcinoma cells. Total RNA and poly (A){sup +} mRNA were isolated from irradiated and non-irradiated HeLa cells. Forward-and reverse-subtracted cDNA libraries were constructed using SSH. Eighty-eight clones of each were used to randomly select differentially expressed genes using reverse Northern blotting (dot blot analysis). Northern blotting was used to verify the screened genes. Of the 176 clones, 10 genes in the forward-subtracted library and 9 genes in the reverse-subtracted library were identified as differentially expressed radiation-induced genes by PCR-select differential screening. Three clones from the forward-subtracted library were confirmed by Northern blotting, and showed increased expression in a dose-dependent manner, including a telomerase catalytic subunit and sodium channel-like protein gene, and an ESTs (expressed sequence tags) gene. We identified differentially expressed radiation-induced genes with low-abundance genes with SSH, but further characterization of theses genes are necessary to clarify the biological functions of them.

  18. Effect of the delta subunit on assembly and proton permeability of the F0 proton channel of Escherichia coli F1F0 ATPase.

    OpenAIRE

    1991-01-01

    During the assembly of the Escherichia coli proton-translocating ATPase, the subunits of F1 interact with F0 to increase the proton permeability of the transmembrane proton channel. We tested the involvement of the delta subunit in this process by partially and completely deleting uncH (delta subunit) from a plasmid carrying the genes for the F0 subunits and delta and testing the effects of those F0 plasmids on the growth of unc+ and unc mutant E. coli strains. We found that the delta subunit...

  19. Cloning and purification of protein kinase CK2 recombinant alpha and beta subunits from the Mediterranean fly Ceratitis capitata.

    Science.gov (United States)

    Kouyanou-Koutsoukou, Sophia; Baier, Andrea; Kolaitis, Regina-Maria; Maniatopoulou, Evanthia; Thanopoulou, Konstantina; Szyszka, Ryszard

    2011-10-01

    The Mediterranean fruit fly Ceratitis capitata is an insect capable of wreaking extensive damage to a wide range of fruit crops. Protein kinase CK2 is a ubiquitous Ser/Thr kinase that is highly conserved among eukaryotes; it is a heterotetramer composed of two catalytic (α) and a dimer of regulatory (β) subunits. We present here the construction of the cDNA molecules of the CK2α and CK2β subunits from the medfly C. capitata by the 5'/3' RACE and RT-PCR methods, respectively. CcCK2α catalytic subunit presents the characteristic and conserved features of a typical protein kinase, similar to the regulatory CcCK2β subunit, that also possess the conserved features of regulatory CK2β subunits, as revealed by comparison of their predicted amino acid sequences with other eukaryotic species. The recombinant CcCK2α and CcCK2β proteins were purified by affinity chromatography to homogeneity, after overexpression in Escherichia coli. CcCK2α is capable to utilize GTP and its activity and is inhibited by polyanions and stimulated by polycations in phosphorylation assays, using purified acidic ribosomal protein P1 as a substrate.

  20. Risk capital allocation with autonomous subunits

    DEFF Research Database (Denmark)

    Hougaard, Jens Leth; Smilgins, Aleksandrs

    2016-01-01

    Risk capital allocation problems have been widely discussed in the academic literature. We consider a set of independent subunits collaborating in order to reduce risk: that is, when subunit portfolios are merged a diversification benefit arises and the risk of the group as a whole is smaller than...

  1. The beta subunit of casein kinase II

    DEFF Research Database (Denmark)

    Boldyreff, B; Piontek, K; Schmidt-Spaniol, I;

    1991-01-01

    cDNAs encoding the beta subunit of pig and mouse CKII were isolated. The porcine cDNA was expressed as a fusion protein in Escherichia coli and used for the production of anti-CKII-beta subunit specific antibodies....

  2. Splice variants of Na(V1.7 sodium channels have distinct β subunit-dependent biophysical properties.

    Directory of Open Access Journals (Sweden)

    Clare Farmer

    Full Text Available Genes encoding the α subunits of neuronal sodium channels have evolutionarily conserved sites of alternative splicing but no functional differences have been attributed to the splice variants. Here, using Na(V1.7 as an exemplar, we show that the sodium channel isoforms are functionally distinct when co-expressed with β subunits. The gene, SCN9A, encodes the α subunit of the Na(V1.7 channel, and contains both sites of alternative splicing that are highly conserved. In conditions where the intrinsic properties of the Na(V1.7 splice variants were similar when expressed alone, co-expression of β1 subunits had different effects on channel availability that were determined by splicing at either site in the α subunit. While the identity of exon 5 determined the degree to which β1 subunits altered voltage-dependence of activation (P = 0.027, the length of exon 11 regulated how far β1 subunits depolarised voltage-dependence of inactivation (P = 0.00012. The results could have a significant impact on channel availability, for example with the long version of exon 11, the co-expression of β1 subunits could lead to nearly twice as large an increase in channel availability compared to channels containing the short version. Our data suggest that splicing can change the way that Na(V channels interact with β subunits. Because splicing is conserved, its unexpected role in regulating the functional impact of β subunits may apply to multiple voltage-gated sodium channels, and the full repertoire of β subunit function may depend on splicing in α subunits.

  3. Mutant U5A cells are complemented by an interferon-alpha beta receptor subunit generated by alternative processing of a new member of a cytokine receptor gene cluster.

    Science.gov (United States)

    Lutfalla, G; Holland, S J; Cinato, E; Monneron, D; Reboul, J; Rogers, N C; Smith, J M; Stark, G R; Gardiner, K; Mogensen, K E

    1995-10-16

    The cellular receptor for the alpha/beta interferons contains at least two components that interact with interferon. The ifnar1 component is well characterized and a putative ifnar2 cDNA has recently been identified. We have cloned the gene for ifnar2 and show that it produces four different transcripts encoding three different polypeptides that are generated by exon skipping, alternative splicing and differential use of polyadenylation sites. One polypeptide is likely to be secreted and two are transmembrane proteins with identical extracellular and transmembrane domains but divergent cytoplasmic tails of 67 and 251 amino acids. A mutant cell line U5A, completely defective in IFN-alpha beta binding and response, has been isolated and characterized. Expression in U5A cells of the polypeptide with the long cytoplasmic domain reconstitutes a functional receptor that restores normal interferon binding, activation of the JAK/STAT signal transduction pathway, interferon-inducible gene expression and antiviral response. The IFNAR2 gene maps at 0.5 kb from the CRFB4 gene, establishing that together IFNAR2, CRFB4, IFNAR1 and AF1 form a cluster of class II cytokine receptor genes on human chromosome 21.

  4. A new light on the meiotic DSB catalytic complex.

    Science.gov (United States)

    Robert, Thomas; Vrielynck, Nathalie; Mézard, Christine; de Massy, Bernard; Grelon, Mathilde

    2016-06-01

    Meiotic recombination is initiated by the formation of programmed DNA double-strand breaks (DSBs). More than 15 years ago, Spo11 was identified as the protein responsible for meiotic DSB formation, notably because of its striking similarities with the A subunit of topoisomerase VI (TopoVI). TopoVI are enzymes that modify DNA topology by generating transient DSBs and are active as heterotetramers, composed of two A and two B subunits. A2 dimers catalyse the DNA cleavage reaction, whereas the B subunits regulate A2 conformation, DNA capture, cleavage and re-ligation. The recent identification in plants and mammals of a B-like TopoVI subunit that interacts with SPO11 and is required for meiotic DSB formation makes us to reconsider our understanding of the meiotic DSB catalytic complex. We provide here an overview of the knowledge on TopoVI structure and mode of action and we compare them with their meiotic counterparts. This allows us to discuss the nature, structure and functions of the meiotic TopoVI-like complex during meiotic DSB formation.

  5. Effect of dexamethasone on skeletal muscle Na+,K+ pump subunit specific expression and K+ homeostasis during exercise in humans

    DEFF Research Database (Denmark)

    Nordsborg, Nikolai; Ovesen, Jakob; Thomassen, Martin;

    2008-01-01

    The effect of dexamethasone on Na(+),K(+) pump subunit expression and muscle exchange of K(+) during exercise in humans was investigated. Nine healthy male subjects completed a randomized double blind placebo controlled protocol, with ingestion of dexamethasone (Dex: 2 x 2 mg per day) or placebo...... (Pla) for 5 days. Na(+),K(+) pump catalytic alpha1 and alpha2 subunit expression was approximately 17% higher (P ...). The results indicate that an increased Na(+),K(+) pump expression per se is of importance for thigh K(+) reuptake at the onset of low and moderate intensity exercise, but less important during high intensity exercise....

  6. Molecular Characterization of Subunit G of the Vacuolar ATPase in Pathogen Dermatophyte Trichophyton rubrum

    Directory of Open Access Journals (Sweden)

    S Rezaie

    2006-06-01

    Full Text Available Trichophyton rubrum is an anthropophilic fungus causing up to 90% of chronic cases of dermatophytosis. Several properties of this fungus have been investigated so far. However, a few studies were carried out in the field of molecular biology of this fungus. In the present study, we tried to identify the subunit G of its vacuolar ATPase (V-ATPase. Pairs of 21 nt primers were designed from highly conserved regions of the V-ATPase subunit G genes in other fungi. Mentioned primers were utilized in PCR using isolated genomic DNA template as well as cytoplasmic RNA of T.rubrum and the PCR and RT-PCR fragments were then sequenced. About 469 nucleotides were sequenced which encoded a polypeptide with 119 amino acids. Nucleotide sequence comparison in gene data banks (NCBI, NIH for both the DNA and its deduced amino acid sequence revealed significant homology with V-ATPase subunit G genes and proteins of other eukaryotic cells. The amino acid sequence of the encoded protein was about 84% identical to the sequence of V-ATPase subunit G from other fungi. In summary, we have cloned the first V-ATPase subunit G of dermatophytes and characterized it as a member of this gene family in other eukaryotic cells.

  7. Arabidopsis Flower and Embryo Developmental Genes are Repressed in Seedlings by Different Combinations of Polycomb Group Proteins in Association with Distinct Sets of Cis-regulatory Elements.

    Science.gov (United States)

    Wang, Hua; Liu, Chunmei; Cheng, Jingfei; Liu, Jian; Zhang, Lei; He, Chongsheng; Shen, Wen-Hui; Jin, Hong; Xu, Lin; Zhang, Yijing

    2016-01-01

    Polycomb repressive complexes (PRCs) play crucial roles in transcriptional repression and developmental regulation in both plants and animals. In plants, depletion of different members of PRCs causes both overlapping and unique phenotypic defects. However, the underlying molecular mechanism determining the target specificity and functional diversity is not sufficiently characterized. Here, we quantitatively compared changes of tri-methylation at H3K27 in Arabidopsis mutants deprived of various key PRC components. We show that CURLY LEAF (CLF), a major catalytic subunit of PRC2, coordinates with different members of PRC1 in suppression of distinct plant developmental programs. We found that expression of flower development genes is repressed in seedlings preferentially via non-redundant role of CLF, which specifically associated with LIKE HETEROCHROMATIN PROTEIN1 (LHP1). In contrast, expression of embryo development genes is repressed by PRC1-catalytic core subunits AtBMI1 and AtRING1 in common with PRC2-catalytic enzymes CLF or SWINGER (SWN). This context-dependent role of CLF corresponds well with the change in H3K27me3 profiles, and is remarkably associated with differential co-occupancy of binding motifs of transcription factors (TFs), including MADS box and ABA-related factors. We propose that different combinations of PRC members distinctively regulate different developmental programs, and their target specificity is modulated by specific TFs.

  8. Arabidopsis Flower and Embryo Developmental Genes are Repressed in Seedlings by Different Combinations of Polycomb Group Proteins in Association with Distinct Sets of Cis-regulatory Elements.

    Directory of Open Access Journals (Sweden)

    Hua Wang

    2016-01-01

    Full Text Available Polycomb repressive complexes (PRCs play crucial roles in transcriptional repression and developmental regulation in both plants and animals. In plants, depletion of different members of PRCs causes both overlapping and unique phenotypic defects. However, the underlying molecular mechanism determining the target specificity and functional diversity is not sufficiently characterized. Here, we quantitatively compared changes of tri-methylation at H3K27 in Arabidopsis mutants deprived of various key PRC components. We show that CURLY LEAF (CLF, a major catalytic subunit of PRC2, coordinates with different members of PRC1 in suppression of distinct plant developmental programs. We found that expression of flower development genes is repressed in seedlings preferentially via non-redundant role of CLF, which specifically associated with LIKE HETEROCHROMATIN PROTEIN1 (LHP1. In contrast, expression of embryo development genes is repressed by PRC1-catalytic core subunits AtBMI1 and AtRING1 in common with PRC2-catalytic enzymes CLF or SWINGER (SWN. This context-dependent role of CLF corresponds well with the change in H3K27me3 profiles, and is remarkably associated with differential co-occupancy of binding motifs of transcription factors (TFs, including MADS box and ABA-related factors. We propose that different combinations of PRC members distinctively regulate different developmental programs, and their target specificity is modulated by specific TFs.

  9. A cysteine in the repetitive domain of a high-molecular-weight glutenin subunit interferes with the mixing properties of wheat dough.

    Science.gov (United States)

    Gao, Xin; Zhang, Qisen; Newberry, Marcus P; Chalmers, Ken J; Mather, Diane E

    2013-03-01

    The quality of wheat (Triticum aestivum L.) for making bread is largely due to the strength and extensibility of wheat dough, which in turn is due to the properties of polymeric glutenin. Polymeric glutenin consists of high- and low-molecular-weight glutenin protein subunits linked by disulphide bonds between cysteine residues. Glutenin subunits differ in their effects on dough mixing properties. The research presented here investigated the effect of a specific, recently discovered, glutenin subunit on dough mixing properties. This subunit, Bx7.1, is unusual in that it has a cysteine in its repetitive domain. With site-directed mutagenesis of the gene encoding Bx7.1, a guanine in the repetitive domain was replaced by an adenine, to provide a mutant gene encoding a subunit (MutBx7.1) in which the repetitive-domain cysteine was replaced by a tyrosine residue. Bx7.1, MutBx7.1 and other Bx-type glutenin subunits were heterologously expressed in Escherichia coli and purified. This made it possible to incorporate each individual subunit into wheat flour and evaluate the effect of the cysteine residue on dough properties. The Bx7.1 subunit affected dough mixing properties differently from the other subunits. These differences are due to the extra cysteine residue, which may interfere with glutenin polymerisation through cross-linkage within the Bx7.1 subunit, causing this subunit to act as a chain terminator.

  10. Genetic Variations in the Kir6.2 Subunit (KCNJ11 of Pancreatic ATP-Sensitive Potassium Channel Gene Are Associated with Insulin Response to Glucose Loading and Early Onset of Type 2 Diabetes in Childhood and Adolescence in Taiwan

    Directory of Open Access Journals (Sweden)

    Yi-Der Jiang

    2014-01-01

    Full Text Available To investigate the role of E23K polymorphism of the KCNJ11 gene on early onset of type 2 diabetes in school-aged children/adolescents in Taiwan, we recruited 38 subjects with type 2 diabetes (ages 18.6 ± 6.6 years; body mass index percentiles 83.3 ± 15.4 and 69 normal controls (ages 17.3 ± 3.8 years; body mass index percentiles 56.7 ± 29.0 from a national surveillance for childhood/adolescent diabetes in Taiwan. We searched for the E23K polymorphism of the KCNJ11 gene. We found that type 2 diabetic subjects had higher carrier rate of E23K polymorphism of KCNJ11 gene than control subjects (P = 0.044. After adjusting for age, gender, body mass index percentiles, and fasting plasma insulin, the E23K polymorphism contributed to an increased risk for type 2 diabetes (P = 0.047. K23-allele-containing genotypes conferring increased plasma insulin level during OGTT in normal subjects. However, the diabetic subjects with the K23-allele-containing genotypes had lower fasting plasma insulin levels after adjustment of age and BMI percentiles. In conclusion, the E23K variant of the KCNJ11 gene conferred higher susceptibility to type 2 diabetes in children/adolescents. Furthermore, in normal glucose-tolerant children/adolescents, K23 allele carriers had a higher insulin response to oral glucose loading.

  11. Molecular cloning and functional expression of the Equine K+ channel KV11.1 (Ether à Go-Go-related/KCNH2 gene) and the regulatory subunit KCNE2 from equine myocardium

    DEFF Research Database (Denmark)

    Pedersen, Philip Juul; Thomsen, Kirsten Brolin; Olander, Emma Rie

    2015-01-01

    intervals on the ECG and increased risk of ventricular tachyarrhythmia and sudden cardiac death-conditions known as congenital or acquired Long QT syndrome (LQTS), respectively. In horses, sudden, unexplained deaths are a well-known problem. We sequenced the cDNA of the KCNH2 and KCNE2 genes using RACE...

  12. Oxidative phosphorylation in Escherichia coli. Characterization of mutant strains in which F1-ATPase contains abnormal beta-subunits.

    Science.gov (United States)

    Senior, A E; Langman, L; Cox, G B; Gibson, F

    1983-02-15

    To facilitate study of the role of the beta-subunit in the membrane-bound proton-translocating ATPase of Escherichia coli, we identified mutant strains from which an F1-ATPase containing abnormal beta-subunits can be purified. Seventeen strains of E. coli, characterized by genetic complementation tests as carrying mutations in the uncD gene (which codes for the beta-subunit), were studied. The majority of these strains (11) were judged to be not useful, as their membranes lacked ATPase activity, and were either proton-permeable as prepared or remained proton-impermeable after washing with buffer of low ionic strength. A further two strains were of a type not hitherto reported, in that their membranes had ATPase activity, were proton-impermeable as prepared, and were not rendered proton-permeable by washing in buffer of low ionic strength. Presumably in these two strains F1-ATPase is not released in soluble form by this procedure. F1-ATPase of normal molecular size were purified from strains AN1340 (uncD478), AN937 (uncD430), AN938 (uncD431) and AN1543 (uncD484). F1-ATPase from strain AN1340 (uncD478) had 15% of normal specific Mg-dependent ATPase activity and 22% of normal ATP-synthesis activity. The F1-ATPase preparations from strains AN937, AN938 and AN1543 had respectively 1.7%, 1.8% and 0.2% of normal specific Mg-dependent ATPase activity, and each of these preparations had very low ATP-synthesis activity. The yield of F1-ATPase from the four strains described was almost twice that obtained from a normal haploid strain. The kinetics of Ca-dependent ATPase activity were unusual in each of the four F1-ATPase preparations. It is likely that these four mutant uncD F1-ATPase preparations will prove valuable for further experimental study of the F1-ATPase catalytic mechanism.

  13. cAMP response element binding protein (CREB activates transcription via two distinct genetic elements of the human glucose-6-phosphatase gene

    Directory of Open Access Journals (Sweden)

    Stefano Luisa

    2005-01-01

    Full Text Available Abstract Background The enzyme glucose-6-phosphatase catalyzes the dephosphorylation of glucose-6-phosphatase to glucose, the final step in the gluconeogenic and glycogenolytic pathways. Expression of the glucose-6-phosphatase gene is induced by glucocorticoids and elevated levels of intracellular cAMP. The effect of cAMP in regulating glucose-6-phosphatase gene transcription was corroborated by the identification of two genetic motifs CRE1 and CRE2 in the human and murine glucose-6-phosphatase gene promoter that resemble cAMP response elements (CRE. Results The cAMP response element is a point of convergence for many extracellular and intracellular signals, including cAMP, calcium, and neurotrophins. The major CRE binding protein CREB, a member of the basic region leucine zipper (bZIP family of transcription factors, requires phosphorylation to become a biologically active transcriptional activator. Since unphosphorylated CREB is transcriptionally silent simple overexpression studies cannot be performed to test the biological role of CRE-like sequences of the glucose-6-phosphatase gene. The use of a constitutively active CREB2/CREB fusion protein allowed us to uncouple the investigation of target genes of CREB from the variety of signaling pathways that lead to an activation of CREB. Here, we show that this constitutively active CREB2/CREB fusion protein strikingly enhanced reporter gene transcription mediated by either CRE1 or CRE2 derived from the glucose-6-phosphatase gene. Likewise, reporter gene transcription was enhanced following expression of the catalytic subunit of cAMP-dependent protein kinase (PKA in the nucleus of transfected cells. In contrast, activating transcription factor 2 (ATF2, known to compete with CREB for binding to the canonical CRE sequence 5'-TGACGTCA-3', did not transactivate reporter genes containing CRE1, CRE2, or both CREs derived from the glucose-6-phosphatase gene. Conclusions Using a constitutively active CREB2

  14. 霍乱毒素A亚单位基因的原核表达及单克隆抗体的制备%Prokaryotic expression of cholera toxin subunit A gene and preparation of monoclonal antibody

    Institute of Scientific and Technical Information of China (English)

    陈健; 韦雨杏; 解晓燕; 朱召芹; 万延民; 徐建青

    2014-01-01

    目的 原核表达并纯化霍乱毒素A亚单位(cholera toxin subunitA,CTA)基因,建立分泌CTA单克隆抗体的杂交瘤细胞系,并制备单克隆抗体.方法 构建原核表达质粒pET30a-CTA,转化大肠埃希菌(E.coli)BL21(DE3),IPTG诱导表达6h,表达产物经12% SDS-PAGE分析后,用Ni2+亲和层析柱纯化;将纯化的重组CTA蛋白免疫C57BL/6小鼠,运用杂交瘤细胞技术制备抗CTA的单抗,采用Western blot法及体外阻断实验检测抗CTA单抗的特异性及对CT毒性作用的体外阻断.结果 原核表达质粒pET30a-CTA经PCR及双酶切鉴定,证明构建正确;表达的重组CTA蛋白在相对分子质量25 000 ~ 35 000之间可见明显的目的蛋白条带,纯化后的重组CTA蛋白纯度达90%以上;获得1株可分泌抗CTA单抗的杂交瘤细胞株G6C3-D10,可与CTA蛋白特异性结合,在相对分子质量25 000 ~ 35 000之间可见明显的特异条带;杂交瘤培养上清中CTA特异性抗体滴度为54,与CT混合后,可明显抑制CT所诱导的细胞内环腺苷酸(cyclic adenosine monophosphate,cAMP)水平的升高(P<0.01).结论 原核表达并纯化了CTA基因,获得了能够分泌具有阻断CT毒性作用的单抗的细胞株,为CTA毒性作用和佐剂机制的进一步研究提供了参考.

  15. Cleft Lip Repair: The Hybrid Subunit Method.

    Science.gov (United States)

    Tollefson, Travis T

    2016-04-01

    The unilateral cleft lip repair is one of the most rewarding and challenging of plastic surgery procedures. Surgeons have introduced a variety of straight line, geometric, and rotation-advancement designs, while in practice the majority of North American surgeons have been using hybrids of the rotation-advancement techniques. The anatomic subunit approach was introduced in 2005 by Fisher and has gained popularity, with early adopters of the design touting its simplicity and effectiveness. The objectives of this article are to summarize the basic tenets of respecting the philtral subunit, accurate measurement and planning, and tips for transitioning to this subunit approach.

  16. Mutant U5A cells are complemented by an interferon-alpha beta receptor subunit generated by alternative processing of a new member of a cytokine receptor gene cluster.

    OpenAIRE

    Lutfalla, G; Holland, S J; Cinato, E; Monneron, D; Reboul, J.; Rogers, N C; J. M. Smith; Stark, G R; Gardiner, K.; Mogensen, K E

    1995-01-01

    The cellular receptor for the alpha/beta interferons contains at least two components that interact with interferon. The ifnar1 component is well characterized and a putative ifnar2 cDNA has recently been identified. We have cloned the gene for ifnar2 and show that it produces four different transcripts encoding three different polypeptides that are generated by exon skipping, alternative splicing and differential use of polyadenylation sites. One polypeptide is likely to be secreted and two ...

  17. The PorX Response Regulator of the Porphyromonas gingivalis PorXY Two-Component System Does Not Directly Regulate the Type IX Secretion Genes but Binds the PorL Subunit

    Science.gov (United States)

    Vincent, Maxence S.; Durand, Eric; Cascales, Eric

    2016-01-01

    The Type IX secretion system (T9SS) is a versatile multi-protein complex restricted to bacteria of the Bacteriodetes phylum and responsible for the secretion or cell surface exposition of diverse proteins that participate to S-layer formation, gliding motility or pathogenesis. The T9SS is poorly characterized but a number of proteins involved in the assembly of the secretion apparatus in the oral pathogen Porphyromonas gingivalis have been identified based on genome substractive analyses. Among these proteins, PorY, and PorX encode typical two-component system (TCS) sensor and CheY-like response regulator respectively. Although the porX and porY genes do not localize at the same genetic locus, it has been proposed that PorXY form a bona fide TCS. Deletion of porX in P. gingivalis causes a slight decrease of the expression of a number of other T9SS genes, including sov, porT, porP, porK, porL, porM, porN, and porY. Here, we show that PorX and the soluble cytoplasmic domain of PorY interact. Using electrophoretic mobility shift, DNA-protein co-purification and heterologous host expression assays, we demonstrate that PorX does not bind T9SS gene promoters and does not directly regulate expression of the T9SS genes. Finally, we show that PorX interacts with the cytoplasmic domain of PorL, a component of the T9SS membrane core complex and propose that the CheY-like PorX protein might be involved in the dynamics of the T9SS. PMID:27630829

  18. The PorX Response Regulator of the Porphyromonas gingivalis PorXY Two-Component System Does Not Directly Regulate the Type IX Secretion Genes but Binds the PorL Subunit.

    Science.gov (United States)

    Vincent, Maxence S; Durand, Eric; Cascales, Eric

    2016-01-01

    The Type IX secretion system (T9SS) is a versatile multi-protein complex restricted to bacteria of the Bacteriodetes phylum and responsible for the secretion or cell surface exposition of diverse proteins that participate to S-layer formation, gliding motility or pathogenesis. The T9SS is poorly characterized but a number of proteins involved in the assembly of the secretion apparatus in the oral pathogen Porphyromonas gingivalis have been identified based on genome substractive analyses. Among these proteins, PorY, and PorX encode typical two-component system (TCS) sensor and CheY-like response regulator respectively. Although the porX and porY genes do not localize at the same genetic locus, it has been proposed that PorXY form a bona fide TCS. Deletion of porX in P. gingivalis causes a slight decrease of the expression of a number of other T9SS genes, including sov, porT, porP, porK, porL, porM, porN, and porY. Here, we show that PorX and the soluble cytoplasmic domain of PorY interact. Using electrophoretic mobility shift, DNA-protein co-purification and heterologous host expression assays, we demonstrate that PorX does not bind T9SS gene promoters and does not directly regulate expression of the T9SS genes. Finally, we show that PorX interacts with the cytoplasmic domain of PorL, a component of the T9SS membrane core complex and propose that the CheY-like PorX protein might be involved in the dynamics of the T9SS.

  19. Formation of fluorescent proteins by the attachment of phycoerythrobilin to R-phycoerythrin alpha and beta apo-subunits.

    Science.gov (United States)

    Isailovic, Dragan; Sultana, Ishrat; Phillips, Gregory J; Yeung, Edward S

    2006-11-01

    Formation of fluorescent proteins was explored after incubation of recombinant apo-subunits of phycobiliprotein R-phycoerythrin with phycoerythrobilin chromophore. Alpha and beta apo-subunit genes of R-phycoerythrin from red algae Polisiphonia boldii were cloned in plasmid pET-21d(+). Hexahistidine-tagged alpha and beta apo-subunits were expressed in Escherichia coli. Although expressed apo-subunits formed inclusion bodies, fluorescent holo-subunits were constituted after incubation of E. coli cells with phycoerythrobilin. Holo-subunits contained both phycoerythrobilin and urobilin chromophores. Fluorescence and differential interference contrast microscopy showed polar location of holo-subunit inclusion bodies in bacterial cells. Cells containing fluorescent holo-subunits were several times brighter than control cells as found by fluorescence microscopy and flow cytometry. The addition of phycoerythrobilin to cells did not show cytotoxic effects, in contrast to expression of proteins in inclusion bodies. In an attempt to improve solubility, R-phycoerythrin apo-subunits were fused to maltose-binding protein and incubated with phycoerythrobilin both in vitro and in vivo. Highly fluorescent soluble fusion proteins containing phycoerythrobilin as the sole chromophore were formed. Fusion proteins were localized by fluorescence microscopy either throughout E. coli cells or at cell poles. Flow cytometry showed that cells containing fluorescent fusion proteins were up to 10 times brighter than control cells. Results indicate that fluorescent proteins formed by attachment of phycoerythrobilin to expressed apo-subunits of phycobiliproteins can be used as fluorescent probes for analysis of cells by microscopy and flow cytometry. A unique property of these fluorescent reporters is their utility in both properly folded (soluble) subunits and subunits aggregated in inclusion bodies.

  20. Exchangeability of the b subunit of the Cl(-)-translocating ATPase of Acetabularia acetabulum with the beta subunit of E. coli F1-ATPase: construction of the chimeric beta subunits and complementation studies.

    Science.gov (United States)

    Ikeda, M; Kadowaki, H; Ikeda, H; Moritani, C; Kanazawa, H

    1997-11-10

    The gene encoding the b subunit of the Cl(-)-translocating ATPase (aclB) was isolated from total RNA and poly(A)+ RNA of Acetabularia acetabulum and sequenced (total nucleotides of 3038 bp and an open reading frame with 478 amino acids). The deduced amino acid sequence showed high similarity to the beta subunit of the F type ATPases, but was different in the N-terminal 120 amino acids. The role of the N-terminal region was investigated using an F -ATPase beta-less mutant of E. coli, JP17. The JP17 strain expressing the aclB could not grow under conditions permitting oxidative phosphorylation, although ACLB was detected in the membrane fraction. The beta subunit was divided into three portions: amino acid position from 1 to 95 (portion A), 96 to 161 (portion B) and 162 to the C-terminus (portion C). The corresponding regions of ACLB were designated as portions A' (from 1 to 106), B' (from 107 to 172) and C' (from 173 to 478). Chimeric proteins with combinations of A-B'-C', A-B-C' and A'-B-C restored the function as the beta subunit in E. coli F0F1-complex, but those with combinations of A'-B'-C and A-B'-C had no function as the beta subunit. These findings suggested that portion B plays an important role in the assembly and function of the beta subunit in the F0F1-complex, while portion B' of ACLB exhibited inhibitory effects on assembly and function. In addition, portion A was also important for interaction of the beta subunit with the alpha subunit in E. coli F0F1-complex. These findings also suggested that the b subunit of the Cl(-)-translocating ATPase of A. acetabulum has a different function in the Cl(-)-translocating ATPase complex, although the primary structure resembled to the beta subunit of the F1-ATPase.

  1. Novel Catalytic Membrane Reactors

    Energy Technology Data Exchange (ETDEWEB)

    Stuart Nemser, PhD

    2010-10-01

    There are many industrial catalytic organic reversible reactions with amines or alcohols that have water as one of the products. Many of these reactions are homogeneously catalyzed. In all cases removal of water facilitates the reaction and produces more of the desired chemical product. By shifting the reaction to right we produce more chemical product with little or no additional capital investment. Many of these reactions can also relate to bioprocesses. Given the large number of water-organic compound separations achievable and the ability of the Compact Membrane Systems, Inc. (CMS) perfluoro membranes to withstand these harsh operating conditions, this is an ideal demonstration system for the water-of-reaction removal using a membrane reactor. Enhanced reaction synthesis is consistent with the DOE objective to lower the energy intensity of U.S. industry 25% by 2017 in accord with the Energy Policy Act of 2005 and to improve the United States manufacturing competitiveness. The objective of this program is to develop the platform technology for enhancing homogeneous catalytic chemical syntheses.

  2. Mutations in the BLOC-1 Subunits Dysbindin and Muted Generate Divergent and Dosage-dependent Phenotypes*

    Science.gov (United States)

    Larimore, Jennifer; Zlatic, Stephanie A.; Gokhale, Avanti; Tornieri, Karine; Singleton, Kaela S.; Mullin, Ariana P.; Tang, Junxia; Talbot, Konrad; Faundez, Victor

    2014-01-01

    Post-mortem analysis has revealed reduced levels of the protein dysbindin in the brains of those suffering from the neurodevelopmental disorder schizophrenia. Consequently, mechanisms controlling the cellular levels of dysbindin and its interacting partners may participate in neurodevelopmental processes impaired in that disorder. To address this question, we studied loss of function mutations in the genes encoding dysbindin and its interacting BLOC-1 subunits. We focused on BLOC-1 mutants affecting synapse composition and function in addition to their established systemic pigmentation, hematological, and lung phenotypes. We tested phenotypic homogeneity and gene dosage effects in the mouse null alleles muted (Bloc1s5mu/mu) and dysbindin (Bloc1s8sdy/sdy). Transcripts of NMDA receptor subunits and GABAergic interneuron markers, as well as expression of BLOC-1 subunit gene products, were affected differently in the brains of Bloc1s5mu/mu and Bloc1s8sdy/sdy mice. Unlike Bloc1s8sdy/sdy, elimination of one or two copies of Bloc1s5 generated indistinguishable pallidin transcript phenotypes. We conclude that monogenic mutations abrogating the expression of a protein complex subunit differentially affect the expression of other complex transcripts and polypeptides as well as their downstream effectors. We propose that the genetic disruption of different subunits of protein complexes and combinations thereof diversifies phenotypic presentation of pathway deficiencies, contributing to the wide phenotypic spectrum and complexity of neurodevelopmental disorders. PMID:24713699

  3. The PorX response regulator of the Porphyromonas gingivalis PorXY two-component system does not directly regulate the Type IX secretion genes but binds the PorL subunit.

    Directory of Open Access Journals (Sweden)

    Maxence S Vincent

    2016-08-01

    Full Text Available The Type IX secretion system (T9SS is a versatile multi-protein complex restricted to bacteria of the Bacteriodetes phylum and responsible for the secretion of surface attachment of diverse proteins that participate to S-layer formation, gliding motility or pathogenesis. The T9SS is poorly characterized but a number of proteins involved in the assembly of the secretion apparatus in the oral pathogen Porphyromonas gingivalis have been identified based on genome substractive analyses. Among these proteins, PorY and PorX encode typical two-component system (TCS sensor and CheY-like response regulator respectively. Although the porX and porY genes do not localize at the same genetic locus, it has been proposed that PorXY form a bona fide TCS. Deletion of the porX in P. gingivalis causes a slight decrease of the expression of a number of other T9SS genes, including sov, porT, porP, porK, porL, porM, porN and porY. Here, we show that PorX and the soluble cytoplasmic domain of PorY interact. Using electrophoretic mobility shift, DNA-protein co-purification and heterologous host expression assays, we showed that PorX does not bind and does not directly regulate expression of the T9SS genes. Finally, we show that PorX interacts with the cytoplasmic domain of PorL, a component of the T9SS membrane core complex and propose that the CheY-like PorX protein might be involved in the dynamics of the T9SS.

  4. 黄河裸裂尻鱼细胞色素C氧化酶Ⅰ、Ⅱ和Ⅲ亚基基因的克隆及序列特征分析%Cloning and Sequence Analysis of Genes Encoding mtDNA Cytochrome C Oxidase Subunits I, I and Ⅲ in Schizopygopsis pylzovi

    Institute of Scientific and Technical Information of China (English)

    晁燕; 祁得林; 申志新; 王国杰; 杨成

    2011-01-01

    The complete sequences of genes encoding cytochrome C oxidase subunits I, li and ill were cloned in Schizopygopsis pylzovi by RT-PCR method. The sequence analysis showed that the complete sequence of CO I was 1 551 bp, which consisted of the open reading frame (ORF) encoding 516 amino acid residues; the CO Ⅱ gene was 691 bp and contained a 690-nucleotide ORF encoding 230 amino acid residues; and the CO Ⅲ gene was 786 bp, which was composed of the ORF encoding 261 amino acid residues. The homologous analysis of sequences showed high similarity both in DNA and corresponding amino acid sequences of CO I, CO Ⅱ and CO Ⅲ between S. pylzovi and other 9 species from family Cyprinidae. Although there were sequence differences in subunit genes among different species, the corresponding amino acids tended to be consistent, suggesting functional stability of cytochrome C oxidase in electron transfer of mitochondrial respiration chain. S. pylzovi and Sinocyclocheilus grahami clustered together in the phylogenetic trees based on gene and amino acid sequences of CO I , and gene sequences of CO Ⅱ and CO Ⅲ, coupled with the same types, positions and numbers of functional sites in CO I and CO Ⅱ , suggesting that there is a relatively close relationship between them, which is consistent with the conclusion that the subfamily Schizothoracinae is originated from one of the primitive Barbinae fishes widely distributed in warm drainages in Qinghai-Tibetan Plateau during the Tertiary Period.%采用RT-PCR技术克隆获得了黄河裸裂尻鱼(Schizopygopsis pylzovi)CO Ⅰ、Ⅱ、Ⅲ基因的编码序列,并对此进行了初步分析.结果表明,黄河裸裂尻鱼CO I基因全长为1 551 bp,开放阅读框(ORF)由基因全长组成,编码516个氨基酸;COⅡ基因全长为691 bp,开放阅读框为690 bp,编码230个氨基酸;COⅢ基因全长为786 bp,其开放阅读框也是由基因全长组成,编码261个氨基酸.序列同源性分析显示

  5. In Silico Identification and Characterization of N-Terminal Acetyltransferase Genes of Poplar (Populus trichocarpa

    Directory of Open Access Journals (Sweden)

    Hang-Yong Zhu

    2014-01-01

    Full Text Available N-terminal acetyltransferase (Nats complex is responsible for protein N-terminal acetylation (Nα-acetylation, which is one of the most common covalent modifications of eukaryotic proteins. Although genome-wide investigation and characterization of Nat catalytic subunits (CS and auxiliary subunits (AS have been conducted in yeast and humans they remain unexplored in plants. Here we report on the identification of eleven genes encoding eleven putative Nat CS polypeptides, and five genes encoding five putative Nat AS polypeptides in Populus. We document that the expansion of Nat CS genes occurs as duplicated blocks distributed across 10 of the 19 poplar chromosomes, likely only as a result of segmental duplication events. Based on phylogenetic analysis, poplar Nat CS were assigned to six subgroups, which corresponded well to the Nat CS types (CS of Nat A–F, being consistent with previous reports in humans and yeast. In silico analysis of microarray data showed that in the process of normal development of the poplar, their Nat CS and AS genes are commonly expressed at one relatively low level but share distinct tissue-specific expression patterns. This exhaustive survey of Nat genes in poplar provides important information to assist future studies on their functional role in poplar.

  6. HYDROGEN TRANSFER IN CATALYTIC CRACKING

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Hydrogen transfer is an important secondary reaction of catalytic cracking reactions, which affects product yield distribution and product quality. It is an exothermic reaction with low activation energy around 43.3 kJ/mol. Catalyst properties and operation parameters in catalytic cracking greatly influence the hydrogen transfer reaction. Satisfactory results are expected through careful selection of proper catalysts and operation conditions.

  7. Waved with open eyelids 2 (woe2 is a novel spontaneous mouse mutation in the protein phosphatase 1, regulatory (inhibitor subunit 13 like (Ppp1r13l gene

    Directory of Open Access Journals (Sweden)

    Toonen Joseph

    2012-08-01

    Full Text Available Abstract Background Waved with open eyelids 2 (woe2 is a novel autosomal recessive mouse mutation that arose spontaneously in our animal facility. Upon initial evaluation, mutant mice exhibited eyelids open at birth (EOB and wavy fur phenotypes. The goals of this study were to phenotypically characterize the woe2 mice and to identify the gene harboring the mutation responsible for the woe2 phenotype. Results Histological analysis of woe2 embryos identified the failure of embryonic eyelid closure. Clinical and histological analysis of woe2 adult eyes identified severe corneal opacities, abnormalities of the anterior segment of the eye, and the absence of meibomian glands. Abnormalities in the fur texture and the absence of meibomian glands prompted us to evaluate other epidermal appendages: skin, teeth, and nails--as well as lacrimal, mammary, salivary, sebaceous and sweat glands. No obvious morphological differences between WT and woe2 mice were identified in these tissues. However, the analysis of woe2 identified cardiac abnormalities. Positional cloning of the woe2 locus identified a 1308 bp deletion in the Ppp1r13l gene. The deletion resulted in an aberrant Ppp1r13lΔexon9-11 transcript that lacks exons 9, 10 and 11 resulting in a premature stop and a loss of 223 amino acids from the C-terminal end of the putative mutant PPP1R13L protein. Immunohistological analysis during eye development identified expression of PPP1R13L in the palpebral epidermis, palpebral and bulbar conjunctiva, corneal epithelium and meibomian glands. Conclusions The woe2 mouse harbors a novel deletion within the Ppp1r13l gene, likely resulting in a complete loss of PPP1R13L function. Results from this study provide evidence that PPP1R13L has an essential role in embryonic eyelid closure as well in development of meibomian glands and the anterior segment of the eye. The woe2 mice are a useful model for investigation of the role of PPP1R13L, especially during ocular and

  8. Luminal starch substrate "brake" on maltase-glucoamylase activity is located within the glucoamylase subunit.

    Science.gov (United States)

    Quezada-Calvillo, Roberto; Sim, Lyann; Ao, Zihua; Hamaker, Bruce R; Quaroni, Andrea; Brayer, Gary D; Sterchi, Erwin E; Robayo-Torres, Claudia C; Rose, David R; Nichols, Buford L

    2008-04-01

    The detailed mechanistic aspects for the final starch digestion process leading to effective alpha-glucogenesis by the 2 mucosal alpha-glucosidases, human sucrase-isomaltase complex (SI) and human maltase-glucoamylase (MGAM), are poorly understood. This is due to the structural complexity and vast variety of starches and their intermediate digestion products, the poorly understood enzyme-substrate interactions occurring during the digestive process, and the limited knowledge of the structure-function properties of SI and MGAM. Here we analyzed the basic catalytic properties of the N-terminal subunit of MGAM (ntMGAM) on the hydrolysis of glucan substrates and compared it with those of human native MGAM isolated by immunochemical methods. In relation to native MGAM, ntMGAM displayed slower activity against maltose to maltopentose (G5) series glucose oligomers, as well as maltodextrins and alpha-limit dextrins, and failed to show the strong substrate inhibitory "brake" effect caused by maltotriose, maltotetrose, and G5 on the native enzyme. In addition, the inhibitory constant for acarbose was 2 orders of magnitude higher for ntMGAM than for native MGAM, suggesting lower affinity and/or fewer binding configurations of the active site in the recombinant enzyme. The results strongly suggested that the C-terminal subunit of MGAM has a greater catalytic efficiency due to a higher affinity for glucan substrates and larger number of binding configurations to its active site. Our results show for the first time, to our knowledge, that the C-terminal subunit of MGAM is responsible for the MGAM peptide's "glucoamylase" activity and is the location of the substrate inhibitory brake. In contrast, the membrane-bound ntMGAM subunit contains the poorly inhibitable "maltase" activity of the internally duplicated enzyme.

  9. AMPKα2基因克隆及其野生型和突变型真核表达载体的构建%Cloning of activating adenosine monophosphate-activated protein kinase alpha 2 subunit gene and construction of its wild-type and mutant eukaryotic expression vectors

    Institute of Scientific and Technical Information of China (English)

    罗招凡; 李芳萍; 丁鹤林; 程桦

    2009-01-01

    背景:实验表明,通过激活单磷酸腺苷激活的蛋白激酶(AMP-Activated Protein Kinase, AMPK)α2可以增加胰岛素的敏感性和骨骼肌葡萄糖的摄取,其有望成为预防和治疗2型糖尿病的新的生理和药理作用靶点.目的:克隆人的AMPKα2基因,并构建其野生型和突变型真核表达载体.设计:单一样本观察.时间及地点:实验于2007-04/2008-01在中山大学附属第二医院临床分子生物实验室完成.材料:QuikChange Ⅱ Site-Directed Mutagenesis Kit为Stratagene公司产品.真核表达载体pcDNA3.1(+),大肠杆菌DH5α为实验室保存.人骨胳肌组织来源于中山大学附属第二医院手术截肢患者,获患者知情同意,新鲜取材后液氮冷冻.方法:采用反转录一聚合酶链反应技术从人骨骼肌扩增AMPKα2基因,并将其克隆到T载体,通过测序对其进行鉴定.采用Quickchange定点诱变试剂盒对AMPKα2基因进行体外定点诱变,并将其野生和突变的编码基因亚克隆到真核表达载体pcDNA3.1中,通过酶切和测序进行鉴定.主要观察指标:①目的基因的克隆.②定点诱变.③真核表达质粒的构建.结果:成功克隆了AMPKα2基因,大小约1 700 bp,与已发表的AMPKα2同源性为99%,GenBank录入号EF056019.成功将AMPKα2第45位Lysine(AAA)突变为Arginine(AGA),成功构建了野生型和突变型pcDNA-AMPK α2重组质粒.结论:实验成功克隆了AMPKα2基因,构建了其野生型和突变型真核表达载体.%BACKGROUND: The experimental results showed that insulin sensitivity and glucose uptake in skeletal muscle could be improved by activating adenosine monophosphate-activated protein kinase a2 (AMPKα2). AMPKa2 is expected to become a new physiological and pharmacological target for the prevention and treatment of type 2 diabetes mellitus. OBJECTIVE: To clone human AMPKa2 subunit gene and to construct its wild-type and mutant eukaryotic expression vectors. DESIGN: A single sample observation

  10. Catalytic quantum error correction

    CERN Document Server

    Brun, T; Hsieh, M H; Brun, Todd; Devetak, Igor; Hsieh, Min-Hsiu

    2006-01-01

    We develop the theory of entanglement-assisted quantum error correcting (EAQEC) codes, a generalization of the stabilizer formalism to the setting in which the sender and receiver have access to pre-shared entanglement. Conventional stabilizer codes are equivalent to dual-containing symplectic codes. In contrast, EAQEC codes do not require the dual-containing condition, which greatly simplifies their construction. We show how any quaternary classical code can be made into a EAQEC code. In particular, efficient modern codes, like LDPC codes, which attain the Shannon capacity, can be made into EAQEC codes attaining the hashing bound. In a quantum computation setting, EAQEC codes give rise to catalytic quantum codes which maintain a region of inherited noiseless qubits. We also give an alternative construction of EAQEC codes by making classical entanglement assisted codes coherent.

  11. An increased expression of Ca(2+) channel alpha(1A) subunit immunoreactivity in deep cerebellar neurons of rolling mouse Nagoya.

    Science.gov (United States)

    Sawada, K; Sakata-Haga, H; Ando, M; Takeda, N; Fukui, Y

    2001-12-01

    Rolling mouse Nagoya (RMN) is an ataxic mutant and carries a mutation in the gene coding for the alpha(1A) subunit of the P/Q-type Ca(2+) channel. We examined the immunohistochemical expression of the alpha(1A) subunit in deep cerebellar nuclei of RMN. The antibody used recognized residues 865-883 of the mouse alpha(1A) subunit not overlapping the altered sequences in RMN. In RMN, many neurons exhibited definite alpha(1A) subunit-staining in the medial nucleus, interposed nucleus, and lateral nucleus of deep cerebellar nuclei. The number of positive neurons in these nuclei was significantly higher in RMN than in controls. Increased expression of the alpha(1A) subunit in deep cerebellar neurons might compensate for the altered function of the P/Q-type Ca(2+) channel of RMN.

  12. ATP synthase from Escherichia coli: Mechanism of rotational catalysis, and inhibition with the ε subunit and phytopolyphenols.

    Science.gov (United States)

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

    2016-02-01

    ATP synthases (FoF1) are found ubiquitously in energy-transducing membranes of bacteria, mitochondria, and chloroplasts. These enzymes couple proton transport and ATP synthesis or hydrolysis through subunit rotation, which has been studied mainly by observing single molecules. In this review, we discuss the mechanism of rotational catalysis of ATP synthases, mainly that from Escherichia coli, emphasizing the high-speed and stochastic rotation including variable rates and an inhibited state. Single molecule studies combined with structural information of the bovine mitochondrial enzyme and mutational analysis have been informative as to an understanding of the catalytic site and the interaction between rotor and stator subunits. We discuss the similarity and difference in structure and inhibitory regulation of F1 from bovine and E. coli. Unlike the crystal structure of bovine F1 (α3β3γ), that of E. coli contains a ε subunit, which is a known inhibitor of bacterial and chloroplast F1 ATPases. The carboxyl terminal domain of E. coli ε (εCTD) interacts with the catalytic and rotor subunits (β and γ, respectively), and then inhibits rotation. The effects of phytopolyphenols on F1-ATPase are also discussed: one of them, piceatannol, lowered the rotational speed by affecting rotor/stator interactions.

  13. Catalytic Combustion of Gasified Waste

    Energy Technology Data Exchange (ETDEWEB)

    Kusar, Henrik

    2003-09-01

    This thesis concerns catalytic combustion for gas turbine application using a low heating-value (LHV) gas, derived from gasified waste. The main research in catalytic combustion focuses on methane as fuel, but an increasing interest is directed towards catalytic combustion of LHV fuels. This thesis shows that it is possible to catalytically combust a LHV gas and to oxidize fuel-bound nitrogen (NH{sub 3}) directly into N{sub 2} without forming NO{sub x} The first part of the thesis gives a background to the system. It defines waste, shortly describes gasification and more thoroughly catalytic combustion. The second part of the present thesis, paper I, concerns the development and testing of potential catalysts for catalytic combustion of LHV gases. The objective of this work was to investigate the possibility to use a stable metal oxide instead of noble metals as ignition catalyst and at the same time reduce the formation of NO{sub x} In paper II pilot-scale tests were carried out to prove the potential of catalytic combustion using real gasified waste and to compare with the results obtained in laboratory scale using a synthetic gas simulating gasified waste. In paper III, selective catalytic oxidation for decreasing the NO{sub x} formation from fuel-bound nitrogen was examined using two different approaches: fuel-lean and fuel-rich conditions. Finally, the last part of the thesis deals with deactivation of catalysts. The various deactivation processes which may affect high-temperature catalytic combustion are reviewed in paper IV. In paper V the poisoning effect of low amounts of sulfur was studied; various metal oxides as well as supported palladium and platinum catalysts were used as catalysts for combustion of a synthetic gas. In conclusion, with the results obtained in this thesis it would be possible to compose a working catalytic system for gas turbine application using a LHV gas.

  14. Modulatory mechanisms and multiple functions of somatodendritic A-type K+ channel auxiliary subunits

    Directory of Open Access Journals (Sweden)

    Henry Hungtao Jerng

    2014-03-01

    Full Text Available Auxiliary subunits are non-conducting, modulatory components of the multi-protein ion channel complexes that underlie normal neuronal signaling. They interact with the pore-forming α-subunits to modulate surface distribution, ion conductance, and channel gating properties. For the somatodendritic subthreshold A-type potassium (ISA channel based on Kv4 α-subunits, two types of auxiliary subunits have been extensively studied: Kv channel-interacting proteins (KChIPs and dipeptidyl peptidase-like proteins (DPLPs. KChIPs are cytoplasmic calcium-binding proteins that interact with intracellular portions of the Kv4 subunits, whereas DPLPs are type II transmembrane proteins that associate with the Kv4 channel core. Both KChIPs and DPLPs genes contain multiple start sites that are used by various neuronal populations to drive the differential expression of functionally distinct N-terminal variants. In turn, these N-terminal variants generate tremendous functional diversity across the nervous system. Here, we focus our review on (1 the molecular mechanism underlying the unique properties of different N-terminal variants, (2 the shaping of native ISA properties by the concerted actions of KChIPs and DPLP variants, and (3 the surprising ways that KChIPs and DPLPs coordinate the activity of multiple channels to fine-tune neuronal excitability. Unlocking the unique contributions of different auxiliary subunit N-terminal variants may provide an important opportunity to develop novel targeted therapeutics to treat numerous neurological disorders.

  15. Mutations of the catalytic subunit of RAB3GAP cause Warburg Micro syndrome

    DEFF Research Database (Denmark)

    Aligianis, Irene A; Johnson, Colin A; Gissen, Paul;

    2005-01-01

    Warburg Micro syndrome (WARBM1) is a severe autosomal recessive disorder characterized by developmental abnormalities of the eye and central nervous system and by microgenitalia. We identified homozygous inactivating mutations in RAB3GAP, encoding RAB3 GTPase activating protein, a key regulator...

  16. Mutations of the catalytic subunit of RAB3GAP cause Warburg Micro syndrome

    NARCIS (Netherlands)

    Aligianis, IA; Johnson, CA; Gissen, P; Chen, DR; Hampshire, D; Hoffmann, K; Maina, EN; Morgan, NV; Tee, L; Morton, J; Ainsworth, [No Value; Horn, D; Rosser, E; Cole, TRP; Stolte-Dijkstra, [No Value; Fieggen, K; Clayton-Smith, J; Megarbane, A; Shield, JP; Newbury-Ecob, R; Dobyns, WB; Graham, JM; Kjaer, KW; Warburg, M; Bond, J; Trembath, RC; Harris, LW; Takai, Y; Mundlos, S; Tannahill, D; Woods, CG; Maher, ER

    2005-01-01

    Warburg Micro syndrome (WARBM1) is a severe autosomal recessive disorder characterized by developmental abnormalities of the eye and central nervous system and by microgenitalia. We identified homozygous inactivating mutations in RAB3GAP, encoding RAB3 GTPase activating protein, a key regulator of t

  17. An Alternate Splicing Variant of the Human Telomerase Catalytic Subunit Inhibits Telomerase Activity

    Directory of Open Access Journals (Sweden)

    Xiaoming Yi

    2000-09-01

    Full Text Available Telomerase, a cellular reverse transcriptase, adds telomeric repeats to chromosome ends. In normal human somatic cells, telomerase is repressed and telomeres progressively shorten, leading to proliferative senescence. Introduction of the telomerase (hTERT cDNA is sufficient to produce telomerase activity and immortalize normal human cells, suggesting that the repression of telomerase activity is transcriptional. The telomerase transcript has been shown to have at least six alternate splicing sites (four insertion sites and two deletion sites, and variants containing both or either of the deletion sites are present during development and in a panel of cancer cell lines we surveyed. One deletion (β site and all four insertions cause premature translation terminations, whereas the other deletion (α site is 36 by and lies within reverse transcriptase (RT motif A, suggesting that this deletion variant may be a candidate as a dominant-negative inhibitor of telomerase. We have cloned three alternately spliced hTERT variants that contain the α,β or both α and,β deletion sites. These alternate splicing variants along with empty vector and wild-type hTERT were introduced into normal human fibroblasts and several telomerase-positive immortal and tumor cell lines. Expression of the α site deletion variant (hTERT α− construct was confirmed by Western blotting. We found that none of the three alternate splicing variants reconstitutes telomerase activity in fibroblasts. However, hTERT α− inhibits telomerase activities in telomerase-positive cells, causes telomere shortening and eventually cell death. This alternately spliced dominant-negative variant may be important in understanding telomerase regulation during development, differentiation and in cancer progression.

  18. Auxiliary Subunits: Shepherding AMPA Receptors to the Plasma Membrane

    Directory of Open Access Journals (Sweden)

    Simon C. Haering

    2014-08-01

    Full Text Available Ionotropic glutamate receptors (iGluRs are tetrameric ligand-gated cation channels that mediate excitatory signal transmission in the central nervous system (CNS of vertebrates. The members of the iGluR subfamily of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA receptors (AMPARs mediate most of the fast excitatory signal transmission, and their abundance in the postsynaptic membrane is a major determinant of the strength of excitatory synapses. Therefore, regulation of AMPAR trafficking to the postsynaptic membrane is an important constituent of mechanisms involved in learning and memory formation, such as long-term potentiation (LTP and long-term depression (LTD. Auxiliary subunits play a critical role in the facilitation and regulation of AMPAR trafficking and function. The currently identified auxiliary subunits of AMPARs are transmembrane AMPA receptor regulatory proteins (TARPs, suppressor of lurcher (SOL, cornichon homologues (CNIHs, synapse differentiation-induced gene I (SynDIG I, cysteine-