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Author (up) Dingley, A.J.; Lorenzen, I.; Grotzinger, J. file  url
openurl 
  Title NMR analysis of viral protein structures Type Journal Article
  Year 2008 Publication Methods in Molecular Biology (Clifton, N.J.) Abbreviated Journal Methods Mol Biol  
  Volume 451 Issue Pages 441-462  
  Keywords Cloning, Molecular; DNA, Complementary; Indicators and Reagents; Magnetic Resonance Spectroscopy--methods; Models, Molecular; Protein Conformation; Recombinant Proteins--chemistry; Solutions; Viral Proteins--chemistry, genetics, isolation & purification  
  Abstract Nuclear magnetic resonance (NMR) spectroscopy is a powerful tool to study the three-dimensional structure of proteins and nucleic acids at atomic resolution. Since the NMR data can be recorded in solution, conditions such as pH, salt concentration, and temperature can be adjusted so as to closely mimic the biomacromolecules natural milieu. In addition to structure determination, NMR applications can investigate time-dependent phenomena, such as dynamic features of the biomacromolecules, reaction kinetics, molecular recognition, or protein folding. The advent of higher magnetic field strengths, new technical developments, and the use of either uniform or selective isotopic labeling techniques, currently allows NMR users the opportunity to investigate the tertiary structure of biomacromolecules of approximately 50 kDa. This chapter will outline the basic protocol for structure determination of proteins by NMR spectroscopy. In general, there are four main stages: (i) preparation of a homogeneous protein sample, (ii) the recording of the NMR data sets, (iii) assignment of the spectra to each NMR observable atom in the protein, and (iv) generation of structures using computer software and the correctly assigned NMR data.  
  Call Number Serial 782  
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Author (up) Gregg-Jolly, L.A.; Ornston, L.N. file  url
openurl 
  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) 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) Van de Ven, W.J.; Creemers, J.W.; Roebroek, A.J. file  url
openurl 
  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 3.4.21.61), 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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Author (up) Yanisch-Perron, C.; Vieira, J.; Messing, J. file  url
openurl 
  Title Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors Type Journal Article
  Year 1985 Publication Gene Abbreviated Journal Gene  
  Volume 33 Issue 1 Pages 103-119  
  Keywords Base Sequence; *Cloning, Molecular; Coliphages/*genetics; Conjugation, Genetic; DNA Restriction Enzymes/metabolism; DNA, Single-Stranded/genetics; Escherichia coli/metabolism; *Genetic Vectors; Methylation; Mutation; Plasmids; Recombination, Genetic  
  Abstract Three kinds of improvements have been introduced into the M13-based cloning systems. (1) New Escherichia coli host strains have been constructed for the E. coli bacteriophage M13 and the high-copy-number pUC-plasmid cloning vectors. Mutations introduced into these strains improve cloning of unmodified DNA and of repetitive sequences. A new suppressorless strain facilitates the cloning of selected recombinants. (2) The complete nucleotide sequences of the M13mp and pUC vectors have been compiled from a number of sources, including the sequencing of selected segments. The M13mp18 sequence is revised to include the G-to-T substitution in its gene II at position 6 125 bp (in M13) or 6967 bp in M13mp18. (3) M13 clones suitable for sequencing have been obtained by a new method of generating unidirectional progressive deletions from the polycloning site using exonucleases HI and VII.  
  Call Number Serial 274  
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Author (up) Yen, C.J.; Beamer, B.A.; Negri, C.; Silver, K.; Brown, K.A.; Yarnall, D.P.; Burns, D.K.; Roth, J.; Shuldiner, A.R. file  url
doi  openurl
  Title Molecular scanning of the human peroxisome proliferator activated receptor gamma (hPPAR gamma) gene in diabetic Caucasians: identification of a Pro12Ala PPAR gamma 2 missense mutation Type Journal Article
  Year 1997 Publication Biochemical and Biophysical Research Communications Abbreviated Journal Biochem Biophys Res Commun  
  Volume 241 Issue 2 Pages 270-274  
  Keywords Adult; Aged; Aged, 80 and over; Amino Acid Sequence; Base Sequence; Cloning, Molecular; Diabetes Mellitus, Type 2/*genetics; European Continental Ancestry Group/*genetics; Exons; Female; Gene Frequency; Genetic Testing/methods; Humans; Male; Middle Aged; Molecular Sequence Data; *Mutation; Obesity/genetics; Polymerase Chain Reaction; Polymorphism, Restriction Fragment Length; Polymorphism, Single-Stranded Conformational; Receptors, Cytoplasmic and Nuclear/*genetics; Sequence Analysis, DNA; Transcription Factors/*genetics; United States  
  Abstract Peroxisome proliferator activated receptor-gamma (PPAR gamma) is a nuclear receptor that regulates adipocyte differentiation, and possibly lipid metabolism and insulin sensitivity. As such, PPAR gamma is a promising candidate gene for several human disorders including obesity and type 2 diabetes mellitus. Screening for mutations in the entire coding region of the PPAR gamma gene (both gamma 1 and gamma 2 isoforms) was performed with DNA of 26 diabetic Caucasians with or without obesity. Two base substitutions were identified: a silent mutation at nucleotide 1431 (CACHis-->CATHis) and a missense mutation (CCGPro-->GCGAla) at codon 12 of PPAR gamma 2. The allele frequency of the Pro12Ala PPAR gamma 2 variant was 0.12 in Caucasian Americans, 0.10 in Mexican Americans, 0.08 in Samoans, 0.03 in African Americans, 0.02 in Nauruans, and 0.01 in Chinese. We conclude that the Pro12Ala PPAR gamma 2 gene variant is present in diverse populations. Further studies of the Pro12Ala variant will determine its relevance to obesity, insulin resistance, and type 2 diabetes.  
  Call Number Serial 252  
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Author (up) Zakin, M.M.; Greene, R.C.; Dautry-Varsat, A.; Duchange, N.; Ferrara, P.; Py, M.C.; Margarita, D.; Cohen, G.N. file  url
openurl 
  Title Construction and physical mapping of plasmids containing the metJBLF gene cluster of E. coli K12 Type Journal Article
  Year 1982 Publication Molecular & General Genetics : MGG Abbreviated Journal Mol Gen Genet  
  Volume 187 Issue 1 Pages 101-106  
  Keywords Bacteriophage lambda/genetics; Base Sequence; Cloning, Molecular; DNA, Bacterial/genetics; DNA, Recombinant; DNA, Viral/genetics; Escherichia coli/*genetics; *Genes, Bacterial; *Plasmids  
  Abstract In vitro recombination techniques were used to clone the E. coli metJBLF gene cluster in a plasmid vector. Several chimeric plasmids were obtained, analyzed by restriction mapping and characterized genetically. The combined results establish that the met gene cluster is contained on an approximately 5.6 kilobase segment of bacterial DNA with metL between metB and metF. The origin of metL was localized precisely by its DNA sequence and its transcription direction was established.  
  Call Number Serial 266  
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Author (up) Zhang, L.-M.; Wang, M.; Prosser, J.I.; Zheng, Y.-M.; He, J.-Z. file  url
doi  openurl
  Title Altitude ammonia-oxidizing bacteria and archaea in soils of Mount Everest Type Journal Article
  Year 2009 Publication FEMS Microbiology Ecology Abbreviated Journal FEMS Microbiol Ecol  
  Volume 70 Issue 2 Pages 52-61  
  Keywords Altitude; Ammonia/*metabolism; Archaea/classification/*genetics; Bacteria/classification/*genetics; Biodiversity; Cloning, Molecular; DNA, Archaeal/genetics; DNA, Bacterial/genetics; Ecosystem; Genes, Archaeal; Genes, Bacterial; Oxidoreductases/genetics; Sequence Analysis, DNA; Soil/analysis; *Soil Microbiology  
  Abstract To determine the abundance and distribution of bacterial and archaeal ammonia oxidizers in alpine and permafrost soils, 12 soils at altitudes of 4000-6550 m above sea level (m a.s.l.) were collected from the northern slope of the Mount Everest (Tibetan Plateau), where the permanent snow line is at 5800-6000 m a.s.l. Communities were characterized by real-time PCR and clone sequencing by targeting on amoA genes, which putatively encode ammonia monooxygenase subunit A. Archaeal amoA abundance was greater than bacterial amoA abundance in lower altitude soils (<or=5400 m a.s.l.), but this situation was reversed in higher altitude soils (>or=5700 m a.s.l.). Both archaeal and bacterial amoA abundance decreased abruptly in higher altitude soils. Communities shifted from a Nitrosospira amoA cluster 3a-dominated ammonia-oxidizing bacteria community in lower altitude soils to communities dominated by a newly designated Nitrosospira ME and cluster 2-related groups and Nitrosomonas cluster 6 in higher altitude soils. All archaeal amoA sequences fell within soil and sediment clusters, and the proportions of the major archaeal amoA clusters changed between the lower altitude and the higher altitude soils. These findings imply that the shift in the relative abundance and community structure of archaeal and bacterial ammonia oxidizers may result from selection of organisms adapted to altitude-dependent environmental factors in elevated soils.  
  Call Number Serial 222  
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