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Author (up) Cho, J.H.; Bandyopadhyay, J.; Lee, J.; Park, C.S.; Ahnn, J. file  url
  Title Two isoforms of sarco/endoplasmic reticulum calcium ATPase (SERCA) are essential in Caenorhabditis elegans Type Journal Article
  Year 2000 Publication Gene Abbreviated Journal Gene  
  Volume 261 Issue 2 Pages 211-219  
  Keywords Alternative Splicing; Amino Acid Sequence; Animals; Caenorhabditis elegans/embryology/enzymology/*genetics; Calcium-Transporting ATPases/*genetics/metabolism; Embryo, Nonmammalian/drug effects/enzymology; Embryonic Development; Gene Expression Regulation, Enzymologic; Green Fluorescent Proteins; Isoenzymes/genetics/metabolism; Luminescent Proteins/genetics/metabolism; Microscopy, Fluorescence; Molecular Sequence Data; Phenotype; Promoter Regions, Genetic/genetics; RNA, Double-Stranded/administration & dosage/genetics; Recombinant Fusion Proteins/genetics/metabolism; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Sequence Homology, Amino Acid; Tissue Distribution  
  Abstract SERCA (Sarco/Endoplasmic Reticulum Calcium ATPase), a membrane bound Ca(2+)- /Mg(2+)- dependent ATPase that sequesters Ca(2+) into the SR/ER lumen, is one of the essential components for the maintenance of intracellular Ca(2+) homeostasis. Here we describe the identification and functional characterization of a C. elegans SERCA gene (ser-1). ser-1 is a single gene alternatively spliced at its carboxyl terminus to form two isoforms (SER-1A and SER-1B) and displays a high homology (70% identity, 80% similarity) with mammalian SERCAs. Green fluorescent protein (GFP) and whole-mount immunostaining analyses reveal that SER-1 expresses in neuronal cells, body-wall muscles, pharyngeal and vulval muscles, excretory cells, and vulva epithelial cells. Furthermore, SER-1::GFP expresses during embryonic stages and the expression is maintained through the adult stages. Double-stranded RNA injection (also known as RNAi) targeted to each SER-1 isoform results in severe phenotypic defects: ser-1A(RNAi) animals show embryonic lethality, whereas ser-1B(RNAi) results in L1 larval arrest phenotype. These findings suggest that both isoforms of C. elegans SERCA, like in mammals, are essential for embryonic development and post-embryonic growth and survival.  
  Call Number Serial 451  
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Author (up) Gregg-Jolly, L.A.; Ornston, L.N. file  url
  Title Properties of Acinetobacter calcoaceticus recA and its contribution to intracellular gene conversion Type Journal Article
  Year 1994 Publication Molecular Microbiology Abbreviated Journal Mol Microbiol  
  Volume 12 Issue 6 Pages 985-992  
  Keywords Acinetobacter calcoaceticus/*genetics/metabolism; Amino Acid Sequence; *Bacterial Proteins; Base Sequence; Cloning, Molecular; DNA Transposable Elements; *DNA-Binding Proteins; Escherichia coli/genetics; Gene Conversion/*physiology; Genes, Bacterial/*genetics; Genetic Complementation Test; Genomic Library; Molecular Sequence Data; Mutation/physiology; Rec A Recombinases/chemistry/*genetics/metabolism; Restriction Mapping; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Transformation, Bacterial  
  Abstract The Acinetobacter calcoaceticus pcaJ and catJ genes, nearly identical in DNA sequence, differ in transcriptional control and are separated by more than 20 kb of chromosomal DNA. The pcaJ3125 mutation is repaired frequently in organisms containing the wild-type catJ gene. This high-frequency repair is eliminated in strains lacking the catJ gene, which suggests that recombination between the homologous catJ and pcaJ genes contributes to the high-frequency repair of the pcaJ3125 mutation. We report here that the high-frequency repair also requires a functional recA gene. The A. calcoaceticus recA gene was cloned in Escherichia coli by complementation of a recA mutation in the host strain. The nucleotide sequence of a 1506 bp DNA fragment containing A. calcoaceticus recA was determined. The amino acid sequences of RecA from E. coli and A. calcoaceticus shared 71% identity. The DNA sequences differed in that a consensus binding site for binding of LexA repressor, represented upstream from recA in E. coli, is not evident in the corresponding region of the A. calcoaceticus DNA sequence. A Tn5 insertion was introduced into the A. calcoaceticus recA gene. Selection for Tn5-encoded kanamycin resistance allowed the inactivated recA gene to be recombined by natural transformation into the A. calcoaceticus chromosome. Strains that had acquired the mutant gene were sensitive to both MMS and u.v. light, were deficient in natural transformation, and failed to carry out catJ-dependent high-frequency repair of the pcaJ3125 mutation.  
  Call Number Serial 299  
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Author (up) Hamada, T.; Sakube, Y.; Ahnn, J.; Kim, D.H.; Kagawa, H. file  url
  Title Molecular dissection, tissue localization and Ca2+ binding of the ryanodine receptor of Caenorhabditis elegans Type Journal Article
  Year 2002 Publication Journal of Molecular Biology Abbreviated Journal J Mol Biol  
  Volume 324 Issue 1 Pages 123-135  
  Keywords Amino Acid Sequence; Animals; Binding Sites; Caenorhabditis elegans/genetics/*metabolism; Caenorhabditis elegans Proteins/genetics/*metabolism; Calcium/*metabolism; Cross Reactions; Gene Expression Regulation, Developmental; Molecular Sequence Data; Muscles/*metabolism; Mutation; Recombinant Proteins/genetics/immunology/metabolism; Ryanodine Receptor Calcium Release Channel/genetics/immunology/*metabolism; Sequence Homology, Amino Acid  
  Abstract The ryanodine receptor of Caenorhabditis elegans (CeRyR) which contains 5,071 amino acid residues, is encoded by a single gene, ryr-1/unc-68. The unc-68(kh30) mutation, isolated in an animal showing abnormal response to the anesthetic ketamine, has the substitution Ser1444Asn in CeRyR, predicted to be a phosphorylation site. To elucidate the function of the region of CeRyR, and to determine the localization of CeRyR in this animal, ten region-peptides were produced in Escherichia coli by using expression plasmids and eight antisera were raised against these fusion peptides. One antibody against the region corresponding to the kh30 mutation site enabled detection of CeRyR from mutant animals both in Western analysis and in situ. Specificity of this antiserum was demonstrated using Western analysis, which showed the full size and the partial size bands in wild-type and in the Tc1-induced deletion mutant animals, respectively, but no corresponding bands in unc-68 null mutant animals. CeRyR was detected in I-bands of muscle sarcomeres by double immunostaining. CeRyR was found in the body wall, pharyngeal, vulval, anal and sex muscles of adult worms and also found to be present in embryonic muscle, but not in non-muscle cells. Two EF-hand motifs and the C terminus were demonstrated to be Ca(2+) binding regions. On the basis of these results, we propose a model for the functional domains of CeRyR, which agrees well with the model of mammalian skeletal RyR, which is based on proteolysis and cross-linking analysis. We discuss the usefulness and limitations of the molecular dissection approach, which uses peptides and peptide-specific antibodies to determine the local structure and function of individual domains within a large molecule.  
  Call Number Serial 447  
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Author (up) Harris, A.K.P.; Williamson, N.R.; Slater, H.; Cox, A.; Abbasi, S.; Foulds, I.; Simonsen, H.T.; Leeper, F.J.; Salmond, G.P.C. file  url
  Title The Serratia gene cluster encoding biosynthesis of the red antibiotic, prodigiosin, shows species- and strain-dependent genome context variation Type Journal Article
  Year 2004 Publication Microbiology (Reading, England) Abbreviated Journal Microbiology  
  Volume 150 Issue Pt 11 Pages 3547-3560  
  Keywords Bacterial Proteins/genetics; Blotting, Southern; DNA Fingerprinting; DNA, Bacterial/chemistry/isolation & purification; DNA-Binding Proteins/genetics; Gene Order; *Genes, Bacterial; *Genetic Variation; Molecular Sequence Data; Multigene Family; Open Reading Frames; Peptide Synthases/genetics; Polyketide Synthases/genetics; Prodigiosin/*biosynthesis; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Serratia/*genetics/*metabolism; Streptomyces coelicolor/genetics  
  Abstract The prodigiosin biosynthesis gene cluster (pig cluster) from two strains of Serratia (S. marcescens ATCC 274 and Serratia sp. ATCC 39006) has been cloned, sequenced and expressed in heterologous hosts. Sequence analysis of the respective pig clusters revealed 14 ORFs in S. marcescens ATCC 274 and 15 ORFs in Serratia sp. ATCC 39006. In each Serratia species, predicted gene products showed similarity to polyketide synthases (PKSs), non-ribosomal peptide synthases (NRPSs) and the Red proteins of Streptomyces coelicolor A3(2). Comparisons between the two Serratia pig clusters and the red cluster from Str. coelicolor A3(2) revealed some important differences. A modified scheme for the biosynthesis of prodigiosin, based on the pathway recently suggested for the synthesis of undecylprodigiosin, is proposed. The distribution of the pig cluster within several Serratia sp. isolates is demonstrated and the presence of cryptic clusters in some strains shown. The pig cluster of Serratia marcescens ATCC 274 is flanked by cueR and copA homologues and this configuration is demonstrated in several S. marcescens strains, whilst these genes are contiguous in strains lacking the pig cluster.  
  Call Number Serial 1607  
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Author (up) Huang, Y.; Lu, Z.; Liu, N.; Chen, Y. file  url
  Title Identification of important residues in diketoreductase from Acinetobacter baylyi by molecular modeling and site-directed mutagenesis Type Journal Article
  Year 2012 Publication Biochimie Abbreviated Journal Biochimie  
  Volume 94 Issue 2 Pages 471-478  
  Keywords 3-Hydroxyacyl CoA Dehydrogenases/chemistry/genetics/metabolism; Acinetobacter/*enzymology/genetics; Amino Acid Motifs; Amino Acid Substitution; Bacterial Proteins/*chemistry/genetics/metabolism; Binding Sites; Biocatalysis; Esters/*metabolism; Humans; Hydrogen-Ion Concentration; Hydroxymethylglutaryl-CoA Reductase Inhibitors/metabolism; Kinetics; Molecular Dynamics Simulation; Molecular Sequence Data; Mutagenesis, Site-Directed; NAD/metabolism; Oxidation-Reduction; Oxidoreductases/*chemistry/genetics/metabolism; Protein Structure, Tertiary; Recombinant Proteins/chemistry/genetics/metabolism; Sequence Homology, Amino Acid; Stereoisomerism  
  Abstract Diketoreductase (DKR) from Acinetobacter baylyi exhibits a unique property of double reduction of a beta, delta-diketo ester with excellent stereoselectivity, which can serve as an efficient biocatalyst for the preparation of an important chiral intermediate for cholesterol lowering statin drugs. Taken the advantage of high homology between DKR and human heart 3-hydroxyacyl-CoA dehydrogenase (HAD), a molecular model was created to compare the tertiary structures of DKR and HAD. In addition to the possible participation of His-143 in the enzyme catalysis by pH profile, three key amino acid residues, Ser-122, His-143 and Glu-155, were identified and mutated to explore the possibility of involving in the catalytic process. The catalytic activities for mutants S122A/C, H143A/K and E155Q were below detectable level, while their binding affinities to the diketo ester substrate and cofactor NADH did not change obviously. The experimental results were further supported by molecular docking, suggesting that Ser-122 and His-143 were essential for the proton transfer to the carbonyl functional groups of the substrate. Moreover, Glu-155 was crucial for maintaining the proper orientation and protonation of the imidazole ring of His-143 for efficient catalysis.  
  Call Number Serial 1415  
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Author (up) Moon, J.L.; Banbula, A.; Oleksy, A.; Mayo, J.A.; Travis, J. file  url
  Title Isolation and characterization of a highly specific serine endopeptidase from an oral strain of Staphylococcus epidermidis Type Journal Article
  Year 2001 Publication Biological Chemistry Abbreviated Journal Biol Chem  
  Volume 382 Issue 7 Pages 1095-1099  
  Keywords Amino Acid Sequence; Animals; Cattle; Complement C--chemistry; Electrophoresis, Polyacrylamide Gel; Fibrinogen--chemistry; Humans; Keratins--chemistry; Molecular Sequence Data; Molecular Weight; Mouth Diseases--microbiology; Protease Inhibitors--chemistry; Sequence Homology, Amino Acid; Serine Endopeptidases--chemistry, isolation & purification, metabolism; Staphylococcal Infections--microbiology; Staphylococcus epidermidis--enzymology  
  Abstract Infection by Staphylococcus epidermidis, an opportunistic pathogen, has become a major problem due to the increased use of implanted medical devices and the growing number of patients who are therapeutically or infectiously immunosuppressed. These infections appear to proceed via modulation of the coagulation and complement systems. In this communication we describe the purification and characterization of a novel extracellular proteinase from an oral strain of S. epidermidis that can degrade fibrinogen, complement protein C5, and several other proteins. This proteinase has a strong preference for cleavage after glutamic acid residues, but not after aspartic acid. The S. epidermidis enzyme may be a multifunctional protein which not only provides this organism with both the ability to evade the complement defense system and to dysregulate the coagulation cascade, but also supplies nutrients for its growth through the degradation of Glu-rich proteins.  
  Call Number Serial 52  
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Author (up) Sakube, Y.; Ando, H.; Kagawa, H. file  url
doi  openurl
  Title An abnormal ketamine response in mutants defective in the ryanodine receptor gene ryr-1 (unc-68) of Caenorhabditis elegans Type Journal Article
  Year 1997 Publication Journal of Molecular Biology Abbreviated Journal J Mol Biol  
  Volume 267 Issue 4 Pages 849-864  
  Keywords Amino Acid Sequence; Anesthetics, Dissociative/*pharmacology; Animals; Animals, Genetically Modified; Aspartic Acid/pharmacology; Caenorhabditis elegans/*drug effects/genetics/physiology; Calcium Channels/analysis/*genetics/metabolism; Cloning, Molecular; Gene Expression; Genes, Helminth/*genetics; Ketamine/*pharmacology; Molecular Sequence Data; Muscle Contraction/drug effects; Muscle Proteins/analysis/*genetics/metabolism; Mutation; N-Methylaspartate/pharmacology; Organ Specificity; Phosphorylation; Receptors, N-Methyl-D-Aspartate/agonists/antagonists & inhibitors; Recombinant Fusion Proteins/analysis; Ryanodine Receptor Calcium Release Channel; Sequence Analysis, DNA; Sequence Homology, Amino Acid  
  Abstract To characterize excitation-contraction coupling in Caenorhabditis elegans, we applied two approaches. First, we isolated a mutant having abnormal responses to ketamine, an anesthetic in vertebrates. The novel mutation unc-68(kh30) (isolated as kra-1(kh30)), exhibited strict ketamine-dependent convulsions followed by paralysis. Second, we cloned the C. elegans ryanodine receptor gene ryr-1 that is located near the center of chromosome V. ryr-1 consists of 46 exons, which encode a predicted protein of 5071 amino acid residues that is homologous to Drosophila and vertebrate ryanodine receptors. ryr-1 promoter/lacZ plasmids were expressed in body-wall and pharyngeal muscles. Non-muscle cell expression may be seen with a truncated promoter. In addition, we show that the unc-68/kra-1(kh30) mutation is a Ser1444 Asn substitution at a putative protein kinase C phosphorylation site in ryr-1, and that unc-68(e540) contains a splice acceptor mutation that creates a premature stop codon in the ryr-1 gene. We confirmed that unc-68(e540) is a mutation in ryr-1 by injecting the complete ryr-1 gene into unc-68(e540) animals and recovering wild-type progeny. Results presented here will be useful in studying the structure and function of ryanodine receptors in excitation-contraction coupling and in understanding the evolution of ryanodine receptor tissue specificity.  
  Call Number Serial 448  
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Author (up) Shanks, R.M.Q.; Lahr, R.M.; Stella, N.A.; Arena, K.E.; Brothers, K.M.; Kwak, D.H.; Liu, X.; Kalivoda, E.J. file  url
  Title A Serratia marcescens PigP homolog controls prodigiosin biosynthesis, swarming motility and hemolysis and is regulated by cAMP-CRP and HexS Type Journal Article
  Year 2013 Publication PloS one Abbreviated Journal PLoS One  
  Volume 8 Issue 3 Pages e57634  
  Keywords Bacterial Proteins/*genetics/metabolism; Depsipeptides/*biosynthesis/genetics/pharmacology; Erythrocytes/drug effects; *Gene Expression Regulation, Bacterial; Genetic Complementation Test; Hemolysis/drug effects; Hexosyltransferases/genetics/metabolism; Humans; Membrane Proteins/genetics/metabolism; Movement/drug effects; Mutation; Operon; Prodigiosin/*biosynthesis; Sequence Homology, Amino Acid; Serratia marcescens/*genetics/metabolism; Signal Transduction; Transcription Factors/*genetics/metabolism  
  Abstract Swarming motility and hemolysis are virulence-associated determinants for a wide array of pathogenic bacteria. The broad host-range opportunistic pathogen Serratia marcescens produces serratamolide, a small cyclic amino-lipid, that promotes swarming motility and hemolysis. Serratamolide is negatively regulated by the transcription factors HexS and CRP. Positive regulators of serratamolide production are unknown. Similar to serratamolide, the antibiotic pigment, prodigiosin, is regulated by temperature, growth phase, HexS, and CRP. Because of this co-regulation, we tested the hypothesis that a homolog of the PigP transcription factor of the atypical Serratia species ATCC 39006, which positively regulates prodigiosin biosynthesis, is also a positive regulator of serratamolide production in S. marcescens. Mutation of pigP in clinical, environmental, and laboratory strains of S. marcescens conferred pleiotropic phenotypes including the loss of swarming motility, hemolysis, and severely reduced prodigiosin and serratamolide synthesis. Transcriptional analysis and electrophoretic mobility shift assays place PigP in a regulatory pathway with upstream regulators CRP and HexS. The data from this study identifies a positive regulator of serratamolide production, describes novel roles for the PigP transcription factor, shows for the first time that PigP directly regulates the pigment biosynthetic operon, and identifies upstream regulators of pigP. This study suggests that PigP is important for the ability of S. marcescens to compete in the environment.  
  Call Number Serial 1612  
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Author (up) Van de Ven, W.J.; Creemers, J.W.; Roebroek, A.J. file  url
  Title Furin: the prototype mammalian subtilisin-like proprotein-processing enzyme. Endoproteolytic cleavage at paired basic residues of proproteins of the eukaryotic secretory pathway Type Journal Article
  Year 1991 Publication Enzyme Abbreviated Journal Enzyme  
  Volume 45 Issue 5-6 Pages 257-270  
  Keywords Animals; Binding Sites; Catalysis; Cloning, Molecular; Drosophila melanogaster; Furin; Humans; Invertebrate Hormones/genetics/metabolism; Mice; Models, Molecular; Multigene Family; Protein Conformation; Protein Precursors/*metabolism; *Protein Processing, Post-Translational; Sequence Homology, Amino Acid; Substrate Specificity; Subtilisins/genetics/*metabolism  
  Abstract Furin, the translational product of the recently discovered fur gene, appears to be the first known mammalian member of the subtilisin family of serine proteases and the first known mammalian proprotein-processing enzyme with cleavage selectivity for paired basic amino acid residues. Structurally and functionally, it resembles the prohormone-processing enzyme, kexin (EC, which is encoded by the KEX2 gene of yeast Saccharomyces cerevisiae. Most likely, furin is primarily involved in the processing of precursors of proteins that are secreted via the constitutive secretory pathway. Here, we review the discovery of the fur gene and describe the isolation of cDNA clones corresponding to human and mouse fur and to two fur-like genes of Drosophila melanogaster, Dfur1 and Dfur2. We also compare the structural organization of the various deduced furin proteins to that of yeast kexin, and of other members of the subtilisin family of serine proteases. Furthermore, the biosynthesis of biologically active human and mouse furin is evaluated. Finally, the cleavage specificity for paired basic amino acid residues of human and mouse furin is demonstrated by the correct processing of the precursor for von Willebrand factor.  
  Call Number Serial 524  
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