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Author (up) Baylis, H.A.; Vazquez-Manrique, R.P. file  url
  Title Genetic analysis of IP3 and calcium signalling pathways in C. elegans Type Journal Article
  Year 2012 Publication Biochimica et Biophysica Acta Abbreviated Journal Biochim Biophys Acta  
  Volume 1820 Issue 8 Pages 1253-1268  
  Keywords Animals; Caenorhabditis elegans--genetics, metabolism, physiology; Caenorhabditis elegans Proteins--genetics, metabolism; Calcium Signaling; Inositol 1,4,5-Trisphosphate Receptors--genetics, metabolism; Inositol Phosphates--physiology; Mutagenesis; Phenotype; Protein Interaction Maps; RNA Interference; Reverse Genetics  
  Abstract BACKGROUND: The nematode, Caenorhabditis elegans is an established model system that is particularly well suited to genetic analysis. C. elegans is easily manipulated and we have an in depth knowledge of many aspects of its biology. Thus, it is an attractive system in which to pursue integrated studies of signalling pathways. C. elegans has a complement of calcium signalling molecules similar to that of other animals. SCOPE OF REVIEW: We focus on IP3 signalling. We describe how forward and reverse genetic approaches, including RNAi, have resulted in a tool kit which enables the analysis of IP3/Ca2+ signalling pathways. The importance of cell and tissue specific manipulation of signalling pathways and the use of epistasis analysis are highlighted. We discuss how these tools have increased our understanding of IP3 signalling in specific developmental, physiological and behavioural roles. Approaches to imaging calcium signals in C. elegans are considered. MAJOR CONCLUSIONS: A wide selection of tools is available for the analysis of IP3/Ca2+ signalling in C. elegans. This has resulted in detailed descriptions of the function of IP3/Ca2+ signalling in the animal's biology. Nevertheless many questions about how IP3 signalling regulates specific processes remain. GENERAL SIGNIFICANCE: Many of the approaches described may be applied to other calcium signalling systems. C. elegans offers the opportunity to dissect pathways, perform integrated studies and to test the importance of the properties of calcium signalling molecules to whole animal function, thus illuminating the function of calcium signalling in animals. This article is part of a Special Issue entitled Biochemical, biophysical and genetic approaches to intracellular calcium signalling.  
  Call Number Serial 258  
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Author (up) Davis, G.W.; Goodman, C.S. file  url
  Title Synapse-specific control of synaptic efficacy at the terminals of a single neuron Type Journal Article
  Year 1998 Publication Nature Abbreviated Journal Nature  
  Volume 392 Issue 6671 Pages 82-86  
  Keywords Animals; Cell Adhesion Molecules, Neuronal/genetics/metabolism; Drosophila/embryology/genetics/physiology; Motor Neurons/*physiology; Muscles/innervation/*physiology; Mutagenesis; Neuromuscular Junction/*physiology; *Synapses  
  Abstract The regulation of synaptic efficacy is essential for the proper functioning of neural circuits. If synaptic gain is set too high or too low, cells are either activated inappropriately or remain silent. There is extra complexity because synapses are not static, but form, retract, expand, strengthen, and weaken throughout life. Homeostatic regulatory mechanisms that control synaptic efficacy presumably exist to ensure that neurons remain functional within a meaningful physiological range. One of the best defined systems for analysis of the mechanisms that regulate synaptic efficacy is the neuromuscular junction. It has been shown, in organisms ranging from insects to humans, that changes in synaptic efficacy are tightly coupled to changes in muscle size during development. It has been proposed that a signal from muscle to motor neuron maintains this coupling. Here we show, by genetically manipulating muscle innervation, that there are two independent mechanisms by which muscle regulates synaptic efficacy at the terminals of single motor neurons. Increased muscle innervation results in a compensatory, target-specific decrease in presynaptic transmitter release, implying a retrograde regulation of presynaptic release. Decreased muscle innervation results in a compensatory increase in quantal size.  
  Call Number Serial 1320  
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Author (up) Delattre, M.; Felix, M.A. file  url
  Title Microevolutionary studies in nematodes: a beginning Type Journal Article
  Year 2001 Publication BioEssays : News and Reviews in Molecular, Cellular and Developmental Biology Abbreviated Journal Bioessays  
  Volume 23 Issue 9 Pages 807-819  
  Keywords Animals; *Biological Evolution; Caenorhabditis elegans/genetics; Drosophila/genetics; Genetic Techniques; Genetic Variation; Genotype; Mutagenesis; Nematoda/*classification/*genetics; *Polymorphism, Genetic  
  Abstract Comparisons between related species often allow the detailed genetic analysis of evolutionary processes. Here we advocate the use of the nematode Caenorhabditis elegans (and several other rhabditid species) as model systems for microevolutionary studies. Compared to Drosophila species, which have been a mainstay of such studies, C. elegans has a self-fertilizing mode of reproduction, a shorter life cycle and a convenient cell-level analysis of phenotypic variation. Data concerning its population genetics and ecology are still scarce, however. We review molecular, behavioral and developmental intraspecific polymorphisms for populations of C. elegans, Oscheius sp. 1 and Pristionchus pacificus. Focusing on vulval development, which has been well characterized in several species, we discuss relationships between patterns of variations: (1) for a given genotype (developmental variants), (2) after mutagenesis (mutability), (3) in different populations of the same species (polymorphisms) and (4) between closely related species. These studies have revealed that evolutionary variations between sister species affect those characters that show phenotypic developmental variants, that are mutable and that are polymorphic within species.  
  Call Number Serial 1024  
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Author (up) Heijink, A.M.; Krajewska, M.; van Vugt, M.A.T.M. file  url
  Title The DNA damage response during mitosis Type Journal Article
  Year 2013 Publication Mutation Research Abbreviated Journal Mutat Res  
  Volume 750 Issue 1-2 Pages 45-55  
  Keywords Animals; Cell Cycle/genetics; Cell Cycle Proteins/physiology; DNA Breaks, Double-Stranded; DNA Damage/*physiology; DNA Repair/physiology; Humans; Mitosis/*genetics; Models, Biological; Signal Transduction/genetics; Adaptation; Cell cycle; Checkpoint; Mitosis; Mutagenesis; Recovery  
  Abstract Cells are equipped with a cell-intrinsic signaling network called the DNA damage response (DDR). This signaling network recognizes DNA lesions and initiates various downstream pathways to coordinate a cell cycle arrest with the repair of the damaged DNA. Alternatively, the DDR can mediate clearance of affected cells that are beyond repair through apoptosis or senescence. The DDR can be activated in response to DNA damage throughout the cell cycle, although the extent of DDR signaling is different in each cell cycle phase. Especially in response to DNA double strand breaks, only a very marginal response was observed during mitosis. Early on it was recognized that cells which are irradiated during mitosis continued division without repairing broken chromosomes. Although these initial observations indicated diminished DNA repair and lack of an acute DNA damage-induced cell cycle arrest, insight into the mechanistic re-wiring of DDR signaling during mitosis was only recently provided. Different mechanisms appear to be at play to inactivate specific signaling axes of the DDR network in mitosis. Importantly, mitotic cells not simply inactivate the entire DDR, but appear to mark their DNA damage for repair after mitotic exit. Since the treatment of cancer frequently involves agents that induce DNA damage as well as agents that block mitotic progression, it is clinically relevant to obtain a better understanding of how cancer cells deal with DNA damage during interphase versus mitosis. In this review, the molecular details concerning DDR signaling during mitosis as well as the consequences of encountering DNA damage during mitosis for cellular fate are discussed.  
  Call Number Serial 919  
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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) Sale, J.E.; Lehmann, A.R.; Woodgate, R. file  url
  Title Y-family DNA polymerases and their role in tolerance of cellular DNA damage Type Journal Article
  Year 2012 Publication Nature Reviews. Molecular Cell Biology Abbreviated Journal Nat Rev Mol Cell Biol  
  Volume 13 Issue 3 Pages 141-152  
  Keywords Animals; Bacterial Proteins--chemistry, metabolism, physiology; Catalytic Domain; DNA Damage; DNA Repair; DNA Replication; Humans; Mutagenesis; Nucleotidyltransferases--chemistry, metabolism, physiology; Protein Binding; Protein Structure, Tertiary  
  Abstract The past 15 years have seen an explosion in our understanding of how cells replicate damaged DNA and how this can lead to mutagenesis. The Y-family DNA polymerases lie at the heart of this process, which is commonly known as translesion synthesis. This family of polymerases has unique features that enable them to synthesize DNA past damaged bases. However, as they exhibit low fidelity when copying undamaged DNA, it is essential that they are only called into play when they are absolutely required. Several layers of regulation ensure that this is achieved.  
  Call Number Serial 456  
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Author (up) Short, C.A.; Cao, A.T.; Wingfield, M.A.; Doers, M.E.; Jobe, E.M.; Wang, N.; Levandoski, M.M. file  url
  Title Subunit interfaces contribute differently to activation and allosteric modulation of neuronal nicotinic acetylcholine receptors Type Journal Article
  Year 2015 Publication Neuropharmacology Abbreviated Journal Neuropharmacology  
  Volume 91 Issue Pages 157-168  
  Keywords Animals; Binding Sites; Cysteine/chemistry; Neurons/*metabolism; Nicotinic Agonists/*metabolism; Oxidation-Reduction; Protein Subunits/*chemistry/*metabolism; Rats; Receptors, Nicotinic/*chemistry/*metabolism; Xenopus; Allosteric regulation; Cys-loop receptors; Cysteine-mediating cross-linking; Interface; Mutagenesis site specific; Nicotinic acetylcholine receptors; Oocyte  
  Abstract Neuronal nicotinic acetylcholine receptors (nAChRs) are widely distributed in the nervous system and are implicated in many normal and pathological processes. The structural determinants of allostery in nAChRs are not well understood. One class of nAChR allosteric modulators, including the small molecule morantel (Mor), acts from a site that is structurally homologous to the canonical agonist site but exists in the beta(+)/alpha(-) subunit interface. We hypothesized that all nAChR subunits move with respect to each other during channel activation and allosteric modulation. We therefore studied five pairs of residues predicted to span the interfaces of alpha3beta2 receptors, one at the agonist interface and four at the modulator interface. Substituting cysteines in these positions, we used disulfide trapping to perturb receptor function. The pair alpha3Y168-beta2D190, involving the C loop region of the beta2 subunit, mediates modulation and agonist activation, because evoked currents were reduced up to 50% following oxidation (H2O2) treatment. The pair alpha3S125-beta2Q39, below the canonical site, is also involved in channel activation, in accord with previous studies of the muscle-type receptor; however, the pair is differentially sensitive to ACh activation and Mor modulation (currents decreased 60% and 80%, respectively). The pairs alpha3Q37-beta2A127 and alpha3E173-beta2R46, both in the non-canonical interface, showed increased currents following oxidation, suggesting that subunit movements are not symmetrical. Together, our results from disulfide trapping and further mutation analysis indicate that subunit interface movement is important for allosteric modulation of nAChRs, but that the two types of interfaces contribute unequally to receptor activation.  
  Call Number Serial 1890  
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Author (up) Snapper, S.B.; Melton, R.E.; Mustafa, S.; Kieser, T.; Jacobs, W.R.J. file  url
  Title Isolation and characterization of efficient plasmid transformation mutants of Mycobacterium smegmatis Type Journal Article
  Year 1990 Publication Molecular Microbiology Abbreviated Journal Mol Microbiol  
  Volume 4 Issue 11 Pages 1911-1919  
  Keywords DNA, Bacterial/isolation & purification; Escherichia coli/genetics; *Mutagenesis; Mycobacterium/*genetics/growth & development/isolation & purification; Phenotype; *Plasmids; Restriction Mapping; Transformation, Bacterial  
  Abstract Recent development of vectors and methodologies to introduce recombinant DNA into members of the genus Mycobacterium has provided new approaches for investigating these important bacteria. While most pathogenic mycobacteria are slow-growing, Mycobacterium smegmatis is a fast-growing, non-pathogenic species that has been used for many years as a host for mycobacteriophage propagation and, recently, as a host for the introduction of recombinant DNA. Its use as a cloning host for the analysis of mycobacterial genes has been limited by its inability to be efficiently transformed with plasmid vectors. This work describes the isolation and characterization of mutants of M. smegmatis that can be transformed, using electroporation, at efficiencies 10(4) to 10(5) times greater than those of the parent strain, yielding more than 10(5) transformants per microgram of plasmid DNA. The mutations conferring this efficient plasmid transformation (Ept) phenotype do not affect phage transfection or the integration of DNA into the M. smegmatis chromosome, but seem to be specific for plasmid transformation. Such Ept mutants have been used to characterize plasmid DNA sequences essential for replication of the Mycobacterium fortuitum plasmid pAL5000 in mycobacteria by permitting the transformation of a library of hybrid plasmid constructs. Efficient plasmid transformation of M. smegmatis will facilitate the analysis of mycobacterial gene function, expression and replication and thus aid in the development of BCG as a multivalent recombinant vaccine vector and in the genetic analysis of the virulence determinants of pathogenic mycobacteria.  
  Call Number Serial 174  
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Author (up) von Schantz, L.; Hakansson, M.; Logan, D.T.; Walse, B.; Osterlin, J.; Nordberg-Karlsson, E.; Ohlin, M. file  url
  Title Structural basis for carbohydrate-binding specificity--a comparative assessment of two engineered carbohydrate-binding modules Type Journal Article
  Year 2012 Publication Glycobiology Abbreviated Journal Glycobiology  
  Volume 22 Issue 7 Pages 948-961  
  Keywords Amino Acid Sequence; Amino Acid Substitution; Binding Sites; Calorimetry; Crystallography, X-Ray; Glucans--chemistry; Glycoside Hydrolases--chemistry, genetics, isolation & purification; Hydrogen Bonding; Hydrophobic and Hydrophilic Interactions; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Oligosaccharides--chemistry; Protein Binding; Protein Engineering; Protein Structure, Secondary; Protein Structure, Tertiary; Recombinant Proteins--chemistry, genetics, isolation & purification; Surface Properties; Thermodynamics; Titrimetry; Xylans--chemistry  
  Abstract Detection, immobilization and purification of carbohydrates can be done using molecular probes that specifically bind to targeted carbohydrate epitopes. Carbohydrate-binding modules (CBMs) are discrete parts of carbohydrate-hydrolyzing enzymes that can be engineered to bind and detect specifically a number of carbohydrates. Design and engineering of CBMs have benefited greatly from structural studies that have helped us to decipher the basis for specificity in carbohydrate-protein interactions. However, more studies are needed to predict which modifications in a CBM would generate probes with predetermined binding properties. In this report, we present the crystal structures of two highly related engineered CBMs with different binding specificity profiles: X-2, which is specific for xylans and the L110F mutant of X-2, which binds xyloglucans and beta-glucans in addition to xylans. The structures of the modules were solved both in the apo form and complexed with oligomers of xylose, as well as with an oligomer of glucose in the case of X-2 L110F. The mutation, leucine to phenylalanine, converting the specific module into a cross-reactive one, introduces a crucial hydrogen-pi interaction that allows the mutant to retain glucan-based ligands. The cross-reactivity of X-2 L110F is furthermore made possible by the plasticity of the protein, in particular, of residue R142, which permits accommodation of an extra hydroxymethyl group present in cellopentaose and not xylopentaose. Altogether, this study shows, in structural detail, altered protein-carbohydrate interactions that have high impact on the binding properties of a carbohydrate probe but are introduced through simple mutagenesis.  
  Call Number Serial 458  
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