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Sample records for mutagenesis reveal sr-bi

  1. Knockdown expression and hepatic deficiency reveal anatheroprotective role for SR-BI in liver and peripheral tissues

    SciTech Connect

    Huby, Thierry; Doucet, Chantal; Dachet, Christiane; Ouzilleau,Betty; Ueda, Yukihiko; Afzal, Veena; Rubin, Edward; Chapman, M. John; Lesnik, Philippe

    2006-07-18

    Scavenger receptor SR-BI has been implicated inHDL-dependent atheroprotective mechanisms. We report the generation of anSR-BI conditional knockout mouse model in which SR-BI gene targeting byloxP site insertion produced a hypomorphic allele (hypomSR-BI).Attenuated SR-BI expression in hypomSR-BI mice resulted in 2-foldelevation in plasma total cholesterol (TC) levels. Cre-mediated SR-BIgene inactivation of the hypomorphic SR-BI allele in hepatocytes(hypomSR-BI-KOliver) was associated with high plasma TC concentrations,increased plasma free cholesterol/TC (FC/TC) ratio, and alipoprotein-cholesterol profile typical of SR-BI-/- mice. Plasma TClevels were increased 2-fold in hypomSR-BI and control mice fed anatherogenic diet, whereas hypomSR-BI-KOliver and SR-BI-/- mice developedsevere hypercholesterolemia due to accumulation of FC-rich, VLDL-sizedparticles. Atherosclerosis in hypomSR-BI mice was enhanced (2.5-fold)compared with that in controls, but to a much lower degree than inhypomSR-BI-KOliver (32-fold) and SR-BI-/- (48-fold) mice. The lattermodels did not differ in either plasma lipid levels or in the capacity ofVLDL-sized lipoproteins to induce macrophage cholesterol loading.However, reduced atherosclerosis in hypomSR-BI-KOliver mice wasassociated with decreased lesional macrophage content as compared withthat in SR-BI-/- mice. These data imply that, in addition to its majoratheroprotective role in liver, SR-BI may exert an antiatherogenic rolein extrahepatic tissues.

  2. Knockdown expression and hepatic deficiency reveal an atheroprotective role for SR-BI in liver and peripheral tissues

    PubMed Central

    Huby, Thierry; Doucet, Chantal; Dachet, Christiane; Ouzilleau, Betty; Ueda, Yukihiko; Afzal, Veena; Rubin, Edward; Chapman, M. John; Lesnik, Philippe

    2006-01-01

    Scavenger receptor SR-BI has been implicated in HDL-dependent atheroprotective mechanisms. We report the generation of an SR-BI conditional knockout mouse model in which SR-BI gene targeting by loxP site insertion produced a hypomorphic allele (hypomSR-BI). Attenuated SR-BI expression in hypomSR-BI mice resulted in 2-fold elevation in plasma total cholesterol (TC) levels. Cre-mediated SR-BI gene inactivation of the hypomorphic SR-BI allele in hepatocytes (hypomSR-BI–KOliver) was associated with high plasma TC concentrations, increased plasma free cholesterol/TC (FC/TC) ratio, and a lipoprotein-cholesterol profile typical of SR-BI–/– mice. Plasma TC levels were increased 2-fold in hypomSR-BI and control mice fed an atherogenic diet, whereas hypomSR-BI–KOliver and SR-BI–/– mice developed severe hypercholesterolemia due to accumulation of FC-rich, VLDL-sized particles. Atherosclerosis in hypomSR-BI mice was enhanced (2.5-fold) compared with that in controls, but to a much lower degree than in hypomSR-BI–KOliver (32-fold) and SR-BI–/– (48-fold) mice. The latter models did not differ in either plasma lipid levels or in the capacity of VLDL-sized lipoproteins to induce macrophage cholesterol loading. However, reduced atherosclerosis in hypomSR-BI–KOliver mice was associated with decreased lesional macrophage content as compared with that in SR-BI–/– mice. These data imply that, in addition to its major atheroprotective role in liver, SR-BI may exert an antiatherogenic role in extrahepatic tissues. PMID:16964311

  3. Low-density lipoprotein upregulate SR-BI through Sp1 Ser702 phosphorylation in hepatic cells.

    PubMed

    Yang, Fan; Du, Yu; Zhang, Jin; Jiang, Zhibo; Wang, Li; Hong, Bin

    2016-09-01

    Scavenger receptor class B type I (SR-BI) is one of the key proteins in the process of reverse cholesterol transport (RCT), and its major function is to uptake high density lipoprotein (HDL) cholesterol from plasma into liver cells. The regulation of SR-BI expression is important for controlling serum lipid content and reducing the risks of cardiovascular diseases. Here we found that SR-BI expression was significantly increased by LDL in vivo and in vitro, and the transcription factor specific protein 1 (Sp1) plays a critical role in this process. Results from co-immunoprecipitation experiments indicate that the activation of SR-BI was associated with Sp1-recruited protein complexes in the promoter region of SR-BI, where histone acetyltransferase p300 was recruited and histone deacetylase HDAC1 was dismissed. As a result, histone acetylation increased, leading to activation of SR-BI transcription. With further investigation, we found that LDL phosphorylated Sp1 through ERK1/2 pathway, which affected Sp1 protein complexes formation in SR-BI promoter. Using mass spectrometry and site directed mutagenesis, a new Sp1 phosphorylation site Ser702 was defined to be associated with Sp1-HDAC1 interaction and may be important in SR-BI activation, shedding light on the knowledge of delicate mechanism of hepatic HDL receptor SR-BI gene modulation by LDL. PMID:27320013

  4. A novel Bi-based oxybromide SrBiO{sub 2}Br: Synthesis, optical property and photocatalytic activity

    SciTech Connect

    He, Ying; Huang, Hongwei Zhang, Yihe Li, Xiaowei; Tian, Na; Guo, Yuxi; Luo, Yi

    2015-04-15

    Highlights: • SrBiO{sub 2}Br was first explored as a novel photocatalyst. • SrBiO{sub 2}Br has been successfully synthesized by a solid state reaction. • We systematically synthesized SrBiO{sub 2}Br in different temperature. • SrBiO{sub 2}Br calcinated at 700 °C exhibited the highest photocatalytic activity. - Abstract: A novel Bi-based photocatalyst SrBiO{sub 2}Br with layered structure was successfully synthesized via a solid state reaction method. The as-prepared samples were characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM) and UV–vis diffuse reflectance spectra (DRS). SrBiO{sub 2}Br has an indirect-transition optical band-gap of 2.58 eV. Density functional calculations revealed that conduction band (CB) were composed of the Bi 6p and Br 4s orbitals, and valence band (VB) were occupied by Br 4p and O 2p. The photodecomposition of rhodamine-B (RhB) experiments demonstrated SrBiO{sub 2}Br can be used as photocatalysts under ultraviolet (UV) light and visible light irradiation (λ > 400 nm). The results revealed that SrBiO{sub 2}Br calcinated at 700 °C exhibited the highest photocatalytic activity among the obtained SrBiO{sub 2}Br samples.

  5. Synthesis and Characterization of SrBi

    SciTech Connect

    Wang, Ying C.; Hoffmann, Roald; DiSalvo, Francis J.

    2001-01-01

    SrBi{sub 2}Se{sub 4} was synthesized at 945 C and its structure was determined using single-crystal X-ray diffraction data obtained at 165 K. SrBi{sub 2}Se{sub 4}is isotypic to 12-Ba Bi{sub 2}Se{sub 4} and Eu1.1 Bi{sub 2}Se{sub 4}. The compound crystallizes in P6{sub 3}/m (Z=12) with a=25.970(2) {angstrom} and c=4.2437(3) {angstrom}. Final R{sub 1}=0.0630 and w R{sub 2}=0.1246 (I > 2{sigma}(I)). The coordination environments of Bi are distorted Se octahedra. These octahedra build up a uniaxial three-dimensional network with tunnels along the z direction, which are filled by Sr{sup 2+}. There is also a second tunnel along the z direction which is partially occupied by Bi atoms. The coordination spheres of Sr are bicapped trigonal prisms of Se. Transport measurements indicate that SrBi{sub 2}Se{sub 4}is semiconducting. This work adds one high-symmetry compound to the family of complex chalcogenides, in which low-symmetry compounds are common.

  6. Photoluminescence of pyrochlore phase in SrBi2Ta2O9 thin films

    NASA Astrophysics Data System (ADS)

    Wang, Y. P.; Ning, H. F.; Zhou, L.; Shen, J. K.; Liu, Z. G.

    2003-07-01

    SrBi2Ta2O9 thin films were prepared by pulsed laser deposition at different substrate temperatures. Photoluminescence (PL) has been detected at room temperature from the pyrochlore phase in the SrBi2Ta2O9 film deposited at 850 °C. The PL shows five luminescence bands of 330, 365, 407, 490, and 600 nm. And the PL excitation shows six excitation bands of 278, 330, 365, 407, 490, and 600 nm. The one-to-one correspondence of PL and PL excitation spectra reveals a band-to-band excitation and a multienergy-gap structure in the pyrochlore phase in SrBi2Ta2O9 films.

  7. Structure of LIMP-2 provides functional insights with implications for SR-BI and CD36.

    PubMed

    Neculai, Dante; Schwake, Michael; Ravichandran, Mani; Zunke, Friederike; Collins, Richard F; Peters, Judith; Neculai, Mirela; Plumb, Jonathan; Loppnau, Peter; Pizarro, Juan Carlos; Seitova, Alma; Trimble, William S; Saftig, Paul; Grinstein, Sergio; Dhe-Paganon, Sirano

    2013-12-01

    Members of the CD36 superfamily of scavenger receptor proteins are important regulators of lipid metabolism and innate immunity. They recognize normal and modified lipoproteins, as well as pathogen-associated molecular patterns. The family consists of three members: SR-BI (which delivers cholesterol to the liver and steroidogenic organs and is a co-receptor for hepatitis C virus), LIMP-2/LGP85 (which mediates lysosomal delivery of β-glucocerebrosidase and serves as a receptor for enterovirus 71 and coxsackieviruses) and CD36 (a fatty-acid transporter and receptor for phagocytosis of effete cells and Plasmodium-infected erythrocytes). Notably, CD36 is also a receptor for modified lipoproteins and β-amyloid, and has been implicated in the pathogenesis of atherosclerosis and of Alzheimer's disease. Despite their prominent roles in health and disease, understanding the function and abnormalities of the CD36 family members has been hampered by the paucity of information about their structure. Here we determine the crystal structure of LIMP-2 and infer, by homology modelling, the structure of SR-BI and CD36. LIMP-2 shows a helical bundle where β-glucocerebrosidase binds, and where ligands are most likely to bind to SR-BI and CD36. Remarkably, the crystal structure also shows the existence of a large cavity that traverses the entire length of the molecule. Mutagenesis of SR-BI indicates that the cavity serves as a tunnel through which cholesterol(esters) are delivered from the bound lipoprotein to the outer leaflet of the plasma membrane. We provide evidence supporting a model whereby lipidic constituents of the ligands attached to the receptor surface are handed off to the membrane through the tunnel, accounting for the selective lipid transfer characteristic of SR-BI and CD36. PMID:24162852

  8. Contributions of a disulfide bond and a reduced cysteine side chain to the intrinsic activity of the high-density lipoprotein receptor SR-BI.

    PubMed

    Yu, Miao; Lau, Thomas Y; Carr, Steven A; Krieger, Monty

    2012-12-18

    The high-density lipoprotein (HDL) receptor scavenger receptor class B, type I (SR-BI), binds HDL and mediates selective cholesteryl ester uptake. SR-BI's structure and mechanism are poorly understood. We used mass spectrometry to assign the two disulfide bonds in SR-BI that connect cysteines within the conserved Cys(321)-Pro(322)-Cys(323) (CPC) motif and connect Cys(280) to Cys(334). We used site-specific mutagenesis to evaluate the contributions of the CPC motif and the side chain of extracellular Cys(384) to HDL binding and lipid uptake. The effects of CPC mutations on activity were context-dependent. Full wild-type (WT) activity required Pro(322) and Cys(323) only when Cys(321) was present. Reduced intrinsic activities were observed for CXC and CPX, but not XXC, XPX, or XXX mutants (X ≠ WT residue). Apparently, a free thiol side chain at position 321 that cannot form an intra-CPC disulfide bond with Cys(323) is deleterious, perhaps because of aberrant disulfide bond formation. Pro(322) may stabilize an otherwise strained CPC disulfide bond, thus supporting WT activity, but this disulfide bond is not absolutely required for normal activity. C(384)X (X = S, T, L, Y, G, or A) mutants exhibited altered activities that varied with the side chain's size: larger side chains phenocopied WT SR-BI treated with its thiosemicarbazone inhibitor BLT-1 (enhanced binding, weakened uptake); smaller side chains produced almost inverse effects (increased uptake:binding ratio). C(384)X mutants were BLT-1-resistant, supporting the proposal that Cys(384)'s thiol interacts with BLT-1. We discuss the implications of our findings on the functions of the extracellular loop cysteines in SR-BI and compare our results to those presented by other laboratories. PMID:23205738

  9. Contributions of a disulfide bond and a reduced cysteine side chain to the intrinsic activity of the HDL receptor SR-BI

    PubMed Central

    Yu, Miao; Lau, Thomas Y.; Carr, Steven A.; Krieger, Monty

    2013-01-01

    The high density lipoprotein (HDL) receptor, scavenger receptor class B, type I (SR-BI), binds HDL and mediates selective cholesteryl ester uptake. SR-BI's structure and mechanism are poorly understood. We used mass spectrometry to assign the two disulfide bonds in SR-BI that connect cysteines within the conserved Cys321-Pro322-Cys323 (CPC) motif and connect Cys280 to Cys334. We used site-specific mutagenesis to evaluate the contributions of the CPC motif and the side chain of extracellular Cys384 to HDL binding and lipid uptake. The effects of CPC mutations on activity were context dependent. Full wild-type (WT) activity required Pro322 and Cys323 only when Cys321 was present. Reduced intrinsic activities were observed for CXC and CPX, but not XXC, XPX or XXX mutants (X≠WT residue). Apparently, a free thiol side chain at position 321 that cannot form an intra-CPC disulfide bond with Cys323 is deleterious, perhaps because of aberrant disulfide bond formation. Pro322 may stabilize an otherwise strained CPC disulfide bond, thus supporting WT activity, but this disulfide bond is not absolutely required for activity. C384X (X=S,T,L,Y,G,A) mutants exhibited altered activities that varied with the side chain's size: larger side chains phenocopied WT SR-BI treated with its thiosemicarbazone inhibitor BLT-1 (increased binding, decreased uptake); smaller side chains produced almost inverse effects (increased uptake:binding ratio). C384X mutants were BLT-1 resistant, supporting the proposal that Cys384's thiol interacts with BLT-1. We discuss the implications of our findings on the functions of the extracellular loop cysteines in SR-BI and compare our results to those presented by other laboratories. PMID:23205738

  10. The Role of Siglec-1 and SR-BI Interaction in the Phagocytosis of Oxidized Low Density Lipoprotein by Macrophages

    PubMed Central

    Li, Chang; Zhu, Lin; Wu, Li-juan; Zhong, Ren-qian

    2013-01-01

    Background Macrophages play a proatherosclerotic role in atherosclerosis via oxLDL uptake. As an adhesion molecular of I-type lectins, Siglec-1 is highly expressed on circulating monocytes and plaque macrophages of atherosclerotic patients, but the exact role of Siglec-1 has not been elucidated. Methods In this study, oxLDL was used to stimulate Siglec-1 and some oxLDL receptors (SR-BI, CD64, CD32B, LOX-1 and TLR-4) expression on bone marrow-derived macrophages, whereas small interfering RNA was used to down-regulate Siglec-1. Meanwhile, an ELISA-based assay for Siglec-1-oxLDL interaction was performed, and co-immunoprecipitation (co-IP) and laser scanning confocal microscopy (LSCM) were used to determine the role of Siglec-1 in oxLDL uptake by macrophages. Results We found that oxLDL could up-regulate the expression of various potential oxLDL receptors, including Siglec-1, in a dose-dependent manner. Moreover, down-regulation of Siglec-1 could attenuate oxLDL uptake by Oil red O staining. LSCM revealed that Siglec-1 and CD64/SR-BI may colocalize on oxLDL-stimulated macrophage surface, whereas co-IP showed that Siglec-1 and SR-BI can be immunoprecipitated by each other. However, no direct interaction between Siglec-1 and oxLDL was found in the in vitro protein interaction system. Conclusions Thus, Siglec-1 can interact with SR-BI in the phagocytosis of oxLDL by macrophages, rather than act as an independent receptor for oxLDL. PMID:23520536

  11. Difference in expression patterns of placental cholesterol transporters, ABCA1 and SR-BI, in Meishan and Yorkshire pigs with different placental efficiency

    PubMed Central

    Hong, Linjun; Xu, Xiangdong; Huang, Ji; Lei, Minggang; Xu, Dequan; Zhao, Shuhong; Yu, Mei

    2016-01-01

    Cholesterol is a key cell membrane component and precursor of steroid hormones. The maternal cholesterol is an important exogenous cholesterol source for the developing embryos and its transportation is mediated by ABCA1 and SR-BI. Here we reported that during the peri-implantation period in pigs, ABCA1 was expressed by uterine luminal epithelium (LE) and interestingly, its expression was more abundantly in LE on mesometrial side of uterus. However, SR-BI was expressed primarily by LE, glandular epithelial cells (GE) and trophoblast cells (Tr). During the placentation period, the expression levels of ABCA1 and SR-BI proteins at epithelial bilayer and placental areolae were significantly higher in Chinese Meishan pigs compared to Yorkshire pigs. Consisitently, mRNA levels of HMGCR, the rate-limiting enzyme for cholesterol synthesis, were significantly higher in Meishan placentas than in Yorkshire placentas. Our findings revealed the routes of transplacental cholesterol transport mediated by ABCA1 and SR-BI in pigs and indicated that ABCA1 related pathway may participate in anchoring the conceptus to the mesometrial side of uterus. Additionally, an ABCA1 dependent compensatory mechanism related to the placental efficiency in response to the smaller placenta size in Meishan pigs was suggested. PMID:26852751

  12. Difference in expression patterns of placental cholesterol transporters, ABCA1 and SR-BI, in Meishan and Yorkshire pigs with different placental efficiency.

    PubMed

    Hong, Linjun; Xu, Xiangdong; Huang, Ji; Lei, Minggang; Xu, Dequan; Zhao, Shuhong; Yu, Mei

    2016-01-01

    Cholesterol is a key cell membrane component and precursor of steroid hormones. The maternal cholesterol is an important exogenous cholesterol source for the developing embryos and its transportation is mediated by ABCA1 and SR-BI. Here we reported that during the peri-implantation period in pigs, ABCA1 was expressed by uterine luminal epithelium (LE) and interestingly, its expression was more abundantly in LE on mesometrial side of uterus. However, SR-BI was expressed primarily by LE, glandular epithelial cells (GE) and trophoblast cells (Tr). During the placentation period, the expression levels of ABCA1 and SR-BI proteins at epithelial bilayer and placental areolae were significantly higher in Chinese Meishan pigs compared to Yorkshire pigs. Consisitently, mRNA levels of HMGCR, the rate-limiting enzyme for cholesterol synthesis, were significantly higher in Meishan placentas than in Yorkshire placentas. Our findings revealed the routes of transplacental cholesterol transport mediated by ABCA1 and SR-BI in pigs and indicated that ABCA1 related pathway may participate in anchoring the conceptus to the mesometrial side of uterus. Additionally, an ABCA1 dependent compensatory mechanism related to the placental efficiency in response to the smaller placenta size in Meishan pigs was suggested. PMID:26852751

  13. Epitaxial growth of (001)-oriented and (110)-oriented SrBi2Ta2O9 thin films

    NASA Astrophysics Data System (ADS)

    Lettieri, J.; Jia, Y.; Urbanik, M.; Weber, C. I.; Maria, J.-P.; Schlom, D. G.; Li, H.; Ramesh, R.; Uecker, R.; Reiche, P.

    1998-11-01

    Epitaxial SrBi2Ta2O9 thin films have been grown with (001) and (110) orientations by pulsed laser deposition on (001) LaAlO3-Sr2AlTaO6 and (100) LaSrAlO4 substrates, respectively. Four-circle x-ray diffraction and transmission electron microscopy reveal nearly phase pure epitaxial films. Minimization of surface mesh mismatch between the film and substrate (i.e., choice of appropriate substrate material and orientation) was used to stabilize the desired orientations and achieve epitaxial growth.

  14. Synthesis and characterization of rare-earth doped SrBi{sub 2}Nb{sub 2}O{sub 9} phase in lithium borate based nanocrystallized glasses

    SciTech Connect

    Harihara Venkataraman, B.; Fujiwara, Takumi; Komatsu, Takayuki

    2009-06-15

    Glass composites comprising of un-doped and samarium-doped SrBi{sub 2}Nb{sub 2}O{sub 9} nanocrystallites are fabricated in the glass system 16.66SrO-16.66[(1-x)Bi{sub 2}O{sub 3}-xSm{sub 2}O{sub 3}]-16.66Nb{sub 2}O{sub 5}-50Li{sub 2}B{sub 4}O{sub 7} (0<=x<=0.5, in mol%) via the melt quenching technique. The glassy nature of the as-quenched samples is established by differential thermal analyses. Transmission electron microscopic studies reveal the presence of about 15 nm sized spherical crystallites of the fluorite-like SrBi{sub 1.9}Sm{sub 0.1}Nb{sub 2}O{sub 9} phase in the samples heat treated at 530 deg. C. The formation of layered perovskite-type un-doped and samarium-doped SrBi{sub 2}Nb{sub 2}O{sub 9} nanocrystallites with an orthorhombic structure through the intermediate fluorite phase is confirmed by X-ray powder diffraction and micro-Raman spectroscopic studies. The influence of samarium doping on the lattice parameters, lattice distortions, and the Raman peak positions of SrBi{sub 2}Nb{sub 2}O{sub 9} perovskite phase is clarified. The dielectric constants of the perovskite SrBi{sub 2}Nb{sub 2}O{sub 9} and SrBi{sub 1.9}Sm{sub 0.1}Nb{sub 2}O{sub 9} nanocrystals are relatively larger than those of the corresponding fluorite-like phase and the precursor glass. - Graphical Abstract: This figure shows the XRD patterns at room temperature for the as-quenched and heat treated samples in Sm{sub 2}O{sub 3}-doped (x=0.1) glass. Based on these results, it is concluded that the formation of samarium-doped perovskite SBN phase takes place via an intermediate fluorite-like phase in the crystallization of this glass.

  15. Ferroelectric domain structures in SrBi2Nb2O9 epitaxial thin films: Electron microscopy and phase-field simulations

    NASA Astrophysics Data System (ADS)

    Li, Y. L.; Chen, L. Q.; Asayama, G.; Schlom, D. G.; Zurbuchen, M. A.; Streiffer, S. K.

    2004-06-01

    Ferroelectric domain structures of (001)SrBi2Nb2O9 epitaxial films, investigated using both transmission electron microscopy and phase-field simulations, are reported. Experiment and numerical simulation both reveal that the domain structures consist of irregularly shaped domains with curved domain walls. It is shown that the elastic contribution to domain structures can be neglected in SrBi2Nb2O9 due to its small ferroelastic distortion, less than 0.0018%. Two-beam dark-field imaging using reflections unique to domains of each of the two 90° polarization axes reveal the domain structure. Phase-field simulation is based on the elastic and electrostatic solutions obtained for thin films under different mechanical and electric boundary conditions. The effects of ferroelastic distortion and dielectric constant on ferroelectric domains are systematically analyzed. It is demonstrated that electrostatic interactions which favor straight domain walls are not sufficient to overcome the domain wall energy which favors curved domains in SrBi2Nb2O9.

  16. Features of MotA proton channel structure revealed by tryptophan-scanning mutagenesis.

    PubMed Central

    Sharp, L L; Zhou, J; Blair, D F

    1995-01-01

    The MotA protein of Escherichia coli is a component of the flagellar motors that functions in transmembrane proton conduction. Here, we report several features of MotA structure revealed by use of a mutagenesis-based approach. Single tryptophan residues were introduced at many positions within the four hydrophobic segments of MotA, and the effects on function were measured. Function was disrupted according to a periodic pattern that implies that the membrane-spanning segments are alpha-helices and that identifies the lipid-facing parts of each helix. The results support a hypothesis for MotA structure and mechanism in which water molecules form most of the proton-conducting pathway. The success of this approach in studying MotA suggests that it could be useful in structure-function studies of other integral membrane proteins. Images Fig. 1 PMID:7644518

  17. Identification of the Molecular Target of Small Molecule Inhibitors of HDL Receptor SR-BI Activity†,‡,§

    PubMed Central

    Nieland, Thomas J. F.; Shaw, Jared T.; Jaipuri, Firoz A.; Duffner, Jay L.; Koehler, Angela N.; Banakos, Sotirios; Zannis, Vassilis I.; Kirchhausen, Tomas; Krieger, Monty

    2009-01-01

    Scavenger receptor, class B, type I (SR-BI), controls high-density lipoprotein (HDL) metabolism by mediating cellular selective uptake of lipids from HDL without the concomitant degradation of the lipoprotein particle. We previously identified in a high-throughput chemical screen of intact cells five compounds (BLT-1–5) that inhibit SR-BI-dependent lipid transport from HDL, but do not block HDL binding to SR-BI on the cell surface. Although these BLTs are widely used to examine the diverse functions of SR-BI, their direct target(s), SR-BI itself or some other component of the SR-BI pathway, has not been identified. Here we show that SR-BI in the context of a membrane lipid environment is the target of BLT-1, -3, -4, and -5. The analysis using intact cells and an in vitro system of purified SR-BI reconstituted into liposomes was aided by information derived from structure–activity relationship (SAR) analysis of the most potent of these BLTs, the thiosemicarbazone BLT-1. We found that the sulfur atom of BLT-1 was crucially important for its inhibitory activity, because changing it to an oxygen atom resulted in the isostructural, but essentially inactive, semicarbazone derivative BLT-1sc. SAR analysis also established the importance of BLT-1’s hydrophobic tail. BLTs and their corresponding inactive compounds can be used to explore the mechanism and function of SR-BI-mediated selective lipid uptake in diverse mammalian experimental models. Consequently, BLTs may help determine the therapeutic potential of SR-BI-targeted pharmaceutical drugs. PMID:18067275

  18. Influence of Zr4+ doping on structural and electrical properties of SrBi4Ti4O15 ceramic

    NASA Astrophysics Data System (ADS)

    Nayak, P.; Badapanda, T.; Panigrahi, S.

    2015-06-01

    This article reports a systematic study of doping effects on the structural and electrical properties of layer structured strontium bismuth titanate ceramic. In this study monophasic SrBi4Ti4-xZrxO15 with x=0.00, 0.05, 0.10, 0.15, 0.20, 0.25 ceramics were synthesized from the solid-state reaction route. X-ray diffraction analysis shows that the Zr-modified SBT ceramics have a pure four-layer Aurivillius phase structure. Dielectric properties revealed that the diffuseness of phase transition increases where as corresponding permittivity value decrease with increasing Zr content. Piezoelectric properties of SBTZ ceramics were improved by the modification of Zirconium ion. Moreover, the reason behind for improvement of piezoelectric properties of modified SBTZ ceramics was also discussed.

  19. Effect of annealing on the charge-voltage characteristics of SrBi2(TaxNb1-x)2O9 films

    NASA Astrophysics Data System (ADS)

    Morozovsky, N. V.; Semchenko, A. V.; Sidsky, V. V.; Kolos, V. V.; Turtsevich, A. S.; Eliseev, E. A.; Morozovska, A. N.

    2015-05-01

    The effect of changes of the Nb content and annealing on charge-voltage and current-voltage characteristics of film structures Pt/SrBi2(Ta1-xNbx)2O9/Pt/TiO2/SiO2/Si-substrate with х=0, 0.1, 0.2 was studied theoretically and experimentally. Theoretical modeling, which takes into account the mobile charged donors impact on the features of charge-voltage and current-voltage characteristics of ferroelectric-semiconductor films, revealed the changes of conductivity value and ferroelectric parameters. The results of theoretical analysis and experimental results are in qualitative agreement.

  20. Novel gene function revealed by mouse mutagenesis screens for models of age-related disease

    PubMed Central

    Potter, Paul K.; Bowl, Michael R.; Jeyarajan, Prashanthini; Wisby, Laura; Blease, Andrew; Goldsworthy, Michelle E.; Simon, Michelle M.; Greenaway, Simon; Michel, Vincent; Barnard, Alun; Aguilar, Carlos; Agnew, Thomas; Banks, Gareth; Blake, Andrew; Chessum, Lauren; Dorning, Joanne; Falcone, Sara; Goosey, Laurence; Harris, Shelley; Haynes, Andy; Heise, Ines; Hillier, Rosie; Hough, Tertius; Hoslin, Angela; Hutchison, Marie; King, Ruairidh; Kumar, Saumya; Lad, Heena V.; Law, Gemma; MacLaren, Robert E.; Morse, Susan; Nicol, Thomas; Parker, Andrew; Pickford, Karen; Sethi, Siddharth; Starbuck, Becky; Stelma, Femke; Cheeseman, Michael; Cross, Sally H.; Foster, Russell G.; Jackson, Ian J.; Peirson, Stuart N.; Thakker, Rajesh V.; Vincent, Tonia; Scudamore, Cheryl; Wells, Sara; El-Amraoui, Aziz; Petit, Christine; Acevedo-Arozena, Abraham; Nolan, Patrick M.; Cox, Roger; Mallon, Anne-Marie; Brown, Steve D. M.

    2016-01-01

    Determining the genetic bases of age-related disease remains a major challenge requiring a spectrum of approaches from human and clinical genetics to the utilization of model organism studies. Here we report a large-scale genetic screen in mice employing a phenotype-driven discovery platform to identify mutations resulting in age-related disease, both late-onset and progressive. We have utilized N-ethyl-N-nitrosourea mutagenesis to generate pedigrees of mutagenized mice that were subject to recurrent screens for mutant phenotypes as the mice aged. In total, we identify 105 distinct mutant lines from 157 pedigrees analysed, out of which 27 are late-onset phenotypes across a range of physiological systems. Using whole-genome sequencing we uncover the underlying genes for 44 of these mutant phenotypes, including 12 late-onset phenotypes. These genes reveal a number of novel pathways involved with age-related disease. We illustrate our findings by the recovery and characterization of a novel mouse model of age-related hearing loss. PMID:27534441

  1. High-throughput mutagenesis reveals functional determinants for DNA targeting by activation-induced deaminase.

    PubMed

    Gajula, Kiran S; Huwe, Peter J; Mo, Charlie Y; Crawford, Daniel J; Stivers, James T; Radhakrishnan, Ravi; Kohli, Rahul M

    2014-09-01

    Antibody maturation is a critical immune process governed by the enzyme activation-induced deaminase (AID), a member of the AID/APOBEC DNA deaminase family. AID/APOBEC deaminases preferentially target cytosine within distinct preferred sequence motifs in DNA, with specificity largely conferred by a small 9-11 residue protein loop that differs among family members. Here, we aimed to determine the key functional characteristics of this protein loop in AID and to thereby inform our understanding of the mode of DNA engagement. To this end, we developed a methodology (Sat-Sel-Seq) that couples saturation mutagenesis at each position across the targeting loop, with iterative functional selection and next-generation sequencing. This high-throughput mutational analysis revealed dominant characteristics for residues within the loop and additionally yielded enzymatic variants that enhance deaminase activity. To rationalize these functional requirements, we performed molecular dynamics simulations that suggest that AID and its hyperactive variants can engage DNA in multiple specific modes. These findings align with AID's competing requirements for specificity and flexibility to efficiently drive antibody maturation. Beyond insights into the AID-DNA interface, our Sat-Sel-Seq approach also serves to further expand the repertoire of techniques for deep positional scanning and may find general utility for high-throughput analysis of protein function. PMID:25064858

  2. High-throughput mutagenesis reveals functional determinants for DNA targeting by activation-induced deaminase

    PubMed Central

    Gajula, Kiran S.; Huwe, Peter J.; Mo, Charlie Y.; Crawford, Daniel J.; Stivers, James T.; Radhakrishnan, Ravi; Kohli, Rahul M.

    2014-01-01

    Antibody maturation is a critical immune process governed by the enzyme activation-induced deaminase (AID), a member of the AID/APOBEC DNA deaminase family. AID/APOBEC deaminases preferentially target cytosine within distinct preferred sequence motifs in DNA, with specificity largely conferred by a small 9–11 residue protein loop that differs among family members. Here, we aimed to determine the key functional characteristics of this protein loop in AID and to thereby inform our understanding of the mode of DNA engagement. To this end, we developed a methodology (Sat-Sel-Seq) that couples saturation mutagenesis at each position across the targeting loop, with iterative functional selection and next-generation sequencing. This high-throughput mutational analysis revealed dominant characteristics for residues within the loop and additionally yielded enzymatic variants that enhance deaminase activity. To rationalize these functional requirements, we performed molecular dynamics simulations that suggest that AID and its hyperactive variants can engage DNA in multiple specific modes. These findings align with AID's competing requirements for specificity and flexibility to efficiently drive antibody maturation. Beyond insights into the AID-DNA interface, our Sat-Sel-Seq approach also serves to further expand the repertoire of techniques for deep positional scanning and may find general utility for high-throughput analysis of protein function. PMID:25064858

  3. Novel gene function revealed by mouse mutagenesis screens for models of age-related disease.

    PubMed

    Potter, Paul K; Bowl, Michael R; Jeyarajan, Prashanthini; Wisby, Laura; Blease, Andrew; Goldsworthy, Michelle E; Simon, Michelle M; Greenaway, Simon; Michel, Vincent; Barnard, Alun; Aguilar, Carlos; Agnew, Thomas; Banks, Gareth; Blake, Andrew; Chessum, Lauren; Dorning, Joanne; Falcone, Sara; Goosey, Laurence; Harris, Shelley; Haynes, Andy; Heise, Ines; Hillier, Rosie; Hough, Tertius; Hoslin, Angela; Hutchison, Marie; King, Ruairidh; Kumar, Saumya; Lad, Heena V; Law, Gemma; MacLaren, Robert E; Morse, Susan; Nicol, Thomas; Parker, Andrew; Pickford, Karen; Sethi, Siddharth; Starbuck, Becky; Stelma, Femke; Cheeseman, Michael; Cross, Sally H; Foster, Russell G; Jackson, Ian J; Peirson, Stuart N; Thakker, Rajesh V; Vincent, Tonia; Scudamore, Cheryl; Wells, Sara; El-Amraoui, Aziz; Petit, Christine; Acevedo-Arozena, Abraham; Nolan, Patrick M; Cox, Roger; Mallon, Anne-Marie; Brown, Steve D M

    2016-01-01

    Determining the genetic bases of age-related disease remains a major challenge requiring a spectrum of approaches from human and clinical genetics to the utilization of model organism studies. Here we report a large-scale genetic screen in mice employing a phenotype-driven discovery platform to identify mutations resulting in age-related disease, both late-onset and progressive. We have utilized N-ethyl-N-nitrosourea mutagenesis to generate pedigrees of mutagenized mice that were subject to recurrent screens for mutant phenotypes as the mice aged. In total, we identify 105 distinct mutant lines from 157 pedigrees analysed, out of which 27 are late-onset phenotypes across a range of physiological systems. Using whole-genome sequencing we uncover the underlying genes for 44 of these mutant phenotypes, including 12 late-onset phenotypes. These genes reveal a number of novel pathways involved with age-related disease. We illustrate our findings by the recovery and characterization of a novel mouse model of age-related hearing loss. PMID:27534441

  4. Linear and nonlinear optical properties of SrBi4Ti4O15 thin films

    NASA Astrophysics Data System (ADS)

    Rambabu, A.; Reddy, E. Sivanagi; Hamad, Syed; Raju, K. C. James; Rao, S. Venugopal

    2016-05-01

    Polycrystalline SrBi4Ti4O15 thin films with good morphology and layered perovskite structure were fabricated on fused silica substrates using r f magnetron sputtering system at various oxygen mixing percentages (25 and 50). The crystallite sizes of the particles are in 17-28 nm range. The Nonlinear optical properties were investigated by using Z-scan method at a wavelength of 800 nm with 2 ps duration pulses. The films exhibit the fast and giant optical nonlinearities having the two-photon absorption coefficient (β) with magnitude of 10-8-10-9 cm/W and the nonlinear refraction coefficient of ˜10-12 cm2/W. These results indicate SrBi4Ti4O15 thin films are promising candidates for applications in nonlinear optical and optical signal processing devices.

  5. Scavenger receptor class B type I (SR-BI) is involved in vitamin E transport across the enterocyte

    PubMed Central

    Reboul, Emmanuelle; Klein, Alexis; Bietrix, Florence; Gleize, Béatrice; Malezet-Desmoulins, Christiane; Schneider, Martina; Margotat, Alain; Lagrost, Laurent; Collet, Xavier; Borel, Patrick

    2006-01-01

    Although cellular uptake of vitamin E was initially described as a passive process, recent studies in the liver and brain have shown that SR-BI is involved in this phenomenon. As SR-BI is expressed at high levels in the intestine, the present study addressed the involvement of SR-BI in vitamin E trafficking across enterocytes. Apical uptake and efflux of the main dietary forms of vitamin E was examined using Caco-2 TC-7 cell monolayers as a model of human intestinal epithelium. RRR-γ-tocopherol bioavailability was compared between wild-type mice and mice overexpressing SR-BI in the intestine. The effect of vitamin E on enterocyte SR-BI mRNA levels was measured by real-time quantitative RT-PCR. Concentration-dependent curves for vitamin E uptake were similar for RRR-α-, RRR-γ- and DL-α-tocopherol. RRR-α-tocopherol transport was dependent on incubation temperature, with a 60% reduction in absorption at 4°C compared to 37°C (p<0.05). Vitamin E flux in enterocytes was directed from the apical to the basal side, with a relative 10-fold reduction in the transfer process when measured in the opposite direction (p<0.05). Co-incubation with cholesterol, γ-tocopherol or lutein significantly impaired α-tocopherol absorption. Anti-human SR-BI antibodies and BLT1 (a chemical inhibitor of lipid transport via SR-BI) blocked up to 80% of vitamin E uptake and up to 30 % of apical vitamin E efflux (p<0.05), and similar results were obtained for RRR-γ-tocopherol. SR-BI mRNA levels were not significantly modified after a 24-hour incubation of Caco-2 cells with vitamin E. Finally, RRR-γ-tocopherol bioavailability was 2.7-fold higher in mice overexpressing SR-BI than in wild-type mice (p<0.05). The present data show for the first time that vitamin E intestinal absorption is, at least partly, mediated by SR-BI. PMID:16380385

  6. A Sleeping Beauty mutagenesis screen reveals a tumor suppressor role for Ncoa2/Src-2 in liver cancer.

    PubMed

    O'Donnell, Kathryn A; Keng, Vincent W; York, Brian; Reineke, Erin L; Seo, Daekwan; Fan, Danhua; Silverstein, Kevin A T; Schrum, Christina T; Xie, Wei Rose; Mularoni, Loris; Wheelan, Sarah J; Torbenson, Michael S; O'Malley, Bert W; Largaespada, David A; Boeke, Jef D

    2012-05-22

    The Sleeping Beauty (SB) transposon mutagenesis system is a powerful tool that facilitates the discovery of mutations that accelerate tumorigenesis. In this study, we sought to identify mutations that cooperate with MYC, one of the most commonly dysregulated genes in human malignancy. We performed a forward genetic screen with a mouse model of MYC-induced liver cancer using SB-mediated mutagenesis. We sequenced insertions in 63 liver tumor nodules and identified at least 16 genes/loci that contribute to accelerated tumor development. RNAi-mediated knockdown in a liver progenitor cell line further validate three of these genes, Ncoa2/Src-2, Zfx, and Dtnb, as tumor suppressors in liver cancer. Moreover, deletion of Ncoa2/Src-2 in mice predisposes to diethylnitrosamine-induced liver tumorigenesis. These findings reveal genes and pathways that functionally restrain MYC-mediated liver tumorigenesis and therefore may provide targets for cancer therapy. PMID:22556267

  7. Impairment of the ABCA1 and SR-BI-mediated cholesterol efflux pathways and HDL anti-inflammatory activity in Alzheimer's disease.

    PubMed

    Khalil, Abdelouahed; Berrougui, Hicham; Pawelec, Graham; Fulop, Tamas

    2012-01-01

    The aim of our study was to investigate the effect of Alzheimer's disease (AD) on the cholesterol efflux capacity and anti-inflammatory activity of HDL. HDL and apoA-I were isolated from 20 healthy subjects and from 39 AD patients. Our results showed that serum- and HDL-mediated cholesterol efflux is significantly impaired in AD patients. This impairment of serum and HDL cholesterol efflux capacity was significantly inversely correlated to the AD severity as evaluated by MMSE scores. Results obtained from SR-BI-enriched Fu5AH and ABCA1-enriched J774 cells revealed that AD impaired the interaction of HDL and apoA-I with both the ABCA1 transporter and SR-BI receptor. Purified apoA-I from AD patients also failed to remove free excess cholesterol from ABCA1-enriched J774 macrophages. Interestingly, the decrease in plasma α-tocopherol content and the increase in MDA formation and HDL relative electrophoretic mobility indicated that AD patients had higher levels of oxidative stress. The anti-inflammatory activity of HDL was also significantly lower in AD patients as measured by the level of ICAM-1 expression. In conclusion, our study provides evidence for the first time that the functionality of HDL is impaired in AD and that this alteration might be caused by AD-associated oxidative stress and inflammation. PMID:22178419

  8. Structural, magnetic, and dielectric studies of the Aurivillius compounds SrBi5Ti4MnO18 and SrBi5Ti4Mn0.5Co0.5O18

    NASA Astrophysics Data System (ADS)

    Yuan, B.; Yang, J.; Song, D. P.; Zuo, X. Z.; Tang, X. W.; Zhu, X. B.; Dai, J. M.; Song, W. H.; Sun, Y. P.

    2015-01-01

    We have successfully synthesized the Aurivillius compounds SrBi5Ti4MnO18 and SrBi5Ti4Mn0.5Co0.5O18 using a modified Pechini method. Both samples have an orthorhombic structure with the space group B2cb. The valence state of Mn is suggested to be +3 and the doped Co ions exist in the form of Co2+ and Co3+ based on the results of x-ray photoelectron spectroscopy. The sample SrBi5Ti4MnO18 exhibits a dominant paramagnetic state with the existence of superparamagnetic state as evidenced by the electron paramagnetic resonance results, whereas SrBi5Ti4Mn0.5Co0.5O18 undergoes a ferrimagnetic transition at 161 K originating from the antiferromagnetic coupling of Co-based and Mn-based sublattices, and a ferromagnetic transition at 45 K arising from the Mn3+-O-Co3+ (low spin) interaction. The sample SrBi5Ti4Mn0.5Co0.5O18 exhibits two dielectric anomalies. One corresponds to a relaxor-like dielectric relaxation which follows the Vogel-Fulcher function and the other dielectric relaxation obeys the Arrhenius law arising from the collective motion of oxygen vacancies. In addition, the sample SrBi5Ti4Mn0.5Co0.5O18 exhibits a magnetodielectric effect caused by the Maxwell-Wagner effect because of the conductivity of the sample. This is demonstrated by the fact that the activation energy in dielectric loss process is close to that for dc conductivity and the magnetodielectric effect is sensitive to the measured frequency.

  9. Microarray analyses reveal that plant mutagenesis may induce more transcriptomic changes than transgene insertion

    PubMed Central

    Batista, Rita; Saibo, Nelson; Lourenço, Tiago; Oliveira, Maria Margarida

    2008-01-01

    Controversy regarding genetically modified (GM) plants and their potential impact on human health contrasts with the tacit acceptance of other plants that were also modified, but not considered as GM products (e.g., varieties raised through conventional breeding such as mutagenesis). What is beyond the phenotype of these improved plants? Should mutagenized plants be treated differently from transgenics? We have evaluated the extent of transcriptome modification occurring during rice improvement through transgenesis versus mutation breeding. We used oligonucleotide microarrays to analyze gene expression in four different pools of four types of rice plants and respective controls: (i) a γ-irradiated stable mutant, (ii) the M1 generation of a 100-Gy γ-irradiated plant, (iii) a stable transgenic plant obtained for production of an anticancer antibody, and (iv) the T1 generation of a transgenic plant produced aiming for abiotic stress improvement, and all of the unmodified original genotypes as controls. We found that the improvement of a plant variety through the acquisition of a new desired trait, using either mutagenesis or transgenesis, may cause stress and thus lead to an altered expression of untargeted genes. In all of the cases studied, the observed alteration was more extensive in mutagenized than in transgenic plants. We propose that the safety assessment of improved plant varieties should be carried out on a case-by-case basis and not simply restricted to foods obtained through genetic engineering. PMID:18303117

  10. Microarray analyses reveal that plant mutagenesis may induce more transcriptomic changes than transgene insertion.

    PubMed

    Batista, Rita; Saibo, Nelson; Lourenço, Tiago; Oliveira, Maria Margarida

    2008-03-01

    Controversy regarding genetically modified (GM) plants and their potential impact on human health contrasts with the tacit acceptance of other plants that were also modified, but not considered as GM products (e.g., varieties raised through conventional breeding such as mutagenesis). What is beyond the phenotype of these improved plants? Should mutagenized plants be treated differently from transgenics? We have evaluated the extent of transcriptome modification occurring during rice improvement through transgenesis versus mutation breeding. We used oligonucleotide microarrays to analyze gene expression in four different pools of four types of rice plants and respective controls: (i) a gamma-irradiated stable mutant, (ii) the M1 generation of a 100-Gy gamma-irradiated plant, (iii) a stable transgenic plant obtained for production of an anticancer antibody, and (iv) the T1 generation of a transgenic plant produced aiming for abiotic stress improvement, and all of the unmodified original genotypes as controls. We found that the improvement of a plant variety through the acquisition of a new desired trait, using either mutagenesis or transgenesis, may cause stress and thus lead to an altered expression of untargeted genes. In all of the cases studied, the observed alteration was more extensive in mutagenized than in transgenic plants. We propose that the safety assessment of improved plant varieties should be carried out on a case-by-case basis and not simply restricted to foods obtained through genetic engineering. PMID:18303117

  11. [Serum amyloid A promotes the inflammatory response via p38-MAPK/SR-BI pathway in THP-1 macrophages].

    PubMed

    Zhu, Ming-Yan; Wang, Yan; Wang, Yu; Peng, Feng-Ling; Ou, Han-Xiao; Zheng, Xiang; Shi, Jin-Feng; Zeng, Gao-Feng; Mo, Zhong-Cheng

    2016-06-25

    To investigate the effect and mechanism of serum amyloid A (SAA) on the expression of scavenger receptor class B type I (SR-BI) and inflammatory response in THP-1 macrophages, the human THP-1 cells were treated with SAA and p38-MAPK agonist (anisomycin) or p38-MAPK inhibitor (SB203580). Then, the expressions of SR-BI, phosphorylated p38-MAPK and inflammatory factors (MCP-1, TNF-α, IL-1β) were examined by real-time quantitative PCR, Western blotting and ELISA, respectively. The results showed that, compared with control group, SAA increased the levels of inflammatory factors (MCP-1, TNF-α, IL-1β), down-regulated the expressions of SR-BI, and up-regulated the expression of phosphorylated p38-MAPK protein in a concentration- and time-dependent manner in THP-1 cells (P < 0.05). After treatment with SAA and p38-MAPK agonist (anisomycin) in THP-1 cells, the expression of SR-BI was down-regulated, and the levels of inflammatory factors and phosphorylated p38-MAPK protein expression were increased, compared with the group only treated by SAA (P < 0.05). In contrast, the SR-BI expression was up-regulated, whereas inflammatory factors and phosphorylated p38-MAPK protein expressions were decreased after the cells were treated with SAA and p38-MAPK inhibitor (SB203580) (P < 0.05). The results suggest that SAA-promoted inflammatory response in THP-1 macrophages may be through the phosphorylation of p38-MAPK and inhibition of SR-BI expression. PMID:27350202

  12. A directed mutagenesis screen in Drosophila melanogaster reveals new mutants that influence hedgehog signaling.

    PubMed Central

    Haines, N; van den Heuvel, M

    2000-01-01

    The Hedgehog signaling pathway has been recognized as essential for patterning processes in development of metazoan animal species. The signaling pathway is, however, not entirely understood. To start to address this problem, we set out to isolate new mutations that influence Hedgehog signaling. We performed a mutagenesis screen for mutations that dominantly suppress Hedgehog overexpression phenotypes in the Drosophila melanogaster wing. We isolated four mutations that influence Hedgehog signaling. These were analyzed in the amenable wing system using genetic and molecular techniques. One of these four mutations affects the stability of the Hedgehog expression domain boundary, also known as the organizer in the developing wing. Another mutation affects a possible Hedgehog autoregulation mechanism, which stabilizes the same boundary. PMID:11102373

  13. Mos1 mutagenesis reveals a diversity of mechanisms affecting response of Caenorhabditis elegans to the bacterial pathogen Microbacterium nematophilum.

    PubMed

    Yook, Karen; Hodgkin, Jonathan

    2007-02-01

    A specific host-pathogen interaction exists between Caenorhabditis elegans and the gram-positive bacterium Microbacterium nematophilum. This bacterium is able to colonize the rectum of susceptible worms and induces a defensive tail-swelling response in the host. Previous mutant screens have identified multiple loci that affect this interaction. Some of these loci correspond to known genes, but many bus genes [those with a bacterially unswollen (Bus) mutant phenotype] have yet to be cloned. We employed Mos1 transposon mutagenesis as a means of more rapidly cloning bus genes and identifying new mutants with altered pathogen response. This approach revealed new infection-related roles for two well-characterized and much-studied genes, egl-8 and tax-4. It also allowed the cloning of a known bus gene, bus-17, which encodes a predicted galactosyltransferase, and of a new bus gene, bus-19, which encodes a novel, albeit ancient, protein. The results illustrate advantages and disadvantages of Mos1 transposon mutagenesis in this system. PMID:17151260

  14. The Roles of Cytochrome b559 in Assembly and Photoprotection of Photosystem II Revealed by Site-Directed Mutagenesis Studies

    PubMed Central

    Chu, Hsiu-An; Chiu, Yi-Fang

    2016-01-01

    Cytochrome b559 (Cyt b559) is one of the essential components of the Photosystem II reaction center (PSII). Despite recent accomplishments in understanding the structure and function of PSII, the exact physiological function of Cyt b559 remains unclear. Cyt b559 is not involved in the primary electron transfer pathway in PSII but may participate in secondary electron transfer pathways that protect PSII against photoinhibition. Site-directed mutagenesis studies combined with spectroscopic and functional analysis have been used to characterize Cyt b559 mutant strains and their mutant PSII complex in higher plants, green algae, and cyanobacteria. These integrated studies have provided important in vivo evidence for possible physiological roles of Cyt b559 in the assembly and stability of PSII, protecting PSII against photoinhibition, and modulating photosynthetic light harvesting. This mini-review presents an overview of recent important progress in site-directed mutagenesis studies of Cyt b559 and implications for revealing the physiological functions of Cyt b559 in PSII. PMID:26793230

  15. Influence of Zr{sup 4+} doping on structural and electrical properties of SrBi{sub 4}Ti{sub 4}O{sub 15} ceramic

    SciTech Connect

    Nayak, P. Panigrahi, S.; Badapanda, T.

    2015-06-24

    This article reports a systematic study of doping effects on the structural and electrical properties of layer structured strontium bismuth titanate ceramic. In this study monophasic SrBi{sub 4}Ti{sub 4-x}Zr{sub x}O{sub 15} with x=0.00, 0.05, 0.10, 0.15, 0.20, 0.25 ceramics were synthesized from the solid-state reaction route. X-ray diffraction analysis shows that the Zr-modified SBT ceramics have a pure four-layer Aurivillius phase structure. Dielectric properties revealed that the diffuseness of phase transition increases where as corresponding permittivity value decrease with increasing Zr content. Piezoelectric properties of SBTZ ceramics were improved by the modification of Zirconium ion. Moreover, the reason behind for improvement of piezoelectric properties of modified SBTZ ceramics was also discussed.

  16. Sleeping Beauty mutagenesis reveals cooperating mutations and pathways in pancreatic adenocarcinoma

    PubMed Central

    Mann, Karen M.; Ward, Jerrold M.; Yew, Christopher Chin Kuan; Kovochich, Anne; Dawson, David W.; Black, Michael A.; Brett, Benjamin T.; Sheetz, Todd E.; Dupuy, Adam J.; Chang, David K.; Biankin, Andrew V.; Waddell, Nicola; Kassahn, Karin S.; Grimmond, Sean M.; Rust, Alistair G.; Adams, David J.; Jenkins, Nancy A.; Copeland, Neal G.

    2012-01-01

    Pancreatic cancer is one of the most deadly cancers affecting the Western world. Because the disease is highly metastatic and difficult to diagnosis until late stages, the 5-y survival rate is around 5%. The identification of molecular cancer drivers is critical for furthering our understanding of the disease and development of improved diagnostic tools and therapeutics. We have conducted a mutagenic screen using Sleeping Beauty (SB) in mice to identify new candidate cancer genes in pancreatic cancer. By combining SB with an oncogenic Kras allele, we observed highly metastatic pancreatic adenocarcinomas. Using two independent statistical methods to identify loci commonly mutated by SB in these tumors, we identified 681 loci that comprise 543 candidate cancer genes (CCGs); 75 of these CCGs, including Mll3 and Ptk2, have known mutations in human pancreatic cancer. We identified point mutations in human pancreatic patient samples for another 11 CCGs, including Acvr2a and Map2k4. Importantly, 10% of the CCGs are involved in chromatin remodeling, including Arid4b, Kdm6a, and Nsd3, and all SB tumors have at least one mutated gene involved in this process; 20 CCGs, including Ctnnd1, Fbxo11, and Vgll4, are also significantly associated with poor patient survival. SB mutagenesis provides a rich resource of mutations in potential cancer drivers for cross-comparative analyses with ongoing sequencing efforts in human pancreatic adenocarcinoma. PMID:22421440

  17. Sleeping Beauty mutagenesis reveals cooperating mutations and pathways in pancreatic adenocarcinoma.

    PubMed

    Mann, Karen M; Ward, Jerrold M; Yew, Christopher Chin Kuan; Kovochich, Anne; Dawson, David W; Black, Michael A; Brett, Benjamin T; Sheetz, Todd E; Dupuy, Adam J; Chang, David K; Biankin, Andrew V; Waddell, Nicola; Kassahn, Karin S; Grimmond, Sean M; Rust, Alistair G; Adams, David J; Jenkins, Nancy A; Copeland, Neal G

    2012-04-17

    Pancreatic cancer is one of the most deadly cancers affecting the Western world. Because the disease is highly metastatic and difficult to diagnosis until late stages, the 5-y survival rate is around 5%. The identification of molecular cancer drivers is critical for furthering our understanding of the disease and development of improved diagnostic tools and therapeutics. We have conducted a mutagenic screen using Sleeping Beauty (SB) in mice to identify new candidate cancer genes in pancreatic cancer. By combining SB with an oncogenic Kras allele, we observed highly metastatic pancreatic adenocarcinomas. Using two independent statistical methods to identify loci commonly mutated by SB in these tumors, we identified 681 loci that comprise 543 candidate cancer genes (CCGs); 75 of these CCGs, including Mll3 and Ptk2, have known mutations in human pancreatic cancer. We identified point mutations in human pancreatic patient samples for another 11 CCGs, including Acvr2a and Map2k4. Importantly, 10% of the CCGs are involved in chromatin remodeling, including Arid4b, Kdm6a, and Nsd3, and all SB tumors have at least one mutated gene involved in this process; 20 CCGs, including Ctnnd1, Fbxo11, and Vgll4, are also significantly associated with poor patient survival. SB mutagenesis provides a rich resource of mutations in potential cancer drivers for cross-comparative analyses with ongoing sequencing efforts in human pancreatic adenocarcinoma. PMID:22421440

  18. Alanine Scanning Mutagenesis of Anti-TRAP (AT) Reveals Residues Involved in Binding to TRAP

    PubMed Central

    Chen, Yanling; Gollnick, Paul

    2008-01-01

    SUMMARY The trp RNA-binding attenuation protein (TRAP) regulates expression of the tryptophan biosynthetic (trp) genes in response to changes in intracellular levels of free L-tryptophan in many gram positive bacteria. When activated by binding tryptophan, TRAP binds to the mRNAs of several genes involved in tryptophan metabolism, and down-regulates transcription or translation of these genes. Anti-TRAP (AT) is an antagonist of TRAP that binds to tryptophan-activated TRAP and prevents it from binding to its RNA targets, and thereby up-regulates trp gene expression. The crystal structure shows that AT is a cone-shaped trimer (AT3) with the N-terminal residues of the three subunits assembled at the apex of the cone and that these trimers can further assemble into a dodecameric (AT12) structure. Using alanine-scanning mutagenesis we found four residues, all located on the “top” region of AT3, which are essential for binding to TRAP. Fluorescent labeling experiments further suggest that the top region of AT is in close juxtaposition to TRAP in the AT-TRAP complex. In vivo studies confirmed the importance of these residues on the top of AT in regulating TRAP mediated gene regulation. PMID:18334255

  19. Surface and finite size effects impact on the phase diagrams, polar, and dielectric properties of (Sr,Bi)Ta2O9 ferroelectric nanoparticles

    NASA Astrophysics Data System (ADS)

    Eliseev, E. A.; Semchenko, A. V.; Fomichov, Y. M.; Glinchuk, M. D.; Sidsky, V. V.; Kolos, V. V.; Pleskachevsky, Yu. M.; Silibin, M. V.; Morozovsky, N. V.; Morozovska, A. N.

    2016-05-01

    In the framework of the thermodynamic approach Landau-Ginzburg-Devonshire (LGD) combined with the equations of electrostatics, we investigated the effect of polarization surface screening on finite size effects of the phase diagrams, polar, and dielectric properties of ferroelectric nanoparticles of different shapes. We obtained and analyzed the analytical results for the dependences of the ferroelectric phase transition temperature, critical size, spontaneous polarization, and thermodynamic coercive field on the shape and size of the nanoparticles. The pronounced size effect of these characteristics on the scaling parameter, the ratio of the particle characteristic size to the length of the surface screening, was revealed. Also our modeling predicts a significant impact of the flexo-chemical effect (that is a joint action of flexoelectric effect and chemical pressure) on the temperature of phase transition, polar, and dielectric properties of nanoparticles when their chemical composition deviates from the stoichiometric one. We showed on the example of the stoichiometric nanosized SrBi2Ta2O9 particles that except the vicinity of the critical size, where the system splitting into domains has an important role, results of analytical calculation of the spontaneous polarization have a little difference from the numerical ones. We revealed a strong impact of the flexo-chemical effect on the phase transition temperature, polar, and dielectric properties of SryBi2+xTa2O9 nanoparticles when the ratio Sr/Bi deviates from the stoichiometric value of 0.5 within the range from 0.35 to 0.65. From the analysis of experimental data, we derived the parameters of the theory, namely, the coefficients of expansion of the LGD functional, the contribution of flexo-chemical effect, and the length of the surface screening.

  20. Properties of the Mechanosensitive Channel MscS Pore Revealed by Tryptophan Scanning Mutagenesis

    PubMed Central

    2015-01-01

    Bacterial mechanosensitive channels gate when the transmembrane turgor rises to levels that compromise the structural integrity of the cell wall. Gating creates a transient large diameter pore that allows hydrated solutes to pass from the cytoplasm at rates close to those of diffusion. In the closed conformation, the channel limits transmembrane solute movement, even that of protons. In the MscS crystal structure (Protein Data Bank entry 2oau), a narrow, hydrophobic opening is visible in the crystal structure, and it has been proposed that a vapor lock created by the hydrophobic seals, L105 and L109, is the barrier to water and ions. Tryptophan scanning mutagenesis has proven to be a highly valuable tool for the analysis of channel structure. Here Trp residues were introduced along the pore-forming TM3a helix and in selected other parts of the protein. Mutants were investigated for their expression, stability, and activity and as fluorescent probes of the physical properties along the length of the pore. Most Trp mutants were expressed at levels similar to that of the parent (MscS YFF) and were stable as heptamers in detergent in the presence and absence of urea. Fluorescence data suggest a long hydrophobic region with low accessibility to aqueous solvents, extending from L105/L109 to G90. Steady-state fluorescence anisotropy data are consistent with significant homo-Förster resonance energy transfer between tryptophan residues from different subunits within the narrow pore. The data provide new insights into MscS structure and gating. PMID:26126964

  1. Genome-wide mutagenesis reveals that ORF7 is a novel VZV skin-tropic factor.

    PubMed

    Zhang, Zhen; Selariu, Anca; Warden, Charles; Huang, Grace; Huang, Ying; Zaccheus, Oluleke; Cheng, Tong; Xia, Ningshao; Zhu, Hua

    2010-01-01

    The Varicella Zoster Virus (VZV) is a ubiquitous human alpha-herpesvirus that is the causative agent of chicken pox and shingles. Although an attenuated VZV vaccine (v-Oka) has been widely used in children in the United States, chicken pox outbreaks are still seen, and the shingles vaccine only reduces the risk of shingles by 50%. Therefore, VZV still remains an important public health concern. Knowledge of VZV replication and pathogenesis remains limited due to its highly cell-associated nature in cultured cells, the difficulty of generating recombinant viruses, and VZV's almost exclusive tropism for human cells and tissues. In order to circumvent these hurdles, we cloned the entire VZV (p-Oka) genome into a bacterial artificial chromosome that included a dual-reporter system (GFP and luciferase reporter genes). We used PCR-based mutagenesis and the homologous recombination system in the E. coli to individually delete each of the genome's 70 unique ORFs. The collection of viral mutants obtained was systematically examined both in MeWo cells and in cultured human fetal skin organ samples. We use our genome-wide deletion library to provide novel functional annotations to 51% of the VZV proteome. We found 44 out of 70 VZV ORFs to be essential for viral replication. Among the 26 non-essential ORF deletion mutants, eight have discernable growth defects in MeWo. Interestingly, four ORFs were found to be required for viral replication in skin organ cultures, but not in MeWo cells, suggesting their potential roles as skin tropism factors. One of the genes (ORF7) has never been described as a skin tropic factor. The global profiling of the VZV genome gives further insights into the replication and pathogenesis of this virus, which can lead to improved prevention and therapy of chicken pox and shingles. PMID:20617166

  2. Targeted mutagenesis of zebrafish antithrombin III triggers disseminated intravascular coagulation and thrombosis, revealing insight into function

    PubMed Central

    Liu, Yang; Kretz, Colin A.; Maeder, Morgan L.; Richter, Catherine E.; Tsao, Philip; Vo, Andy H.; Huarng, Michael C.; Rode, Thomas; Hu, Zhilian; Mehra, Rohit; Olson, Steven T.; Joung, J. Keith

    2014-01-01

    Pathologic blood clotting is a leading cause of morbidity and mortality in the developed world, underlying deep vein thrombosis, myocardial infarction, and stroke. Genetic predisposition to thrombosis is still poorly understood, and we hypothesize that there are many additional risk alleles and modifying factors remaining to be discovered. Mammalian models have contributed to our understanding of thrombosis, but are low throughput and costly. We have turned to the zebrafish, a tool for high-throughput genetic analysis. Using zinc finger nucleases, we show that disruption of the zebrafish antithrombin III (at3) locus results in spontaneous venous thrombosis in larvae. Although homozygous mutants survive into early adulthood, they eventually succumb to massive intracardiac thrombosis. Characterization of null fish revealed disseminated intravascular coagulation in larvae secondary to unopposed thrombin activity and fibrinogen consumption, which could be rescued by both human and zebrafish at3 complementary DNAs. Mutation of the human AT3-reactive center loop abolished the ability to rescue, but the heparin-binding site was dispensable. These results demonstrate overall conservation of AT3 function in zebrafish, but reveal developmental variances in the ability to tolerate excessive clot formation. The accessibility of early zebrafish development will provide unique methods for dissection of the underlying mechanisms of thrombosis. PMID:24782510

  3. Expression of the Scavenger Receptor Class B type I (SR-BI) family in Drosophila melanogaster.

    PubMed

    Herboso, Leire; Talamillo, Ana; Pérez, Coralia; Barrio, Rosa

    2011-01-01

    In mammals, cholesterol is transformed into steroid hormones in the adrenal gland, the ovaries or the testes. The Scavenger Receptors Class B Type I (SR-BI) are membrane proteins that belong to the CD36 family and participate in the selective uptake of high density lipoprotein cholesteryl ester in the mammalian steroidogenic tissues. Fourteen members of the CD36 family have been identified in Diptera, although their expression patterns remain uncharacterized. Using in situ hybridization we have characterized the expression patterns of the fourteen SR-BIs in Drosophila melanogaster. We analyzed three different developmental larval stages prior to and during the peak of the insect steroid hormone ecdysone, which triggers the larval to pupal transition. We focused on the steroidogenic tissues, such as the prothoracic gland, the ovaries and the testes, and extended our analysis to non-steroidogenic tissues, such as the fat body, salivary glands, the gut, the gastric caeca or the central nervous system. Our results show highly regulated expression patterns, with three genes crq, pes and Snmp being upregulated in steroidogenic tissues at the onset of pupariation when steroidogenesis is crucial. This study underlines the importance of the transport of cholesterol and steroids in the process of ecdysone synthesis. PMID:21948708

  4. Reaction Mechanism of Glutamate Carboxypeptidase II Revealed by Mutagenesis, X-ray Crystallography, and Computational Methods

    SciTech Connect

    Klusak, Vojtech; Barinka, Cyril; Plechanovova, Anna; Mlcochova, Petra; Konvalinka, Jan; Rulisek, Lubomir; Lubkowski, Jacek

    2009-05-29

    Glutamate carboxypeptidase II (GCPII, EC 3.4.17.21) is a zinc-dependent exopeptidase and an important therapeutic target for neurodegeneration and prostate cancer. The hydrolysis of N-acetyl-l-aspartyl-l-glutamate (N-Ac-Asp-Glu), the natural dipeptidic substrate of the GCPII, is intimately involved in cellular signaling within the mammalian nervous system, but the exact mechanism of this reaction has not yet been determined. To investigate peptide hydrolysis by GCPII in detail, we constructed a mutant of human GCPII [GCPII(E424A)], in which Glu424, a putative proton shuttle residue, is substituted with alanine. Kinetic analysis of GCPII(E424A) using N-Ac-Asp-Glu as substrate revealed a complete loss of catalytic activity, suggesting the direct involvement of Glu424 in peptide hydrolysis. Additionally, we determined the crystal structure of GCPII(E424A) in complex with N-Ac-Asp-Glu at 1.70 {angstrom} resolution. The presence of the intact substrate in the GCPII(E424A) binding cavity substantiates our kinetic data and allows a detailed analysis of GCPII/N-Ac-Asp-Glu interactions. The experimental data are complemented by the combined quantum mechanics/molecular mechanics calculations (QM/MM) which enabled us to characterize the transition states, including the associated reaction barriers, and provided detailed information concerning the GCPII reaction mechanism. The best estimate of the reaction barrier was calculated to be {Delta}G {approx} 22({+-}5) kcal{center_dot}mol{sup -1}, which is in a good agreement with the experimentally observed reaction rate constant (k{sub cat} {approx} 1 s{sup -1}). Combined together, our results provide a detailed and consistent picture of the reaction mechanism of this highly interesting enzyme at the atomic level.

  5. Multidrug resistance P-glycoprotein dampens SR-BI cholesteryl ester uptake from high density lipoproteins in human leukemia cells

    PubMed Central

    Spolitu, Stefano; Uda, Sabrina; Deligia, Stefania; Frau, Alessandra; Collu, Maria; Angius, Fabrizio; Batetta, Barbara

    2016-01-01

    Tumor cells are characterised by a high content of cholesterol esters (CEs), while tumor-bearing patients show low levels of high-density lipoproteins (HDLs). The origin and significance of high CE levels in cancer cell biology has not been completely clarified. Recent evidence that lymphoblastic cells selectively acquire exogenous CE from HDL via the scavenger receptor SR-BI has drawn attention to the additional membrane proteins involved in this pathway. P-glycopotein-MDR1 (P-gp) is a product of the MDR1 gene and confers resistance to antitumor drugs. Its possible role in plasma membrane cholesterol trafficking and CE metabolism has been suggested. In the present study this aspect was investigated in a lymphoblastic cell line selected for MDR1 resistance. CEM were made resistant by stepwise exposure to low (LR) and high (HR) doses of vincristine (VCR). P-gp activity (3H-vinblastine), CE content, CE and triglycerides (TG) synthesis (14C-oleate), neutral lipids and Dil-HDL uptake (fluorescence), SR-BI, ABCA1 and P-gp protein expression (western blotting) were determined. To better evaluate the relationship between CE metabolism and P-gp activity, the ACAT inhibitor Sandoz-58035 and the P-gp inhibitors progesterone, cyclosporine and verapamil were used. CE content and synthesis were similar in the parental and resistant cells. However, in the latter population, SR-BI protein expression increased, whereas CE-HDL uptake decreased. These changes correlated with the degree of VCR-resistance. As well as reverting MDR1-resistance, the inhibitors of P-gp activity induced the CE-HDL/SR-BI pathway by reactivating membrane cholesterol trafficking. Indeed, CE-HDL uptake, SRBI expression and CE content increased, whereas there was a decrease in cholesterol esterification. These results demonstrated that P-gp overexpression impairs anticancer drug uptake as well as the SR-BI mediated selective CE-HDL uptake. This suggests that these membrane proteins act in an opposite manner on

  6. Multidrug resistance P-glycoprotein dampens SR-BI cholesteryl ester uptake from high density lipoproteins in human leukemia cells.

    PubMed

    Spolitu, Stefano; Uda, Sabrina; Deligia, Stefania; Frau, Alessandra; Collu, Maria; Angius, Fabrizio; Batetta, Barbara

    2016-01-01

    Tumor cells are characterised by a high content of cholesterol esters (CEs), while tumor-bearing patients show low levels of high-density lipoproteins (HDLs). The origin and significance of high CE levels in cancer cell biology has not been completely clarified. Recent evidence that lymphoblastic cells selectively acquire exogenous CE from HDL via the scavenger receptor SR-BI has drawn attention to the additional membrane proteins involved in this pathway. P-glycopotein-MDR1 (P-gp) is a product of the MDR1 gene and confers resistance to antitumor drugs. Its possible role in plasma membrane cholesterol trafficking and CE metabolism has been suggested. In the present study this aspect was investigated in a lymphoblastic cell line selected for MDR1 resistance. CEM were made resistant by stepwise exposure to low (LR) and high (HR) doses of vincristine (VCR). P-gp activity ((3)H-vinblastine), CE content, CE and triglycerides (TG) synthesis ((14)C-oleate), neutral lipids and Dil-HDL uptake (fluorescence), SR-BI, ABCA1 and P-gp protein expression (western blotting) were determined. To better evaluate the relationship between CE metabolism and P-gp activity, the ACAT inhibitor Sandoz-58035 and the P-gp inhibitors progesterone, cyclosporine and verapamil were used. CE content and synthesis were similar in the parental and resistant cells. However, in the latter population, SR-BI protein expression increased, whereas CE-HDL uptake decreased. These changes correlated with the degree of VCR-resistance. As well as reverting MDR1-resistance, the inhibitors of P-gp activity induced the CE-HDL/SR-BI pathway by reactivating membrane cholesterol trafficking. Indeed, CE-HDL uptake, SRBI expression and CE content increased, whereas there was a decrease in cholesterol esterification. These results demonstrated that P-gp overexpression impairs anticancer drug uptake as well as the SR-BI mediated selective CE-HDL uptake. This suggests that these membrane proteins act in an opposite

  7. Mechanism of Porcine Liver Xanthine Oxidoreductase Mediated N-Oxide Reduction of Cyadox as Revealed by Docking and Mutagenesis Studies

    PubMed Central

    Hao, Haihong; Dai, Menghong; Wang, Xu; Huang, Lingli; Liu, Zhenli; Yuan, Zonghui

    2013-01-01

    Xanthine oxidoreductase (XOR) is a cytoplasmic molybdenum-containing oxidoreductase, catalyzing both endogenous purines and exogenous compounds. It is suggested that XOR in porcine hepatocytes catalyzes the N-oxide reduction of quinoxaline 1,4-di-N-oxides (QdNOs). To elucidate the molecular mechanism underlying this metabolism, the cDNA of porcine XOR was cloned and heterologously expressed in Spodoptera frugiperda insect cells. The bovine XOR, showing sequence identity of 91% to porcine XOR, was employed as template for homology modeling. By docking cyadox, a representative compound of QdNOs, into porcine XOR model, eight amino acid residues, Gly47, Asn352, Ser360, Arg427, Asp430, Asp431, Ser1227 and Lys1230, were located at distances of less than 4Å to cyadox. Site-directed mutagenesis was performed to analyze their catalytic functions. Compared with wild type porcine XOR, G47A, S360P, D431A, S1227A, and K1230A displayed altered kinetic parameters in cyadox reduction, similarly to that in xanthine oxidation, indicating these mutations influenced electron-donating process of xanthine before subsequent electron transfer to cyadox to fulfill the N-oxide reduction. Differently, R427E and D430H, both located in the 424–434 loop, exhibited a much lower Km and a decreased Vmax respectively in cyadox reduction. Arg427 may be related to the substrate binding of porcine XOR to cyadox, and Asp430 is suggested to be involved in the transfer of electron to cyadox. This study initially reveals the possible catalytic mechanism of porcine XOR in cyadox metabolism, providing with novel insights into the structure-function relationship of XOR in the reduction of exogenous di-N-oxides. PMID:24040113

  8. Transposon Mutagenesis Paired with Deep Sequencing of Caulobacter crescentus under Uranium Stress Reveals Genes Essential for Detoxification and Stress Tolerance

    PubMed Central

    Yung, Mimi C.; Park, Dan M.; Overton, K. Wesley; Blow, Matthew J.; Hoover, Cindi A.; Smit, John; Murray, Sean R.; Ricci, Dante P.; Christen, Beat; Bowman, Grant R.

    2015-01-01

    ABSTRACT The ubiquitous aquatic bacterium Caulobacter crescentus is highly resistant to uranium (U) and facilitates U biomineralization and thus holds promise as an agent of U bioremediation. To gain an understanding of how C. crescentus tolerates U, we employed transposon (Tn) mutagenesis paired with deep sequencing (Tn-seq) in a global screen for genomic elements required for U resistance. Of the 3,879 annotated genes in the C. crescentus genome, 37 were found to be specifically associated with fitness under U stress, 15 of which were subsequently tested through mutational analysis. Systematic deletion analysis revealed that mutants lacking outer membrane transporters (rsaFa and rsaFb), a stress-responsive transcription factor (cztR), or a ppGpp synthetase/hydrolase (spoT) exhibited a significantly lower survival rate under U stress. RsaFa and RsaFb, which are homologues of TolC in Escherichia coli, have previously been shown to mediate S-layer export. Transcriptional analysis revealed upregulation of rsaFa and rsaFb by 4- and 10-fold, respectively, in the presence of U. We additionally show that rsaFa mutants accumulated higher levels of U than the wild type, with no significant increase in oxidative stress levels. Our results suggest a function for RsaFa and RsaFb in U efflux and/or maintenance of membrane integrity during U stress. In addition, we present data implicating CztR and SpoT in resistance to U stress. Together, our findings reveal novel gene targets that are key to understanding the molecular mechanisms of U resistance in C. crescentus. IMPORTANCE Caulobacter crescentus is an aerobic bacterium that is highly resistant to uranium (U) and has great potential to be used in U bioremediation, but its mechanisms of U resistance are poorly understood. We conducted a Tn-seq screen to identify genes specifically required for U resistance in C. crescentus. The genes that we identified have previously remained elusive using other omics approaches and thus

  9. Reaction Mechanism of N-Acetylneuraminic Acid Lyase Revealed by a Combination of Crystallography, QM/MM Simulation, and Mutagenesis

    PubMed Central

    2014-01-01

    N-Acetylneuraminic acid lyase (NAL) is a Class I aldolase that catalyzes the reversible condensation of pyruvate with N-acetyl-d-mannosamine (ManNAc) to yield the sialic acid N-acetylneuraminic acid (Neu5Ac). Aldolases are finding increasing use as biocatalysts for the stereospecific synthesis of complex molecules. Incomplete understanding of the mechanism of catalysis in aldolases, however, can hamper development of new enzyme activities and specificities, including control over newly generated stereocenters. In the case of NAL, it is clear that the enzyme catalyzes a Bi-Uni ordered condensation reaction in which pyruvate binds first to the enzyme to form a catalytically important Schiff base. The identity of the residues required for catalysis of the condensation step and the nature of the transition state for this reaction, however, have been a matter of conjecture. In order to address, this we crystallized a Y137A variant of the E. coli NAL in the presence of Neu5Ac. The three-dimensional structure shows a full length sialic acid bound in the active site of subunits A, B, and D, while in subunit C, discontinuous electron density reveals the positions of enzyme-bound pyruvate and ManNAc. These ‘snapshot’ structures, representative of intermediates in the enzyme catalytic cycle, provided an ideal starting point for QM/MM modeling of the enzymic reaction of carbon–carbon bond formation. This revealed that Tyr137 acts as the proton donor to the aldehyde oxygen of ManNAc during the reaction, the activation barrier is dominated by carbon–carbon bond formation, and proton transfer from Tyr137 is required to obtain a stable Neu5Ac-Lys165 Schiff base complex. The results also suggested that a triad of residues, Tyr137, Ser47, and Tyr110 from a neighboring subunit, are required to correctly position Tyr137 for its function, and this was confirmed by site-directed mutagenesis. This understanding of the mechanism and geometry of the transition states along the C

  10. Optical Temperature Sensor Through Upconversion Emission from the Er3+ Doped SrBi8Ti7O27 Ferroelectrics

    NASA Astrophysics Data System (ADS)

    Zou, Hua; Wang, Xusheng; Hu, Yifeng; Zhu, Xiaoqing; Sui, Yongxing; Song, Zhitang

    2016-06-01

    Er doped SrBi8Ti7O27 (SBT) ferroelectric ceramics were prepared by a solid-state reaction technique. By Er doping, the intensive green upconversion emissions were recorded under 980 nm diode laser excitation with 20 mW. The fluorescence spectrum was investigated in the temperature range of 150-580 K. By the fluorescence intensity ratio technique, the green emission band was studied as a function of temperature with a maximum sensing sensitivity of 0.0028 at 510 K. These results indicate that the Er doped SBT ferroelectric ceramics are promising multifunctional sensing materials.

  11. Thickness-Shear Vibration Mode Characteristics of SrBi4Ti4O15-Based Ceramics

    NASA Astrophysics Data System (ADS)

    Oka, Hitoshi; Hirose, Masakazu; Tsukada, Takeo; Watanabe, Yasuo; Nomura, Takeshi

    2000-09-01

    Dielectric and piezoelectric properties have been investigated in bismuth layer-structure compounds SrBi4Ti4O15 (SBT) and BaBi4Ti4O15 (BBT)-based solid solutions. Lanthanum-substituted and manganese-added SBT and BBT form solid solutions for all levels of Ba substitution. The Curie temperature and coercive field strength decrease monotonously with the amount of Ba substitution. The mechanical quality factor, Qm, of the thickness-shear vibration mode also decreases. The value of elastic compliance increases with the amount of Ba substitution, but conversely its temperature dependency decreases.

  12. Upconversion luminescence, ferroelectrics and piezoelectrics of Er Doped SrBi{sub 4}Ti{sub 4}O{sub 15}

    SciTech Connect

    Peng Dengfeng; Zou Hua; Wang Xusheng; Yao Xi; Xu Chaonan; Lin Jian; Sun Tiantuo

    2012-12-15

    Er{sup 3+} doped SrBi{sub 4}Ti{sub 4}O{sub 15} (SBT) bismuth layered-structure ferroelectric ceramics were synthesized by the traditional solid-state method, and their upconversion photoluminescent (UC) properties were investigated as a function of Er{sup 3+} concentration and incident pump power. Green (555 nm) and red (670 nm) emission bands were obtained under 980 nm excitation at room temperature, which corresponded to the radiative transitions from {sup 4}S{sub 3/2}, and {sup 4}F{sub 9/2} to {sup 4}I{sub 15/2}, respectively. The emission color of the samples could be changed with moderating the doping concentrations. The dependence of UC intensity on pumping power indicated a two-photon emission process. Studies on dielectric properties indicated that the introduction of Er increased the ferroelectric-paraelectric phase transition temperature (Tc) of SBT, thus making this ceramic suitable for piezoelectric sensor applications at higher temperatures. Piezoelectric measurement showed that the doped SBT had a relative higher piezoelectric constant d{sub 33} compared with the non-doped ceramics. The thermal annealing behaviors of the doped sample revealed a stable piezoelectric property. The doped SBT showed bright UC emission while simultaneously having increased Tc and d{sub 33}. As a multifunctional material, Er doped SBT ferroelectric oxide showed great potential in application of sensor, future optical-electro integration and coupling devices.

  13. Ferrimagnetic and spin-glass transition in the Aurivillius compound SrBi5Ti4Cr0.5Co0.5O18

    NASA Astrophysics Data System (ADS)

    Yuan, B.; Yang, J.; Zuo, X. Z.; Song, D. P.; Tang, X. W.; Zhu, X. B.; Dai, J. M.; Song, W. H.; Sun, Y. P.

    2015-06-01

    Single-phase polycrystalline SrBi5Ti4CrO18 and SrBi5Ti4Cr0.5Co0.5O18 were synthesized by a modified Pechini method. Both samples have an orthorhombic structure with the space group B2cb. The valence state of Cr is suggested to be +3 and the Co ions exist in the form of Co2+ and Co3+ based on the results of x-ray photoelectron spectroscopy. The sample SrBi5Ti4CrO18 exhibits the paramagnetic state, whereas SrBi5Ti4Cr0.5Co0.5O18 undergoes a ferrimagnetic transition at 89 K originating from the antiferromagnetic coupling of Cr-based and Co-based sublattices. In addition, SrBi5Ti4Cr0.5Co0.5O18 shows a typical spin-glass behavior below 89 K with zν = 6.02 and τ0 = (1.75 ± 0.33) × 10-14 s as evidenced by the results of the frequency dependence of ac susceptibility and magnetic relaxation measurements. In particular, both the dielectric constant and dielectric loss of SrBi5Ti4Cr0.5Co0.5O18 exhibit the characteristics of dielectric relaxation around 89 K with the activation energy of (0.14 ± 0.02) eV, which can be ascribed to the electron hopping of Co2+-VO-Co3+ through the bridging oxygen vacancies.

  14. Electrical properties of ferroelectric-gate FETs with SrBi2Ta2O9 formed using MOCVD technique

    NASA Astrophysics Data System (ADS)

    Yan, Kang; Takahashi, Mitsue; Sakai, Shigeki

    2012-09-01

    Ferroelectric-gate field-effect transistors (FeFETs) with a Pt/SrBi2Ta2O9/Hf-Al-O/Si gate stack were fabricated using the metal-organic chemical vapor deposition (MOCVD) technique to prepare the SrBi2Ta2O9 (SBT) ferroelectric layer. A good threshold voltage ( V th) distribution was found for more than 90 n-channel FeFETs in one chip with a 170 nm SBT layer owing to the good film uniformity of the SBT layer deposited by MOCVD. The average memory window (Vw^{av}) and the standard deviations ( σ thl, σ thr) of the left- and right-side branches of the drain-gate voltage curves of the FeFETs yielded a Vw^{av}/(σ_{thl} + σ_{thr}) value of 5.45, indicating that the FeFETs can be adapted for large-scale-integration. The electric field, the energy band profile in the gate stack, and the gate leakage current were also investigated at high gate voltages. We found that the effect of Fowler-Nordheim tunneling appeared under these conditions. Because of the tunneling injection and trapping of electrons into the gate insulators, the operation voltage ranges of the FeFETs were limited by this tunneling.

  15. Hypolipidemic action of the SERM acolbifene is associated with decreased liver MTP and increased SR-BI and LDL receptors.

    PubMed

    Lemieux, Christian; Gélinas, Yves; Lalonde, Josée; Labrie, Fernand; Cianflone, Katherine; Deshaies, Yves

    2005-06-01

    This study aimed to identify the mechanisms of the hypolipidemic action of the selective estrogen receptor modulator (SERM) acolbifene (ACOL). Four weeks of treatment with ACOL reduced fasting and postprandial plasma triglycerides (TGs), an effect associated with lower VLDL-TG secretion rate (-25%), and decreased mRNA of microsomal triglyceride transfer protein (MTP; -29%). ACOL increased liver TG concentration (+100%) and amplified the feeding-induced increase in the master lipogenic regulators sterol-regulatory element binding protein-1a (SREBP-1a) and SREBP-1c. ACOL decreased total, HDL, and non-HDL cholesterol (CHOL) by 50%. SREBP-2 mRNA and HMG-CoA reductase activity were minimally affected by ACOL. However, in the fasted state, liver concentration of scavenger receptor class B type I (SR-BI) protein, but not mRNA, was 3-fold higher in ACOL-treated than in control animals and correlated with plasma HDL-CHOL levels (r = 0.80, P < 0.002). Liver LDL receptor (LDLR) protein, but not mRNA, was increased 2-fold by ACOL, independently of the nutritional status. This study demonstrates that ACOL possesses the unique ability among SERMs to reduce VLDL-TG secretion, likely by reducing MTP expression, and strongly suggests that the robust hypocholesterolemic action of ACOL is related to increased removal of CHOL from the circulation as a consequence of enhanced liver SR-BI and LDLR abundance. PMID:15741653

  16. SR-BI protects against endotoxemia in mice through its roles in glucocorticoid production and hepatic clearance

    PubMed Central

    Cai, Lei; Ji, Ailing; de Beer, Frederick C.; Tannock, Lisa R.; van der Westhuyzen, Deneys R.

    2007-01-01

    Septic shock results from an uncontrolled inflammatory response, mediated primarily by LPS. Cholesterol transport plays an important role in the host response to LPS, as LPS is neutralized by lipoproteins and adrenal cholesterol uptake is required for antiinflammatory glucocorticoid synthesis. In this study, we show that scavenger receptor B-I (SR-BI), an HDL receptor that mediates HDL cholesterol ester uptake into cells, is required for the normal antiinflammatory response to LPS-induced endotoxic shock. Despite elevated plasma HDL levels, SR-BI–null mice displayed an uncontrollable inflammatory cytokine response and a markedly higher lethality rate than control mice in response to LPS. In addition, SR-BI–null mice showed a lack of inducible glucocorticoid synthesis in response to LPS, bacterial infection, stress, or ACTH. Glucocorticoid insufficiency in SR-BI–null mice was due to primary adrenal malfunction resulting from deficient cholesterol delivery from HDL. Furthermore, corticosterone supplementation decreased the sensitivity of SR-BI–null mice to LPS. Plasma from control and SR-BI–null mice exhibited a similar ability to neutralize LPS, whereas SR-BI–null mice showed decreased plasma clearance of LPS into the liver and hepatocytes compared with normal mice. We conclude that SR-BI in mice is required for the antiinflammatory response to LPS-induced endotoxic shock, likely through its essential role in facilitating glucocorticoid production and LPS hepatic clearance. PMID:18064300

  17. Large enhancement of superconducting transition temperature of SrBi3 induced by Na substitution for Sr

    PubMed Central

    Iyo, Akira; Yanagi, Yousuke; Kinjo, Tatsuya; Nishio, Taichiro; Hase, Izumi; Yanagisawa, Takashi; Ishida, Shigeyuki; Kito, Hijiri; Takeshita, Nao; Oka, Kunihiko; Yoshida, Yoshiyuki; Eisaki, Hiroshi

    2015-01-01

    The Matthias rule, which is an empirical correlation between the superconducting transition temperature (Tc) and the average number of valence electrons per atom (n) in alloys and intermetallic compounds, has been used in the past as a guiding principle to search for new superconductors with higher Tc. The intermetallic compound SrBi3 (AuCu3 structure) exhibits a Tc of 5.6 K. An ab-initio electronic band structure calculation for SrBi3 predicted that Tc increases on decreasing the Fermi energy, i.e., on decreasing n, because of a steep increase in the density of states. In this study, we demonstrated that high-pressure (~ 3 GPa) and low-temperature ( < 350 °C) synthesis conditions enables the substitution of Na for about 40 at.% of Sr. With a consequent decrease in n, the Tc of (Sr,Na)Bi3 increases to 9.0 K. A new high-Tc peak is observed in the oscillatory dependence of Tc on n in compounds with the AuCu3 structure. We have shown that the oscillatory dependence of Tc is in good agreement with the band structure calculation. Our experiments reaffirm the importance of controlling the number of electrons in intermetallic compounds. PMID:25965162

  18. Novel structure--function information on biogenic amine transporters revealed by site-directed mutagenesis and alkylation.

    PubMed

    Reith, Maarten E A

    2013-07-01

    The study reported by Wenge and Bönisch in this issue provides critical structural information regarding extracellular loop 2 (EL2) of the human norepinephrine transporter (NET). A systematic search among all 10 cysteine and 13 histidine residues in NET led to His222 in EL2 as the target for N-ethylmaleimide: its alkylation interferes with [(3)H]nisoxetine binding, indicating the part of EL2 containing His 222 reaches back into the protein interior where it prevents access by nisoxetine to its binding site. Thus, EL2 in human NET does much more than conformationally assisting substrate translocation. The present study underscores the importance of site-directed mutagenesis approaches to elucidate structural features that cannot be deduced from crystals of homolog proteins. In the case of NET, the closest crystal structure is that of the homolog LeuT, but EL2 is difficult to align with 22 less loop residues in LeuT than in NET. The present results could only be achieved by the systematic mutagenesis study of all cysteines and all histidines in NET. PMID:23532308

  19. Highly localized strain fields due to planar defects in epitaxial SrBi2Nb2O9 thin films

    NASA Astrophysics Data System (ADS)

    Boulle, A.; Guinebretière, R.; Dauger, A.

    2005-04-01

    Thin films of (00l) oriented SrBi2Nb2O9 epitaxially grown on SrTiO3 by sol-gel spin coating have been studied by means of high-resolution x-ray diffraction reciprocal space mapping. It is shown that these materials contain highly localized heterogeneous strain fields due to imperfect stacking faults (i.e., faults that do not propagate throughout the crystallites building up the film). In the film plane, the strain fields are confined to 11 nm wide regions and characterized by a vertical displacement of 0.18c (where c is the cell parameter) showing that the stacking faults are mainly composed of one additional (or missing) perovskite layer. Prolonged thermal annealing at 700 °C strongly reduces the density of stacking faults and yields a more uniform strain distribution within the film volume without inducing significant grain growth.

  20. Effect of La-substitution on the structure, dielectric and ferroelectric properties of Nb modified SrBi{sub 8}Ti{sub 7}O{sub 27} ceramics

    SciTech Connect

    Parida, Geetanjali Bera, J.

    2015-08-15

    Graphical abstract: The ferroelectric properties of Nb modified Bi{sub 4}Ti{sub 3}O{sub 12}–SrBi{sub 4}Ti{sub 4}O{sub 15} intergrowth ceramics increases significantly when Bi is substituted by La. - Highlights: • La{sup 3+} substitution for Bi{sup 3+} in Nb doped Bi{sub 4}Ti{sub 3}O{sub 12}–SrBi{sub 4}Ti{sub 4}O{sub 15} ferroelectrics is reported. • The orthorhombic distortion of the structure decreased with the increasing La. • La acts as a grain growth inhibitor in the ceramics. • The remnant polarization of the ferroelectrics increased significantly with La substitution. - Abstract: The effect of La substitution on the electrical properties of SrLa{sub x}Bi{sub 8−x}Ti{sub 6.88}Nb{sub 0}.{sub 12}O{sub 27} intergrowth Aurivillius phase ferroelectric ceramic was investigated. La content ‘x’ was ranging from 0.0 to 1.0 in a step of 0.2. The ceramic phase was synthesized through a modified oxalate route. X-ray diffraction was used to identify the phase and to investigate the change in lattice parameter and microstrain with the substitution. La-substitution does not affect the crystal structure of the intergrowth. Microstructural investigation revealed that the grain size of the ceramic decreases with La addition. The lattice parameters and orthorhombicity of intergrowth structure were found to decrease with increasing La substitution. The temperature dependence of dielectric behavior was investigated in the temperature range 30–700 °C and the frequency of 100 kHz. The remnant polarization 2P{sub r} increased and the Curie temperature T{sub c} decreased with the increase in the La substitution.

  1. Targeted mutagenesis of intergenic regions in the Neisseria gonorrhoeae gonococcal genetic island reveals multiple regulatory mechanisms controlling type IV secretion.

    PubMed

    Ramsey, Meghan E; Bender, Tobias; Klimowicz, Amy K; Hackett, Kathleen T; Yamamoto, Ami; Jolicoeur, Adrienne; Callaghan, Melanie M; Wassarman, Karen M; van der Does, Chris; Dillard, Joseph P

    2015-09-01

    Gonococci secrete chromosomal DNA into the extracellular environment using a type IV secretion system (T4SS). The secreted DNA acts in natural transformation and initiates biofilm development. Although the DNA and its effects are detectable, structural components of the T4SS are present at very low levels, suggestive of uncharacterized regulatory control. We sought to better characterize the expression and regulation of T4SS genes and found that the four operons containing T4SS genes are transcribed at very different levels. Increasing transcription of two of the operons through targeted promoter mutagenesis did not increase DNA secretion. The stability and steady-state levels of two T4SS structural proteins were affected by a homolog of tail-specific protease. An RNA switch was also identified that regulates translation of a third T4SS operon. The switch mechanism relies on two putative stem-loop structures contained within the 5' untranslated region of the transcript, one of which occludes the ribosome binding site and start codon. Mutational analysis of these stem loops supports a model in which induction of an alternative structure relieves repression. Taken together, these results identify multiple layers of regulation, including transcriptional, translational and post-translational mechanisms controlling T4SS gene expression and DNA secretion. PMID:26076069

  2. Functional mutagenesis screens reveal the ‘cap structure’ formation in disulfide-bridge free TASK channels

    PubMed Central

    Goldstein, Matthias; Rinné, Susanne; Kiper, Aytug K.; Ramírez, David; Netter, Michael F.; Bustos, Daniel; Ortiz-Bonnin, Beatriz; González, Wendy; Decher, Niels

    2016-01-01

    Two-pore-domain potassium (K2P) channels have a large extracellular cap structure formed by two M1-P1 linkers, containing a cysteine for dimerization. However, this cysteine is not present in the TASK-1/3/5 subfamily. The functional role of the cap is poorly understood and it remained unclear whether K2P channels assemble in the domain-swapped orientation or not. Functional alanine-mutagenesis screens of TASK-1 and TRAAK were used to build an in silico model of the TASK-1 cap. According to our data the cap structure of disulfide-bridge free TASK channels is similar to that of other K2P channels and is most likely assembled in the domain-swapped orientation. As the conserved cysteine is not essential for functional expression of all K2P channels tested, we propose that hydrophobic residues at the inner leaflets of the cap domains can interact with each other and that this way of stabilizing the cap is most likely conserved among K2P channels. PMID:26794006

  3. PiggyBac Transposon-Mediated Mutagenesis in Rats Reveals a Crucial Role of Bbx in Growth and Male Fertility.

    PubMed

    Wang, Chieh-Ying; Tang, Ming-Chu; Chang, Wen-Chi; Furushima, Kenryo; Jang, Chuan-Wei; Behringer, Richard R; Chen, Chun-Ming

    2016-09-01

    Bobby sox homolog (Bbx) is an evolutionally conserved gene, but its biological function remains elusive. Here, we characterized defects of Bbx mutant rats that were created by PiggyBac-mediated insertional mutagenesis. Smaller body size and male infertility were the two major phenotypes of homozygous Bbx mutants. Bbx expression profile analysis showed that Bbx was more highly expressed in the testis and pituitary gland than in other organs. Histology and hormonal gene expression analysis of control and Bbx-null pituitary glands showed that loss of Bbx appeared to be dispensable for pituitary histogenesis and the expression of major hormones. BBX was localized in the nuclei of postmeiotic spermatids and Sertoli cells in wild-type testes, but absent in mutant testes. An increased presence of aberrant multinuclear giant cells and apoptotic cells was observed in mutant seminiferous tubules. TUNEL-positive cells costained with CREM (round spermatid marker), but not PLZF (spermatogonia marker), gammaH2Ax (meiotic spermatocyte marker), or GATA4 (Sertoli cell marker). Finally, there were drastically reduced numbers and motility of epididymal sperm from Bbx-null rats. These results suggest that loss of BBX induces apoptosis of postmeiotic spermatids and results in spermiogenesis defects and infertility. PMID:27465138

  4. Insertional mutagenesis and deep profiling reveals gene hierarchies and a Myc/p53-dependent bottleneck in lymphomagenesis.

    PubMed

    Huser, Camille A; Gilroy, Kathryn L; de Ridder, Jeroen; Kilbey, Anna; Borland, Gillian; Mackay, Nancy; Jenkins, Alma; Bell, Margaret; Herzyk, Pawel; van der Weyden, Louise; Adams, David J; Rust, Alistair G; Cameron, Ewan; Neil, James C

    2014-02-01

    Retroviral insertional mutagenesis (RIM) is a powerful tool for cancer genomics that was combined in this study with deep sequencing (RIM/DS) to facilitate a comprehensive analysis of lymphoma progression. Transgenic mice expressing two potent collaborating oncogenes in the germ line (CD2-MYC, -Runx2) develop rapid onset tumours that can be accelerated and rendered polyclonal by neonatal Moloney murine leukaemia virus (MoMLV) infection. RIM/DS analysis of 28 polyclonal lymphomas identified 771 common insertion sites (CISs) defining a 'progression network' that encompassed a remarkably large fraction of known MoMLV target genes, with further strong indications of oncogenic selection above the background of MoMLV integration preference. Progression driven by RIM was characterised as a Darwinian process of clonal competition engaging proliferation control networks downstream of cytokine and T-cell receptor signalling. Enhancer mode activation accounted for the most efficiently selected CIS target genes, including Ccr7 as the most prominent of a set of chemokine receptors driving paracrine growth stimulation and lymphoma dissemination. Another large target gene subset including candidate tumour suppressors was disrupted by intragenic insertions. A second RIM/DS screen comparing lymphomas of wild-type and parental transgenics showed that CD2-MYC tumours are virtually dependent on activation of Runx family genes in strong preference to other potent Myc collaborating genes (Gfi1, Notch1). Ikzf1 was identified as a novel collaborating gene for Runx2 and illustrated the interface between integration preference and oncogenic selection. Lymphoma target genes for MoMLV can be classified into (a) a small set of master regulators that confer self-renewal; overcoming p53 and other failsafe pathways and (b) a large group of progression genes that control autonomous proliferation in transformed cells. These findings provide insights into retroviral biology, human cancer genetics

  5. High-density lipoprotein inhibits ox-LDL-induced adipokine secretion by upregulating SR-BI expression and suppressing ER Stress pathway.

    PubMed

    Song, Guohua; Wu, Xia; Zhang, Pu; Yu, Yang; Yang, Mingfeng; Jiao, Peng; Wang, Ni; Song, Haiming; Wu, You; Zhang, Xiangjian; Liu, Huaxia; Qin, Shucun

    2016-01-01

    Endoplasmic reticulum stress (ERS) in adipocytes can modulate adipokines secretion. The aim of this study was to explore the protective effect of high-density lipoprotein (HDL) on oxidized low-density lipoprotein (ox-LDL)-induced ERS-C/EBP homologous protein (CHOP) pathway-mediated adipokine secretion. Our results showed that serum adipokines, including visfatin, resistin and TNF-α, correlated inversely with serum HDL cholesterol level in patients with abdominal obesity. In vitro, like ERS inhibitor 4-phenylbutyric acid (PBA), HDL inhibited ox-LDL- or tunicamycin (TM, an ERS inducer)-induced increase in visfatin and resistin secretion. Moreover, HDL inhibited ox-LDL-induced free cholesterol (FC) accumulation in whole cell lysate and in the endoplasmic reticulum. Additionally, like PBA, HDL inhibited ox-LDL- or TM-induced activation of ERS response as assessed by the decreased phosphorylation of protein kinase-like ER kinase and eukaryotic translation initiation factor 2α and reduced nuclear translocation of activating transcription factor 6 as well as the downregulation of Bip and CHOP. Furthermore, HDL increased scavenger receptor class B type I (SR-BI) expression and SR-BI siRNA treatment abolished the inhibitory effects of HDL on ox-LDL-induced FC accumulation and CHOP upregulation. These data indicate that HDL may suppress ox-LDL-induced FC accumulation in adipocytes through upregulation of SR-BI, subsequently preventing ox-LDL-induced ER stress-CHOP pathway-mediated adipocyte inflammation. PMID:27468698

  6. High-density lipoprotein inhibits ox-LDL-induced adipokine secretion by upregulating SR-BI expression and suppressing ER Stress pathway

    PubMed Central

    Song, Guohua; Wu, Xia; Zhang, Pu; Yu, Yang; Yang, Mingfeng; Jiao, Peng; Wang, Ni; Song, Haiming; Wu, You; Zhang, Xiangjian; Liu, Huaxia; Qin, Shucun

    2016-01-01

    Endoplasmic reticulum stress (ERS) in adipocytes can modulate adipokines secretion. The aim of this study was to explore the protective effect of high-density lipoprotein (HDL) on oxidized low-density lipoprotein (ox-LDL)-induced ERS-C/EBP homologous protein (CHOP) pathway-mediated adipokine secretion. Our results showed that serum adipokines, including visfatin, resistin and TNF-α, correlated inversely with serum HDL cholesterol level in patients with abdominal obesity. In vitro, like ERS inhibitor 4-phenylbutyric acid (PBA), HDL inhibited ox-LDL- or tunicamycin (TM, an ERS inducer)-induced increase in visfatin and resistin secretion. Moreover, HDL inhibited ox-LDL-induced free cholesterol (FC) accumulation in whole cell lysate and in the endoplasmic reticulum. Additionally, like PBA, HDL inhibited ox-LDL- or TM-induced activation of ERS response as assessed by the decreased phosphorylation of protein kinase-like ER kinase and eukaryotic translation initiation factor 2α and reduced nuclear translocation of activating transcription factor 6 as well as the downregulation of Bip and CHOP. Furthermore, HDL increased scavenger receptor class B type I (SR-BI) expression and SR-BI siRNA treatment abolished the inhibitory effects of HDL on ox-LDL-induced FC accumulation and CHOP upregulation. These data indicate that HDL may suppress ox-LDL-induced FC accumulation in adipocytes through upregulation of SR-BI, subsequently preventing ox-LDL-induced ER stress-CHOP pathway-mediated adipocyte inflammation. PMID:27468698

  7. Mutagenesis of GATA motifs controlling the endoderm regulator elt-2 reveals distinct dominant and secondary cis-regulatory elements.

    PubMed

    Du, Lawrence; Tracy, Sharon; Rifkin, Scott A

    2016-04-01

    Cis-regulatory elements (CREs) are crucial links in developmental gene regulatory networks, but in many cases, it can be difficult to discern whether similar CREs are functionally equivalent. We found that despite similar conservation and binding capability to upstream activators, different GATA cis-regulatory motifs within the promoter of the C. elegans endoderm regulator elt-2 play distinctive roles in activating and modulating gene expression throughout development. We fused wild-type and mutant versions of the elt-2 promoter to a gfp reporter and inserted these constructs as single copies into the C. elegans genome. We then counted early embryonic gfp transcripts using single-molecule RNA FISH (smFISH) and quantified gut GFP fluorescence. We determined that a single primary dominant GATA motif located 527bp upstream of the elt-2 start codon was necessary for both embryonic activation and later maintenance of transcription, while nearby secondary GATA motifs played largely subtle roles in modulating postembryonic levels of elt-2. Mutation of the primary activating site increased low-level spatiotemporally ectopic stochastic transcription, indicating that this site acts repressively in non-endoderm cells. Our results reveal that CREs with similar GATA factor binding affinities in close proximity can play very divergent context-dependent roles in regulating the expression of a developmentally critical gene in vivo. PMID:26896592

  8. Targeted mutagenesis of the human papillomavirus type 16 E2 transactivation domain reveals separable transcriptional activation and DNA replication functions.

    PubMed

    Sakai, H; Yasugi, T; Benson, J D; Dowhanick, J J; Howley, P M

    1996-03-01

    The E2 gene products of papillomavirus play key roles in viral replication, both as regulators of viral transcription and as auxiliary factors that act with E1 in viral DNA replication. We have carried out a detailed structure-function analysis of conserved amino acids within the N-terminal domain of the human papillomavirus type 16 (HPV16) E2 protein. These mutants were tested for their transcriptional activation activities as well as transient DNA replication and E1 binding activities. Analysis of the stably expressed mutants revealed that the transcriptional activation and replication activities of HPV16 E2 could be dissociated. The 173A mutant was defective for the transcriptional activation function but retained wild-type DNA replication activity, whereas the E39A mutant wild-type transcriptional activation function but was defective in transient DNA replication assays. The E39A mutant was also defective for HPV16 E1 binding in vitro, suggesting that the ability of E2 protein to form a complex with E1 appears to be essential for its function as an auxiliary replication factor. PMID:8627680

  9. Site-Directed Mutagenesis of HgcA and HgcB Reveals Amino Acid Residues Important for Mercury Methylation

    PubMed Central

    Smith, Steven D.; Bridou, Romain; Johs, Alexander; Parks, Jerry M.; Elias, Dwayne A.; Hurt, Richard A.; Brown, Steven D.; Podar, Mircea

    2015-01-01

    Methylmercury is a potent neurotoxin that is produced by anaerobic microorganisms from inorganic mercury by a recently discovered pathway. A two-gene cluster, consisting of hgcA and hgcB, encodes two of the proteins essential for this activity. hgcA encodes a corrinoid protein with a strictly conserved cysteine proposed to be the ligand for cobalt in the corrinoid cofactor, whereas hgcB encodes a ferredoxin-like protein thought to be an electron donor to HgcA. Deletion of either gene eliminates mercury methylation by the methylator Desulfovibrio desulfuricans ND132. Here, site-directed mutants of HgcA and HgcB were constructed to determine amino acid residues essential for mercury methylation. Mutations of the strictly conserved residue Cys93 in HgcA, the proposed ligand for the corrinoid cobalt, to Ala or Thr completely abolished the methylation capacity, but a His substitution produced measurable methylmercury. Mutations of conserved amino acids near Cys93 had various impacts on the methylation capacity but showed that the structure of the putative “cap helix” region harboring Cys93 is crucial for methylation function. In the ferredoxin-like protein HgcB, only one of two conserved cysteines found at the C terminus was necessary for methylation, but either cysteine sufficed. An additional, strictly conserved cysteine, Cys73, was also determined to be essential for methylation. This study supports the previously predicted importance of Cys93 in HgcA for methylation of mercury and reveals additional residues in HgcA and HgcB that facilitate the production of this neurotoxin. PMID:25724962

  10. Site-directed Mutagenesis Reveals Regions Implicated in the Stability and Fiber Formation of Human λ3r Light Chains*

    PubMed Central

    Villalba, Miryam I.; Canul-Tec, Juan C.; Luna-Martínez, Oscar D.; Sánchez-Alcalá, Rosalba; Olamendi-Portugal, Timoteo; Rudiño-Piñera, Enrique; Rojas, Sonia; Sánchez-López, Rosana; Fernández-Velasco, Daniel A.; Becerril, Baltazar

    2015-01-01

    Light chain amyloidosis (AL) is a disease that affects vital organs by the fibrillar aggregation of monoclonal light chains. λ3r germ line is significantly implicated in this disease. In this work, we contrasted the thermodynamic stability and aggregation propensity of 3mJL2 (nonamyloidogenic) and 3rJL2 (amyloidogenic) λ3 germ lines. Because of an inherent limitation (extremely low expression), Cys at position 34 of the 3r germ line was replaced by Tyr reaching a good expression yield. A second substitution (W91A) was introduced in 3r to obtain a better template to incorporate additional mutations. Although the single mutant (C34Y) was not fibrillogenic, the second mutation located at CDR3 (W91A) induced fibrillogenesis. We propose, for the first time, that CDR3 (position 91) affects the stability and fiber formation of human λ3r light chains. Using the double mutant (3rJL2/YA) as template, other variants were constructed to evaluate the importance of those substitutions into the stability and aggregation propensity of λ3 light chains. A change in position 7 (P7D) boosted 3rJL2/YA fibrillogenic properties. Modification of position 48 (I48M) partially reverted 3rJL2/YA fibril aggregation. Finally, changes at positions 8 (P8S) or 40 (P40S) completely reverted fibril formation. These results confirm the influential roles of N-terminal region (positions 7 and 8) and the loop 40–60 (positions 40 and 48) on AL. X-ray crystallography revealed that the three-dimensional topology of the single and double λ3r mutants was not significantly altered. This mutagenic approach helped to identify key regions implicated in λ3 AL. PMID:25505244

  11. Site-Directed Mutagenesis of HgcA and HgcB Reveals Amino Acid Residues Important for Mercury Methylation

    SciTech Connect

    Smith, Steven D.; Bridou, Romain; Johs, Alexander; Parks, Jerry M.; Elias, Dwayne A.; Hurt, Richard A.; Brown, Steven D.; Podar, Mircea; Wall, Judy D.

    2015-02-27

    Methylmercury is a potent neurotoxin that is produced by anaerobic microorganisms from inorganic mercury by a recently discovered pathway. A two-gene cluster, consisting of hgcA and hgcB, encodes two of the proteins essential for this activity. hgcA encodes a corrinoid protein with a strictly conserved cysteine proposed to be the ligand for cobalt in the corrinoid cofactor, whereas hgcB encodes a ferredoxin-like protein thought to be an electron donor to HgcA. Deletion of either gene eliminates mercury methylation by the methylator Desulfovibrio desulfuricans ND132. Here, site-directed mutants of HgcA and HgcB were constructed to determine amino acid residues essential for mercury methylation. Mutations of the strictly conserved residue Cys93 in HgcA, the proposed ligand for the corrinoid cobalt, to Ala or Thr completely abolished the methylation capacity, but a His substitution produced measurable methylmercury. Mutations of conserved amino acids near Cys93 had various impacts on the methylation capacity but showed that the structure of the putative “cap helix” region harboring Cys93 is crucial for methylation function. In the ferredoxin-like protein HgcB, only one of two conserved cysteines found at the C terminus was necessary for methylation, but either cysteine sufficed. An additional, strictly conserved cysteine, Cys73, was also determined to be essential for methylation. Ultimately, this study supports the previously predicted importance of Cys93 in HgcA for methylation of mercury and reveals additional residues in HgcA and HgcB that facilitate the production of this neurotoxin.

  12. Site-Directed Mutagenesis of HgcA and HgcB Reveals Amino Acid Residues Important for Mercury Methylation

    DOE PAGESBeta

    Smith, Steven D.; Bridou, Romain; Johs, Alexander; Parks, Jerry M.; Elias, Dwayne A.; Hurt, Richard A.; Brown, Steven D.; Podar, Mircea; Wall, Judy D.

    2015-02-27

    Methylmercury is a potent neurotoxin that is produced by anaerobic microorganisms from inorganic mercury by a recently discovered pathway. A two-gene cluster, consisting of hgcA and hgcB, encodes two of the proteins essential for this activity. hgcA encodes a corrinoid protein with a strictly conserved cysteine proposed to be the ligand for cobalt in the corrinoid cofactor, whereas hgcB encodes a ferredoxin-like protein thought to be an electron donor to HgcA. Deletion of either gene eliminates mercury methylation by the methylator Desulfovibrio desulfuricans ND132. Here, site-directed mutants of HgcA and HgcB were constructed to determine amino acid residues essential formore » mercury methylation. Mutations of the strictly conserved residue Cys93 in HgcA, the proposed ligand for the corrinoid cobalt, to Ala or Thr completely abolished the methylation capacity, but a His substitution produced measurable methylmercury. Mutations of conserved amino acids near Cys93 had various impacts on the methylation capacity but showed that the structure of the putative “cap helix” region harboring Cys93 is crucial for methylation function. In the ferredoxin-like protein HgcB, only one of two conserved cysteines found at the C terminus was necessary for methylation, but either cysteine sufficed. An additional, strictly conserved cysteine, Cys73, was also determined to be essential for methylation. Ultimately, this study supports the previously predicted importance of Cys93 in HgcA for methylation of mercury and reveals additional residues in HgcA and HgcB that facilitate the production of this neurotoxin.« less

  13. Integration of SrBi2Ta2O9 thin films for high density ferroelectric random access memory

    NASA Astrophysics Data System (ADS)

    Wouters, D. J.; Maes, D.; Goux, L.; Lisoni, J. G.; Paraschiv, V.; Johnson, J. A.; Schwitters, M.; Everaert, J.-L.; Boullart, W.; Schaekers, M.; Willegems, M.; Vander Meeren, H.; Haspeslagh, L.; Artoni, C.; Caputa, C.; Casella, P.; Corallo, G.; Russo, G.; Zambrano, R.; Monchoix, H.; Vecchio, G.; Van Autryve, L.

    2006-09-01

    Ferroelectric random access memory (FeRAM) is an attractive candidate technology for embedded nonvolatile memory, especially in applications where low power and high program speed are important. Market introduction of high-density FeRAM is, however, lagging behind standard complementary metal-oxide semiconductor (CMOS) because of the difficult integration technology. This paper discusses the major integration issues for high-density FeRAM, based on SrBi2Ta2O9 (strontium bismuth tantalate or SBT), in relation to the fabrication of our stacked cell structure. We have worked in the previous years on the development of SBT-FeRAM integration technology, based on a so-called pseudo-three-dimensional (3D) cell, with a capacitor that can be scaled from quasi two-dimensional towards a true three-dimensional capacitor where the sidewalls will importantly contribute to the signal. In the first phase of our integration development, we integrated our FeRAM cell in a 0.35μm CMOS technology. In a second phase, then, possibility of scaling of our cell is demonstrated in 0.18μm technology. The excellent electrical and reliability properties of the small integrated ferroelectric capacitors prove the feasibility of the technology, while the verification of the potential 3D effect confirms the basic scaling potential of our concept beyond that of the single-mask capacitor. The paper outlines the different material and technological challenges, and working solutions are demonstrated. While some issues are specific to our own cell, many are applicable to different stacked FeRAM cell concepts, or will become more general concerns when more developments are moving into 3D structures.

  14. Structural, magnetic, and dielectric studies of the Aurivillius compounds SrBi{sub 5}Ti{sub 4}MnO{sub 18} and SrBi{sub 5}Ti{sub 4}Mn{sub 0.5}Co{sub 0.5}O{sub 18}

    SciTech Connect

    Yuan, B.; Yang, J. Zuo, X. Z.; Tang, X. W.; Zhu, X. B.; Dai, J. M.; Song, W. H.; Song, D. P.; Sun, Y. P.

    2015-01-14

    We have successfully synthesized the Aurivillius compounds SrBi{sub 5}Ti{sub 4}MnO{sub 18} and SrBi{sub 5}Ti{sub 4}Mn{sub 0.5}Co{sub 0.5}O{sub 18} using a modified Pechini method. Both samples have an orthorhombic structure with the space group B2cb. The valence state of Mn is suggested to be +3 and the doped Co ions exist in the form of Co{sup 2+} and Co{sup 3+} based on the results of x-ray photoelectron spectroscopy. The sample SrBi{sub 5}Ti{sub 4}MnO{sub 18} exhibits a dominant paramagnetic state with the existence of superparamagnetic state as evidenced by the electron paramagnetic resonance results, whereas SrBi{sub 5}Ti{sub 4}Mn{sub 0.5}Co{sub 0.5}O{sub 18} undergoes a ferrimagnetic transition at 161 K originating from the antiferromagnetic coupling of Co-based and Mn-based sublattices, and a ferromagnetic transition at 45 K arising from the Mn{sup 3+}-O-Co{sup 3+} (low spin) interaction. The sample SrBi{sub 5}Ti{sub 4}Mn{sub 0.5}Co{sub 0.5}O{sub 18} exhibits two dielectric anomalies. One corresponds to a relaxor-like dielectric relaxation which follows the Vogel-Fulcher function and the other dielectric relaxation obeys the Arrhenius law arising from the collective motion of oxygen vacancies. In addition, the sample SrBi{sub 5}Ti{sub 4}Mn{sub 0.5}Co{sub 0.5}O{sub 18} exhibits a magnetodielectric effect caused by the Maxwell-Wagner effect because of the conductivity of the sample. This is demonstrated by the fact that the activation energy in dielectric loss process is close to that for dc conductivity and the magnetodielectric effect is sensitive to the measured frequency.

  15. Oxygen-vacancy-related dielectric relaxation in SrBi2Ta1.8V0.2O9 ferroelectrics

    NASA Astrophysics Data System (ADS)

    Wu, Yun; Forbess, Mike J.; Seraji, Seana; Limmer, Steven J.; Chou, Tammy P.; Cao, Guozhong

    2001-05-01

    The strontium bismuth tantalate vanadate, SrBi2Ta1.8V0.2O9, (SBTV) layered perovskite ferroelectrics were made by solid state powder sintering. It was found that the SBTV ferroelectrics had the same crystal structure as that of strontium bismuth tantalate, SrBi2Ta2O9 (SBT), but an increased paraferroelectric transition temperature at ˜360 °C as compared to 305 °C for SBT. In addition, SBTV ferroelectrics showed a frequency dispersion at low frequencies and broadened dielectric peaks at the paraferroelectric transition temperature that shifted to a higher temperature with a reduced frequency. However, after a postsintering annealing at 850 °C in air for 60 h, SBTV ferroelectrics showed reduced dielectric constants and tangent loss, particularly at high temperatures. In addition, no frequency dependence of paraferroelectric transition was found in the annealed SBTV ferroelectrics. Furthermore, there was a significant reduction in dc conductivity with annealing. The prior results implied that the dielectric relaxation in the as-sintered SBTV ferroelectrics was most likely due to the oxygen-vacancy-related dielectric relaxation instead of relaxor ferroelectric behavior.

  16. Crystal structure and dielectric properties of ordered perovskites Ba 2BiSbO 6 and BaSrBiSbO 6

    NASA Astrophysics Data System (ADS)

    Mangalam, R. V. K.; Suard, E.; Sundaresan, A.

    2009-01-01

    Neutron powder diffraction studies showed that the ordered perovskites Ba 2BiSbO 6 (BBS) and BaSrBiSbO 6 (BSBS) crystallize in a rhombohedral structure with the space group R3bar. The room-temperature lattice parameters are a=6.0351(2) Å; α=60.202(1)° and a=5.9809(2) Å; α=60.045(2)°, respectively. BBS exhibits a dielectric anomaly near room temperature which may be related to structural transition from the R3bar to low-temperature monoclinic I2/m symmetry. BSBS shows a dielectric anomaly near 723 K which coincides with a phase transition from the rhombohedral to cubic (Fm3barm) structure. In contrast to BBS, BSBS does not undergo structural transition below room temperature.

  17. Photoluminescence, enhanced ferroelectric, and dielectric properties of Pr{sup 3+}-doped SrBi{sub 2}Nb{sub 2}O{sub 9} multifunctional ceramics

    SciTech Connect

    Zou, Hua; Yu, Yao; Li, Jun; Cao, Qiufeng; Wang, Xusheng; Hou, Junwei

    2015-09-15

    Pr{sup 3+}-doped SrBi{sub 2}Nb{sub 2}O{sub 9} (SBN) multifunctional ceramics were synthesized by the conventional solid state method. The photoluminescence (PL) excitation and emission spectra, enhanced ferroelectric and dielectric properties were investigated. The X-ray diffraction (XRD) and FESEM analyses indicated that the samples were simple phase and uniform flake-structure. Under the 250–350 nm ultraviolet (UV) excitations, the red emission was obtained and the optimal emission intensity was investigated when Pr doping level was 0.005 mol. With the increasing of the Pr{sup 3+} ion contents, the ferroelectric properties were enhanced obviously. As a new multifunctional material, the Pr{sup 3+}-doped SBN ceramics could be used for a wide range of application, such as integrated electro-optical devices.

  18. Impact of total ionizing dose irradiation on Pt/SrBi{sub 2}Ta{sub 2}O{sub 9}/HfTaO/Si memory capacitors

    SciTech Connect

    Yan, S. A.; Tang, M. H. E-mail: lizheng@xtu.edu.cn; Xiao, Y. G.; Zhang, W. L.; Zhao, W.; Guo, H. X.; Xiong, Y.; Li, Z. E-mail: lizheng@xtu.edu.cn; Ding, H.; Chen, J. W.; Zhou, Y. C.

    2015-01-05

    In this work, metal-ferroelectric-insulator-semiconductor (MFIS) structure capacitors with SrBi{sub 2}Ta{sub 2}O{sub 9} (300 nm) as ferroelectric thin film and HfTaO (6 nm, 8 nm, 10 nm, and 12 nm) as insulating buffer layer were proposed and investigated. The prepared capacitors were fabricated and characterized before radiation and then subjected to {sup 60}Co gamma irradiation in steps of two dose levels. Significant irradiation-induced degradation of the electrical characteristics was observed. The radiation experimental results indicated that stability and reliability of as-fabricated MFIS capacitors for nonvolatile memory applications could become uncontrollable under strong irradiation dose and/or long irradiation time.

  19. Ferrimagnetic and spin-glass transition in the Aurivillius compound SrBi{sub 5}Ti{sub 4}Cr{sub 0.5}Co{sub 0.5}O{sub 18}

    SciTech Connect

    Yuan, B.; Yang, J. Zuo, X. Z.; Tang, X. W.; Zhu, X. B.; Dai, J. M.; Song, W. H.; Song, D. P.; Sun, Y. P.

    2015-06-21

    Single-phase polycrystalline SrBi{sub 5}Ti{sub 4}CrO{sub 18} and SrBi{sub 5}Ti{sub 4}Cr{sub 0.5}Co{sub 0.5}O{sub 18} were synthesized by a modified Pechini method. Both samples have an orthorhombic structure with the space group B2cb. The valence state of Cr is suggested to be +3 and the Co ions exist in the form of Co{sup 2+} and Co{sup 3+} based on the results of x-ray photoelectron spectroscopy. The sample SrBi{sub 5}Ti{sub 4}CrO{sub 18} exhibits the paramagnetic state, whereas SrBi{sub 5}Ti{sub 4}Cr{sub 0.5}Co{sub 0.5}O{sub 18} undergoes a ferrimagnetic transition at 89 K originating from the antiferromagnetic coupling of Cr-based and Co-based sublattices. In addition, SrBi{sub 5}Ti{sub 4}Cr{sub 0.5}Co{sub 0.5}O{sub 18} shows a typical spin-glass behavior below 89 K with zν = 6.02 and τ{sub 0} = (1.75 ± 0.33) × 10{sup −14} s as evidenced by the results of the frequency dependence of ac susceptibility and magnetic relaxation measurements. In particular, both the dielectric constant and dielectric loss of SrBi{sub 5}Ti{sub 4}Cr{sub 0.5}Co{sub 0.5}O{sub 18} exhibit the characteristics of dielectric relaxation around 89 K with the activation energy of (0.14 ± 0.02) eV, which can be ascribed to the electron hopping of Co{sup 2+}-V{sub O}-Co{sup 3+} through the bridging oxygen vacancies.

  20. A systematic survey of conserved histidines in the core subunits of Photosystem I by site-directed mutagenesis reveals the likely axial ligands of P700.

    PubMed

    Redding, K; MacMillan, F; Leibl, W; Brettel, K; Hanley, J; Rutherford, A W; Breton, J; Rochaix, J D

    1998-01-01

    The Photosystem I complex catalyses the transfer of an electron from lumenal plastocyanin to stromal ferredoxin, using the energy of an absorbed photon. The initial photochemical event is the transfer of an electron from the excited state of P700, a pair of chlorophylls, to a monomer chlorophyll serving as the primary electron acceptor. We have performed a systematic survey of conserved histidines in the last six transmembrane segments of the related polytopic membrane proteins PsaA and PsaB in the green alga Chlamydomonas reinhardtii. These histidines, which are present in analogous positions in both proteins, were changed to glutamine or leucine by site-directed mutagenesis. Double mutants in which both histidines had been changed to glutamine were screened for changes in the characteristics of P700 using electron paramagnetic resonance, Fourier transform infrared and visible spectroscopy. Only mutations in the histidines of helix 10 (PsaA-His676 and PsaB-His656) resulted in changes in spectroscopic properties of P700, leading us to conclude that these histidines are most likely the axial ligands to the P700 chlorophylls. PMID:9427740

  1. Comprehensive mutagenesis of the fimS promoter regulatory switch reveals novel regulation of type 1 pili in uropathogenic Escherichia coli

    PubMed Central

    Zhang, Huibin; Susanto, Teodorus T.; Wan, Yue

    2016-01-01

    Type 1 pili (T1P) are major virulence factors for uropathogenic Escherichia coli (UPEC), which cause both acute and recurrent urinary tract infections. T1P expression therefore is of direct relevance for disease. T1P are phase variable (both piliated and nonpiliated bacteria exist in a clonal population) and are controlled by an invertible DNA switch (fimS), which contains the promoter for the fim operon encoding T1P. Inversion of fimS is stochastic but may be biased by environmental conditions and other signals that ultimately converge at fimS itself. Previous studies of fimS sequences important for T1P phase variation have focused on laboratory-adapted E. coli strains and have been limited in the number of mutations or by alteration of the fimS genomic context. We surmounted these limitations by using saturating genomic mutagenesis of fimS coupled with accurate sequencing to detect both mutations and phase status simultaneously. In addition to the sequences known to be important for biasing fimS inversion, our method also identifies a previously unknown pair of 5′ UTR inverted repeats that act by altering the relative fimA levels to control phase variation. Thus we have uncovered an additional layer of T1P regulation potentially impacting virulence and the coordinate expression of multiple pilus systems. PMID:27035967

  2. The influence of microscopic parameters on the ionic conductivity of SrBi2(Nb1-xVx)2O9-δ(0≤x≤0.3) ceramics

    NASA Astrophysics Data System (ADS)

    Harihara Venkataraman, B.; Varma, K. B. R.

    2005-10-01

    Layered SrBi2(Nb1-xVx)2O9-δ (SBVN) ceramics with x lying in the range 0 0.3 (30 mol%) were fabricated by the conventional sintering technique. The microstructural studies confirmed the truncating effect of V2O5 on the abnormal platy growth of SBN grains. The electrical conductivity studies were centred in the 573 823 K as the Curie temperature lies in this range. The concentration of mobile charge carriers (n), the diffusion constant (D0) and the mean free path (a) were calculated by using Rice and Roth formalism. The conductivity parameters such as ion-hopping rate (ωp) and the charge carrier concentration (K‧) term have been calculated using Almond and West formalism. The aforementioned microscopic parameters were found to be V2O5 content dependent on SrBi2(Nb1-xVx)2O9-δ ceramics.

  3. N-Ethyl-N-Nitrosourea (ENU) Mutagenesis Reveals an Intronic Residue Critical for Caenorhabditis elegans 3' Splice Site Function in Vivo.

    PubMed

    Itani, Omar A; Flibotte, Stephane; Dumas, Kathleen J; Guo, Chunfang; Blumenthal, Thomas; Hu, Patrick J

    2016-01-01

    Metazoan introns contain a polypyrimidine tract immediately upstream of the AG dinucleotide that defines the 3' splice site. In the nematode Caenorhabditis elegans, 3' splice sites are characterized by a highly conserved UUUUCAG/R octamer motif. While the conservation of pyrimidines in this motif is strongly suggestive of their importance in pre-mRNA splicing, in vivo evidence in support of this is lacking. In an N-ethyl-N-nitrosourea (ENU) mutagenesis screen in Caenorhabditis elegans, we have isolated a strain containing a point mutation in the octamer motif of a 3' splice site in the daf-12 gene. This mutation, a single base T-to-G transversion at the -5 position relative to the splice site, causes a strong daf-12 loss-of-function phenotype by abrogating splicing. The resulting transcript is predicted to encode a truncated DAF-12 protein generated by translation into the retained intron, which contains an in-frame stop codon. Other than the perfectly conserved AG dinucleotide at the site of splicing, G at the -5 position of the octamer motif is the most uncommon base in C. elegans 3' splice sites, occurring at closely paired sites where the better match to the splicing consensus is a few bases downstream. Our results highlight both the biological importance of the highly conserved -5 uridine residue in the C. elegans 3' splice site octamer motif as well as the utility of using ENU as a mutagen to study the function of polypyrimidine tracts and other AU- or AT-rich motifs in vivo. PMID:27172199

  4. N-Ethyl-N-Nitrosourea (ENU) Mutagenesis Reveals an Intronic Residue Critical for Caenorhabditis elegans 3′ Splice Site Function in Vivo

    PubMed Central

    Itani, Omar A.; Flibotte, Stephane; Dumas, Kathleen J.; Guo, Chunfang; Blumenthal, Thomas; Hu, Patrick J.

    2016-01-01

    Metazoan introns contain a polypyrimidine tract immediately upstream of the AG dinucleotide that defines the 3′ splice site. In the nematode Caenorhabditis elegans, 3′ splice sites are characterized by a highly conserved UUUUCAG/R octamer motif. While the conservation of pyrimidines in this motif is strongly suggestive of their importance in pre-mRNA splicing, in vivo evidence in support of this is lacking. In an N-ethyl-N-nitrosourea (ENU) mutagenesis screen in Caenorhabditis elegans, we have isolated a strain containing a point mutation in the octamer motif of a 3′ splice site in the daf-12 gene. This mutation, a single base T-to-G transversion at the -5 position relative to the splice site, causes a strong daf-12 loss-of-function phenotype by abrogating splicing. The resulting transcript is predicted to encode a truncated DAF-12 protein generated by translation into the retained intron, which contains an in-frame stop codon. Other than the perfectly conserved AG dinucleotide at the site of splicing, G at the –5 position of the octamer motif is the most uncommon base in C. elegans 3′ splice sites, occurring at closely paired sites where the better match to the splicing consensus is a few bases downstream. Our results highlight both the biological importance of the highly conserved –5 uridine residue in the C. elegans 3′ splice site octamer motif as well as the utility of using ENU as a mutagen to study the function of polypyrimidine tracts and other AU- or AT-rich motifs in vivo. PMID:27172199

  5. Mutagenesis Reveals the Complex Relationships between ATPase Rate and the Chaperone Activities of Escherichia coli Heat Shock Protein 70 (Hsp70/DnaK)*

    PubMed Central

    Chang, Lyra; Thompson, Andrea D.; Ung, Peter; Carlson, Heather A.; Gestwicki, Jason E.

    2010-01-01

    The Escherichia coli 70-kDa heat shock protein, DnaK, is a molecular chaperone that engages in a variety of cellular activities, including the folding of proteins. During this process, DnaK binds its substrates in coordination with a catalytic ATPase cycle. Both the ATPase and protein folding activities of DnaK are stimulated by its co-chaperones, DnaJ and GrpE. However, it is not yet clear how changes in the stimulated ATPase rate of DnaK impact the folding process. In this study, we performed mutagenesis throughout the nucleotide-binding domain of DnaK to generate a collection of mutants in which the stimulated ATPase rates varied from 0.7 to 13.6 pmol/μg/min−1. We found that this range was largely established by differences in the ability of the mutants to be stimulated by one or both of the co-chaperones. Next, we explored how changes in ATPase rate might impact refolding of denatured luciferase in vitro and found that the two activities were poorly correlated. Unexpectedly, we found several mutants that refold luciferase normally in the absence of significant ATP turnover, presumably by increasing the flexibility of DnaK. Finally, we tested whether DnaK mutants could complement growth of ΔdnaK E. coli cells under heat shock and found that the ability to refold luciferase was more predictive of in vivo activity than ATPase rate. This study provides insights into how flexibility and co-chaperone interactions affect DnaK-mediated ATP turnover and protein folding. PMID:20439464

  6. Reduction of the hydrogen degradation in SrBi2(Ta1-xNbx)2O9 by TiN barrier metal

    NASA Astrophysics Data System (ADS)

    Furuya, Akira; Cuchiaro, J. D.

    2000-11-01

    The use of ferroelectric SrBi2(Ta1-xNbx)2O9 (SBTN) as a mainstream form of nonvolatile memory requires that the degradation of its electrical qualities that is caused by annealing in a hydrogen atmosphere be reduced. Titanium nitride (TiN) is a candidate for use as a barrier-metal layer against hydrogen diffusion. The relationship between the degradation in the qualities of SBTN and the quality of the TiN barrier metal has been investigated. TiN when sputtered onto SBTN capacitors creates a good barrier under all sputtering conditions, and maintains the electrical characteristics of the SBTN through annealing in an atmosphere of H2. Higher density TiN films provide more effective protection. The characteristics of the degraded capacitor were investigated in terms of its current-voltage characteristic. Remanent polarization can be recovered from, at least partially, by applying a series of bipolar pulses. This rejuvenation of the electrical qualities indicates that degradation arises from a combination of electrical and structural faults.

  7. Distinct functions of the laminin β LN domain and collagen IV during cardiac extracellular matrix formation and stabilization of alary muscle attachments revealed by EMS mutagenesis in Drosophila

    PubMed Central

    2014-01-01

    Background The Drosophila heart (dorsal vessel) is a relatively simple tubular organ that serves as a model for several aspects of cardiogenesis. Cardiac morphogenesis, proper heart function and stability require structural components whose identity and ways of assembly are only partially understood. Structural components are also needed to connect the myocardial tube with neighboring cells such as pericardial cells and specialized muscle fibers, the so-called alary muscles. Results Using an EMS mutagenesis screen for cardiac and muscular abnormalities in Drosophila embryos we obtained multiple mutants for two genetically interacting complementation groups that showed similar alary muscle and pericardial cell detachment phenotypes. The molecular lesions underlying these defects were identified as domain-specific point mutations in LamininB1 and Cg25C, encoding the extracellular matrix (ECM) components laminin β and collagen IV α1, respectively. Of particular interest within the LamininB1 group are certain hypomorphic mutants that feature prominent defects in cardiac morphogenesis and cardiac ECM layer formation, but in contrast to amorphic mutants, only mild defects in other tissues. All of these alleles carry clustered missense mutations in the laminin LN domain. The identified Cg25C mutants display weaker and largely temperature-sensitive phenotypes that result from glycine substitutions in different Gly-X-Y repeats of the triple helix-forming domain. While initial basement membrane assembly is not abolished in Cg25C mutants, incorporation of perlecan is impaired and intracellular accumulation of perlecan as well as the collagen IV α2 chain is detected during late embryogenesis. Conclusions Assembly of the cardiac ECM depends primarily on laminin, whereas collagen IV is needed for stabilization. Our data underscore the importance of a correctly assembled ECM particularly for the development of cardiac tissues and their lateral connections. The mutational

  8. Site-directed mutagenesis of Gln103 reveals the influence of this residue on the redox properties and stability of MauG.

    PubMed

    Shin, Sooim; Yukl, Erik T; Sehanobish, Esha; Wilmot, Carrie M; Davidson, Victor L

    2014-03-01

    The diheme enzyme MauG catalyzes a six-electron oxidation that is required for the posttranslational modification of a precursor of methylamine dehydrogenase (preMADH) to complete the biosynthesis of its protein-derived cofactor, tryptophan tryptophylquinone (TTQ). Crystallographic and computational studies have implicated Gln103 in stabilizing the Fe(IV)═O moiety of the bis-Fe(IV) state by hydrogen bonding. The role of Gln103 was probed by site-directed mutagenesis. Q103L and Q103E mutations resulted in no expression and very little expression of the protein, respectively. Q103A MauG exhibited oxidative damage when isolated. Q103N MauG was isolated at levels comparable to that of wild-type MauG and exhibited normal activity in catalyzing the biosynthesis of TTQ from preMADH. The crystal structure of the Q103N MauG-preMADH complex suggests that a water may mediate hydrogen bonding between the shorter Asn103 side chain and the Fe(IV)═O moiety. The Q103N mutation caused the two redox potentials associated with the diferric/diferrous redox couple to become less negative, although the redox cooperativity of the hemes of MauG was retained. Upon addition of H2O2, Q103N MauG exhibits changes in the absorbance spectrum in the Soret and near-IR regions consistent with formation of the bis-Fe(IV) redox state. However, the rate of spontaneous return of the spectrum in the Soret region was 4.5-fold greater for Q103N MauG than for wild-type MauG. In contrast, the rate of spontaneous decay of the absorbance at 950 nm, which is associated with charge-resonance stabilization of the high-valence state, was similar for wild-type MauG and Q103N MauG. This suggests that as a consequence of the mutation a different distribution of resonance structures stabilizes the bis-Fe(IV) state. These results demonstrate that subtle changes in the structure of the side chain of residue 103 can significantly affect the overall protein stability of MauG and alter the redox properties of the

  9. Computer Simulation of Mutagenesis.

    ERIC Educational Resources Information Center

    North, J. C.; Dent, M. T.

    1978-01-01

    A FORTRAN program is described which simulates point-substitution mutations in the DNA strands of typical organisms. Its objective is to help students to understand the significance and structure of the genetic code, and the mechanisms and effect of mutagenesis. (Author/BB)

  10. 2004 Mutagenesis Gordon Conference

    SciTech Connect

    Dr. Sue Jinks-Robertson

    2005-09-16

    Mutations are genetic alterations that drive biological evolution and cause many, if not all, human diseases. Mutation originates via two distinct mechanisms: ''vertical'' variation is de novo change of one or few bases, whereas ''horizontal'' variation occurs by genetic recombination, which creates new mosaics of pre-existing sequences. The Mutagenesis Conference has traditionally focused on the generation of mutagenic intermediates during normal DNA synthesis or in response to environmental insults, as well as the diverse repair mechanisms that prevent the fixation of such intermediates as permanent mutations. While the 2004 Conference will continue to focus on the molecular mechanisms of mutagenesis, there will be increased emphasis on the biological consequences of mutations, both in terms of evolutionary processes and in terms of human disease. The meeting will open with two historical accounts of mutation research that recapitulate the intellectual framework of this field and thereby place the current research paradigms into perspective. The two introductory keynote lectures will be followed by sessions on: (1) mutagenic systems, (2) hypermutable sequences, (3) mechanisms of mutation, (4) mutation avoidance systems, (5) mutation in human hereditary and infectious diseases, (6) mutation rates in evolution and genotype-phenotype relationships, (7) ecology, mutagenesis and the modeling of evolution and (8) genetic diversity of the human population and models for human mutagenesis. The Conference will end with a synthesis of the meeting as the keynote closing lecture.

  11. Inhibition of intestinal absorption of cholesterol by ezetimibe or bile acids by SC-435 alters lipoprotein metabolism and extends the lifespan of SR-BI/apoE double knockout mice.

    PubMed

    Braun, Anne; Yesilaltay, Ayce; Acton, Susan; Broschat, Kay O; Krul, Elaine S; Napawan, Nida; Stagliano, Nancy; Krieger, Monty

    2008-05-01

    SR-BI/apoE double knockout (dKO) mice exhibit many features of human coronary heart disease (CHD), including hypercholesterolemia, occlusive coronary atherosclerosis, cardiac hypertrophy, myocardial infarctions, cardiac dysfunction and premature death. Ezetimibe is a FDA-approved, intestinal cholesterol absorption inhibitor that lowers plasma LDL cholesterol in humans and animals and inhibits aortic root atherosclerosis in apoE KO mice, but has not been proven to reduce CHD. Three-week-ezetimibe treatment of dKO mice (0.005% (w/w) in standard chow administered from weaning) resulted in a 35% decrease in cholesterol in IDL/LDL-size lipoproteins, but not in VLDL- and HDL-size lipoproteins. Ezetimibe treatment significantly reduced aortic root (57%) and coronary arterial (68%) atherosclerosis, cardiomegaly (24%) and cardiac fibrosis (57%), and prolonged the lives of the mice (27%). This represents the first demonstration of beneficial effects of ezetimibe treatment on CHD. The dKO mice were similarly treated with SC-435 (0.01% (w/w)), an apical sodium codependent bile acid transporter (ASBT) inhibitor, that blocks intestinal absorption of bile acids, lowers plasma cholesterol in animals, and reduces aortic root atherosclerosis in apoE KO mice. The effects of SC-435 treatment were similar to those of ezetimibe: 37% decrease in ILD/LDL-size lipoprotein cholesterol and 57% prolongation in median lifespan. Thus, inhibition of intestinal absorption of either cholesterol (ezetimibe) or bile acids (SC-435) significantly reduced plasma IDL/LDL-size lipoprotein cholesterol levels and improved survival of SR-BI/apoE dKO mice. The SR-BI/apoE dKO murine model of atherosclerotic occlusive, arterial CHD appears to provide a useful system to evaluate compounds that modulate cholesterol homeostasis and atherosclerosis. PMID:18054357

  12. Procollagen C-endopeptidase Enhancer Protein 2 (PCPE2) Reduces Atherosclerosis in Mice by Enhancing Scavenger Receptor Class B1 (SR-BI)-mediated High-density Lipoprotein (HDL)-Cholesteryl Ester Uptake.

    PubMed

    Pollard, Ricquita D; Blesso, Christopher N; Zabalawi, Manal; Fulp, Brian; Gerelus, Mark; Zhu, Xuewei; Lyons, Erica W; Nuradin, Nebil; Francone, Omar L; Li, Xiang-An; Sahoo, Daisy; Thomas, Michael J; Sorci-Thomas, Mary G

    2015-06-19

    Studies in human populations have shown a significant correlation between procollagen C-endopeptidase enhancer protein 2 (PCPE2) single nucleotide polymorphisms and plasma HDL cholesterol concentrations. PCPE2, a 52-kDa glycoprotein located in the extracellular matrix, enhances the cleavage of C-terminal procollagen by bone morphogenetic protein 1 (BMP1). Our studies here focused on investigating the basis for the elevated concentration of enlarged plasma HDL in PCPE2-deficient mice to determine whether they protected against diet-induced atherosclerosis. PCPE2-deficient mice were crossed with LDL receptor-deficient mice to obtain LDLr(-/-), PCPE2(-/-) mice, which had elevated HDL levels compared with LDLr(-/-) mice with similar LDL concentrations. We found that LDLr(-/-), PCPE2(-/-) mice had significantly more neutral lipid and CD68+ infiltration in the aortic root than LDLr(-/-) mice. Surprisingly, in light of their elevated HDL levels, the extent of aortic lipid deposition in LDLr(-/-), PCPE2(-/-) mice was similar to that reported for LDLr(-/-), apoA-I(-/-) mice, which lack any apoA-I/HDL. Furthermore, LDLr(-/-), PCPE2(-/-) mice had reduced HDL apoA-I fractional clearance and macrophage to fecal reverse cholesterol transport rates compared with LDLr(-/-) mice, despite a 2-fold increase in liver SR-BI expression. PCPE2 was shown to enhance SR-BI function by increasing the rate of HDL-associated cholesteryl ester uptake, possibly by optimizing SR-BI localization and/or conformation. We conclude that PCPE2 is atheroprotective and an important component of the reverse cholesterol transport HDL system. PMID:25947382

  13. A comprehensive alanine-scanning mutagenesis study reveals roles for salt bridges in the structure and activity of Pseudomonas aeruginosa elastase.

    PubMed

    Bian, Fei; Yue, Shousong; Peng, Zhenying; Zhang, Xiaowei; Chen, Gao; Yu, Jinhui; Xuan, Ning; Bi, Yuping

    2015-01-01

    The relationship between salt bridges and stability/enzymatic activity is unclear. We studied this relationship by systematic alanine-scanning mutation analysis using the typical M4 family metalloprotease Pseudomonas aeruginosa elastase (PAE, also known as pseudolysin) as a model. Structural analysis revealed seven salt bridges in the PAE structure. We constructed ten mutants for six salt bridges. Among these mutants, six (Asp189Ala, Arg179Ala, Asp201Ala, Arg205Ala, Arg245Ala and Glu249Ala) were active and four (Asp168Ala, Arg198Ala, Arg253Ala, and Arg279Ala) were inactive. Five mutants were purified, and their catalytic efficiencies (kcat/Km), half-lives (t1/2) and thermal unfolding curves were compared with those of PAE. Mutants Asp189Ala and Arg179Ala both showed decreased thermal stabilities and increased activities, suggesting that the salt bridge Asp189-Arg179 stabilizes the protein at the expense of catalytic efficiency. In contrast, mutants Asp201Ala and Arg205Ala both showed slightly increased thermal stability and slightly decreased activity, suggesting that the salt bridge Asp201-Arg205 destabilizes the protein. Mutant Glu249Ala is related to a C-terminal salt bridge network and showed both decreased thermal stability and decreased activity. Furthermore, Glu249Ala showed a thermal unfolding curve with three discernable states [the native state (N), the partially unfolded state (I) and the unfolded state (U)]. In comparison, there were only two discernable states (N and U) in the thermal unfolding curve of PAE. These results suggest that Glu249 is important for catalytic efficiency, stability and unfolding cooperativity. This study represents a systematic mutational analyses of salt bridges in the model metalloprotease PAE and provides important insights into the structure-function relationship of enzymes. PMID:25815820

  14. ENU mutagenesis reveals that Notchless homolog 1 (Drosophila) affects Cdkn1a and several members of the Wnt pathway during murine pre-implantation development

    PubMed Central

    2012-01-01

    Background Our interests lie in determining the genes and genetic pathways that are important for establishing and maintaining maternal-fetal interactions during pregnancy. Mutation analysis targeted to a 34 Mb domain flanked by Trp53 and Wnt3 demonstrates that this region of mouse chromosome 11 contains a large number of essential genes. Two mutant alleles (l11Jus1 and l11Jus4), which fall into the same complementation group, survive through implantation but fail prior to gastrulation. Results Through a positional cloning strategy, we discovered that these homozygous mutant alleles contain non-conservative missense mutations in the Notchless homolog 1 (Drosophila) (Nle1) gene. NLE1 is a member of the large WD40-repeat protein family, and is thought to signal via the canonical NOTCH pathway in vertebrates. However, the phenotype of the Nle1 mutant mice is much more severe than single Notch receptor mutations or even in animals in which NOTCH signaling is blocked. To test the hypothesis that NLE1 functions in multiple signaling pathways during pre-implantation development, we examined expression of multiple Notch downstream target genes, as well as select members of the Wnt pathway in wild-type and mutant embryos. We did not detect altered expression of any primary members of the Notch pathway or in Notch downstream target genes. However, our data reveal that Cdkn1a, a NOTCH target, was upregulated in Nle1 mutants, while several members of the Wnt pathway are downregulated. In addition, we found that Nle1 mutant embryos undergo caspase-mediated apoptosis as hatched blastocysts, but not as morulae or blastocysts. Conclusions Taken together, these results uncover potential novel functions for NLE1 in the WNT and CDKN1A pathways during embryonic development in mammals. PMID:23231322

  15. Structure and Mutagenesis of the Parainfluenza Virus 5 Hemagglutinin-Neuraminidase Stalk Domain Reveals a Four-Helix Bundle and the Role of the Stalk in Fusion Promotion

    SciTech Connect

    Bose, Sayantan; Welch, Brett D.; Kors, Christopher A.; Yuan, Ping; Jardetzky, Theodore S.; Lamb, Robert A.

    2014-10-02

    Paramyxovirus entry into cells requires the fusion protein (F) and a receptor binding protein (hemagglutinin-neuraminidase [HN], H, or G). The multifunctional HN protein of some paramyxoviruses, besides functioning as the receptor (sialic acid) binding protein (hemagglutinin activity) and the receptor-destroying protein (neuraminidase activity), enhances F activity, presumably by lowering the activation energy required for F to mediate fusion of viral and cellular membranes. Before or upon receptor binding by the HN globular head, F is believed to interact with the HN stalk. Unfortunately, until recently none of the receptor binding protein crystal structures have shown electron density for the stalk domain. Parainfluenza virus 5 (PIV5) HN exists as a noncovalent dimer-of-dimers on the surface of cells, linked by a single disulfide bond in the stalk. Here we present the crystal structure of the PIV5-HN stalk domain at a resolution of 2.65 {angstrom}, revealing a four-helix bundle (4HB) with an upper (N-terminal) straight region and a lower (C-terminal) supercoiled part. The hydrophobic core residues are a mix of an 11-mer repeat and a 3- to 4-heptad repeat. To functionally characterize the role of the HN stalk in F interactions and fusion, we designed mutants along the PIV5-HN stalk that are N-glycosylated to physically disrupt F-HN interactions. By extensive study of receptor binding, neuraminidase activity, oligomerization, and fusion-promoting functions of the mutant proteins, we found a correlation between the position of the N-glycosylation mutants on the stalk structure and their neuraminidase activities as well as their abilities to promote fusion.

  16. Mutagenesis Study Reveals the Rim of Catalytic Entry Site of HDAC4 and -5 as the Major Binding Surface of SMRT Corepressor.

    PubMed

    Kim, Gwang Sik; Jung, Ha-Eun; Kim, Jeong-Sun; Lee, Young Chul

    2015-01-01

    Histone deacetylases (HDACs) play a pivotal role in eukaryotic gene expression by modulating the levels of acetylation of chromatin and related transcription factors. In contrast to class I HDACs (HDAC1, -2, -3 and -8), the class IIa HDACs (HDAC4, -5, -7 and -9) harbor cryptic deacetylases activity and recruit the SMRT-HDAC3 complex to repress target genes in vivo. In this regard, the specific interaction between the HDAC domain of class IIa HDACs and the C-terminal region of SMRT repression domain 3 (SRD3c) is known to be critical, but the molecular basis of this interaction has not yet been addressed. Here, we used an extensive mutant screening system, named the "partitioned one- plus two-hybrid system", to isolate SRD3c interaction-defective (SRID) mutants over the entire catalytic domains of HDAC4 (HDAC4c) and -5. The surface presentation of the SRID mutations on the HDAC4c structure revealed that most of the mutations were mapped to the rim surface of the catalytic entry site, strongly suggesting this mutational hot-spot region as the major binding surface of SRD3c. Notably, among the HDAC4c surface residues required for SRD3c binding, some residues (C667, C669, C751, D759, T760 and F871) are present only in class IIa HDACs, providing the molecular basis for the specific interactions between SRD3c and class IIa enzymes. To investigate the functional consequence of SRID mutation, the in vitro HDAC activities of HDAC4 mutants immuno-purified from HEK293 cells were measured. The levels of HDAC activity of the HDAC4c mutants were substantially decreased compared to wild-type. Consistent with this, SRID mutations of HDAC4c prevented the association of HDAC4c with the SMRT-HDAC3 complex in vivo. Our findings may provide structural insight into the binding interface of HDAC4 and -5 with SRD3c, as a novel target to design modulators specific to these enzymes. PMID:26161557

  17. Interfacial conditions and electrical properties of the SrBi 2Ta 2O 9/ZrO 2/Si (MFIS) structure according to the heat treatment of the ZrO 2 buffer layer

    NASA Astrophysics Data System (ADS)

    Park, Chul-Ho; Kim, Jae-Hyun; Kim, Min-Cheol; Son, Young-Gook; Won, Mi-Sook

    2005-08-01

    The possibility of the ZrO 2 buffer layer as the insulator for the metal-ferroelectric-insulator-semiconductor (MFIS) structure was investigated. ZrO 2 and SrBi 2Ta 2O 9 (SBT) thin films were deposited on the p-type Si(1 1 1) wafer by the rf magnetron-sputtering method. According to the process with and without the post-annealing of the ZrO 2 buffer layer, the diffusion amount of Sr, Bi, Ta elements show slight difference through the glow discharge spectrometer (GDS) analysis. From X-ray photoelectron spectroscopy (XPS) results, we could confirm that the post-annealing process affects the chemical binding condition of the interface between the ZrO 2 thin film and the Si substrate, which results in the chemical stability of the ZrO 2 thin film. The electrical properties of the MFIS structure were relatively improved by the post-annealing ZrO 2 buffer layer. The window memory of the Pt/SBT (260 nm, 800 °C)/ZrO 2 (20 nm) structure increases from 0.75 to 2.2 V. This memory window is sufficient for the practical application of the NDRO-FRAM operating at low voltage.

  18. Optimization of Combinatorial Mutagenesis

    NASA Astrophysics Data System (ADS)

    Parker, Andrew S.; Griswold, Karl E.; Bailey-Kellogg, Chris

    Protein engineering by combinatorial site-directed mutagenesis evaluates a portion of the sequence space near a target protein, seeking variants with improved properties (stability, activity, immunogenicity, etc.). In order to improve the hit-rate of beneficial variants in such mutagenesis libraries, we develop methods to select optimal positions and corresponding sets of the mutations that will be used, in all combinations, in constructing a library for experimental evaluation. Our approach, OCoM (Optimization of Combinatorial Mutagenesis), encompasses both degenerate oligonucleotides and specified point mutations, and can be directed accordingly by requirements of experimental cost and library size. It evaluates the quality of the resulting library by one- and two-body sequence potentials, averaged over the variants. To ensure that it is not simply recapitulating extant sequences, it balances the quality of a library with an explicit evaluation of the novelty of its members. We show that, despite dealing with a combinatorial set of variants, in our approach the resulting library optimization problem is actually isomorphic to single-variant optimization. By the same token, this means that the two-body sequence potential results in an NP-hard optimization problem. We present an efficient dynamic programming algorithm for the one-body case and a practically-efficient integer programming approach for the general two-body case. We demonstrate the effectiveness of our approach in designing libraries for three different case study proteins targeted by previous combinatorial libraries - a green fluorescent protein, a cytochrome P450, and a beta lactamase. We found that OCoM worked quite efficiently in practice, requiring only 1 hour even for the massive design problem of selecting 18 mutations to generate 107 variants of a 443-residue P450. We demonstrate the general ability of OCoM in enabling the protein engineer to explore and evaluate trade-offs between quality and

  19. Transposon Mutagenesis of the Plant-Associated Bacillus amyloliquefaciens ssp. plantarum FZB42 Revealed That the nfrA and RBAM17410 Genes Are Involved in Plant-Microbe-Interactions

    PubMed Central

    Dietel, Kristin; Beator, Barbara; Dolgova, Olga; Fan, Ben; Bleiss, Wilfrid; Ziegler, Jörg; Schmid, Michael; Hartmann, Anton; Borriss, Rainer

    2014-01-01

    Bacillus amyloliquefaciens ssp. plantarum FZB42 represents the prototype of Gram-positive plant growth promoting and biocontrol bacteria. In this study, we applied transposon mutagenesis to generate a transposon library, which was screened for genes involved in multicellular behavior and biofilm formation on roots as a prerequisite of plant growth promoting activity. Transposon insertion sites were determined by rescue-cloning followed by DNA sequencing. As in B. subtilis, the global transcriptional regulator DegU was identified as an activator of genes necessary for swarming and biofilm formation, and the DegU-mutant of FZB42 was found impaired in efficient root colonization. Direct screening of 3,000 transposon insertion mutants for plant-growth-promotion revealed the gene products of nfrA and RBAM_017140 to be essential for beneficial effects exerted by FZB42 on plants. We analyzed the performance of GFP-labeled wild-type and transposon mutants in the colonization of lettuce roots using confocal laser scanning microscopy. While the wild-type strain heavily colonized root surfaces, the nfrA mutant did not colonize lettuce roots, although it was not impaired in growth in laboratory cultures, biofilm formation and swarming motility on agar plates. The RBAM17410 gene, occurring in only a few members of the B. subtilis species complex, was directly involved in plant growth promotion. None of the mutant strains were affected in producing the plant growth hormone auxin. We hypothesize that the nfrA gene product is essential for overcoming the stress caused by plant response towards bacterial root colonization. PMID:24847778

  20. Maximizing mutagenesis with solubilized CRISPR-Cas9 ribonucleoprotein complexes.

    PubMed

    Burger, Alexa; Lindsay, Helen; Felker, Anastasia; Hess, Christopher; Anders, Carolin; Chiavacci, Elena; Zaugg, Jonas; Weber, Lukas M; Catena, Raul; Jinek, Martin; Robinson, Mark D; Mosimann, Christian

    2016-06-01

    CRISPR-Cas9 enables efficient sequence-specific mutagenesis for creating somatic or germline mutants of model organisms. Key constraints in vivo remain the expression and delivery of active Cas9-sgRNA ribonucleoprotein complexes (RNPs) with minimal toxicity, variable mutagenesis efficiencies depending on targeting sequence, and high mutation mosaicism. Here, we apply in vitro assembled, fluorescent Cas9-sgRNA RNPs in solubilizing salt solution to achieve maximal mutagenesis efficiency in zebrafish embryos. MiSeq-based sequence analysis of targeted loci in individual embryos using CrispRVariants, a customized software tool for mutagenesis quantification and visualization, reveals efficient bi-allelic mutagenesis that reaches saturation at several tested gene loci. Such virtually complete mutagenesis exposes loss-of-function phenotypes for candidate genes in somatic mutant embryos for subsequent generation of stable germline mutants. We further show that targeting of non-coding elements in gene regulatory regions using saturating mutagenesis uncovers functional control elements in transgenic reporters and endogenous genes in injected embryos. Our results establish that optimally solubilized, in vitro assembled fluorescent Cas9-sgRNA RNPs provide a reproducible reagent for direct and scalable loss-of-function studies and applications beyond zebrafish experiments that require maximal DNA cutting efficiency in vivo. PMID:27130213

  1. Optimization of combinatorial mutagenesis.

    PubMed

    Parker, Andrew S; Griswold, Karl E; Bailey-Kellogg, Chris

    2011-11-01

    Protein engineering by combinatorial site-directed mutagenesis evaluates a portion of the sequence space near a target protein, seeking variants with improved properties (e.g., stability, activity, immunogenicity). In order to improve the hit-rate of beneficial variants in such mutagenesis libraries, we develop methods to select optimal positions and corresponding sets of the mutations that will be used, in all combinations, in constructing a library for experimental evaluation. Our approach, OCoM (Optimization of Combinatorial Mutagenesis), encompasses both degenerate oligonucleotides and specified point mutations, and can be directed accordingly by requirements of experimental cost and library size. It evaluates the quality of the resulting library by one- and two-body sequence potentials, averaged over the variants. To ensure that it is not simply recapitulating extant sequences, it balances the quality of a library with an explicit evaluation of the novelty of its members. We show that, despite dealing with a combinatorial set of variants, in our approach the resulting library optimization problem is actually isomorphic to single-variant optimization. By the same token, this means that the two-body sequence potential results in an NP-hard optimization problem. We present an efficient dynamic programming algorithm for the one-body case and a practically-efficient integer programming approach for the general two-body case. We demonstrate the effectiveness of our approach in designing libraries for three different case study proteins targeted by previous combinatorial libraries--a green fluorescent protein, a cytochrome P450, and a beta lactamase. We found that OCoM worked quite efficiently in practice, requiring only 1 hour even for the massive design problem of selecting 18 mutations to generate 10⁷ variants of a 443-residue P450. We demonstrate the general ability of OCoM in enabling the protein engineer to explore and evaluate trade-offs between quality and

  2. Intrinsic relationship between electronic structures and phase transition of SrBi{sub 2−x}Nd{sub x}Nb{sub 2}O{sub 9} ceramics from ultraviolet ellipsometry at elevated temperatures

    SciTech Connect

    Duan, Z. H.; Jiang, K.; Xu, L. P.; Li, Y. W.; Hu, Z. G. Chu, J. H.

    2014-02-07

    The ferroelectric orthorhombic to paraelectric tetragonal phase transition of SrBi{sub 2−x}Nd{sub x}Nb{sub 2}O{sub 9} (x = 0, 0.05, 0.1, and 0.2) layer-structured ceramics has been investigated by temperature-dependent spectroscopic ellipsometry. Based on the analysis of dielectric functions from 0 to 500 °C with double Tauc-Lorentz dispersion model, the interband transitions located at ultraviolet region have shown an abrupt variation near the Curie temperature. The changes of dielectric functions are mainly due to the thermal-optical and/or photoelastic effect. Moreover, the characteristic alteration in interband transitions can be ascribed to distortion of NbO{sub 6} octahedron and variation of hybridization between Bi 6s and O 2p states during the structure transformation.

  3. Chlordecone Altered Hepatic Disposition of [14C]Cholesterol and Plasma Cholesterol Distribution but not SR-BI or ABCG8 Proteins in Livers of C57BL/6 Mice

    PubMed Central

    Lee, Junga; Scheri, Richard C.; Curtis, Lawrence R.

    2011-01-01

    Organochlorine (OC) insecticides continue to occur in tissues of humans and wildlife throughout the world although they were banned in the United States a few decades ago. Low doses of the OC insecticide chlordecone (CD) alter hepatic disposition of lipophilic xenobiotics and perturb lipid homeostasis in rainbow trout, mice and rats. CD pretreatment altered tissue and hepatic subcellular distribution of exogenous [14C]cholesterol (CH) equivalents 4 and 16 h after a bolus intraperitoneal (ip) injection of 5 ml corn oil/kg that contained 10 mg CH/kg. CD pretreatment altered tissue distribution of exogenously administered [14C]CH by decreased hepatic and renal accumulation, and increased biliary excretion up to 300%. Biliary excretion of polar [14C]CH metabolites was not altered by CD. CD pretreatment decreased subcellular distribution of [14C]CH equivalents in hepatic cytosol and microsomes and lipoprotein-rich fraction-to-homogenate ratio. CD pretreatment increased the ratio of [14C]CH equivalents in high density lipoprotein (HDL) to that in plasma and reduced [14C]CH equivalents in the non-HDL fraction 4 h after a bolus lipid dose. CD pretreatment increased plasma non-HDL total CH by 80% 4 h after a bolus lipid dose. Scavenger receptor class B type I (SR-BI) and ATPbinding cassette transporter G8 (ABCG8) proteins were quantified by western blotting in hepatic membranes from control and CD treated mice. Liver membrane contents of SR-BI and ABCG8 proteins were unchanged by CD pretreatment. The data demonstrated that a single dose of CD altered CH homeostasis and lipoprotein metabolism. PMID:18387646

  4. Environmental stress induces trinucleotide repeat mutagenesis in human cells.

    PubMed

    Chatterjee, Nimrat; Lin, Yunfu; Santillan, Beatriz A; Yotnda, Patricia; Wilson, John H

    2015-03-24

    The dynamic mutability of microsatellite repeats is implicated in the modification of gene function and disease phenotype. Studies of the enhanced instability of long trinucleotide repeats (TNRs)-the cause of multiple human diseases-have revealed a remarkable complexity of mutagenic mechanisms. Here, we show that cold, heat, hypoxic, and oxidative stresses induce mutagenesis of a long CAG repeat tract in human cells. We show that stress-response factors mediate the stress-induced mutagenesis (SIM) of CAG repeats. We show further that SIM of CAG repeats does not involve mismatch repair, nucleotide excision repair, or transcription, processes that are known to promote TNR mutagenesis in other pathways of instability. Instead, we find that these stresses stimulate DNA rereplication, increasing the proportion of cells with >4 C-value (C) DNA content. Knockdown of the replication origin-licensing factor CDT1 eliminates both stress-induced rereplication and CAG repeat mutagenesis. In addition, direct induction of rereplication in the absence of stress also increases the proportion of cells with >4C DNA content and promotes repeat mutagenesis. Thus, environmental stress triggers a unique pathway for TNR mutagenesis that likely is mediated by DNA rereplication. This pathway may impact normal cells as they encounter stresses in their environment or during development or abnormal cells as they evolve metastatic potential. PMID:25775519

  5. Environmental stress induces trinucleotide repeat mutagenesis in human cells

    PubMed Central

    Chatterjee, Nimrat; Lin, Yunfu; Santillan, Beatriz A.; Yotnda, Patricia; Wilson, John H.

    2015-01-01

    The dynamic mutability of microsatellite repeats is implicated in the modification of gene function and disease phenotype. Studies of the enhanced instability of long trinucleotide repeats (TNRs)—the cause of multiple human diseases—have revealed a remarkable complexity of mutagenic mechanisms. Here, we show that cold, heat, hypoxic, and oxidative stresses induce mutagenesis of a long CAG repeat tract in human cells. We show that stress-response factors mediate the stress-induced mutagenesis (SIM) of CAG repeats. We show further that SIM of CAG repeats does not involve mismatch repair, nucleotide excision repair, or transcription, processes that are known to promote TNR mutagenesis in other pathways of instability. Instead, we find that these stresses stimulate DNA rereplication, increasing the proportion of cells with >4 C-value (C) DNA content. Knockdown of the replication origin-licensing factor CDT1 eliminates both stress-induced rereplication and CAG repeat mutagenesis. In addition, direct induction of rereplication in the absence of stress also increases the proportion of cells with >4C DNA content and promotes repeat mutagenesis. Thus, environmental stress triggers a unique pathway for TNR mutagenesis that likely is mediated by DNA rereplication. This pathway may impact normal cells as they encounter stresses in their environment or during development or abnormal cells as they evolve metastatic potential. PMID:25775519

  6. GERM-LINE SPECIFIC FACTORS IN CHEMICAL MUTAGENESIS

    EPA Science Inventory

    Chemical mutagenesis test results ave not revealed evidence of germ-line specific mutagens. owever, conventional assays have indicated that there are male-female differences in mutagenic response, as well as quantitative/qualitative differences in induced mutations which depend u...

  7. Forward and reverse mutagenesis in C. elegans

    PubMed Central

    Kutscher, Lena M.; Shaham, Shai

    2014-01-01

    Mutagenesis drives natural selection. In the lab, mutations allow gene function to be deciphered. C. elegans is highly amendable to functional genetics because of its short generation time, ease of use, and wealth of available gene-alteration techniques. Here we provide an overview of historical and contemporary methods for mutagenesis in C. elegans, and discuss principles and strategies for forward (genome-wide mutagenesis) and reverse (target-selected and gene-specific mutagenesis) genetic studies in this animal. PMID:24449699

  8. Optogenetic mutagenesis in Caenorhabditis elegans

    PubMed Central

    Noma, Kentaro; Jin, Yishi

    2015-01-01

    Reactive oxygen species (ROS) can modify and damage DNA. Here we report an optogenetic mutagenesis approach that is free of toxic chemicals and easy to perform by taking advantage of a genetically encoded ROS generator. This method relies on the potency of ROS generation by His-mSOG, the mini singlet oxygen generator, miniSOG, fused to a histone. Caenorhabditis elegans expressing His-mSOG in the germline behave and reproduce normally, without photoinduction. Following exposure to blue light, the His-mSOG animals produce progeny with a wide range of heritable phenotypes. We show that optogenetic mutagenesis by His-mSOG induces a broad spectrum of mutations including single-nucleotide variants (SNVs), chromosomal deletions, as well as integration of extrachromosomal transgenes, which complements those derived from traditional chemical or radiation mutagenesis. The optogenetic mutagenesis expands the toolbox for forward genetic screening and also provides direct evidence that nuclear ROS can induce heritable and specific genetic mutations. PMID:26632265

  9. A model for optical and electrical polarization fatigue in SrBi{sub 2}Ta{sub 2}O{sub 9} and Pb(Zr,Ti)O{sub 3}

    SciTech Connect

    Al-Shareef, H.N.; Dimos, D.; Warren, W.L.; Tuttle, B.A.

    1996-06-01

    Polarization fatigue, the decrease in switchable polarization with electric field cycling, has received considerable attention lately because ferroelectric thin films are being evaluated for use in nonvolatile memory applications. Here, the authors find that significant polarization fatigue (> 90%) can be induced in SrBi{sub 2}Ta{sub 2}O{sub 9} (SBT) thin films using (a) broad-band optical illumination combined with a bias near the switching threshold and (b) electric field cycling under broadband optical illumination. In the latter case, the extent of polarization fatigue increases with decreasing cycling voltage. In either case, the optically fatigued SBT capacitors can be fully rejuvenated by applying a saturating dc bias with light or by electric field cycling without light, which suggests a field-assisted recovery mechanism. A similar behavior was observed in Pb(Zr,Ti)O{sub 3} (PZT) films with LSCO electrodes. Based on these results, they suggest that polarization fatigue in ferroelectrics is essentially a dynamic competition between domain wall pinning due to electronic charge trapping, and field-assisted unpinning of the domain walls. Thus, domain wall pinning is not necessarily absent in nominally fatigue-free systems. Instead, these systems are ones in which domain wall unpinning occurs at least as rapidly as any pinning. Factors which may affect the pinning and unpinning rates will be discussed.

  10. Characterization of the dielectric properties and alternating current conductivity of the SrBi5-xLaxTi4FeO18 (x=0, 0.2) compound

    NASA Astrophysics Data System (ADS)

    Almodovar, N. S.; Portelles, J.; Raymond, O.; Heiras, J.; Siqueiros, J. M.

    2007-12-01

    Lanthanum-doped bismuth layer-structured ferroelectric ceramics SrBi5-xLaxTi4FeO18 (x =0,0.2) were prepared by the solid-state reaction method. X-ray diffraction patterns indicate that single phases were formed. Hysteresis loops at room temperature (20 °C) show that the La-doped ceramic presents a slightly lower spontaneous polarization than the undoped compound. Measurements of relative permittivity and dielectric loss versus temperature were performed from room temperature to 700 °C in the 100 Hz-1 MHz frequency range. Three anomalies were observed in the thermal behavior of the relative permittivity in both samples. Anomalies around the temperatures of 465 and 430 °C have been identified as the ferroelectric-paraelectric transition temperatures for the x =0 and 0.2 compounds, respectively. The sizable shift of the transition temperatures toward lower temperatures with the La doping is interpreted as a manifestation of the La ion incorporation into the crystal structure. From the conductivity studies, the activation energies as functions of frequency for three different temperature zones are obtained. It is found that activation energies are strongly frequency dependent, particularly in the low-frequency region. The frequency dependence of the conductivity at different temperatures was analyzed using Jonscher's power law and the Almond-West conductivity formalism.

  11. Site-directed mutagenesis reveals a conservation of the copper-binding site and the crucial role of His24 in CopH from Cupriavidus metallidurans CH34.

    PubMed

    Sendra, Véronique; Gambarelli, Serge; Bersch, Beate; Covès, Jacques

    2009-12-01

    CopH is a periplasmic copper-binding protein from Cupriavidus metallidurans CH34 that contains two histidine residues. Both His24 and His26 contribute to the formation of two high-affinity copper-binding sites in wild-type CopH and are likely involved in a 2N2O coordination sphere in the equatorial plane. We have used site-directed mutagenesis, and a series of spectroscopic and calorimetric studies to further characterize the copper-binding sites in CopH. While His24 plays a predominant role in copper affinity, one Cu-binding site was lost when either histidine residue was mutated. However, as shown by NMR and EPR, the mutation of the His residues does not affect the structural organization of the Cu-binding site nor the number of nitrogen ligands involved in copper ligation. In the absence of structural data, we propose a model that conciliates most of the spectroscopic data recorded during this study. PMID:19857899

  12. Novel full color greenish-white-emitting SrBi()3∶Eu phosphor for white light-emitting diodes

    NASA Astrophysics Data System (ADS)

    Liu, Yufeng; Ding, Yanjun; Peng, Zhimin

    2014-01-01

    The full color greenish-white-emitting phosphors Eu-doped Sr3Bi( have been successfully synthesized by the conventional solid-state reaction, and its photoluminescence properties have been investigated for the first time. The emission spectra of Sr3Bi(∶Eu phosphors exhibit a greenish-white-emitting by combining blue, green, and red emissions, which are originated from the 5d→4f transition of the Eu. The excitation spectra reveal broad strong bands from 300 to 400 nm, which match well with the readily available emissions from near-ultraviolet light-emitting diode (LED) chips. The correlated color temperature, color rendering index (CRI), and Commission Internationale de I'Eclairage chromaticity coordinates of the entitled phosphors excited by 330 and 365 nm are also investigated. The experimental results indicate that the Eu-doped Sr3Bi( phosphors are a promising innovative greenish-white-emitting phosphor for white LEDs.

  13. High dielectric permittivity in the microwave region of SrBi2Nb2O9 (SBN) added La2O3, PbO and Bi2O3, obtained by mechanical alloying

    NASA Astrophysics Data System (ADS)

    Rocha, M. J. S.; Silva, P. M. O.; Theophilo, K. R. B.; Sancho, E. O.; Paula, P. V. L.; Silva, M. A. S.; Honorato, S. B.; Sombra, A. S. B.

    2012-08-01

    This paper presents the microwave dielectric properties and a structural study of SrBi2Nb2O9 (SBN) added La2O3, PbO or Bi2O3 obtained by a solid state procedure. High-energy mechanical milling was used to reduce the particle size, which allows for a better shaping of the green body and an increased reactivity. The mechanical milling activation process produced a reduced sintering temperature in the material, decreasing the loss of the volatile elements and controlling the growth of the grain that is produced when a high temperature is required to obtain dense ceramics. The incorporation of La3+, or Pb2+, or Bi3+ of different amounts (0, 3, 5, 10 and 15 wt%) was used to improve the densification without changing the crystal structure, since with a low doping content these ions can occupy the A site of the perovskite blocks; they can also occupy the Bi3+ sites in Bi2O3 layers. A single orthorhombic phase was formed after calcination at 800 °C for 2 h. X-ray diffraction, Fourier transformation, infrared and Raman spectroscopy have been carried out in order to investigate the effects of doping on SBN. The dielectric permittivity (ɛ‧r) and loss in the microwave region (2-4 GHz) of SBN ceramics with additions of Bi2O3, La2O3 and PbO were studied. Higher values of permittivity (ɛr‧ = 154.6) have been obtained for the SBN added La (15 wt%) a lower loss (tg δ = 0.01531) was also achieved in the SBN added La (15 wt%) sample with PVA and TEOS, respectively. The samples that showed the highest dielectric permittivities were all lanthanum doped, all with values of permittivity above 90. A comparative study associated with different types of binders was completed (with glycerin, PVA and TEOS). This procedure allowed us to obtain phases at lower temperatures than usually appear in the literature. The microwave dielectric properties (permittivity and loss) in the region 2-4 GHz, were studied for all samples. The structural and microwave dielectric properties of SBN show a

  14. MECHANISM OF CHEMICAL MUTAGENESIS IV.

    PubMed Central

    Lorkiewicz, Z.; Szybalski, Waclaw

    1961-01-01

    Lorkiewicz, Z. (University of Wisconsin, Madison), and Waclaw Szybalski. Mechanism of chemical mutagenesis. IV. Reaction between triethylene melamine and nucleic acid components. J. Bacteriol. 82: 195–201. 1961.—Triethylene melamine interacts primarily with phosphorylated intracellular deoxyribonucleic acid (DNA) precursors and not with DNA. It was found by direct chemical and chromatographic analysis that only pyrimidine precursors of nucleic acids are attacked by triethylene melamine. In the course of the triethylene melamine-deoxycytidine reaction the mutagenicity of the reaction mixture is lost, but the mutagenicity of the triethylene melamine-thymidine reaction products significantly increases above that of the reaction substrates. Several steps are postulated to explain the mechanism of the triethylene melamine-initiated mutagenic reaction: (i) Reaction I, semireversible uptake of triethylene melamine; (ii) reaction II, chemical interaction between triethylene melamine and intracellular thymidine mono- or triphosphate with the production of a functional analogue of the latter; (iii) incorporation of this fraudulent analogue into the newly formed DNA strand; (iv) occurrence of self-perpetuating errors in the sequence of natural bases during subsequent rounds of replication of the analogue-containing DNA strand. It is postulated that the mechanism of mutagenic responses to different types of mutagens can fit either a simplified (mutagenic base analogues) or extended version (radiation) of this schema. PMID:16561917

  15. Highly Efficient Targeted Mutagenesis in Mice Using TALENs

    PubMed Central

    Panda, Sudeepta Kumar; Wefers, Benedikt; Ortiz, Oskar; Floss, Thomas; Schmid, Bettina; Haass, Christian; Wurst, Wolfgang; Kühn, Ralf

    2013-01-01

    Targeted mouse mutants are instrumental for the analysis of gene function in health and disease. We recently provided proof-of-principle for the fast-track mutagenesis of the mouse genome, using transcription activator-like effector nucleases (TALENs) in one-cell embryos. Here we report a routine procedure for the efficient production of disease-related knockin and knockout mutants, using improved TALEN mRNAs that include a plasmid-coded poly(A) tail (TALEN-95A), circumventing the problematic in vitro polyadenylation step. To knock out the C9orf72 gene as a model of frontotemporal lobar degeneration, TALEN-95A mutagenesis induced sequence deletions in 41% of pups derived from microinjected embryos. Using TALENs together with mutagenic oligodeoxynucleotides, we introduced amyotrophic lateral sclerosis patient-derived missense mutations in the fused in sarcoma (Fus) gene at a rate of 6.8%. For the simple identification of TALEN-induced mutants and their progeny we validate high-resolution melt analysis (HRMA) of PCR products as a sensitive and universal genotyping tool. Furthermore, HRMA of off-target sites in mutant founder mice revealed no evidence for undesired TALEN-mediated processing of related genomic sequences. The combination of TALEN-95A mRNAs for enhanced mutagenesis and of HRMA for simplified genotyping enables the accelerated, routine production of new mouse models for the study of genetic disease mechanisms. PMID:23979585

  16. Systematic Mutagenesis of the Escherichia coli Genome†

    PubMed Central

    Kang, Yisheng; Durfee, Tim; Glasner, Jeremy D.; Qiu, Yu; Frisch, David; Winterberg, Kelly M.; Blattner, Frederick R.

    2004-01-01

    A high-throughput method has been developed for the systematic mutagenesis of the Escherichia coli genome. The system is based on in vitro transposition of a modified Tn5 element, the Sce-poson, into linear fragments of each open reading frame. The transposon introduces both positive (kanamycin resistance) and negative (I-SceI recognition site) selectable markers for isolation of mutants and subsequent allele replacement, respectively. Reaction products are then introduced into the genome by homologous recombination via the λRed proteins. The method has yielded insertion alleles for 1976 genes during a first pass through the genome including, unexpectedly, a number of known and putative essential genes. Sce-poson insertions can be easily replaced by markerless mutations by using the I-SceI homing endonuclease to select against retention of the transposon as demonstrated by the substitution of amber and/or in-frame deletions in six different genes. This allows a Sce-poson-containing gene to be specifically targeted for either designed or random modifications, as well as permitting the stepwise engineering of strains with multiple mutations. The promiscuous nature of Tn5 transposition also enables a targeted gene to be dissected by using randomly inserted Sce-posons as shown by a lacZ allelic series. Finally, assessment of the insertion sites by an iterative weighted matrix algorithm reveals that these hyperactive Tn5 complexes generally recognize a highly degenerate asymmetric motif on one end of the target site helping to explain the randomness of Tn5 transposition. PMID:15262929

  17. Economical analysis of saturation mutagenesis experiments.

    PubMed

    Acevedo-Rocha, Carlos G; Reetz, Manfred T; Nov, Yuval

    2015-01-01

    Saturation mutagenesis is a powerful technique for engineering proteins, metabolic pathways and genomes. In spite of its numerous applications, creating high-quality saturation mutagenesis libraries remains a challenge, as various experimental parameters influence in a complex manner the resulting diversity. We explore from the economical perspective various aspects of saturation mutagenesis library preparation: We introduce a cheaper and faster control for assessing library quality based on liquid media; analyze the role of primer purity and supplier in libraries with and without redundancy; compare library quality, yield, randomization efficiency, and annealing bias using traditional and emergent randomization schemes based on mixtures of mutagenic primers; and establish a methodology for choosing the most cost-effective randomization scheme given the screening costs and other experimental parameters. We show that by carefully considering these parameters, laboratory expenses can be significantly reduced. PMID:26190439

  18. Economical analysis of saturation mutagenesis experiments

    PubMed Central

    Acevedo-Rocha, Carlos G.; Reetz, Manfred T.; Nov, Yuval

    2015-01-01

    Saturation mutagenesis is a powerful technique for engineering proteins, metabolic pathways and genomes. In spite of its numerous applications, creating high-quality saturation mutagenesis libraries remains a challenge, as various experimental parameters influence in a complex manner the resulting diversity. We explore from the economical perspective various aspects of saturation mutagenesis library preparation: We introduce a cheaper and faster control for assessing library quality based on liquid media; analyze the role of primer purity and supplier in libraries with and without redundancy; compare library quality, yield, randomization efficiency, and annealing bias using traditional and emergent randomization schemes based on mixtures of mutagenic primers; and establish a methodology for choosing the most cost-effective randomization scheme given the screening costs and other experimental parameters. We show that by carefully considering these parameters, laboratory expenses can be significantly reduced. PMID:26190439

  19. CHALLENGES FOR THE FUTURE IN ENVIRONMENTAL MUTAGENESIS

    EPA Science Inventory

    CHALLENGES FOR THE FUTURE IN ENVIRONMENTAL MUTAGENESIS
    Michael D. Waters
    US Environmental Protection Agency, MD-51A, Research Triangle Park, NC 27711 USA

    Our rapidly growing understanding of the structure of the human genome is forming the basis for numerous new...

  20. Faux Mutagenesis: Teaching Troubleshooting through Controlled Failure

    ERIC Educational Resources Information Center

    Hartberg, Yasha

    2006-01-01

    By shifting pedagogical goals from obtaining successful mutations to teaching students critical troubleshooting skills, it has been possible to introduce site-directed mutagenesis into an undergraduate teaching laboratory. Described in this study is an inexpensive laboratory exercise in which students follow a slightly modified version of…

  1. MAMMALIAN CELL MUTAGENESIS, BANBURY CONFERENCE (JOURNAL VERSION)

    EPA Science Inventory

    A conference on mammalian cell mutagenesis was held at the Banbury Center, Cold Spring Harbor, NY, USA, March 22-25, 1987. The objective of the conference was to provide a forum for discussions concerning the genetic, biochemical, and molecular basis of induced mutations in stand...

  2. An APOBEC Cytidine Deaminase Mutagenesis Pattern is Widespread in Human Cancers

    PubMed Central

    Roberts, Steven A.; Lawrence, Michael S.; Klimczak, Leszek J.; Grimm, Sara A.; Fargo, David; Stojanov, Petar; Kiezun, Adam; Kryukov, Gregory V.; Carter, Scott L.; Saksena, Gordon; Harris, Shawn; Shah, Ruchir R.; Resnick, Michael A.; Getz, Gad; Gordenin, Dmitry A.

    2013-01-01

    Recent studies indicate that a subclass of APOBEC cytidine deaminases, which convert cytosine to uracil during RNA editing and retrovirus or retrotransposon restriction, may induce mutation clusters in human tumors. We show here that throughout cancer genomes APOBEC mutagenesis is pervasive and correlates with APOBEC mRNA levels. Mutation clusters in whole-genome and exome datasets conformed to stringent criteria indicative of an APOBEC mutation pattern. Applying these criteria to 954,247 mutations in 2,680 exomes of 14 cancer types, mostly from TCGA, revealed significant presence of the APOBEC mutation pattern in bladder, cervical, breast, head and neck and lung cancers, reaching 68% of all mutations in some samples. Within breast cancer, the HER2E subtype was clearly enriched with tumors displaying the APOBEC mutation pattern, suggesting this type of mutagenesis is functionally linked with cancer development. The APOBEC mutation pattern also extended to cancer-associated genes, implying that ubiquitous APOBEC mutagenesis is carcinogenic. PMID:23852170

  3. REPLACR-mutagenesis, a one-step method for site-directed mutagenesis by recombineering

    PubMed Central

    Trehan, Ashutosh; Kiełbus, Michał; Czapinski, Jakub; Stepulak, Andrzej; Huhtaniemi, Ilpo; Rivero-Müller, Adolfo

    2016-01-01

    Mutagenesis is an important tool to study gene regulation, model disease-causing mutations and for functional characterisation of proteins. Most of the current methods for mutagenesis involve multiple step procedures. One of the most accurate methods for genetically altering DNA is recombineering, which uses bacteria expressing viral recombination proteins. Recently, the use of in vitro seamless assembly systems using purified enzymes for multiple-fragment cloning as well as mutagenesis is gaining ground. Although these in vitro isothermal reactions are useful when cloning multiple fragments, for site-directed mutagenesis it is unnecessary. Moreover, the use of purified enzymes in vitro is not only expensive but also more inaccurate than the high-fidelity recombination inside bacteria. Here we present a single-step method, named REPLACR-mutagenesis (Recombineering of Ends of linearised PLAsmids after PCR), for creating mutations (deletions, substitutions and additions) in plasmids by in vivo recombineering. REPLACR-mutagenesis only involves transformation of PCR products in bacteria expressing Red/ET recombineering proteins. Modifications in a variety of plasmids up to bacterial artificial chromosomes (BACs; 144 kb deletion) have been achieved by this method. The presented method is more robust, involves fewer steps and is cost-efficient. PMID:26750263

  4. REPLACR-mutagenesis, a one-step method for site-directed mutagenesis by recombineering.

    PubMed

    Trehan, Ashutosh; Kiełbus, Michał; Czapinski, Jakub; Stepulak, Andrzej; Huhtaniemi, Ilpo; Rivero-Müller, Adolfo

    2016-01-01

    Mutagenesis is an important tool to study gene regulation, model disease-causing mutations and for functional characterisation of proteins. Most of the current methods for mutagenesis involve multiple step procedures. One of the most accurate methods for genetically altering DNA is recombineering, which uses bacteria expressing viral recombination proteins. Recently, the use of in vitro seamless assembly systems using purified enzymes for multiple-fragment cloning as well as mutagenesis is gaining ground. Although these in vitro isothermal reactions are useful when cloning multiple fragments, for site-directed mutagenesis it is unnecessary. Moreover, the use of purified enzymes in vitro is not only expensive but also more inaccurate than the high-fidelity recombination inside bacteria. Here we present a single-step method, named REPLACR-mutagenesis (Recombineering of Ends of linearised PLAsmids after PCR), for creating mutations (deletions, substitutions and additions) in plasmids by in vivo recombineering. REPLACR-mutagenesis only involves transformation of PCR products in bacteria expressing Red/ET recombineering proteins. Modifications in a variety of plasmids up to bacterial artificial chromosomes (BACs; 144 kb deletion) have been achieved by this method. The presented method is more robust, involves fewer steps and is cost-efficient. PMID:26750263

  5. Final report [DNA Repair and Mutagenesis - 1999

    SciTech Connect

    Walker, Graham C.

    2001-05-30

    The meeting, titled ''DNA Repair and Mutagenesis: Mechanism, Control, and Biological Consequences'', was designed to bring together the various sub-disciplines that collectively comprise the field of DNA Repair and Mutagenesis. The keynote address was titled ''Mutability Doth Play Her Cruel Sports to Many Men's Decay: Variations on the Theme of Translesion Synthesis.'' Sessions were held on the following themes: Excision repair of DNA damage; Transcription and DNA excision repair; UmuC/DinB/Rev1/Rad30 superfamily of DNA polymerases; Cellular responses to DNA damage, checkpoints, and damage tolerance; Repair of mismatched bases, mutation; Genome-instability, and hypermutation; Repair of strand breaks; Replicational fidelity, and Late-breaking developments; Repair and mutation in challenging environments; and Defects in DNA repair: consequences for human disease and aging.

  6. The Parasol Protocol for computational mutagenesis.

    PubMed

    Aronica, P G A; Verma, C; Popovic, B; Leatherbarrow, R J; Gould, I R

    2016-07-01

    To aid in the discovery and development of peptides and proteins as therapeutic agents, a virtual screen can be used to predict trends and direct workflow. We have developed the Parasol Protocol, a dynamic method implemented using the AMBER MD package, for computational site-directed mutagenesis. This tool can mutate between any pair of amino acids in a computationally expedient, automated manner. To demonstrate the potential of this methodology, we have employed the protocol to investigate a test case involving stapled peptides, and have demonstrated good agreement with experiment. PMID:27255759

  7. Fluorometric method of quantitative cell mutagenesis

    DOEpatents

    Dolbeare, F.A.

    1980-12-12

    A method for assaying a cell culture for mutagenesis is described. A cell culture is stained first with a histochemical stain, and then a fluorescent stain. Normal cells in the culture are stained by both the histochemical and fluorescent stains, while abnormal cells are stained only by the fluorescent stain. The two stains are chosen so that the histochemical stain absorbs the wavelengths that the fluorescent stain emits. After the counterstained culture is subjected to exciting light, the fluorescence from the abnormal cells is detected.

  8. Fluorometric method of quantitative cell mutagenesis

    DOEpatents

    Dolbeare, Frank A.

    1982-01-01

    A method for assaying a cell culture for mutagenesis is described. A cell culture is stained first with a histochemical stain, and then a fluorescent stain. Normal cells in the culture are stained by both the histochemical and fluorescent stains, while abnormal cells are stained only by the fluorescent stain. The two stains are chosen so that the histochemical stain absorbs the wavelengths that the fluorescent stain emits. After the counterstained culture is subjected to exciting light, the fluorescence from the abnormal cells is detected.

  9. Effect of the microwave oven on structural, morphological and electrical properties of SrBi{sub 4}Ti{sub 4}O{sub 15} thin films grown on Pt/Ti/SiO{sub 2}/Si substrates by a soft chemical method

    SciTech Connect

    Simoes, A.Z.; Ramirez, M.A. Riccardi, C.S.; Longo, E.; Varela, J.A.

    2008-06-15

    Thin films of SrBi{sub 4}Ti{sub 4}O{sub 15} (SBTi), a prototype of the Bi-layered-ferroelectric oxide family, were obtained by a soft chemical method and crystallized in a domestic microwave oven. For comparison, films were also crystallized in a conventional method at 700 deg. C for 2 h. Structural and morphological characterization of the SBTi thin films were investigated by X-ray diffraction (XRD) and atomic force microscopy (AFM), respectively. Using platinum coated silicon substrates, the ferroelectric properties of the films were determined. Remanent polarization P{sub r} and a coercive field E{sub c} values of 5.1 {mu}C/cm{sup 2} and 135 kV/cm for the film thermally treated in the microwave oven and 5.4 {mu}C/cm{sup 2} and 85 kV/cm for the film thermally treated in conventional furnace were found. The films thermally treated in the conventional furnace exhibited excellent fatigue-free characteristics up to 10{sup 10} switching cycles indicating that SBTi thin films are a promising material for use in non-volatile memories.

  10. Mutagenesis as a Genetic Research Strategy

    PubMed Central

    Falk, Raphael

    2010-01-01

    Morgan's three students (Muller, Sturtevant, and Bridges) introduced reductionist empirical methods to the study of the chromosomal theory of heredity. Herman J. Muller concentrated on mutations, namely changes in the heterocatalytic properties of genes, without losing their autocatalytic (self-replication) properties. Experimental induction of mutations allowed quantitative analyses of genes' parameters, but hopes to deduce their chemicophysical character were never fulfilled. Once the model for DNA structure was proposed, the reductionist notions of mutation analysis were successfully applied to the molecular genes. However, it was soon realized that the concept of the particulate gene was inadequate. The more the molecular analysis of the genome advanced, the clearer it became that the entities of heredity must be conceived within systems' perspectives, for which special tools for handling large number of variables were developed. Analytic mutagenesis, however, continues to be a major strategy for the study of the cellular and chromosomal mechanisms that control mutation inductions. PMID:20713742

  11. Mutagenesis assays of human amniotic fluid

    SciTech Connect

    Everson, R.B.; Milne, K.L.; Warbuton, D.; McClamrock, H.D.; Buchanan, P.D.

    1985-01-01

    Extracts of amniocentesis samples from 144 women were tested for the presence of mutagenic substances using tester strain TA1538 in the Ames Salmonella/mammalian-microsome mutagenicity test. Because the volume of amniotic fluid in these samples was limited (generally less than 10 ml), the authors investigated modifications of this mutagenesis assay that could increase its ability to detect effects from small quantities of test material. Using mutagenicity in samples of urine from smokers as a model, it appeared that improved ability to detect small amounts of mutagen could be obtained by reducing volumes of media and reagents while keeping the amount of test sample constant. Tests of amniotic fluid extracts by this modified procedure showed small increases in revertants, about 50% above dimethylsulfoxide solvent control values. The increases suggest the presence of small amounts of mutagenic material in many of the amniotic fluid samples. At the doses employed, mutagenic activity in these samples was not associated with maternal smoking.

  12. Homemade Site Directed Mutagenesis of Whole Plasmids

    PubMed Central

    Laible, Mark; Boonrod, Kajohn

    2009-01-01

    Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. With this method the cloned target gene can be altered by substitution, deletion or insertion of a few bases directly into a plasmid. It works by simply amplifying the whole plasmid, in a non PCR-based thermocycling reaction. During the reaction mutagenic primers, carrying the desired mutation, are integrated into the newly synthesized plasmid. In this video tutorial we demonstrate an easy and cost effective way to introduce base substitutions into a plasmid. The protocol works with standard reagents and is independent from commercial kits, which often are very expensive. Applying this protocol can reduce the total cost of a reaction to an eighth of what it costs using some of the commercial kits. In this video we also comment on critical steps during the process and give detailed instructions on how to design the mutagenic primers. PMID:19488024

  13. A threshold of endogenous stress is required to engage cellular response to protect against mutagenesis.

    PubMed

    Saintigny, Yannick; Chevalier, François; Bravard, Anne; Dardillac, Elodie; Laurent, David; Hem, Sonia; Dépagne, Jordane; Radicella, J Pablo; Lopez, Bernard S

    2016-01-01

    Endogenous stress represents a major source of genome instability, but is in essence difficult to apprehend. Incorporation of labeled radionuclides into DNA constitutes a tractable model to analyze cellular responses to endogenous attacks. Here we show that incorporation of [(3)H]thymidine into CHO cells generates oxidative-induced mutagenesis, but, with a peak at low doses. Proteomic analysis showed that the cellular response differs between low and high levels of endogenous stress. In particular, these results confirmed the involvement of proteins implicated in redox homeostasis and DNA damage signaling pathways. Induced-mutagenesis was abolished by the anti-oxidant N-acetyl cysteine and plateaued, at high doses, upon exposure to L-buthionine sulfoximine, which represses cellular detoxification. The [(3)H]thymidine-induced mutation spectrum revealed mostly base substitutions, exhibiting a signature specific for low doses (GC > CG and AT > CG). Consistently, the enzymatic activity of the base excision repair protein APE-1 is induced at only medium or high doses. Collectively, the data reveal that a threshold of endogenous stress must be reached to trigger cellular detoxification and DNA repair programs; below this threshold, the consequences of endogenous stress escape cellular surveillance, leading to high levels of mutagenesis. Therefore, low doses of endogenous local stress can jeopardize genome integrity more efficiently than higher doses. PMID:27406380

  14. A threshold of endogenous stress is required to engage cellular response to protect against mutagenesis

    PubMed Central

    Saintigny, Yannick; Chevalier, François; Bravard, Anne; Dardillac, Elodie; Laurent, David; Hem, Sonia; Dépagne, Jordane; Radicella, J. Pablo; Lopez, Bernard S.

    2016-01-01

    Endogenous stress represents a major source of genome instability, but is in essence difficult to apprehend. Incorporation of labeled radionuclides into DNA constitutes a tractable model to analyze cellular responses to endogenous attacks. Here we show that incorporation of [3H]thymidine into CHO cells generates oxidative-induced mutagenesis, but, with a peak at low doses. Proteomic analysis showed that the cellular response differs between low and high levels of endogenous stress. In particular, these results confirmed the involvement of proteins implicated in redox homeostasis and DNA damage signaling pathways. Induced-mutagenesis was abolished by the anti-oxidant N-acetyl cysteine and plateaued, at high doses, upon exposure to L-buthionine sulfoximine, which represses cellular detoxification. The [3H]thymidine-induced mutation spectrum revealed mostly base substitutions, exhibiting a signature specific for low doses (GC > CG and AT > CG). Consistently, the enzymatic activity of the base excision repair protein APE-1 is induced at only medium or high doses. Collectively, the data reveal that a threshold of endogenous stress must be reached to trigger cellular detoxification and DNA repair programs; below this threshold, the consequences of endogenous stress escape cellular surveillance, leading to high levels of mutagenesis. Therefore, low doses of endogenous local stress can jeopardize genome integrity more efficiently than higher doses. PMID:27406380

  15. Targeted mutagenesis of an odorant receptor co-receptor using TALEN in Ostrinia furnacalis.

    PubMed

    Yang, Bin; Fujii, Takeshi; Ishikawa, Yukio; Matsuo, Takashi

    2016-03-01

    Genome editing using transcription activator-like effector nuclease (TALEN) has been applied for various model organisms but not yet for agricultural pest insects. In this study, TALEN-mediated mutagenesis of the gene encoding odorant receptor co-receptor (Orco) of an important agricultural pest Ostrinia furnacalis (OfurOrco) was carried out. Of the two pairs of TALEN constructs designed, one generated somatic and germline mutations at rates of 70.8% and 20.8%, respectively. Physiological and behavioral analyses using a gas chromatograph-electroantennographic detector system and a wind tunnel, respectively, revealed that antennal responses to sex pheromone components were decreased to trace levels, and behavioral responses were abolished in OfurOrco mutants. This study demonstrated that TALEN-mediated mutagenesis is applicable to pest insects, and these results will open the way for a better understanding of chemosensory systems in wild insects. PMID:26689645

  16. Heat shock and herpes virus: enhanced reactivation without untargeted mutagenesis

    SciTech Connect

    Lytle, C.D.; Carney, P.G.

    1988-01-01

    Enhanced reactivation of Ultraviolet-irradiated virus has been reported to occur in heat-shocked host cells. Since enhanced virus reactivation is often accompanied by untargeted mutagenesis, we investigated whether such mutagenesis would occur for herpes simplex virus (HSV) in CV-1 monkey kidney cells subjected to heat shock. In addition to expressing enhanced reactivation, the treated cells were transiently more susceptible to infection by unirradiated HSV. No mutagenesis of unirradiated HSV was found whether infection occurred at the time of increased susceptibility to infection or during expression of enhanced viral reactivation.

  17. Protein engineering: single or multiple site-directed mutagenesis.

    PubMed

    Hsieh, Pei-Chung; Vaisvila, Romualdas

    2013-01-01

    Site-directed mutagenesis techniques are invaluable tools in molecular biology to study the structural and functional properties of a protein. To expedite the time required and simplify methods for mutagenesis, we recommend two protocols in this chapter. The first method for single site-directed mutagenesis, which includes point mutations, insertions, or deletions, can be achieved by an inverse PCR strategy with mutagenic primers and the high-fidelity Phusion(®) DNA Polymerase to introduce a site-directed mutation with exceptional efficiency. The second method is for engineering multiple mutations into a gene of interest. This can be completed in one step by PCR with mutagenic primers and by assembling all mutagenized PCR products using the Gibson Assembly™ Master Mix. This method allows multiple nucleotides to be changed simultaneously, which not only saves time but also reagents compared to traditional methods of mutagenesis. PMID:23423897

  18. Symposium on molecular and cellular mechanisms of mutagenesis

    SciTech Connect

    Not Available

    1981-01-01

    These proceedings contain abstracts only of the 21 papers presented at the Sympsoium. The papers dealt with molecular mechanisms of mutagenesis and cellular responses to chemical and physical mutagenic agents. (ERB)

  19. Mutagenesis during plant responses to UVB radiation.

    PubMed

    Holá, M; Vágnerová, R; Angelis, K J

    2015-08-01

    We tested an idea that induced mutagenesis due to unrepaired DNA lesions, here the UV photoproducts, underlies the impact of UVB irradiation on plant phenotype. For this purpose we used protonemal culture of the moss Physcomitrella patens with 50% of apical cells, which mimics actively growing tissue, the most vulnerable stage for the induction of mutations. We measured the UVB mutation rate of various moss lines with defects in DNA repair (pplig4, ppku70, pprad50, ppmre11), and in selected clones resistant to 2-Fluoroadenine, which were mutated in the adenosine phosphotrasferase gene (APT), we analysed induced mutations by sequencing. In parallel we followed DNA break repair and removal of cyclobutane pyrimidine dimers with a half-life τ = 4 h 14 min determined by comet assay combined with UV dimer specific T4 endonuclease V. We show that UVB induces massive, sequence specific, error-prone bypass repair that is responsible for a high mutation rate owing to relatively slow, though error-free, removal of photoproducts by nucleotide excision repair (NER). PMID:25542779

  20. Codon compression algorithms for saturation mutagenesis.

    PubMed

    Pines, Gur; Pines, Assaf; Garst, Andrew D; Zeitoun, Ramsey I; Lynch, Sean A; Gill, Ryan T

    2015-05-15

    Saturation mutagenesis is employed in protein engineering and genome-editing efforts to generate libraries that span amino acid design space. Traditionally, this is accomplished by using degenerate/compressed codons such as NNK (N = A/C/G/T, K = G/T), which covers all amino acids and one stop codon. These solutions suffer from two types of redundancy: (a) different codons for the same amino acid lead to bias, and (b) wild type amino acid is included within the library. These redundancies increase library size and downstream screening efforts. Here, we present a dynamic approach to compress codons for any desired list of amino acids, taking into account codon usage. This results in a unique codon collection for every amino acid to be mutated, with the desired redundancy level. Finally, we demonstrate that this approach can be used to design precise oligo libraries amendable to recombineering and CRISPR-based genome editing to obtain a diverse population with high efficiency. PMID:25303315

  1. Signature-tagged mutagenesis of Vibrio vulnificus

    PubMed Central

    YAMAMOTO, Mai; KASHIMOTO, Takashige; TONG, Ping; XIAO, Jianbo; SUGIYAMA, Michiko; INOUE, Miyuki; MATSUNAGA, Rie; HOSOHARA, Kohei; NAKATA, Kazue; YOKOTA, Kenji; OGUMA, Keiji; YAMAMOTO, Koichiro

    2015-01-01

    Vibrio vulnificus is the causative agent of primary septicemia, wound infection and gastroenteritis in immunocompromised people. In this study, signature-tagged mutagenesis (STM) was applied to identify the virulence genes of V. vulnificus. Using STM, 6,480 mutants in total were constructed and divided into 81 sets (INPUT pools); each mutant in a set was assigned a different tag. Each INPUT pool was intraperitoneally injected into iron-overloaded mice, and in vivo surviving mutants were collected from blood samples from the heart (OUTPUT pools). From the genomic DNA of mixed INPUT or OUTPUT pools, digoxigenin-labeled DNA probes against the tagged region were prepared and used for dot hybridization. Thirty tentatively attenuated mutants, which were hybridized clearly with INPUT probes but barely with OUTPUT probes, were negatively selected. Lethal doses of 11 of the 30 mutants were reduced to more than 1/100; of these, the lethal doses of 2 were reduced to as low as 1/100,000. Transposon-inserted genes in the 11 attenuated mutants were those for IMP dehydrogenase, UDP-N-acetylglucosamine-2-epimerase, aspartokinase, phosphoribosylformylglycinamidine cyclo-ligase, malate Na (+) symporter and hypothetical protein. When mice were immunized with an attenuated mutant strain into which IMP dehydrogenase had been inserted with a transposon, they were protected against V. vulnificus infection. In this study, we demonstrated that the STM method can be used to search for the virulence genes of V. vulnificus. PMID:25755021

  2. Isolation of rare recombinants without using selectable markers for one-step seamless BAC mutagenesis

    PubMed Central

    Lyozin, George T.; Kosaka, Yasuhiro; Demarest, Bradley L.; Yost, H. Joseph; Kuehn, Michael R.; Brunelli, Luca

    2014-01-01

    Current laboratory methods to isolate rare (1:10,000 to 1:100,000) bacterial artificial chromosome (BAC) recombinants require selectable markers. Seamless BAC mutagenesis needs two steps: isolation of rare recombinants using selectable markers, followed by marker removal through counterselection. Here we illustrate founder principle-driven enrichment (FPE), a simple method developed to rapidly isolate rare recombinants without using selectable markers, allowing one-step seamless BAC mutagenesis. As proof-of-principle, we isolated 1:100,000 seamless fluorescent protein-modified Nodal BACs via FPE and confirmed BAC functionality by generating fluorescent reporter mice. We also isolated small indel P1-phage derived artificial chromosome (PAC) and BAC recombinants. Statistical analysis revealed that 1:100,000 recombinants can be isolated running <40 PCRs and we developed a web-based calculator to optimize FPE. By eliminating the need for selection-counterselection, this work highlights a straightforward and low-cost approach to BAC mutagenesis, providing a tool for seamless recombineering pipelines in functional genomics. PMID:25028895

  3. Mouse ENU Mutagenesis to Understand Immunity to Infection: Methods, Selected Examples, and Perspectives

    PubMed Central

    Caignard, Grégory; Eva, Megan M.; van Bruggen, Rebekah; Eveleigh, Robert; Bourque, Guillaume; Malo, Danielle; Gros, Philippe; Vidal, Silvia M.

    2014-01-01

    Infectious diseases are responsible for over 25% of deaths globally, but many more individuals are exposed to deadly pathogens. The outcome of infection results from a set of diverse factors including pathogen virulence factors, the environment, and the genetic make-up of the host. The completion of the human reference genome sequence in 2004 along with technological advances have tremendously accelerated and renovated the tools to study the genetic etiology of infectious diseases in humans and its best characterized mammalian model, the mouse. Advancements in mouse genomic resources have accelerated genome-wide functional approaches, such as gene-driven and phenotype-driven mutagenesis, bringing to the fore the use of mouse models that reproduce accurately many aspects of the pathogenesis of human infectious diseases. Treatment with the mutagen N-ethyl-N-nitrosourea (ENU) has become the most popular phenotype-driven approach. Our team and others have employed mouse ENU mutagenesis to identify host genes that directly impact susceptibility to pathogens of global significance. In this review, we first describe the strategies and tools used in mouse genetics to understand immunity to infection with special emphasis on chemical mutagenesis of the mouse germ-line together with current strategies to efficiently identify functional mutations using next generation sequencing. Then, we highlight illustrative examples of genes, proteins, and cellular signatures that have been revealed by ENU screens and have been shown to be involved in susceptibility or resistance to infectious diseases caused by parasites, bacteria, and viruses. PMID:25268389

  4. PiggyBac transposon mutagenesis: a tool for cancer gene discovery in mice.

    PubMed

    Rad, Roland; Rad, Lena; Wang, Wei; Cadinanos, Juan; Vassiliou, George; Rice, Stephen; Campos, Lia S; Yusa, Kosuke; Banerjee, Ruby; Li, Meng Amy; de la Rosa, Jorge; Strong, Alexander; Lu, Dong; Ellis, Peter; Conte, Nathalie; Yang, Fang Tang; Liu, Pentao; Bradley, Allan

    2010-11-19

    Transposons are mobile DNA segments that can disrupt gene function by inserting in or near genes. Here, we show that insertional mutagenesis by the PiggyBac transposon can be used for cancer gene discovery in mice. PiggyBac transposition in genetically engineered transposon-transposase mice induced cancers whose type (hematopoietic versus solid) and latency were dependent on the regulatory elements introduced into transposons. Analysis of 63 hematopoietic tumors revealed that PiggyBac is capable of genome-wide mutagenesis. The PiggyBac screen uncovered many cancer genes not identified in previous retroviral or Sleeping Beauty transposon screens, including Spic, which encodes a PU.1-related transcription factor, and Hdac7, a histone deacetylase gene. PiggyBac and Sleeping Beauty have different integration preferences. To maximize the utility of the tool, we engineered 21 mouse lines to be compatible with both transposon systems in constitutive, tissue- or temporal-specific mutagenesis. Mice with different transposon types, copy numbers, and chromosomal locations support wide applicability. PMID:20947725

  5. CRISPR/Cas9-mediated targeted gene mutagenesis in Spodoptera litura.

    PubMed

    Bi, Hong-Lun; Xu, Jun; Tan, An-Jiang; Huang, Yong-Ping

    2016-06-01

    Custom-designed nuclease technologies such as the clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) system provide attractive genome editing tools for insect functional genetics. The targeted gene mutagenesis mediated by the CRISPR/Cas9 system has been achieved in several insect orders including Diptera, Lepidoptera and Coleoptera. However, little success has been reported in agricultural pests due to the lack of genomic information and embryonic microinjection techniques in these insect species. Here we report that the CRISPR/Cas9 system induced efficient gene mutagenesis in an important Lepidopteran pest Spodoptera litura. We targeted the S. litura Abdominal-A (Slabd-A) gene which is an important embryonic development gene and plays a significant role in determining the identities of the abdominal segments of insects. Direct injection of Cas9 messenger RNA and Slabd-A-specific single guide RNA (sgRNA) into S. litura embryos successfully induced the typical abd-A deficient phenotype, which shows anomalous segmentation and ectopic pigmentation during the larval stage. A polymerase chain reaction-based analysis revealed that the Cas9/sgRNA complex effectively induced a targeted mutagenesis in S. litura. These results demonstrate that the CRISPR/Cas9 system is a powerful tool for genome manipulation in Lepidopteran pests such as S. litura. PMID:27061764

  6. Sleeping Beauty mutagenesis in a mouse medulloblastoma model defines networks that discriminate between human molecular subgroups.

    PubMed

    Genovesi, Laura A; Ng, Ching Ging; Davis, Melissa J; Remke, Marc; Taylor, Michael D; Adams, David J; Rust, Alistair G; Ward, Jerrold M; Ban, Kenneth H; Jenkins, Nancy A; Copeland, Neal G; Wainwright, Brandon J

    2013-11-12

    The Sleeping Beauty (SB) transposon mutagenesis screen is a powerful tool to facilitate the discovery of cancer genes that drive tumorigenesis in mouse models. In this study, we sought to identify genes that functionally cooperate with sonic hedgehog signaling to initiate medulloblastoma (MB), a tumor of the cerebellum. By combining SB mutagenesis with Patched1 heterozygous mice (Ptch1(lacZ/+)), we observed an increased frequency of MB and decreased tumor-free survival compared with Ptch1(lacZ/+) controls. From an analysis of 85 tumors, we identified 77 common insertion sites that map to 56 genes potentially driving increased tumorigenesis. The common insertion site genes identified in the mutagenesis screen were mapped to human orthologs, which were used to select probes and corresponding expression data from an independent set of previously described human MB samples, and surprisingly were capable of accurately clustering known molecular subgroups of MB, thereby defining common regulatory networks underlying all forms of MB irrespective of subgroup. We performed a network analysis to discover the likely mechanisms of action of subnetworks and used an in vivo model to confirm a role for a highly ranked candidate gene, Nfia, in promoting MB formation. Our analysis implicates candidate cancer genes in the deregulation of apoptosis and translational elongation, and reveals a strong signature of transcriptional regulation that will have broad impact on expression programs in MB. These networks provide functional insights into the complex biology of human MB and identify potential avenues for intervention common to all clinical subgroups. PMID:24167280

  7. Sleeping Beauty mutagenesis in a mouse medulloblastoma model defines networks that discriminate between human molecular subgroups

    PubMed Central

    Genovesi, Laura A.; Ng, Ching Ging; Davis, Melissa J.; Remke, Marc; Taylor, Michael D.; Adams, David J.; Rust, Alistair G.; Ward, Jerrold M.; Ban, Kenneth H.; Jenkins, Nancy A.; Copeland, Neal G.; Wainwright, Brandon J.

    2013-01-01

    The Sleeping Beauty (SB) transposon mutagenesis screen is a powerful tool to facilitate the discovery of cancer genes that drive tumorigenesis in mouse models. In this study, we sought to identify genes that functionally cooperate with sonic hedgehog signaling to initiate medulloblastoma (MB), a tumor of the cerebellum. By combining SB mutagenesis with Patched1 heterozygous mice (Ptch1lacZ/+), we observed an increased frequency of MB and decreased tumor-free survival compared with Ptch1lacZ/+ controls. From an analysis of 85 tumors, we identified 77 common insertion sites that map to 56 genes potentially driving increased tumorigenesis. The common insertion site genes identified in the mutagenesis screen were mapped to human orthologs, which were used to select probes and corresponding expression data from an independent set of previously described human MB samples, and surprisingly were capable of accurately clustering known molecular subgroups of MB, thereby defining common regulatory networks underlying all forms of MB irrespective of subgroup. We performed a network analysis to discover the likely mechanisms of action of subnetworks and used an in vivo model to confirm a role for a highly ranked candidate gene, Nfia, in promoting MB formation. Our analysis implicates candidate cancer genes in the deregulation of apoptosis and translational elongation, and reveals a strong signature of transcriptional regulation that will have broad impact on expression programs in MB. These networks provide functional insights into the complex biology of human MB and identify potential avenues for intervention common to all clinical subgroups. PMID:24167280

  8. Induction of Pectinase Hyper Production by Multistep Mutagenesis Using a Fungal Isolate--Aspergillus flavipes.

    PubMed

    Akbar, Sabika; Prasuna, R Gyana; Khanam, Rasheeda

    2014-04-01

    Aspergillus flavipes, a slow growing pectinase producing ascomycete, was isolated from soil identified and characterised in the previously done preliminary studies. Optimisation studies revealed that Citrus peel--groundnut oil cake [CG] production media is the best media for production of high levels of pectinase up to 39 U/ml using wild strain of A. flavipes. Strain improvement of this isolated strain for enhancement of pectinase production using multistep mutagenesis procedure is the endeavour of this project. For this, the wild strain of A. flavipes was treated with both physical (UV irradiation) and chemical [Colchicine, Ethidium bromide, H2O2] mutagens to obtain Ist generation mutants. The obtained mutants were assayed and differentiated basing on pectinase productivity. The better pectinase producing strains were further subjected to multistep mutagenesis to attain stability in mutants. The goal of this project was achieved by obtaining the best pectinase secreting mutant, UV80 of 45 U/ml compared to wild strain and sister mutants. This fact was confirmed by quantitatively analysing 3rd generation mutants obtained after multistep mutagenesis. PMID:26563068

  9. Cationic Antimicrobial Peptides Promote Microbial Mutagenesis and Pathoadaptation in Chronic Infections

    PubMed Central

    Limoli, Dominique H.; Rockel, Andrea B.; Host, Kurtis M.; Jha, Anuvrat; Kopp, Benjamin T.; Hollis, Thomas; Wozniak, Daniel J.

    2014-01-01

    Acquisition of adaptive mutations is essential for microbial persistence during chronic infections. This is particularly evident during chronic Pseudomonas aeruginosa lung infections in cystic fibrosis (CF) patients. Thus far, mutagenesis has been attributed to the generation of reactive species by polymorphonucleocytes (PMN) and antibiotic treatment. However, our current studies of mutagenesis leading to P. aeruginosa mucoid conversion have revealed a potential new mutagen. Our findings confirmed the current view that reactive oxygen species can promote mucoidy in vitro, but revealed PMNs are proficient at inducing mucoid conversion in the absence of an oxidative burst. This led to the discovery that cationic antimicrobial peptides can be mutagenic and promote mucoidy. Of specific interest was the human cathelicidin LL-37, canonically known to disrupt bacterial membranes leading to cell death. An alternative role was revealed at sub-inhibitory concentrations, where LL-37 was found to induce mutations within the mucA gene encoding a negative regulator of mucoidy and to promote rifampin resistance in both P. aeruginosa and Escherichia coli. The mechanism of mutagenesis was found to be dependent upon sub-inhibitory concentrations of LL-37 entering the bacterial cytosol and binding to DNA. LL-37/DNA interactions then promote translesion DNA synthesis by the polymerase DinB, whose error-prone replication potentiates the mutations. A model of LL-37 bound to DNA was generated, which reveals amino termini α-helices of dimerized LL-37 bind the major groove of DNA, with numerous DNA contacts made by LL-37 basic residues. This demonstrates a mutagenic role for antimicrobials previously thought to be insusceptible to resistance by mutation, highlighting a need to further investigate their role in evolution and pathoadaptation in chronic infections. PMID:24763694

  10. [Rapid site-directed mutagenesis on full-length plasmid DNA by using designed restriction enzyme assisted mutagenesis].

    PubMed

    Zhang, Baozhong; Ran, Duoliang; Zhang, Xin; An, Xiaoping; Shan, Yunzhu; Zhou, Yusen; Tong, Yigang

    2009-02-01

    To use the designed restriction enzyme assisted mutagenesis technique to perform rapid site-directed mutagenesis on double-stranded plasmid DNA. The target amino acid sequence was reversely translated into DNA sequences with degenerate codons, resulting in large amount of silently mutated sequences containing various restriction endonucleases (REs). Certain mutated sequence with an appropriate RE was selected as the target DNA sequence for designing mutation primers. The full-length plasmid DNA was amplified with high-fidelity Phusion DNA polymerase and the amplified product was 5' phosphorylated by T4 polynucleotide kinase and then self-ligated. After transformation into an E. coli host the transformants were rapidly screened by cutting with the designed RE. With this strategy we successfully performed the site-directed mutagenesis on an 8 kb plasmid pcDNA3.1-pIgR and recovered the wild-type amino acid sequence of human polymeric immunoglobulin receptor (pIgR). A novel site-directed mutagenesis strategy based on DREAM was developed which exploited RE as a rapid screening measure. The highly efficient, high-fidelity Phusion DNA polymerase was applied to ensure the efficient and faithful amplification of the full-length sequence of a plasmid of up to 8 kb. This rapid mutagenesis strategy avoids using any commercial site-directed mutagenesis kits, special host strains or isotopes. PMID:19459340

  11. 2012 MUTAGENESIS GORDON RESEARCH CONFERENCE, AUGUST 19-23, 2012

    SciTech Connect

    Demple, Bruce

    2012-08-23

    The delicate balance among cellular pathways that control mutagenic changes in DNA will be the focus of the 2012 Mutagenesis Gordon Research Conference. Mutagenesis is essential for evolution, while genetic stability maintains cellular functions in all organisms from microbes to metazoans. Different systems handle DNA lesions at various times of the cell cycle and in different places within the nucleus, and inappropriate actions can lead to mutations. While mutation in humans is closely linked to disease, notably cancers, mutational systems can also be beneficial. The conference will highlight topics of beneficial mutagenesis, including full establishment of the immune system, cell survival mechanisms, and evolution and adaptation in microbial systems. Equal prominence will be given to detrimental mutation processes, especially those involved in driving cancer, neurological diseases, premature aging, and other threats to human health. Provisional session titles include Branching Pathways in Mutagenesis; Oxidative Stress and Endogenous DNA Damage; DNA Maintenance Pathways; Recombination, Good and Bad; Problematic DNA Structures; Localized Mutagenesis; Hypermutation in the Microbial World; and Mutation and Disease.

  12. Graphene oxide can induce in vitro and in vivo mutagenesis

    NASA Astrophysics Data System (ADS)

    Liu, Yuanyuan; Luo, Yi; Wu, Jing; Wang, Yinsong; Yang, Xiaoying; Yang, Rui; Wang, Baiqi; Yang, Jinrong; Zhang, Ning

    2013-12-01

    Graphene oxide (GO) has attracted enormous interests due to its extraordinary properties. Recent studies have confirmed the cytotoxicity of GO, we further investigate its mutagenic potential in this study. The results showed that GO interfered with DNA replication and induced mutagenesis at molecular level. GO treatments at concentrations of 10 and 100 μg/mL altered gene expression patterns at cellular level, and 101 differentially expressed genes mediated DNA-damage control, cell apoptosis, cell cycle, and metabolism. Intravenous injection of GO at 4 mg/kg for 5 consecutive days clearly induced formation of micronucleated polychromic erythrocytes in mice, and its mutagenesis potential appeared to be comparable to cyclophosphamide, a classic mutagen. In conclusion, GO can induce mutagenesis both in vitro and in vivo, thus extra consideration is required for its biomedical applications.

  13. An algorithm for protein engineering: simulations of recursive ensemble mutagenesis.

    PubMed Central

    Arkin, A P; Youvan, D C

    1992-01-01

    An algorithm for protein engineering, termed recursive ensemble mutagenesis, has been developed to produce diverse populations of phenotypically related mutants whose members differ in amino acid sequence. This method uses a feedback mechanism to control successive rounds of combinatorial cassette mutagenesis. Starting from partially randomized "wild-type" DNA sequences, a highly parallel search of sequence space for peptides fitting an experimenter's criteria is performed. Each iteration uses information gained from the previous rounds to search the space more efficiently. Simulations of the technique indicate that, under a variety of conditions, the algorithm can rapidly produce a diverse population of proteins fitting specific criteria. In the experimental analog, genetic selection or screening applied during recursive ensemble mutagenesis should force the evolution of an ensemble of mutants to a targeted cluster of related phenotypes. Images PMID:1502200

  14. A computer program to display codon changes caused by mutagenesis.

    PubMed

    Sirotkin, K

    1988-04-01

    A FORTRAN program for displaying the correspondence between codon changes and different possible base changes is presented. Changes of both single bases and dimers are considered. The user can specify the mutagenesis spectrum. Additionally, the user can choose whether or not to consider single or double events in a codon and whether or not to consider the possibility that the change of two bases (a dimer) can overlap a codon boundary. Furthermore, a variety of ways may be chosen to display and summarize the codon changes that can result from the specified mutagenesis. A user-supplied sequence or the genetic code table can be analyzed. PMID:3167596

  15. Mutagenesis protocols in Saccharomyces cerevisiae by in vivo overlap extension.

    PubMed

    Alcalde, Miguel

    2010-01-01

    A high recombination frequency and its ease of manipulation has made Saccharomyces cerevisiae a unique model eukaryotic organism to study homologous recombination. Indeed, the well-developed recombination machinery in S. cerevisiae facilitates the construction of mutant libraries for directed evolution experiments. In this context, in vivo overlap extension (IVOE) is a particularly attractive protocol that takes advantage of the eukaryotic apparatus to carry out combinatorial saturation mutagenesis, site-directed recombination or site-directed mutagenesis, avoiding ligation steps and additional PCR reactions that are common to standard in vitro protocols. PMID:20676972

  16. Production and Screening of High Yield Avermectin B1b Mutant of Streptomyces avermitilis 41445 Through Mutagenesis

    PubMed Central

    Siddique, Samia; Syed, Quratulain; Adnan, Ahmad; Qureshi, Fahim Ashraf

    2014-01-01

    Background: Secondary metabolite production from wild strains is very low for economical purpose therefore certain strain improvement strategies are required to achieve hundred times greater yield of metabolites. Most important strain improvement techniques include physical and chemical mutagenesis. Broad spectrum mutagenesis through UV irradiation is the most important and convenient physical method. Objectives: The present study was conducted for enhanced production of avermectin B1b from Streptomyces avermitilis 41445 by mutagenesis using ultraviolet (UV) radiation, ethidium bromide (EB), and ethyl methanesulfonate (EMS) as mutagens. Materials and Methods: S. avermitilis DSM 41445 maintained on yeast extract malt extract glucose medium (YMG) was used as inoculum for SM2 fermentation medium. Spores of S. avermitilis DSM 41445 were exposed to UV radiation for physical broad spectrum mutagenesis and to EMS and EB for chemical mutagenesis. For each mutagen, the lethality rate and mutation rate were calculated along with positive mutation rate. Results: Avermectin B1b-hyper-producing mutant, produced using these three different methods, was selected according to the HPLC results. The mutant obtained after 45 minutes of UV radiation to the spores of S. avermitilis 41445, was found to be the best mutant for the enhanced production of avermectin B1b component (254.14 mg/L). Other avermectin B1b-hyper-producing mutants, were obtained from EMS (1 µL/mL) and EB (30 µL/mL) treatments, and yielded 202.63 mg/L and 199.30 mg/L of B1b, respectively. Conclusions: The hereditary stability analysis of the UV mentioning 45 minutes revealed the UV exposure time for mutants and 3 represented the colony taken from the plate irradiated for 45 minutes mutant showed that the production of avermectin B1b remained constant and no reverse mutation occurred after 15 generations. PMID:25147669

  17. Site-targeted mutagenesis for stabilization of recombinant monoclonal antibody expressed in tobacco (Nicotiana tabacum) plants

    PubMed Central

    Hehle, Verena K.; Paul, Matthew J.; Roberts, Victoria A.; van Dolleweerd, Craig J.; Ma, Julian K.-C.

    2016-01-01

    This study examined the degradation pattern of a murine IgG1κ monoclonal antibody expressed in and extracted from transformed Nicotiana tabacum. Gel electrophoresis of leaf extracts revealed a consistent pattern of recombinant immunoglobulin bands, including intact and full-length antibody, as well as smaller antibody fragments. N-terminal sequencing revealed these smaller fragments to be proteolytic cleavage products and identified a limited number of protease-sensitive sites in the antibody light and heavy chain sequences. No strictly conserved target sequence was evident, although the peptide bonds that were susceptible to proteolysis were predominantly and consistently located within or near to the interdomain or solvent-exposed regions in the antibody structure. Amino acids surrounding identified cleavage sites were mutated in an attempt to increase resistance. Different Guy’s 13 antibody heavy and light chain mutant combinations were expressed transiently in N. tabacum and demonstrated intensity shifts in the fragmentation pattern, resulting in alterations to the full-length antibody-to-fragment ratio. The work strengthens the understanding of proteolytic cleavage of antibodies expressed in plants and presents a novel approach to stabilize full-length antibody by site-directed mutagenesis.—Hehle, V. K., Paul, M. J., Roberts, V. A., van Dolleweerd, C. J., Ma, J. K.-C. Site-targeted mutagenesis for stabilization of recombinant monoclonal antibody expressed in tobacco (Nicotiana tabacum) plants. PMID:26712217

  18. Site-targeted mutagenesis for stabilization of recombinant monoclonal antibody expressed in tobacco (Nicotiana tabacum) plants.

    PubMed

    Hehle, Verena K; Paul, Matthew J; Roberts, Victoria A; van Dolleweerd, Craig J; Ma, Julian K-C

    2016-04-01

    This study examined the degradation pattern of a murine IgG1κ monoclonal antibody expressed in and extracted from transformedNicotiana tabacum Gel electrophoresis of leaf extracts revealed a consistent pattern of recombinant immunoglobulin bands, including intact and full-length antibody, as well as smaller antibody fragments. N-terminal sequencing revealed these smaller fragments to be proteolytic cleavage products and identified a limited number of protease-sensitive sites in the antibody light and heavy chain sequences. No strictly conserved target sequence was evident, although the peptide bonds that were susceptible to proteolysis were predominantly and consistently located within or near to the interdomain or solvent-exposed regions in the antibody structure. Amino acids surrounding identified cleavage sites were mutated in an attempt to increase resistance. Different Guy's 13 antibody heavy and light chain mutant combinations were expressed transiently inN. tabacumand demonstrated intensity shifts in the fragmentation pattern, resulting in alterations to the full-length antibody-to-fragment ratio. The work strengthens the understanding of proteolytic cleavage of antibodies expressed in plants and presents a novel approach to stabilize full-length antibody by site-directed mutagenesis.-Hehle, V. K., Paul, M. J., Roberts, V. A., van Dolleweerd, C. J., Ma, J. K.-C. Site-targeted mutagenesis for stabilization of recombinant monoclonal antibody expressed in tobacco (Nicotiana tabacum) plants. PMID:26712217

  19. Insertional mutagenesis using Tnt1 retrotransposon in potato

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Potato is the third most important food crop in the world. However, genetics and genomics research of potato has lagged behind many major crop species due to its autotetraploidy and a highly heterogeneous genome. Insertional mutagenesis using T-DNA or transposable elements, which is available in sev...

  20. Methods for targetted mutagenesis in gram-positive bacteria

    SciTech Connect

    Yang, Yunfeng

    2014-05-27

    The present invention provides a method of targeted mutagenesis in Gram-positive bacteria. In particular, the present invention provides a method that effectively integrates a suicide integrative vector into a target gene in the chromosome of a Gram-positive bacterium, resulting in inactivation of the target gene.

  1. Coupled mutagenesis screens and genetic mapping in zebrafish.

    PubMed Central

    Rawls, John F; Frieda, Matthew R; McAdow, Anthony R; Gross, Jason P; Clayton, Chad M; Heyen, Candy K; Johnson, Stephen L

    2003-01-01

    Forward genetic analysis is one of the principal advantages of the zebrafish model system. However, managing zebrafish mutant lines derived from mutagenesis screens and mapping the corresponding mutations and integrating them into the larger collection of mutations remain arduous tasks. To simplify and focus these endeavors, we developed an approach that facilitates the rapid mapping of new zebrafish mutations as they are generated through mutagenesis screens. We selected a minimal panel of 149 simple sequence length polymorphism markers for a first-pass genome scan in crosses involving C32 and SJD inbred lines. We also conducted a small chemical mutagenesis screen that identified several new mutations affecting zebrafish embryonic melanocyte development. Using our first-pass marker panel in bulked-segregant analysis, we were able to identify the genetic map positions of these mutations as they were isolated in our screen. Rapid mapping of the mutations facilitated stock management, helped direct allelism tests, and should accelerate identification of the affected genes. These results demonstrate the efficacy of coupling mutagenesis screens with genetic mapping. PMID:12663538

  2. 2002 Gordon Research Conference on Mutagenesis. Final Progress Report

    SciTech Connect

    2002-08-02

    The Gordon Research Conference (GRC) on MUTAGENESIS was held at Bates College from 7/28/02 thru 8/2/02. The Conference was well-attended with 157 participants. The attendees represented the spectrum of endeavor in this field coming from academia, industry, and government laboratories, both U.S. and foreign scientists, senior researchers, young investigators, and students.

  3. Transposon mutagenesis of Bacteroides fragilis using a mariner transposon vector.

    PubMed

    Veeranagouda, Yaligara; Husain, Fasahath; Wexler, Hannah M

    2013-08-01

    The mariner transposon vector pYV07 was tested for use in the mutagenesis of Bacteroides fragilis 638R. The transposon vector efficiently generated mutants in B. fragilis 638R. The transposon disrupted genes were scattered throughout the genome of B. fragilis 638R. This method serves as a powerful tool to study B. fragilis. PMID:23664906

  4. CRISPR/Cas9-targeted mutagenesis in Caenorhabditis elegans.

    PubMed

    Waaijers, Selma; Portegijs, Vincent; Kerver, Jana; Lemmens, Bennie B L G; Tijsterman, Marcel; van den Heuvel, Sander; Boxem, Mike

    2013-11-01

    The generation of genetic mutants in Caenorhabditis elegans has long relied on the selection of mutations in large-scale screens. Directed mutagenesis of specific loci in the genome would greatly speed up analysis of gene function. Here, we adapt the CRISPR/Cas9 system to generate mutations at specific sites in the C. elegans genome. PMID:23979586

  5. What Can a Micronucleus Teach? Learning about Environmental Mutagenesis

    ERIC Educational Resources Information Center

    Linde, Ana R.; Garcia-Vazquez, Eva

    2009-01-01

    The micronucleus test is widely employed in environmental health research. It can also be an excellent tool for learning important concepts in environmental health. In this article we present an inquiry-based laboratory exercise where students explore several theoretical and practical aspects of environmental mutagenesis employing the micronucleus…

  6. Favipiravir elicits antiviral mutagenesis during virus replication in vivo

    PubMed Central

    Arias, Armando; Thorne, Lucy; Goodfellow, Ian

    2014-01-01

    Lethal mutagenesis has emerged as a novel potential therapeutic approach to treat viral infections. Several studies have demonstrated that increases in the high mutation rates inherent to RNA viruses lead to viral extinction in cell culture, but evidence during infections in vivo is limited. In this study, we show that the broad-range antiviral nucleoside favipiravir reduces viral load in vivo by exerting antiviral mutagenesis in a mouse model for norovirus infection. Increased mutation frequencies were observed in samples from treated mice and were accompanied with lower or in some cases undetectable levels of infectious virus in faeces and tissues. Viral RNA isolated from treated animals showed reduced infectivity, a feature of populations approaching extinction during antiviral mutagenesis. These results suggest that favipiravir can induce norovirus mutagenesis in vivo, which in some cases leads to virus extinction, providing a proof-of-principle for the use of favipiravir derivatives or mutagenic nucleosides in the clinical treatment of noroviruses. DOI: http://dx.doi.org/10.7554/eLife.03679.001 PMID:25333492

  7. Targeted mutagenesis in mammalian cells mediated by intracellular triple helix formation.

    PubMed Central

    Wang, G; Levy, D D; Seidman, M M; Glazer, P M

    1995-01-01

    As an alternative to standard gene transfer techniques for genetic manipulation, we have investigated the use of triple helix-forming oligonucleotides to target mutations to selected genes within mammalian cells. By treating monkey COS cells with oligonucleotides linked to psoralen, we have generated targeted mutations in a simian virus 40 (SV40) vector contained within the cells via intracellular triple helix formation. Oligonucleotide entry into the cells and sequence-specific triplex formation within the SV40 DNA deliver the psoralen to the targeted site. Photoactivation of the psoralen by long-wavelength UV light yields adducts and thereby mutations at that site. We engineered into the SV40 vector novel supF mutation reporter genes containing modified polypurine sites amenable to triplex formation. By comparing the abilities of a series of oligonucleotides to target these new sites, we show that targeted mutagenesis in vivo depends on the strength and specificity of the third-strand binding. Oligonucleotides with weak target site binding affinity or with only partial target site homology were ineffective at inducing mutations in the SV40 vectors within the COS cells. We also show that the targeted mutagenesis is dependent on the oligonucleotide concentration and is influenced by the timing of the oligonucleotide treatment and of the UV irradiation of the cells. Frequencies of intracellular targeted mutagenesis in the range of 1 to 2% were observed, depending upon the conditions of the experiment. DNA sequence analysis revealed that most of the mutations were T.A-to-A.T transversions precisely at the targeted psoralen intercalation site. Several deletions encompassing that site were also seen. The ability to target mutations to selected sites within mammalian cells by using modified triplex-forming oligonucleotides may provide a new research tool and may eventually lead to therapeutic applications. PMID:7862165

  8. Illegitimate recombination: An efficient method for random mutagenesis in Mycobacterium avium subsp. hominissuis

    PubMed Central

    2012-01-01

    Background The genus Mycobacterium (M.) comprises highly pathogenic bacteria such as M. tuberculosis as well as environmental opportunistic bacteria called non-tuberculous mycobacteria (NTM). While the incidence of tuberculosis is declining in the developed world, infection rates by NTM are increasing. NTM are ubiquitous and have been isolated from soil, natural water sources, tap water, biofilms, aerosols, dust and sawdust. Lung infections as well as lymphadenitis are most often caused by M. avium subsp. hominissuis (MAH), which is considered to be among the clinically most important NTM. Only few virulence genes from M. avium have been defined among other things due to difficulties in generating M. avium mutants. More efforts in developing new methods for mutagenesis of M. avium and identification of virulence-associated genes are therefore needed. Results We developed a random mutagenesis method based on illegitimate recombination and integration of a Hygromycin-resistance marker. Screening for mutations possibly affecting virulence was performed by monitoring of pH resistance, colony morphology, cytokine induction in infected macrophages and intracellular persistence. Out of 50 randomly chosen Hygromycin-resistant colonies, four revealed to be affected in virulence-related traits. The mutated genes were MAV_4334 (nitroreductase family protein), MAV_5106 (phosphoenolpyruvate carboxykinase), MAV_1778 (GTP-binding protein LepA) and MAV_3128 (lysyl-tRNA synthetase LysS). Conclusions We established a random mutagenesis method for MAH that can be easily carried out and combined it with a set of phenotypic screening methods for the identification of virulence-associated mutants. By this method, four new MAH genes were identified that may be involved in virulence. PMID:22966811

  9. Identification of 17 hearing impaired mouse strains in the TMGC ENU-mutagenesis screen

    SciTech Connect

    Kermany, Mohammad; Parker, Lisan; Guo, Yun-Kai; Miller, Darla R; Swanson, Douglas J; Yoo, Tai-June; Goldowitz, Daniel; Zuo, Jian

    2006-01-01

    The Tennessee Mouse Genome Consortium (TMGC) employed an N-ethyl-N-nitrosourea (ENU)-mutagenesis scheme to identify mouse recessive mutants with hearing phenotypes. We employed auditory brainstem responses (ABR) to click and 8, 16, and 32 kHz stimuli and screened 285 pedigrees (1819 mice of 8-11 weeks old in various mixed genetic backgrounds) each bred to carry a homozygous ENU-induced mutation. To define mutant pedigrees, we measured P12 mice per pedigree in P2 generations and used a criterion where the mean ABR threshold per pedigree was two standard deviations above the mean of all offspring from the same parental strain. We thus identified 17 mutant pedigrees (6%), all exhibiting hearing loss at high frequencies (P16 kHz) with an average threshold elevation of 30-35 dB SPL. Interestingly, four mutants showed sex-biased hearing loss and six mutants displayed wide range frequency hearing loss. Temporal bone histology revealed that six of the first nine mutants displayed cochlear morphological defects: degeneration of spiral ganglia, spiral ligament fibrocytes or inner hair cells (but not outer hair cells) mostly in basal turns. In contrast to other ENU-mutagenesis auditory screens, our screen identified high-frequency, mild and sex-biased hearing defects. Further characterization of these 17 mouse models will advance our understanding of presbycusis and noise-induced hearing loss in humans.

  10. Sleeping Beauty-mediated somatic mutagenesis implicates CSF1 in the formation of high grade astrocytomas

    PubMed Central

    Bender, Aaron M.; Collier, Lara S.; Rodriguez, Fausto J.; Tieu, Christina; Larson, Jon D.; Halder, Chandralekha; Mahlum, Eric; Kollmeyer, Thomas M.; Akagi, Keiko; Sarkar, Gobinda; Largaespada, David A.; Jenkins, Robert B.

    2010-01-01

    The Sleeping Beauty (SB) transposon system has been used as an insertional mutagenesis tool to identify novel cancer genes. To identify glioma-associated genes, we evaluated tumor formation in brain tissue from 117 transgenic mice that had undergone constitutive SB-mediated transposition. Upon analysis, 21 samples (18%) contained neoplastic tissue with features of high grade astrocytomas. These tumors expressed glial markers and were histologically similar to human glioma. Genomic DNA from SB-induced astrocytoma tissue was extracted and transposon insertion sites were identified. Insertions in the growth factor gene Csf1 were found in 13 of the 21 tumors (62%), clustered in introns 5 and 8. Using RT-PCR, we documented increased Csf1 RNAs in tumor versus adjacent normal tissue, with identification of transposon-terminated Csf1 mRNAs in astrocytomas with SB insertions in intron 8. Analysis of human glioblastomas revealed increased levels of Csf1 RNA and protein. Together, these results indicate that SB-insertional mutagenesis can identify high-grade astrocytoma-associated genes, and they imply an important role for CSF1 in the development of these tumors. PMID:20388773

  11. Insertional mutagenesis by transposable elements in the mammalian genome.

    PubMed

    Amariglio, N; Rechavi, G

    1993-01-01

    Several mammalian repetitive transposable genetic elements were characterized in recent years, and their role in mutagenesis is delineated in this review. Two main groups have been described: elements with symmetrical termini such as the murine IAP sequences and the human THE 1 elements and elements characterized by a poly-A rich tail at the 3' end such as the SINE and LINE sequences. The characteristic property of such mobile elements to spread and integrate in the host genome leads to insertional mutagenesis. Both germline and somatic mutations have been documented resulting from the insertion of the various types of mammalian repetitive transposable genetic elements. As foreseen by Barbara McClintock, such genetic events can cause either the activation or the inactivation of specific genes, resulting in their identification via an altered phenotype. Several disease states, such as hemophilia and cancer, are the result of this apparent aspect of genome instability. PMID:8385004

  12. European Community research on environmental mutagenesis and carcinogenesis.

    PubMed Central

    Sors, A I

    1993-01-01

    Within the 12 Member States of the European Community (EC), environmental policy is now formulated primarily at Community level. As a result, the EC has important regulatory responsibilities for the protection of workers, consumers, and the general public from risks that may arise from environmental chemicals, foremost among them potential carcinogens and mutagens. An important part of EC environmental research and development is intended to provide a scientific basis for these regulations as well as increasing understanding of the basic mechanisms involved in environmental carcinogenesis and mutagenesis. This paper contains a brief introduction to EC environment policy and research, followed by an overview of EC chemicals control activities that are of particular relevance to the research and development program. Community-level research on environmental mutagenesis and carcinogenesis is then reviewed in some detail, including the achievements of recent projects, the scientific content of the current program, and perspectives for the future. PMID:8143645

  13. Structural evidence for the rare tautomer hypothesis of spontaneous mutagenesis

    SciTech Connect

    Wang, Weina; Hellinga, Homme W.; Beese, Lorena S.

    2012-05-10

    Even though high-fidelity polymerases copy DNA with remarkable accuracy, some base-pair mismatches are incorporated at low frequency, leading to spontaneous mutagenesis. Using high-resolution X-ray crystallographic analysis of a DNA polymerase that catalyzes replication in crystals, we observe that a C {center_dot} A mismatch can mimic the shape of cognate base pairs at the site of incorporation. This shape mimicry enables the mismatch to evade the error detection mechanisms of the polymerase, which would normally either prevent mismatch incorporation or promote its nucleolytic excision. Movement of a single proton on one of the mismatched bases alters the hydrogen-bonding pattern such that a base pair forms with an overall shape that is virtually indistinguishable from a canonical, Watson-Crick base pair in double-stranded DNA. These observations provide structural evidence for the rare tautomer hypothesis of spontaneous mutagenesis, a long-standing concept that has been difficult to demonstrate directly.

  14. Antiviral Strategies Based on Lethal Mutagenesis and Error Threshold.

    PubMed

    Perales, Celia; Domingo, Esteban

    2016-01-01

    The concept of error threshold derived from quasispecies theory is at the basis of lethal mutagenesis, a new antiviral strategy based on the increase of virus mutation rate above an extinction threshold. Research on this strategy is justified by several inhibitor-escape routes that viruses utilize to ensure their survival. Successive steps in the transition from an organized viral quasispecies into loss of biologically meaningful genomic sequences are dissected. The possible connections between theoretical models and experimental observations on lethal mutagenesis are reviewed. The possibility of using combination of virus-specific mutagenic nucleotide analogues and broad-spectrum, non-mutagenic inhibitors is evaluated. We emphasize the power that quasispecies theory has had to stimulate exploration of new means to combat pathogenic viruses. PMID:26294225

  15. Efficient site-directed saturation mutagenesis using degenerate oligonucleotides.

    PubMed

    Steffens, David L; Williams, John G K

    2007-07-01

    We describe a reliable protocol for constructing single-site saturation mutagenesis libraries consisting of all 20 naturally occurring amino acids at a specific site within a protein. Such libraries are useful for structure-function studies and directed evolution. This protocol extends the utility of Stratagene's QuikChange Site-Directed Mutagenesis Kit, which is primarily recommended for single amino acid substitutions. Two complementary primers are synthesized, containing a degenerate mixture of the four bases at the three positions of the selected codon. These primers are added to starting plasmid template and thermal cycled to produce mutant DNA molecules, which are subsequently transformed into competent bacteria. The protocol does not require purification of mutagenic oligonucleotides or PCR products. This reduces both the cost and turnaround time in high-throughput directed evolution applications. We have utilized this protocol to generate over 200 site-saturation libraries in a DNA polymerase, with a success rate of greater than 95%. PMID:17595310

  16. Minimizing off-Target Mutagenesis Risks Caused by Programmable Nucleases

    PubMed Central

    Ishida, Kentaro; Gee, Peter; Hotta, Akitsu

    2015-01-01

    Programmable nucleases, such as zinc finger nucleases (ZFNs), transcription activator like effector nucleases (TALENs), and clustered regularly interspersed short palindromic repeats associated protein-9 (CRISPR-Cas9), hold tremendous potential for applications in the clinical setting to treat genetic diseases or prevent infectious diseases. However, because the accuracy of DNA recognition by these nucleases is not always perfect, off-target mutagenesis may result in undesirable adverse events in treated patients such as cellular toxicity or tumorigenesis. Therefore, designing nucleases and analyzing their activity must be carefully evaluated to minimize off-target mutagenesis. Furthermore, rigorous genomic testing will be important to ensure the integrity of nuclease modified cells. In this review, we provide an overview of available nuclease designing platforms, nuclease engineering approaches to minimize off-target activity, and methods to evaluate both on- and off-target cleavage of CRISPR-Cas9. PMID:26501275

  17. Specific mutagenesis of a chlorophyll-binding protein. Progress report.

    SciTech Connect

    Eaton-Rye, Dr., Julian; Shen, Gaozhong

    1990-01-01

    During the first phase of the project regarding specific mutagenesis of the chlorophyll-binding protein CP47 in photosystem II (PS II) most of the time has been devoted to (1) establishment of an optimal procedure for the reintroduction of psbB (the gene encoding CP47) carrying a site-directed mutation into the experimental organism, the cyanobacterium Synechocystis sp. PCC 6803, (2) preparations for site-directed mutagenesis, and (3) creation and analysis of chimaeric spinach/cyanobacterial CP47 mutants of Synechocystis. In the coming year, psbB constructs with site-directed mutations in potential chlorophyll-binding regions of CP47 will be introduced into the Synechocystis genome, and site-directed mutants will be characterized according to procedures described in the original project description. In addition, analysis of chimaeric CP47 mutants will be continued.

  18. Oligonucleotide-directed site-specific mutagenesis in Drosophila melanogaster.

    PubMed Central

    Banga, S S; Boyd, J B

    1992-01-01

    An efficient technique has been developed for performing in vivo site-directed mutagenesis in Drosophila melanogaster. This procedure involves directed repair of P-element-induced DNA lesions after injection of a modified DNA sequence into early embryos. An oligonucleotide of 50 base pairs, whose sequence spans the P-element insertion site, mediates base replacement in the endogenous gene. Restriction mapping, DNA sequencing, and polymerase chain reaction analysis demonstrate that base substitutions present in an injected oligonucleotide are incorporated into genomic sequences flanking a P insertion site in the white gene. This analysis suggests that progeny bearing directed mutations are recovered with a frequency of about 0.5 x 10(-3). Because Drosophila remains a premier organism for the analysis of eukaryotic gene regulation, this system should find strong application in that analysis as well as in the analysis of DNA recombination, conversion, repair, and mutagenesis. Images PMID:1311850

  19. Minimizing off-Target Mutagenesis Risks Caused by Programmable Nucleases.

    PubMed

    Ishida, Kentaro; Gee, Peter; Hotta, Akitsu

    2015-01-01

    Programmable nucleases, such as zinc finger nucleases (ZFNs), transcription activator like effector nucleases (TALENs), and clustered regularly interspersed short palindromic repeats associated protein-9 (CRISPR-Cas9), hold tremendous potential for applications in the clinical setting to treat genetic diseases or prevent infectious diseases. However, because the accuracy of DNA recognition by these nucleases is not always perfect, off-target mutagenesis may result in undesirable adverse events in treated patients such as cellular toxicity or tumorigenesis. Therefore, designing nucleases and analyzing their activity must be carefully evaluated to minimize off-target mutagenesis. Furthermore, rigorous genomic testing will be important to ensure the integrity of nuclease modified cells. In this review, we provide an overview of available nuclease designing platforms, nuclease engineering approaches to minimize off-target activity, and methods to evaluate both on- and off-target cleavage of CRISPR-Cas9. PMID:26501275

  20. Transcriptional mutagenesis: causes and involvement in tumor development

    PubMed Central

    Brégeon, Damien; Doetsch, Paul W.

    2013-01-01

    The majority of normal cells in a human do not multiply continuously but are quiescent and devote most of their energy to gene transcription. When DNA damages in the transcribed strand of an active gene are bypassed by an RNA polymerase, they can miscode at the damaged site and produce mutant transcripts. This process known as transcriptional mutagenesis can lead to the production of mutant proteins that could be important in tumor development. PMID:21346784

  1. Nitrosoguanidine and ultraviolet light mutagenesis in Eudorina elegans (chlorophyceae)

    SciTech Connect

    Toby, A.L.; Kemp, C.L.

    1980-06-01

    Reversion of an acetate requiring strain and the induction of sectored colonies are used to establish optimal conditions for nitrosoguanidine and ultraviolet light mutagenesis in Eudorina elegans Ehrenberg. Nitrosoguanidine is more effective in causing reversion of the acetate requiring strain and inducing auxotrophs. Morphogenetic mutants are more readily induced by ultraviolet light. The effectiveness of ultraviolet light as a mutagen is cell cycle dependent whereas the mutagenic action of nitrosoguanidine is not.

  2. A Chemical Mutagenesis Screen Identifies Mouse Models with ERG Defects.

    PubMed

    Charette, Jeremy R; Samuels, Ivy S; Yu, Minzhong; Stone, Lisa; Hicks, Wanda; Shi, Lan Ying; Krebs, Mark P; Naggert, Jürgen K; Nishina, Patsy M; Peachey, Neal S

    2016-01-01

    Mouse models provide important resources for many areas of vision research, pertaining to retinal development, retinal function and retinal disease. The Translational Vision Research Models (TVRM) program uses chemical mutagenesis to generate new mouse models for vision research. In this chapter, we report the identification of mouse models for Grm1, Grk1 and Lrit3. Each of these is characterized by a primary defect in the electroretinogram. All are available without restriction to the research community. PMID:26427409

  3. Metal mutagenesis in transgenic Chinese hamster cell lines.

    PubMed Central

    Klein, C B; Kargacin, B; Su, L; Cosentino, S; Snow, E T; Costa, M

    1994-01-01

    Metals are toxic agents for which genotoxic effects are often difficult to demonstrate. To study metal mutagenesis, we have used two stable hprt/gpt+ transgenic cell lines that were derived from Chinese hamster V79 cells. Both the G12 and G10 cell lines are known to be very sensitive to clastogens such as X-rays and bleomycin, with the mutagenic response of the integrated xanthine guanine phosphoribosyl transferase (gpt) gene in G10 usually exceeding that of the same gene in the transgenic G12 cells. In studies with carcinogenic insoluble nickel compounds, a high level of mutagenesis was found at the gpt locus of G12 cells but not at the endogenous hypoxanthine phosphoribosyl transferase (hprt) locus of V79 cells. We have since demonstrated the similar recovery of a high frequency of viable G12 mutants with other insoluble nickel salts including nickel oxides (black and green). The relative mutant yield for the insoluble nickel compounds (G12 > G10) is the opposite of that obtained with nonmetal clastogens (G10 > G12). In the G12 cells, nickel mutagenesis may be related to the integration of the gpt sequence into a heterochromatic region of the genome. For some of the insoluble nickel compounds, significant inhibition of both cytotoxicity and mutant yield resulted when the G12 cells were pretreated with vitamin E. In comparison with the nickel studies, the mutagenic responses to chromium compounds in these cell lines were not as dramatic. Mutagenesis of the gpt target could not be demonstrated with other metals such as mercury or vanadium. PMID:7843139

  4. Targeted mutagenesis of Arabidopsis thaliana using engineered TAL effector nucleases.

    PubMed

    Christian, Michelle; Qi, Yiping; Zhang, Yong; Voytas, Daniel F

    2013-10-01

    Custom TAL effector nucleases (TALENs) are increasingly used as reagents to manipulate genomes in vivo. Here, we used TALENs to modify the genome of the model plant, Arabidopsis thaliana. We engineered seven TALENs targeting five Arabidopsis genes, namely ADH1, TT4, MAPKKK1, DSK2B, and NATA2. In pooled seedlings expressing the TALENs, we observed somatic mutagenesis frequencies ranging from 2-15% at the intended targets for all seven TALENs. Somatic mutagenesis frequencies as high as 41-73% were observed in individual transgenic plant lines expressing the TALENs. Additionally, a TALEN pair targeting a tandemly duplicated gene induced a 4.4-kb deletion in somatic cells. For the most active TALEN pairs, namely those targeting ADH1 and NATA2, we found that TALEN-induced mutations were transmitted to the next generation at frequencies of 1.5-12%. Our work demonstrates that TALENs are useful reagents for achieving targeted mutagenesis in this important plant model. PMID:23979944

  5. Development of an inducible transposon system for efficient random mutagenesis in Clostridium acetobutylicum

    PubMed Central

    Zhang, Ying; Xu, Shu; Chai, Changsheng; Yang, Sheng; Jiang, Weihong; Minton, Nigel P.; Gu, Yang

    2016-01-01

    Clostridium acetobutylicum is an industrially important Gram-positive organism, which is capable of producing economically important chemicals in the ABE (Acetone, Butanol and Ethanol) fermentation process. Renewed interests in the ABE process necessitate the availability of additional genetics tools to facilitate the derivation of a greater understanding of the underlying metabolic and regulatory control processes in operation through forward genetic strategies. In this study, a xylose inducible, mariner-based, transposon system was developed and shown to allow high-efficient random mutagenesis in the model strain ATCC 824. Of the thiamphenicol resistant colonies obtained, 91.9% were shown to be due to successful transposition of the catP-based mini-transposon element. Phenotypic screening of 200 transposon clones revealed a sporulation-defective clone with an insertion in spo0A, thereby demonstrating that this inducible transposon system can be used for forward genetic studies in C. acetobutylicum. PMID:27001972

  6. Systematic analysis of the kalimantacin assembly line NRPS module using an adapted targeted mutagenesis approach.

    PubMed

    Uytterhoeven, Birgit; Appermans, Kenny; Song, Lijiang; Masschelein, Joleen; Lathouwers, Thomas; Michiels, Chris W; Lavigne, Rob

    2016-04-01

    Kalimantacin is an antimicrobial compound with strong antistaphylococcal activity that is produced by a hybrid trans-acyltransferase polyketide synthase/nonribosomal peptide synthetase system in Pseudomonas fluorescens BCCM_ID9359. We here present a systematic analysis of the substrate specificity of the glycine-incorporating adenylation domain from the kalimantacin biosynthetic assembly line by a targeted mutagenesis approach. The specificity-conferring code was adapted for use in Pseudomonas and mutated adenylation domain active site sequences were introduced in the kalimantacin gene cluster, using a newly adapted ligation independent cloning method. Antimicrobial activity screens and LC-MS analyses revealed that the production of the kalimantacin analogues in the mutated strains was abolished. These results support the idea that further insight in the specificity of downstream domains in nonribosomal peptide synthetases and polyketide synthases is required to efficiently engineer these strains in vivo. PMID:26666990

  7. Development of an inducible transposon system for efficient random mutagenesis in Clostridium acetobutylicum.

    PubMed

    Zhang, Ying; Xu, Shu; Chai, Changsheng; Yang, Sheng; Jiang, Weihong; Minton, Nigel P; Gu, Yang

    2016-04-01

    Clostridium acetobutylicum is an industrially important Gram-positive organism, which is capable of producing economically important chemicals in the ABE (Acetone, Butanol and Ethanol) fermentation process. Renewed interests in the ABE process necessitate the availability of additional genetics tools to facilitate the derivation of a greater understanding of the underlying metabolic and regulatory control processes in operation through forward genetic strategies. In this study, a xylose inducible, mariner-based, transposon system was developed and shown to allow high-efficient random mutagenesis in the model strain ATCC 824. Of the thiamphenicol resistant colonies obtained, 91.9% were shown to be due to successful transposition of the catP-based mini-transposon element. Phenotypic screening of 200 transposon clones revealed a sporulation-defective clone with an insertion in spo0A, thereby demonstrating that this inducible transposon system can be used for forward genetic studies in C. acetobutylicum. PMID:27001972

  8. Genes Associated with Desiccation and Osmotic Stress in Listeria monocytogenes as Revealed by Insertional Mutagenesis

    PubMed Central

    Hingston, Patricia A.; Piercey, Marta J.

    2015-01-01

    Listeria monocytogenes is a foodborne pathogen whose survival in food processing environments may be associated with its tolerance to desiccation. To probe the molecular mechanisms used by this bacterium to adapt to desiccation stress, a transposon library of 11,700 L. monocytogenes mutants was screened, using a microplate assay, for strains displaying increased or decreased desiccation survival (43% relative humidity, 15°C) in tryptic soy broth (TSB). The desiccation phenotypes of selected mutants were subsequently assessed on food-grade stainless steel (SS) coupons in TSB plus 1% glucose (TSB-glu). Single transposon insertions in mutants exhibiting a change in desiccation survival of >0.5 log CFU/cm2 relative to that of the wild type were determined by sequencing arbitrary PCR products. Strain morphology, motility, and osmotic stress survival (in TSB-glu plus 20% NaCl) were also analyzed. The initial screen selected 129 desiccation-sensitive (DS) and 61 desiccation-tolerant (DT) mutants, out of which secondary screening on SS confirmed 15 DT and 15 DS mutants. Among the DT mutants, seven immotile and flagellum-less strains contained transposons in genes involved in flagellum biosynthesis (fliP, flhB, flgD, flgL) and motor control (motB, fliM, fliY), while others harbored transposons in genes involved in membrane lipid biosynthesis, energy production, potassium uptake, and virulence. The genes that were interrupted in the 15 DS mutants included those involved in energy production, membrane transport, protein metabolism, lipid biosynthesis, oxidative damage control, and putative virulence. Five DT and 14 DS mutants also demonstrated similar significantly (P < 0.05) different survival relative to that of the wild type when exposed to osmotic stress, demonstrating that some genes likely have similar roles in allowing the organism to survive the two water stresses. PMID:26025900

  9. Extensive site-directed mutagenesis reveals interconnected functional units in the alkaline phosphatase active site.

    PubMed

    Sunden, Fanny; Peck, Ariana; Salzman, Julia; Ressl, Susanne; Herschlag, Daniel

    2015-01-01

    Enzymes enable life by accelerating reaction rates to biological timescales. Conventional studies have focused on identifying the residues that have a direct involvement in an enzymatic reaction, but these so-called 'catalytic residues' are embedded in extensive interaction networks. Although fundamental to our understanding of enzyme function, evolution, and engineering, the properties of these networks have yet to be quantitatively and systematically explored. We dissected an interaction network of five residues in the active site of Escherichia coli alkaline phosphatase. Analysis of the complex catalytic interdependence of specific residues identified three energetically independent but structurally interconnected functional units with distinct modes of cooperativity. From an evolutionary perspective, this network is orders of magnitude more probable to arise than a fully cooperative network. From a functional perspective, new catalytic insights emerge. Further, such comprehensive energetic characterization will be necessary to benchmark the algorithms required to rationally engineer highly efficient enzymes. PMID:25902402

  10. Genes Associated with Desiccation and Osmotic Stress in Listeria monocytogenes as Revealed by Insertional Mutagenesis.

    PubMed

    Hingston, Patricia A; Piercey, Marta J; Truelstrup Hansen, Lisbeth

    2015-08-15

    Listeria monocytogenes is a foodborne pathogen whose survival in food processing environments may be associated with its tolerance to desiccation. To probe the molecular mechanisms used by this bacterium to adapt to desiccation stress, a transposon library of 11,700 L. monocytogenes mutants was screened, using a microplate assay, for strains displaying increased or decreased desiccation survival (43% relative humidity, 15°C) in tryptic soy broth (TSB). The desiccation phenotypes of selected mutants were subsequently assessed on food-grade stainless steel (SS) coupons in TSB plus 1% glucose (TSB-glu). Single transposon insertions in mutants exhibiting a change in desiccation survival of >0.5 log CFU/cm(2) relative to that of the wild type were determined by sequencing arbitrary PCR products. Strain morphology, motility, and osmotic stress survival (in TSB-glu plus 20% NaCl) were also analyzed. The initial screen selected 129 desiccation-sensitive (DS) and 61 desiccation-tolerant (DT) mutants, out of which secondary screening on SS confirmed 15 DT and 15 DS mutants. Among the DT mutants, seven immotile and flagellum-less strains contained transposons in genes involved in flagellum biosynthesis (fliP, flhB, flgD, flgL) and motor control (motB, fliM, fliY), while others harbored transposons in genes involved in membrane lipid biosynthesis, energy production, potassium uptake, and virulence. The genes that were interrupted in the 15 DS mutants included those involved in energy production, membrane transport, protein metabolism, lipid biosynthesis, oxidative damage control, and putative virulence. Five DT and 14 DS mutants also demonstrated similar significantly (P < 0.05) different survival relative to that of the wild type when exposed to osmotic stress, demonstrating that some genes likely have similar roles in allowing the organism to survive the two water stresses. PMID:26025900

  11. Structure-based mutagenesis reveals the albumin-binding site of the neonatal Fc receptor

    PubMed Central

    Andersen, Jan Terje; Dalhus, Bjørn; Cameron, Jason; Daba, Muluneh Bekele; Plumridge, Andrew; Evans, Leslie; Brennan, Stephan O.; Gunnarsen, Kristin Støen; Bjørås, Magnar; Sleep, Darrell; Sandlie, Inger

    2012-01-01

    Albumin is the most abundant protein in blood where it has a pivotal role as a transporter of fatty acids and drugs. Like IgG, albumin has long serum half-life, protected from degradation by pH-dependent recycling mediated by interaction with the neonatal Fc receptor, FcRn. Although the FcRn interaction with IgG is well characterized at the atomic level, its interaction with albumin is not. Here we present structure-based modelling of the FcRn–albumin complex, supported by binding analysis of site-specific mutants, providing mechanistic evidence for the presence of pH-sensitive ionic networks at the interaction interface. These networks involve conserved histidines in both FcRn and albumin domain III. Histidines also contribute to intramolecular interactions that stabilize the otherwise flexible loops at both the interacting surfaces. Molecular details of the FcRn–albumin complex may guide the development of novel albumin variants with altered serum half-life as carriers of drugs. PMID:22215085

  12. Mutagenesis of the borage Delta(6) fatty acid desaturase.

    PubMed

    Sayanova, O; Beaudoin, F; Libisch, B; Shewry, P; Napier, J

    2000-12-01

    The consensus sequence of the third histidine box of a range of Delta(5), Delta(6), Delta(8) and sphingolipid desaturases differs from that of the membrane-bound non-fusion Delta(12) and Delta(15) desaturases in the presence of glutamine instead of histidine. We have used site-directed mutagenesis to determine the importance of glutamine and other residues of the third histidine box and created a chimaeric enzyme to determine the ability of the Cyt b(5) fusion domain from the plant sphingolipid desaturase to substitute for the endogenous domain of the Delta(6) desaturase. PMID:11171152

  13. Pollen tetrads in the detection of environmental mutagenesis

    SciTech Connect

    Mulcahy, D.L.

    1981-01-01

    Although pollen is a very sensitive indicator of environmental mutagenesis, it is also sensitive to nonmutagenic environmental stress. By analyzing pollen tetrads, rather than individual pollen grains, it is possible to distinguish between mutagenic and nonmutagenic influences. Another advantage of using pollen tetrads in mutagenicity studies is that it is possible to discriminate between pre- and post-pachytene mutations. This eliminates the mutant sector problem of a single mutational event giving rise to a large number of mutant cells. Methods of analyzing pollen tetrads are described.

  14. Chemical mutagenesis: an emerging issue for public health.

    PubMed Central

    Claxton, L D; Barry, P Z

    1977-01-01

    Chemical mutagens are recognized as prevalent in the environment and a potential threat to the health of future generations. This paper presents an overview of chemical mutagenesis as an issue for public health. Several problems in the determination of risk to human populations are discussed, including difficulties of extrapolating scientific data to humans, the latency period between exposure and recognizable genetic damage, and the large number of chemicals which must be tested. Test systems are described. Possibilities of control through federal regulation are discussed. PMID:911015

  15. Mutagenesis in Newts: Protocol for Iberian Ribbed Newts.

    PubMed

    Hayashi, Toshinori; Takeuchi, Takashi

    2016-01-01

    Newts have the remarkable capability of organ/tissue regeneration, and have been used as a unique experimental model for regenerative biology. The Iberian ribbed newt (Pleurodeles waltl) is suitable as a model animal. We have established methods for artificial insemination and efficient transgenesis using P. waltl newts. In addition to the transgenic technique, development of TALENs enables targeting mutagenesis in the newts. We have reported that TALENs efficiently disrupted targeted genes in newt embryos. In this chapter, we introduce a protocol for TALEN-mediated gene targeting in Iberian ribbed newts. PMID:26443218

  16. Therapeutic genome mutagenesis using synthetic donor DNA and triplex-forming molecules.

    PubMed

    Reza, Faisal; Glazer, Peter M

    2015-01-01

    Genome mutagenesis can be achieved in a variety of ways, though a select few are suitable for therapeutic settings. Among them, the harnessing of intracellular homologous recombination affords the safety and efficacy profile suitable for such settings. Recombinagenic donor DNA and mutagenic triplex-forming molecules co-opt this natural recombination phenomenon to enable the specific, heritable editing and targeting of the genome. Editing the genome is achieved by designing the sequence-specific recombinagenic donor DNA to have base mismatches, insertions, and deletions that will be incorporated into the genome when it is used as a template for recombination. Targeting the genome is similarly achieved by designing the sequence-specific mutagenic triplex-forming molecules to further recruit the recombination machinery thereby upregulating its activity with the recombinagenic donor DNA. This combination of extracellularly introduced, designed synthetic molecules and intercellularly ubiquitous, evolved natural machinery enables the mutagenesis of chromosomes and engineering of whole genomes with great fidelity while limiting nonspecific interactions. Herein, we demonstrate the harnessing of recombinagenic donor DNA and mutagenic triplex-forming molecular technology for potential therapeutic applications. These demonstrations involve, among others, utilizing this technology to correct genes so that they become physiologically functional, to induce dormant yet functional genes in place of non-functional counterparts, to place induced genes under regulatory elements, and to disrupt genes to abrogate a cellular vulnerability. Ancillary demonstrations of the design and synthesis of this recombinagenic and mutagenic molecular technology as well as their delivery and assayed interaction with duplex DNA reveal a potent technological platform for engineering specific changes into the living genome. PMID:25408401

  17. Breeding of New Strains of Mushroom by Basidiospore Chemical Mutagenesis

    PubMed Central

    Lee, Jia; Kang, Hyeon-Woo; Kim, Sang-Woo; Lee, Chang-Yun

    2011-01-01

    Chemical mutagenesis of basidiospores of Hypsizygus marmoreus generated new mushroom strains. The basidospores were treated with methanesulfonate methylester, an alkylating agent, to yield 400 mutant monokaryotic mycelia. Twenty fast-growing mycelia were selected and mated each other by hyphal fusion. Fifty out of the 190 matings were successful (mating rate of 26.3%), judged by the formation of clamp connections. The mutant dikaryons were cultivated to investigate their morphological and cultivation characteristics. Mutant strains No. 3 and No. 5 showed 10% and 6% increase in fruiting body production, respectively. Eight mutant strains showed delayed and reduced primordia formation, resulting in the reduced production yield with prolonged cultivation period. The number of the fruiting bodies of mutant No. 31, which displayed reduced primordial formation, was only 15, compared to the parental number of 65. Another interesting phenotype was a fruiting body with a flattened stipe and pileus. Dikaryons generated by mating with the mutant spore No. 14 produced flat fruiting bodies. Further molecular biological studies will provide details of the mechanism. This work shows that the chemical mutagenesis approach is highly utilizable in the development of mushroom strains as well as in the generation of resources for molecular genetic studies. PMID:22783115

  18. Mechanisms of Base Substitution Mutagenesis in Cancer Genomes

    PubMed Central

    Bacolla, Albino; Cooper, David N.; Vasquez, Karen M.

    2014-01-01

    Cancer genome sequence data provide an invaluable resource for inferring the key mechanisms by which mutations arise in cancer cells, favoring their survival, proliferation and invasiveness. Here we examine recent advances in understanding the molecular mechanisms responsible for the predominant type of genetic alteration found in cancer cells, somatic single base substitutions (SBSs). Cytosine methylation, demethylation and deamination, charge transfer reactions in DNA, DNA replication timing, chromatin status and altered DNA proofreading activities are all now known to contribute to the mechanisms leading to base substitution mutagenesis. We review current hypotheses as to the major processes that give rise to SBSs and evaluate their relative relevance in the light of knowledge acquired from cancer genome sequencing projects and the study of base modifications, DNA repair and lesion bypass. Although gene expression data on APOBEC3B enzymes provide support for a role in cancer mutagenesis through U:G mismatch intermediates, the enzyme preference for single-stranded DNA may limit its activity genome-wide. For SBSs at both CG:CG and YC:GR sites, we outline evidence for a prominent role of damage by charge transfer reactions that follow interactions of the DNA with reactive oxygen species (ROS) and other endogenous or exogenous electron-abstracting molecules. PMID:24705290

  19. Fitness Loss and Library Size Determination in Saturation Mutagenesis

    PubMed Central

    Nov, Yuval

    2013-01-01

    Saturation mutagenesis is a widely used directed evolution technique, in which a large number of protein variants, each having random amino acids in certain predetermined positions, are screened in order to discover high-fitness variants among them. Several metrics for determining the library size (the number of variants screened) have been suggested in the literature, but none of them incorporates the actual fitness of the variants discovered in the experiment. We present the results of an extensive simulation study, which is based on probabilistic models for protein fitness landscape, and which investigates how the result of a saturation mutagenesis experiment – the fitness of the best variant discovered – varies as a function of the library size. In particular, we study the loss of fitness in the experiment: the difference between the fitness of the best variant discovered, and the fitness of the best variant in variant space. Our results are that the existing criteria for determining the library size are conservative, so smaller libraries are often satisfactory. Reducing the library size can save labor, time, and expenses in the laboratory. PMID:23844158

  20. Establishment of a counter-selectable markerless mutagenesis system in Veillonella atypica.

    PubMed

    Zhou, Peng; Li, Xiaoli; Qi, Fengxia

    2015-05-01

    Using an alternative sigma factor ecf3 as target, we successfully established the first markerless mutagenesis system in the Veillonella genus. This system will be a valuable tool for mutagenesis of multiple genes for gene function analysis as well as for gene regulation studies in Veillonella. PMID:25771833

  1. A mouse chromosome 4 balancer ENU-mutagenesis screen isolates eleven lethal lines

    Technology Transfer Automated Retrieval System (TEKTRAN)

    ENU-mutagenesis is a powerful technique to identify genes regulating mammalian development. To functionally annotate the distal region of mouse chromosome 4, we performed an ENU-mutagenesis screen using a balancer chromosome targeted to this region of the genome. We isolated 11 lethal lines that map...

  2. Targeted Mutagenesis in Zebrafish Using Customized Zinc Finger Nucleases

    PubMed Central

    Foley, Jonathan E.; Maeder, Morgan L.; Pearlberg, Joseph; Joung, J. Keith; Peterson, Randall T.; Yeh, Jing-Ruey J.

    2009-01-01

    Zebrafish mutants have traditionally been obtained using random mutagenesis or retroviral insertions, methods that cannot be targeted to a specific gene and require laborious gene mapping and sequencing. Recently, we and others have shown that customized zinc finger nucleases (ZFNs) can introduce targeted frame-shift mutations with high efficiency, thereby enabling directed creation of zebrafish gene mutations. Here we describe a detailed protocol for constructing ZFN expression vectors, for generating and introducing ZFN-encoding RNAs into zebrafish embryos, and for identifying ZFN-generated mutations in targeted genomic sites. All of our vectors and methods are compatible with previously described Zinc Finger Consortium reagents for constructing engineered zinc finger arrays. Using these methods, zebrafish founders carrying targeted mutations can be identified within four months. PMID:20010934

  3. Mutagenesis and differentiation induction in mammalian cells by environmental chemicals

    SciTech Connect

    Friedman, J.; Huberman, E.

    1980-01-01

    These studies indicate that in agreement with the somatic mutation hypothesis, chemical carcinogens: (1) are mutagenic for mammalian cells as tested in the cell-mediated assay; (2) the degree of mutagenicity is correlated with their degree of carcinogenicity; (3) that at least in cases when analyzed carefully the metabolites responsible for mutagenesis are also responsible for initiating the carcinogenic event; and (4) that a cell organ type specificity can be established using the cell-mediated assay. Studies with HL-60 cells and HO melanoma cells and those of others suggest that tumor-promoting phorbol diesters can alter cell differentiation in various cell types and that the degree of the observed alteration in the differentiation properties may be related to the potency of the phorbol esters. Thus these and similar systems may serve as models for both studies and identification of certain types of tumor promoting agents. (ERB)

  4. CRISPR-Cas9 enables conditional mutagenesis of challenging loci

    PubMed Central

    Schick, Joel A.; Seisenberger, Claudia; Beig, Joachim; Bürger, Antje; Iyer, Vivek; Maier, Viola; Perera, Sajith; Rosen, Barry; Skarnes, William C.; Wurst, Wolfgang

    2016-01-01

    The International Knockout Mouse Consortium (IKMC) has produced a genome-wide collection of 15,000 isogenic targeting vectors for conditional mutagenesis in C57BL/6N mice. Although most of the vectors have been used successfully in murine embryonic stem (ES) cells, there remain a set of nearly two thousand genes that have failed to target even after several attempts. Recent attention has turned to the use of new genome editing technology for the generation of mutant alleles in mice. Here, we demonstrate how Cas9-assisted targeting can be combined with the IKMC targeting vector resource to generate conditional alleles in genes that have previously eluded targeting using conventional methods. PMID:27580957

  5. Environmental mutagenesis during the end-Permian ecological crisis.

    PubMed

    Visscher, Henk; Looy, Cindy V; Collinson, Margaret E; Brinkhuis, Henk; van Konijnenburg-van Cittert, Johanna H A; Kürschner, Wolfram M; Sephton, Mark A

    2004-08-31

    During the end-Permian ecological crisis, terrestrial ecosystems experienced preferential dieback of woody vegetation. Across the world, surviving herbaceous lycopsids played a pioneering role in repopulating deforested terrain. We document that the microspores of these lycopsids were regularly released in unseparated tetrads indicative of failure to complete the normal process of spore development. Although involvement of mutation has long been hinted at or proposed in theory, this finding provides concrete evidence for chronic environmental mutagenesis at the time of global ecological crisis. Prolonged exposure to enhanced UV radiation could account satisfactorily for a worldwide increase in land plant mutation. At the end of the Permian, a period of raised UV stress may have been the consequence of severe disruption of the stratospheric ozone balance by excessive emission of hydrothermal organohalogens in the vast area of Siberian Traps volcanism. PMID:15282373

  6. Radiosensitivity Parameters For Lethal Mutagenesis In Caenorhabditis Elegans

    SciTech Connect

    Cucinotta, F.A.; Wilson, J.W.; Katz, R.

    1994-01-01

    For the first time track structure theory has been applied to radiobiological effects in a living organism. Data for lethal mutagenesis in Caenorhabditis elegans, obtained after irradiation with nine different types of ions of atomic number 1-57 and gamma rays have yielded radiosensitivity parameters (E{sub 0}, sigma{sub 0}, Kappa, m = 68 Gy, 2.5 x 10(exp {minus}9) cm (exp 2), 750, 2) comparable with those found for the transformation of C3HT10 1/2 cells (180 Gy, 1.15 x 10(exp {minus}10) cm(exp 2), 750, 2) but remote from those (E{sub 0} and sigma{sub 0} = approx. 2 Gy, approx. 5 x 10(exp {minus}7) cm(exp 2)) for mammalian cell survival.

  7. Mutant fatty acid desaturase and methods for directed mutagenesis

    DOEpatents

    Shanklin, John; Whittle, Edward J.

    2008-01-29

    The present invention relates to methods for producing fatty acid desaturase mutants having a substantially increased activity towards substrates with fewer than 18 carbon atom chains relative to an unmutagenized precursor desaturase having an 18 carbon chain length specificity, the sequences encoding the desaturases and to the desaturases that are produced by the methods. The present invention further relates to a method for altering a function of a protein, including a fatty acid desaturase, through directed mutagenesis involving identifying candidate amino acid residues, producing a library of mutants of the protein by simultaneously randomizing all amino acid candidates, and selecting for mutants which exhibit the desired alteration of function. Candidate amino acids are identified by a combination of methods. Enzymatic, binding, structural and other functions of proteins can be altered by the method.

  8. CRISPR-Cas9 enables conditional mutagenesis of challenging loci.

    PubMed

    Schick, Joel A; Seisenberger, Claudia; Beig, Joachim; Bürger, Antje; Iyer, Vivek; Maier, Viola; Perera, Sajith; Rosen, Barry; Skarnes, William C; Wurst, Wolfgang

    2016-01-01

    The International Knockout Mouse Consortium (IKMC) has produced a genome-wide collection of 15,000 isogenic targeting vectors for conditional mutagenesis in C57BL/6N mice. Although most of the vectors have been used successfully in murine embryonic stem (ES) cells, there remain a set of nearly two thousand genes that have failed to target even after several attempts. Recent attention has turned to the use of new genome editing technology for the generation of mutant alleles in mice. Here, we demonstrate how Cas9-assisted targeting can be combined with the IKMC targeting vector resource to generate conditional alleles in genes that have previously eluded targeting using conventional methods. PMID:27580957

  9. Marker reconstitution mutagenesis: a simple and efficient reverse genetic approach.

    PubMed

    Tang, Xie; Huang, Junqi; Padmanabhan, Anup; Bakka, Kavya; Bao, Yun; Tan, Brenda Yuelin; Cande, W Zacheus; Balasubramanian, Mohan K

    2011-03-01

    A novel reverse genetic approach termed 'marker reconstitution mutagenesis' was designed to generate mutational allelic series in genes of interest. This approach consists of two simple steps which utilize two selective markers. First, using one selective marker, a partial fragment of another selective marker gene is inserted adjacently to a gene of interest by homologous recombination. Second, random mutations are introduced precisely into the gene of interest, together with the reconstitution of the latter selective marker by homologous recombination. This approach was successfully tested for several genes in the fission yeast Schizosaccharomyces pombe. It circumvents the problems encountered with other methods and should be adaptable to any organism that incorporates exogenous DNA by homologous recombination. PMID:21360732

  10. Environmental mutagenesis during the end-Permian ecological crisis

    PubMed Central

    Visscher, Henk; Looy, Cindy V.; Collinson, Margaret E.; Brinkhuis, Henk; van Konijnenburg-van Cittert, Johanna H. A.; Kürschner, Wolfram M.; Sephton, Mark A.

    2004-01-01

    During the end-Permian ecological crisis, terrestrial ecosystems experienced preferential dieback of woody vegetation. Across the world, surviving herbaceous lycopsids played a pioneering role in repopulating deforested terrain. We document that the microspores of these lycopsids were regularly released in unseparated tetrads indicative of failure to complete the normal process of spore development. Although involvement of mutation has long been hinted at or proposed in theory, this finding provides concrete evidence for chronic environmental mutagenesis at the time of global ecological crisis. Prolonged exposure to enhanced UV radiation could account satisfactorily for a worldwide increase in land plant mutation. At the end of the Permian, a period of raised UV stress may have been the consequence of severe disruption of the stratospheric ozone balance by excessive emission of hydrothermal organohalogens in the vast area of Siberian Traps volcanism. PMID:15282373

  11. Cationic Peptides Facilitate Iron-induced Mutagenesis in Bacteria

    PubMed Central

    Rodríguez-Rojas, Alexandro; Makarova, Olga; Müller, Uta; Rolff, Jens

    2015-01-01

    Pseudomonas aeruginosa is the causative agent of chronic respiratory infections and is an important pathogen of cystic fibrosis patients. Adaptive mutations play an essential role for antimicrobial resistance and persistence. The factors that contribute to bacterial mutagenesis in this environment are not clear. Recently it has been proposed that cationic antimicrobial peptides such as LL-37 could act as mutagens in P. aeruginosa. Here we provide experimental evidence that mutagenesis is the product of a joint action of LL-37 and free iron. By estimating mutation rate, mutant frequencies and assessing mutational spectra in P. aeruginosa treated either with LL-37, iron or a combination of both we demonstrate that mutation rate and mutant frequency were increased only when free iron and LL-37 were present simultaneously. Colistin had the same effect. The addition of an iron chelator completely abolished this mutagenic effect, suggesting that LL-37 enables iron to enter the cells resulting in DNA damage by Fenton reactions. This was also supported by the observation that the mutational spectrum of the bacteria under LL-37-iron regime showed one of the characteristic Fenton reaction fingerprints: C to T transitions. Free iron concentration in nature and within hosts is kept at a very low level, but the situation in infected lungs of cystic fibrosis patients is different. Intermittent bleeding and damage to the epithelial cells in lungs may contribute to the release of free iron that in turn leads to generation of reactive oxygen species and deterioration of the respiratory tract, making it more susceptible to the infection. PMID:26430769

  12. Cationic Peptides Facilitate Iron-induced Mutagenesis in Bacteria.

    PubMed

    Rodríguez-Rojas, Alexandro; Makarova, Olga; Müller, Uta; Rolff, Jens

    2015-10-01

    Pseudomonas aeruginosa is the causative agent of chronic respiratory infections and is an important pathogen of cystic fibrosis patients. Adaptive mutations play an essential role for antimicrobial resistance and persistence. The factors that contribute to bacterial mutagenesis in this environment are not clear. Recently it has been proposed that cationic antimicrobial peptides such as LL-37 could act as mutagens in P. aeruginosa. Here we provide experimental evidence that mutagenesis is the product of a joint action of LL-37 and free iron. By estimating mutation rate, mutant frequencies and assessing mutational spectra in P. aeruginosa treated either with LL-37, iron or a combination of both we demonstrate that mutation rate and mutant frequency were increased only when free iron and LL-37 were present simultaneously. Colistin had the same effect. The addition of an iron chelator completely abolished this mutagenic effect, suggesting that LL-37 enables iron to enter the cells resulting in DNA damage by Fenton reactions. This was also supported by the observation that the mutational spectrum of the bacteria under LL-37-iron regime showed one of the characteristic Fenton reaction fingerprints: C to T transitions. Free iron concentration in nature and within hosts is kept at a very low level, but the situation in infected lungs of cystic fibrosis patients is different. Intermittent bleeding and damage to the epithelial cells in lungs may contribute to the release of free iron that in turn leads to generation of reactive oxygen species and deterioration of the respiratory tract, making it more susceptible to the infection. PMID:26430769

  13. Precision Targeted Mutagenesis via Cas9 Paired Nickases in Rice

    PubMed Central

    Mikami, Masafumi; Toki, Seiichi; Endo, Masaki

    2016-01-01

    Recent reports of CRISPR- (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) mediated heritable mutagenesis in plants highlight the need for accuracy of the mutagenesis directed by this system. Off-target mutations are an important issue when considering functional gene analysis, as well as the molecular breeding of crop plants with large genome size, i.e. with many duplicated genes, and where the whole-genome sequence is still lacking. In mammals, off-target mutations can be suppressed by using Cas9 paired nickases together with paired guide RNAs (gRNAs). However, the performance of Cas9 paired nickases has not yet been fully assessed in plants. Here, we analyzed on- and off-target mutation frequency in rice calli and regenerated plants using Cas9 nuclease or Cas9 nickase with paired gRNAs. When Cas9 paired nickases were used, off-target mutations were fully suppressed in rice calli and regenerated plants. However, on-target mutation frequency also decreased compared with that induced by the Cas9 paired nucleases system. Since the gRNA sequence determines specific binding of Cas9 protein–gRNA ribonucleoproteins at the targeted sequence, the on-target mutation frequency of Cas9 paired nickases depends on the design of paired gRNAs. Our results suggest that a combination of gRNAs that can induce mutations at high efficiency with Cas9 nuclease should be used together with Cas9 nickase. Furthermore, we confirmed that a combination of gRNAs containing a one nucleotide (1 nt) mismatch toward the target sequence could not induce mutations when expressed with Cas9 nickase. Our results clearly show the effectiveness of Cas9 paired nickases in delivering on-target specific mutations. PMID:26936792

  14. Association of elevated mutagenesis in the spleen with genetic susceptibility to induced plasmacytoma development in mice.

    PubMed

    Felix, K; Kelliher, K; Bornkamm, G W; Janz, S

    1998-04-15

    Using the phage lambdaLIZ-based transgenic in vivo mutagenesis assay, mean mutant rates were determined in the spleen of mice exposed to sustained oxidative stress and were found to be increased approximately 3-fold in plasmacytoma-susceptible BALB/c and C.D2-Idh1-Pep3 mice, but not in plasmacytoma-resistant DBA/2N mice. This finding suggests a correlation between the genetic susceptibility to inflammation-induced peritoneal plasmacytomagenesis and the phenotype of increased mutagenesis in lymphoid tissues, raising the possibility that plasmacytoma resistance genes may inhibit tumor development by minimizing oxidative mutagenesis in B cells. PMID:9563470

  15. A Defect in DNA Ligase4 Enhances the Frequency of TALEN-Mediated Targeted Mutagenesis in Rice.

    PubMed

    Nishizawa-Yokoi, Ayako; Cermak, Tomas; Hoshino, Tomoki; Sugimoto, Kazuhiko; Saika, Hiroaki; Mori, Akiko; Osakabe, Keishi; Hamada, Masao; Katayose, Yuichi; Starker, Colby; Voytas, Daniel F; Toki, Seiichi

    2016-02-01

    We have established methods for site-directed mutagenesis via transcription activator-like effector nucleases (TALENs) in the endogenous rice (Oryza sativa) waxy gene and demonstrated stable inheritance of TALEN-induced somatic mutations to the progeny. To analyze the role of classical nonhomologous end joining (cNHEJ) and alternative nonhomologous end joining (altNHEJ) pathways in TALEN-induced mutagenesis in plant cells, we investigated whether a lack of DNA Ligase4 (Lig4) affects the kinetics of TALEN-induced double-strand break repair in rice cells. Deep-sequencing analysis revealed that the frequency of all types of mutations, namely deletion, insertion, combination of insertion with deletion, and substitution, in lig4 null mutant calli was higher than that in a lig4 heterozygous mutant or the wild type. In addition, the ratio of large deletions (greater than 10 bp) and deletions repaired by microhomology-mediated end joining (MMEJ) to total deletion mutations in lig4 null mutant calli was higher than that in the lig4 heterozygous mutant or wild type. Furthermore, almost all insertions (2 bp or greater) were shown to be processed via copy and paste of one or more regions around the TALENs cleavage site and rejoined via MMEJ regardless of genetic background. Taken together, our findings indicate that the dysfunction of cNHEJ leads to a shift in the repair pathway from cNHEJ to altNHEJ or synthesis-dependent strand annealing. PMID:26668331

  16. Sleeping beauty-mediated somatic mutagenesis implicates CSF1 in the formation of high-grade astrocytomas.

    PubMed

    Bender, Aaron M; Collier, Lara S; Rodriguez, Fausto J; Tieu, Christina; Larson, Jon D; Halder, Chandralekha; Mahlum, Eric; Kollmeyer, Thomas M; Akagi, Keiko; Sarkar, Gobinda; Largaespada, David A; Jenkins, Robert B

    2010-05-01

    The Sleeping Beauty (SB) transposon system has been used as an insertional mutagenesis tool to identify novel cancer genes. To identify glioma-associated genes, we evaluated tumor formation in the brain tissue from 117 transgenic mice that had undergone constitutive SB-mediated transposition. Upon analysis, 21 samples (18%) contained neoplastic tissue with features of high-grade astrocytomas. These tumors expressed glial markers and were histologically similar to human glioma. Genomic DNA from SB-induced astrocytoma tissue was extracted and transposon insertion sites were identified. Insertions in the growth factor gene Csf1 were found in 13 of the 21 tumors (62%), clustered in introns 5 and 8. Using reverse transcription-PCR, we documented increased Csf1 RNAs in tumor versus adjacent normal tissue, with the identification of transposon-terminated Csf1 mRNAs in astrocytomas with SB insertions in intron 8. Analysis of human glioblastomas revealed increased levels of Csf1 RNA and protein. Together, these results indicate that SB-insertional mutagenesis can identify high-grade astrocytoma-associated genes and they imply an important role for CSF1 in the development of these tumors. PMID:20388773

  17. Site-directed Mutagenesis Switching a Dimethylallyl Tryptophan Synthase to a Specific Tyrosine C3-Prenylating Enzyme*

    PubMed Central

    Fan, Aili; Zocher, Georg; Stec, Edyta; Stehle, Thilo; Li, Shu-Ming

    2015-01-01

    The tryptophan prenyltransferases FgaPT2 and 7-DMATS (7-dimethylallyl tryptophan synthase) from Aspergillus fumigatus catalyze C4- and C7-prenylation of the indole ring, respectively. 7-DMATS was found to accept l-tyrosine as substrate as well and converted it to an O-prenylated derivative. An acceptance of l-tyrosine by FgaPT2 was also observed in this study. Interestingly, isolation and structure elucidation revealed the identification of a C3-prenylated l-tyrosine as enzyme product. Molecular modeling and site-directed mutagenesis led to creation of a mutant FgaPT2_K174F, which showed much higher specificity toward l-tyrosine than l-tryptophan. Its catalytic efficiency toward l-tyrosine was found to be 4.9-fold in comparison with that of non-mutated FgaPT2, whereas the activity toward l-tryptophan was less than 0.4% of that of the wild-type. To the best of our knowledge, this is the first report on an enzymatic C-prenylation of l-tyrosine as free amino acid and altering the substrate preference of a prenyltransferase by mutagenesis. PMID:25477507

  18. Improving the solubility of anti-LINGO-1 monoclonal antibody Li33 by isotype switching and targeted mutagenesis

    SciTech Connect

    Pepinsky, R. Blake; Silvian, Laura; Berkowitz, Steven A.; Farrington, Graham; Lugovskoy, Alexey; Walus, Lee; Eldredge, John; Capili, Allan; Mi, Sha; Graff, Christilyn; Garber, Ellen

    2010-11-15

    Monoclonal antibodies (Mabs) are a favorite drug platform of the biopharmaceutical industry. Currently, over 20 Mabs have been approved and several hundred others are in clinical trials. The anti-LINGO-1 Mab Li33 was selected from a large panel of antibodies by Fab phage display technology based on its extraordinary biological activity in promoting oligodendrocyte differentiation and myelination in vitro and in animal models of remyelination. However, the Li33 Fab had poor solubility when converted into a full antibody in an immunoglobulin G1 framework. A detailed analysis of the biochemical and structural features of the antibody revealed several possible reasons for its propensity to aggregate. Here, we successfully applied three molecular approaches (isotype switching, targeted mutagenesis of complementarity determining region residues, and glycosylation site insertion mutagenesis) to address the solubility problem. Through these efforts we were able to improve the solubility of the Li33 Mab from 0.3 mg/mL to >50 mg/mL and reduce aggregation to an acceptable level. These strategies can be readily applied to other proteins with solubility issues.

  19. A Defect in DNA Ligase4 Enhances the Frequency of TALEN-Mediated Targeted Mutagenesis in Rice1[OPEN

    PubMed Central

    Cermak, Tomas; Sugimoto, Kazuhiko; Saika, Hiroaki; Mori, Akiko; Osakabe, Keishi; Hamada, Masao; Katayose, Yuichi; Voytas, Daniel F.

    2016-01-01

    We have established methods for site-directed mutagenesis via transcription activator-like effector nucleases (TALENs) in the endogenous rice (Oryza sativa) waxy gene and demonstrated stable inheritance of TALEN-induced somatic mutations to the progeny. To analyze the role of classical nonhomologous end joining (cNHEJ) and alternative nonhomologous end joining (altNHEJ) pathways in TALEN-induced mutagenesis in plant cells, we investigated whether a lack of DNA Ligase4 (Lig4) affects the kinetics of TALEN-induced double-strand break repair in rice cells. Deep-sequencing analysis revealed that the frequency of all types of mutations, namely deletion, insertion, combination of insertion with deletion, and substitution, in lig4 null mutant calli was higher than that in a lig4 heterozygous mutant or the wild type. In addition, the ratio of large deletions (greater than 10 bp) and deletions repaired by microhomology-mediated end joining (MMEJ) to total deletion mutations in lig4 null mutant calli was higher than that in the lig4 heterozygous mutant or wild type. Furthermore, almost all insertions (2 bp or greater) were shown to be processed via copy and paste of one or more regions around the TALENs cleavage site and rejoined via MMEJ regardless of genetic background. Taken together, our findings indicate that the dysfunction of cNHEJ leads to a shift in the repair pathway from cNHEJ to altNHEJ or synthesis-dependent strand annealing. PMID:26668331

  20. Molecular Mechanisms for High Hydrostatic Pressure-Induced Wing Mutagenesis in Drosophila melanogaster.

    PubMed

    Wang, Hua; Wang, Kai; Xiao, Guanjun; Ma, Junfeng; Wang, Bingying; Shen, Sile; Fu, Xueqi; Zou, Guangtian; Zou, Bo

    2015-01-01

    Although High hydrostatic pressure (HHP) as an important physical and chemical tool has been increasingly applied to research of organism, the response mechanisms of organism to HHP have not been elucidated clearly thus far. To identify mutagenic mechanisms of HHP on organisms, here, we treated Drosophila melanogaster (D. melanogaster) eggs with HHP. Approximately 75% of the surviving flies showed significant morphological abnormalities from the egg to the adult stages compared with control flies (p < 0.05). Some eggs displayed abnormal chorionic appendages, some larvae were large and red, and some adult flies showed wing abnormalities. Abnormal wing phenotypes of D. melanogaster induced by HHP were used to investigate the mutagenic mechanisms of HHP on organism. Thus 285 differentially expressed genes associated with wing mutations were identified using Affymetrix Drosophila Genome Array 2.0 and verified with RT-PCR. We also compared wing development-related central genes in the mutant flies with control flies using DNA sequencing to show two point mutations in the vestigial (vg) gene. This study revealed the mutagenic mechanisms of HHP-induced mutagenesis in D. melanogaster and provided a new model for the study of evolution on organisms. PMID:26446369

  1. [KIL-d] Protein Element Confers Antiviral Activity via Catastrophic Viral Mutagenesis.

    PubMed

    Suzuki, Genjiro; Weissman, Jonathan S; Tanaka, Motomasa

    2015-11-19

    Eukaryotic cells are targeted by pathogenic viruses and have developed cell defense mechanisms against viral infection. In yeast, the cellular extrachromosomal genetic element [KIL-d] alters killer activity of M double-stranded RNA killer virus and confers cell resistance against the killer virus. However, its underlying mechanism and the molecular nature of [KIL-d] are unknown. Here, we demonstrate that [KIL-d] is a proteinaceous prion-like aggregate with non-Mendelian cytoplasmic transmission. Deep sequencing analyses revealed that [KIL-d] selectively increases the rate of de novo mutation in the killer toxin gene of the viral genome, producing yeast harboring a defective mutant killer virus with a selective growth advantage over those with WT killer virus. These results suggest that a prion-like [KIL-d] element reprograms the viral replication machinery to induce mutagenesis and genomic inactivation via the long-hypothesized mechanism of "error catastrophe." The findings also support a role for prion-like protein aggregates in cellular defense and adaptation. PMID:26590718

  2. Exploring purine N7 interactions via atomic mutagenesis: The group I ribozyme as a case study

    PubMed Central

    Forconi, Marcello; Benz-Moy, Tara; Gleitsman, Kristin Rule; Ruben, Eliza; Metz, Clyde; Herschlag, Daniel

    2012-01-01

    Atomic mutagenesis has emerged as a powerful tool to unravel specific interactions in complex RNA molecules. An early extensive study of analogs of the exogenous guanosine nucleophile in group I intron self-splicing by Bass and Cech demonstrated structure–function relationships analogous to those seen for protein ligands and provided strong evidence for a well-formed substrate binding site made of RNA. Subsequent functional and structural studies have confirmed these interacting sites and extended our understanding of them, with one notable exception. Whereas 7-methyl guanosine did not affect reactivity in the original study, a subsequent study revealed a deleterious effect of the seemingly more conservative 7-deaza substitution. Here we investigate this paradox, studying these and other analogs with the more thoroughly characterized ribozyme derived from the Tetrahymena group I intron. We found that the 7-deaza substitution lowers binding by ∼20-fold, relative to the cognate exogenous guanosine nucleophile, whereas binding and reaction with 7-methyl and 8-aza-7-deaza substitutions have no effect. These and additional results suggest that there is no functionally important contact between the N7 atom of the exogenous guanosine and the ribozyme. Rather, they are consistent with indirect effects introduced by the N7 substitution on stacking interactions and/or solvation that are important for binding. The set of analogs used herein should be valuable in deciphering nucleic acid interactions and how they change through reaction cycles for other RNAs and RNA/protein complexes. PMID:22543863

  3. Mutational spectrum at GATA1 provides insights into mutagenesis and leukemogenesis in Down syndrome

    PubMed Central

    Cabelof, Diane C.; Patel, Hiral V.; Chen, Qing; van Remmen, Holly; Matherly, Larry H.; Ge, Yubin

    2009-01-01

    Down syndrome (DS) children have a unique genetic susceptibility to develop leukemia, in particular, acute megakaryocytic leukemia (AMkL) associated with somatic GATA1 mutations. The study of this genetic susceptibility with the use of DS as a model of leukemogenesis has broad applicability to the understanding of leukemia in children overall. On the basis of the role of GATA1 mutations in DS AMkL, we analyzed the mutational spectrum of GATA1 mutations to begin elucidating possible mechanisms by which these sequence alterations arise. Mutational analysis revealed a predominance of small insertion/deletion, duplication, and base substitution mutations, including G:C>T:A, G:C>A:T, and A:T>G:C. This mutational spectrum points to potential oxidative stress and aberrant folate metabolism secondary to genes on chromosome 21 (eg, cystathionine-β-synthase, superoxide dismutase) as potential causes of GATA1 mutations. Furthermore, DNA repair capacity evaluated in DS and non-DS patient samples provided evidence that the base excision repair pathway is compromised in DS tissues, suggesting that inability to repair DNA damage also may play a critical role in the unique susceptibility of DS children to develop leukemia. A model of leukemogenesis in DS is proposed in which mutagenesis is driven by cystathionine-β-synthase overexpression and altered folate homeostasis that becomes fixed as the ability to repair DNA damage is compromised. PMID:19633202

  4. Antibacterial activity and mutagenesis of sponge-associated Pseudomonas fluorescens H41.

    PubMed

    Ye, Lumeng; Santos-Gandelman, Juliana F; Hardoim, Cristiane C P; George, Isabelle; Cornelis, Pierre; Laport, Marinella S

    2015-07-01

    Marine sponges (phylum Porifera) are well known to harbour a complex and diverse bacterial community. Some of these sponge-associated bacteria have been shown to be the real producers of secondary metabolites with a wide range of activities from antimicrobials to anticancer agents. Previously, we revealed that the strain Pseudomonas fluorescens H41 isolated from the sponge Haliclona sp. (collected at the coast of Rio de Janeiro, Brazil) showed a strong antimicrobial activity against clinical and marine bacteria. Thus, in this study the genes involved in the antimicrobial activity of P. fluorescens H41 were identified. To this end, a library of mutants was generated via miniTnphoA3 transposon mutagenesis and the resulting clones were characterized for their antimicrobial activity. It was demonstrated that genes involved in the biosynthesis of the pyoverdine siderophore are related to the inhibitory activity of P. fluorescens H41. Therefore, this strain might play an important role in the biocontrol of the host sponge. PMID:25957971

  5. BCL11A enhancer dissection by Cas9-mediated in situ saturating mutagenesis.

    PubMed

    Canver, Matthew C; Smith, Elenoe C; Sher, Falak; Pinello, Luca; Sanjana, Neville E; Shalem, Ophir; Chen, Diane D; Schupp, Patrick G; Vinjamur, Divya S; Garcia, Sara P; Luc, Sidinh; Kurita, Ryo; Nakamura, Yukio; Fujiwara, Yuko; Maeda, Takahiro; Yuan, Guo-Cheng; Zhang, Feng; Orkin, Stuart H; Bauer, Daniel E

    2015-11-12

    Enhancers, critical determinants of cellular identity, are commonly recognized by correlative chromatin marks and gain-of-function potential, although only loss-of-function studies can demonstrate their requirement in the native genomic context. Previously, we identified an erythroid enhancer of human BCL11A, subject to common genetic variation associated with the fetal haemoglobin level, the mouse orthologue of which is necessary for erythroid BCL11A expression. Here we develop pooled clustered regularly interspaced palindromic repeat (CRISPR)-Cas9 guide RNA libraries to perform in situ saturating mutagenesis of the human and mouse enhancers. This approach reveals critical minimal features and discrete vulnerabilities of these enhancers. Despite conserved function of the composite enhancers, their architecture diverges. The crucial human sequences appear to be primate-specific. Through editing of primary human progenitors and mouse transgenesis, we validate the BCL11A erythroid enhancer as a target for fetal haemoglobin reinduction. The detailed enhancer map will inform therapeutic genome editing, and the screening approach described here is generally applicable to functional interrogation of non-coding genomic elements. PMID:26375006

  6. An Assessment of Heavy Ion Irradiation Mutagenesis for Reverse Genetics in Wheat (Triticum aestivum L.)

    PubMed Central

    Fitzgerald, Timothy L.; Powell, Jonathan J.; Stiller, Jiri; Weese, Terri L.; Abe, Tomoko; Zhao, Guangyao; Jia, Jizeng; McIntyre, C. Lynne; Li, Zhongyi; Manners, John M.; Kazan, Kemal

    2015-01-01

    Reverse genetic techniques harnessing mutational approaches are powerful tools that can provide substantial insight into gene function in plants. However, as compared to diploid species, reverse genetic analyses in polyploid plants such as bread wheat can present substantial challenges associated with high levels of sequence and functional similarity amongst homoeologous loci. We previously developed a high-throughput method to identify deletions of genes within a physically mutagenized wheat population. Here we describe our efforts to combine multiple homoeologous deletions of three candidate disease susceptibility genes (TaWRKY11, TaPFT1 and TaPLDß1). We were able to produce lines featuring homozygous deletions at two of the three homoeoloci for all genes, but this was dependent on the individual mutants used in crossing. Intriguingly, despite extensive efforts, viable lines possessing homozygous deletions at all three homoeoloci could not be produced for any of the candidate genes. To investigate deletion size as a possible reason for this phenomenon, we developed an amplicon sequencing approach based on synteny to Brachypodium distachyon to assess the size of the deletions removing one candidate gene (TaPFT1) in our mutants. These analyses revealed that genomic deletions removing the locus are relatively large, resulting in the loss of multiple additional genes. The implications of this work for the use of heavy ion mutagenesis for reverse genetic analyses in wheat are discussed. PMID:25719507

  7. Evaluation of Glucose Dehydrogenase and Pyrroloquinoline Quinine (pqq) Mutagenesis that Renders Functional Inadequacies in Host Plants.

    PubMed

    Naveed, Muhammad; Sohail, Younas; Khalid, Nauman; Ahmed, Iftikhar; Mumtaz, Abdul Samad

    2015-08-01

    The rhizospheric zone abutting plant roots usually clutches a wealth of microbes. In the recent past, enormous genetic resources have been excavated with potential applications in host plant interaction and ancillary aspects. Two Pseudomonas strains were isolated and identified through 16S rRNA and rpoD sequence analyses as P. fluorescens QAU67 and P. putida QAU90. Initial biochemical characterization and their root-colonizing traits indicated their potential role in plant growth promotion. Such aerobic systems, involved in gluconic acid production and phosphate solubilization, essentially require the pyrroloquinoline quinine (PQQ)- dependent glucose dehydrogenase (GDH) in the genome. The PCR screening and amplification of GDH and PQQ and subsequent induction of mutagenesis characterized their possible role as antioxidants as well as in growth promotion, as probed in vitro in lettuce and in vivo in rice, bean, and tomato plants. The results showed significant differences (p < or = 0.05) in parameters of plant height, fresh weight, and dry weight, etc., deciphering a clear and in fact complementary role of GDH and PQQ in plant growth promotion. Our study not only provides direct evidence of the in vivo role of GDH and PQQ in host plants but also reveals their functional inadequacy in the event of mutation at either of these loci. PMID:25839331

  8. BCL11A enhancer dissection by Cas9-mediated in situ saturating mutagenesis

    PubMed Central

    Canver, Matthew C.; Smith, Elenoe C.; Sher, Falak; Pinello, Luca; Sanjana, Neville E.; Shalem, Ophir; Chen, Diane D.; Schupp, Patrick G.; Vinjamur, Divya S.; Garcia, Sara P.; Luc, Sidinh; Kurita, Ryo; Nakamura, Yukio; Fujiwara, Yuko; Maeda, Takahiro; Yuan, Guo-Cheng; Feng, Zhang; Orkin, Stuart H.; Bauer, Daniel E.

    2015-01-01

    Summary Enhancers, critical determinants of cellular identity, are commonly identified by correlative chromatin marks and gain-of-function potential, though only loss-of-function studies can demonstrate their requirement in the native genomic context. Previously we identified an erythroid enhancer of BCL11A, subject to common genetic variation associated with fetal hemoglobin (HbF) level, whose mouse ortholog is necessary for erythroid BCL11A expression. Here we develop pooled CRISPR-Cas9 guide RNA libraries to perform in situ saturating mutagenesis of the human and mouse enhancers. This approach reveals critical minimal features and discrete vulnerabilities of these enhancers. Despite conserved function of the composite enhancers, their architecture diverges. The crucial human sequences appear primate-specific. Through editing of primary human progenitors and mouse transgenesis, we validate the BCL11A erythroid enhancer as a target for HbF reinduction. The detailed enhancer map will inform therapeutic genome editing. The screening approach described here is generally applicable to functional interrogation of noncoding genomic elements. PMID:26375006

  9. Conversed mutagenesis of an inactive peptide to ASIC3 inhibitor for active sites determination.

    PubMed

    Osmakov, Dmitry I; Koshelev, Sergey G; Andreev, Yaroslav A; Dyachenko, Igor A; Bondarenko, Dmitry A; Murashev, Arkadii N; Grishin, Eugene V; Kozlov, Sergey A

    2016-06-15

    Peptide Ugr9-1 from the venom of sea anemone Urticina grebelnyi selectively inhibits the ASIC3 channel and significantly reverses inflammatory and acid-induced pain in vivo. A close homolog peptide Ugr 9-2 does not have these features. To find the pharmacophore residues and explore structure-activity relationships of Ugr 9-1, we performed site-directed mutagenesis of Ugr 9-2 and replaced several positions by the corresponding residues from Ugr 9-1. Mutant peptides Ugr 9-2 T9F and Ugr 9-2 Y12H were able to inhibit currents of the ASIC3 channels 2.2 times and 1.3 times weaker than Ugr 9-1, respectively. Detailed analysis of the spatial models of Ugr 9-1, Ugr 9-2 and both mutant peptides revealed the presence of the basic-aromatic clusters on opposite sides of the molecule, each of which is responsible for the activity. Additionally, Ugr9-1 mutant with truncated N- and C-termini retained similar with the Ugr9-1 action in vitro and was equally potent in vivo model of thermal hypersensitivity. All together, these results are important for studying the structure-activity relationships of ligand-receptor interaction and for the future development of peptide drugs from animal toxins. PMID:26686983

  10. Random and direct mutagenesis to enhance protein secretion in Ashbya gossypii

    PubMed Central

    Ribeiro, Orquídea; Magalhães, Frederico; Aguiar, Tatiana Q; Wiebe, Marilyn G; Penttilä, Merja; Domingues, Lucília

    2013-01-01

    To improve the general secretion ability of the biotechnologically relevant fungus Ashbya gossypii, random mutagenesis with ethyl methane sulfonate (EMS) was performed. The selection and screening strategy followed revealed mutants with improved secretion of heterologous Trichoderma reesei endoglucanase I (EGI), native α-amylase and/or native β-glucosidase. One mutant, S436, presented 1.4- to 2-fold increases in all extracellular enzymatic activities measured, when compared with the parent strain, pointing to a global improvement in protein secretion. Three other mutants exhibited 2- to 3-fold improvements in only one (S397, B390) or two (S466) of the measured activities.   A targeted genetic approach was also followed. Two homologs of the Saccharomyces cerevisiae GAS1, AgGAS1A (AGL351W) and AgGAS1B (AGL352W), were deleted from the A. gossypii genome. For both copies deletion, a new antibiotic marker cassette conferring resistance to phleomycin, BLE3, was constructed. GAS1 encodes an β-1,3-glucanosyltransglycosylase involved in cell wall assembly. Higher permeability of the cell wall was expected to increase the protein secretion capacity. However, total protein secreted to culture supernatants and secreted EGI activity did not increase in the Aggas1AΔ mutants. Deletion of the AgGAS1B copy affected cellular morphology and resulted in severe retardation of growth, similarly to what has been reported for GAS1-defficient yeast. Thus, secretion could not be tested in these mutants. PMID:23644277

  11. Improving the neutral phytase activity from Bacillus amyloliquefaciens DSM 1061 by site-directed mutagenesis.

    PubMed

    Xu, Wei; Shao, Rong; Wang, Zupeng; Yan, Xiuhua

    2015-03-01

    Neutral phytase is used as a feed additive for degradation of anti-nutritional phytate in aquatic feed industry. Site-directed mutagenesis of Bacillus amyloliquefaciens DSM 1061 phytase was performed with an aim to increase its activity. Mutation residues were chosen based on multiple sequence alignments and structure analysis of neutral phytsaes from different microorganisms. The mutation sites on surface (D148E, S197E and N156E) and around the active site (D52E) of phytase were selected. Analysis of the phytase variants showed that the specific activities of mutants D148E and S197E remarkably increased by about 35 and 13% over a temperature range of 40-75 °C at pH 7.0, respectively. The k cat of mutants D148E and S197E were 1.50 and 1.25 times than that of the wild-type phytase, respectively. Both D148E and S197E showed much higher thermostability than that of the wild-type phytase. However, mutants N156E and D52E led to significant loss of specific activity of the enzyme. Structural analysis revealed that these mutations may affect conformation of the active site of phytase. The present mutant phytases D148E and S197E with increased activities and thermostabilities have application potential as additives in aquaculture feed. PMID:25613522

  12. Structural insights from random mutagenesis of Campylobacter jejuni oligosaccharyltransferase PglB

    PubMed Central

    2012-01-01

    Background Protein glycosylation is of fundamental importance in many biological systems. The discovery of N-glycosylation in bacteria and the functional expression of the N-oligosaccharyltransferase PglB of Campylobacter jejuni in Escherichia coli enabled the production of engineered glycoproteins and the study of the underlying molecular mechanisms. A particularly promising application for protein glycosylation in recombinant bacteria is the production of potent conjugate vaccines where polysaccharide antigens of pathogenic bacteria are covalently bound to immunogenic carrier proteins. Results In this study capsular polysaccharides of the clinically relevant pathogen Staphylococcus aureus serotype 5 (CP5) were expressed in Escherichia coli and linked in vivo to a detoxified version of Pseudomonas aeruginosa exotoxin (EPA). We investigated which amino acids of the periplasmic domain of PglB are crucial for the glycosylation reaction using a newly established 96-well screening system enabling the relative quantification of glycoproteins by enzyme-linked immunosorbent assay. A random mutant library was generated by error-prone PCR and screened for inactivating amino acid substitutions. In addition to 15 inactive variants with amino acid changes within the previously known, strictly conserved WWDYG motif of N-oligosaccharyltransferases, 8 inactivating mutations mapped to a flexible loop in close vicinity of the amide nitrogen atom of the acceptor asparagine as revealed in the crystal structure of the homologous enzyme C. lari PglB. The importance of the conserved loop residue H479 for glycosylation was confirmed by site directed mutagenesis, while a change to alanine of the adjacent, non-conserved L480 had no effect. In addition, we investigated functional requirements in the so-called MIV motif of bacterial N-oligosaccharyltransferases. Amino acid residues I571 and V575, which had been postulated to interact with the acceptor peptide, were subjected to cassette

  13. Retroviral Vectors for Analysis of Viral Mutagenesis and Recombination

    PubMed Central

    Rawson, Jonathan M.O.; Mansky, Louis M.

    2014-01-01

    Retrovirus population diversity within infected hosts is commonly high due in part to elevated rates of replication, mutation, and recombination. This high genetic diversity often complicates the development of effective diagnostics, vaccines, and antiviral drugs. This review highlights the diverse vectors and approaches that have been used to examine mutation and recombination in retroviruses. Retroviral vectors for these purposes can broadly be divided into two categories: those that utilize reporter genes as mutation or recombination targets and those that utilize viral genes as targets of mutation or recombination. Reporter gene vectors greatly facilitate the detection, quantification, and characterization of mutants and/or recombinants, but may not fully recapitulate the patterns of mutagenesis or recombination observed in native viral gene sequences. In contrast, the detection of mutations or recombination events directly in viral genes is more biologically relevant but also typically more challenging and inefficient. We will highlight the advantages and disadvantages of the various vectors and approaches used as well as propose ways in which they could be improved. PMID:25254386

  14. Oligonucleotide-directed mutagenesis for precision gene editing.

    PubMed

    Sauer, Noel J; Mozoruk, Jerry; Miller, Ryan B; Warburg, Zachary J; Walker, Keith A; Beetham, Peter R; Schöpke, Christian R; Gocal, Greg F W

    2016-02-01

    Differences in gene sequences, many of which are single nucleotide polymorphisms, underlie some of the most important traits in plants. With humanity facing significant challenges to increase global agricultural productivity, there is an urgent need to accelerate the development of these traits in plants. oligonucleotide-directed mutagenesis (ODM), one of the many tools of Cibus' Rapid Trait Development System (RTDS(™) ) technology, offers a rapid, precise and non-transgenic breeding alternative for trait improvement in agriculture to address this urgent need. This review explores the application of ODM as a precision genome editing technology, with emphasis on using oligonucleotides to make targeted edits in plasmid, episomal and chromosomal DNA of bacterial, fungal, mammalian and plant systems. The process of employing ODM by way of RTDS technology has been improved in many ways by utilizing a fluorescence conversion system wherein a blue fluorescent protein (BFP) can be changed to a green fluorescent protein (GFP) by editing a single nucleotide of the BFP gene (CAC→TAC; H66 to Y66). For example, dependent on oligonucleotide length, applying oligonucleotide-mediated technology to target the BFP transgene in Arabidopsis thaliana protoplasts resulted in up to 0.05% precisely edited GFP loci. Here, the development of traits in commercially relevant plant varieties to improve crop performance by genome editing technologies such as ODM, and by extension RTDS, is reviewed. PMID:26503400

  15. Genome-wide transposon mutagenesis in pathogenic Leptospira species.

    PubMed

    Murray, Gerald L; Morel, Viviane; Cerqueira, Gustavo M; Croda, Julio; Srikram, Amporn; Henry, Rebekah; Ko, Albert I; Dellagostin, Odir A; Bulach, Dieter M; Sermswan, Rasana W; Adler, Ben; Picardeau, Mathieu

    2009-02-01

    Leptospira interrogans is the most common cause of leptospirosis in humans and animals. Genetic analysis of L. interrogans has been severely hindered by a lack of tools for genetic manipulation. Recently we developed the mariner-based transposon Himar1 to generate the first defined mutants in L. interrogans. In this study, a total of 929 independent transposon mutants were obtained and the location of insertion determined. Of these mutants, 721 were located in the protein coding regions of 551 different genes. While sequence analysis of transposon insertion sites indicated that transposition occurred in an essentially random fashion in the genome, 25 unique transposon mutants were found to exhibit insertions into genes encoding 16S or 23S rRNAs, suggesting these genes are insertional hot spots in the L. interrogans genome. In contrast, loci containing notionally essential genes involved in lipopolysaccharide and heme biosynthesis showed few transposon insertions. The effect of gene disruption on the virulence of a selected set of defined mutants was investigated using the hamster model of leptospirosis. Two attenuated mutants with disruptions in hypothetical genes were identified, thus validating the use of transposon mutagenesis for the identification of novel virulence factors in L. interrogans. This library provides a valuable resource for the study of gene function in L. interrogans. Combined with the genome sequences of L. interrogans, this provides an opportunity to investigate genes that contribute to pathogenesis and will provide a better understanding of the biology of L. interrogans. PMID:19047402

  16. Efficient Gene Transfer and Targeted Mutagenesis in Fusobacterium nucleatum

    PubMed Central

    Haake, Susan Kinder; Yoder, Sean; Gerardo, Sharon Hunt

    2006-01-01

    Fusobacterium nucleatum is a Gram-negative anaerobe important in dental biofilm ecology and infectious diseases with significant societal impact. The lack of efficient genetic systems has hampered molecular analyses in this microorganism. We previously reported construction of a shuttle plasmid, pHS17, using the native fusobacterial plasmid pFN1 and an erythromycin resistance cassette. However, the host range of pHS17 was restricted to F. nucleatum, ATCC 10953 and the transformation efficiency was limited. This study was undertaken to improve genetic systems for molecular analysis in F. nucleatum. We identified a second F. nucleatum strain, ATCC 23726, which is transformed with improved efficiency compared to ATCC 10953. Two novel second generation pFN1-based shuttle plasmids, pHS23 and pHS30, were developed and enable transformation of ATCC 23726 at 6.2 x 104 and 1.5 x 106 transformants/microgram of plasmid DNA, respectively. The transformation efficiency of pHS30, which harbors a catP gene conferring resistance to chloramphenicol, was more than 1,000-fold greater than that of pHS17. The improved transformation efficiency facilitated disruption of the chromosomal rnr gene using a suicide plasmid pHS19, the first demonstration of targeted mutagenesis in F. nucleatum. These results provide significant advances in the development of systems for molecular analysis in F. nucleatum. PMID:16115683

  17. Site-directed mutagenesis and gene deletion using reverse genetics.

    PubMed

    Muhl, Daniela; Filloux, Alain

    2014-01-01

    Understanding gene function is far easier when tools are available to engineer a bacterial strain lacking a specific gene and phenotypically compare its behavior with the corresponding parental strain. Such mutants could be selected randomly, either by natural selection under particular stress conditions or by random mutagenesis using transposon delivery as described elsewhere in this book. However, with the advent of the genomic era there are now hundreds of bacterial genomes whose sequence is available, and thus, genes can be identified, chosen, and strategies designed to specifically inactivate them. This can be done by using suicide plasmids and is most convenient when the bacterium of interest is easily amenable to genetic manipulation. The method presented here will describe the use of a suicide vector, pKNG101, which allows the selection of a double-recombination event. The first event results in the integration of the pKNG101 derivative carrying the "mutator" fragment onto the chromosome, and could be selected on plates containing appropriate antibiotics. The pKNG101 carries the sacB gene, which induces death when cells are grown on sucrose. Growth on sucrose plates will thus select the second homologous recombination event, which results in removing the plasmid backbone and leaving behind the mutated target gene. This method has been widely used over the last 20 years to inactivate genes in a wide range of gram-negative bacteria and in particular in Pseudomonas aeruginosa. PMID:24818930

  18. Targeted mutagenesis using CRISPR/Cas system in medaka

    PubMed Central

    Ansai, Satoshi; Kinoshita, Masato

    2014-01-01

    ABSTRACT Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system-based RNA-guided endonuclease (RGEN) has recently emerged as a simple and efficient tool for targeted genome editing. In this study, we showed successful targeted mutagenesis using RGENs in medaka, Oryzias latipes. Somatic and heritable mutations were induced with high efficiency at the targeted genomic sequence on the DJ-1 gene in embryos that had been injected with the single guide RNA (sgRNA) transcribed by a T7 promoter and capped RNA encoding a Cas9 nuclease. The sgRNAs that were designed for the target genomic sequences without the 5′ end of GG required by the T7 promoter induced the targeted mutations. This suggests that the RGEN can target any sequence adjacent to an NGG protospacer adjacent motif (PAM) sequence, which occurs once every 8 bp. The off-target alterations at 2 genomic loci harboring double mismatches in the 18-bp targeting sequences were induced in the RGEN-injected embryos. However, we also found that the off-target effects could be reduced by lower dosages of sgRNA. Taken together, our results suggest that CRISPR/Cas-mediated RGENs may be an efficient and flexible tool for genome editing in medaka. PMID:24728957

  19. Analysis of HIV-2 Vpx by modeling and insertional mutagenesis

    SciTech Connect

    Mahnke, Lisa A. . E-mail: lmahnke@im.wustl.edu; Belshan, Michael; Ratner, Lee . E-mail: lratner@im.wustl.edu

    2006-04-25

    Vpx facilitates HIV-2 nuclear localization by a poorly understood mechanism. We have compared Vpx to an NMR structure HIV-1 Vpr in a central helical domain and probed regions of Vpx by insertional mutagenesis. A predicted loop between helices two and three appears to be unique, overlapping with a known novel nuclear localization signal. Overall, Vpx was found to be surprisingly flexible, tolerating a series of large insertions. We found that insertion within the polyproline-containing C-terminus destabilizes nuclear localization, whereas mutating a second helix in the central domain disrupts viral packaging. Other insertional mutants in the predicted loop and in a linker region between the central domain and the C-terminus may be useful as sites of intramolecular tags as they could be packaged adequately and retained preintegration complex associated integration activity in a serum starvation assay. An unexpected result was found within a previously defined nuclear localization motif near aa 71. This mutant retained robust nuclear localization in a GFP fusion assay and was competent for preintegration complex associated nuclear import. In summary, we have modeled helical content in Vpx and assessed potential sites of intramolecular tags which may prove useful for protein-protein interactions studies.

  20. Lethal Mutagenesis of HIV with Mutagenic Nucleoside Analogs

    NASA Astrophysics Data System (ADS)

    Loeb, Lawrence A.; Essigmann, John M.; Kazazi, Farhad; Zhang, Jue; Rose, Karl D.; Mullins, James I.

    1999-02-01

    The human immunodeficiency virus (HIV) replicates its genome and mutates at exceptionally high rates. As a result, the virus is able to evade immunological and chemical antiviral agents. We tested the hypothesis that a further increase in the mutation rate by promutagenic nucleoside analogs would abolish viral replication. We evaluated deoxynucleoside analogs for lack of toxicity to human cells, incorporation by HIV reverse transcriptase, resistance to repair when incorporated into the DNA strand of an RNA\\cdot DNA hybrid, and mispairing at high frequency. Among the candidates tested, 5-hydroxydeoxycytidine (5-OH-dC) fulfilled these criteria. In seven of nine experiments, the presence of this analog resulted in the loss of viral replicative potential after 9-24 sequential passages of HIV in human CEM cells. In contrast, loss of viral replication was not observed in 28 control cultures passaged in the absence of the nucleoside analog, nor with other analogs tested. Sequence analysis of a portion of the HIV reverse transcriptase gene demonstrated a disproportionate increase in G -> A substitutions, mutations predicted to result from misincorporation of 5-OH-dC into the cDNA during reverse transcription. Thus, "lethal mutagenesis" driven by the class of deoxynucleoside analogs represented by 5-OH-dC could provide a new approach to treating HIV infections and, potentially, other viral infections.

  1. Functional mapping of Cre recombinase by pentapeptide insertional mutagenesis.

    PubMed

    Petyuk, Vladislav; McDermott, Jeffrey; Cook, Malcolm; Sauer, Brian

    2004-08-27

    Cre is a site-specific recombinase from bacteriophage P1. It is a member of the tyrosine integrase family and catalyzes reciprocal recombination between specific 34-bp sites called loxP. To analyze the structure-function relationships of this enzyme, we performed large scale pentapeptide insertional mutagenesis to generate insertions of five amino acids at random positions in the protein. The high density of insertion mutations into Cre allowed us to identify an unexpected degree of functional tolerance to insertions into the 4-5 beta-hairpin and into the loop between helices J and K (both of which contact the DNA in the minor groove) and also into helix A. The phenotypes of the majority of inserts allowed us to confirm a variety of predictions made on the basis of sequence conservation, known three-dimensional structure, and proposed catalytic mechanism. In particular, most insertions into conserved regions or secondary structure elements inactivated Cre, and most insertions located in nonconserved, unstructured regions preserved Cre activity. Less expectedly, the non-conserved and poorly structured loops and linkers between helices A-B, E-F, and M-N did not tolerate insertions, thus identifying these as critical regions for recombinase activity. We purified and characterized in vitro several representatives of these "unexpected" Cre insertion mutants. The role of those regions in the recombination process is discussed. PMID:15218019

  2. Recurrent AAV2-related insertional mutagenesis in human hepatocellular carcinomas.

    PubMed

    Nault, Jean-Charles; Datta, Shalini; Imbeaud, Sandrine; Franconi, Andrea; Mallet, Maxime; Couchy, Gabrielle; Letouzé, Eric; Pilati, Camilla; Verret, Benjamin; Blanc, Jean-Frédéric; Balabaud, Charles; Calderaro, Julien; Laurent, Alexis; Letexier, Mélanie; Bioulac-Sage, Paulette; Calvo, Fabien; Zucman-Rossi, Jessica

    2015-10-01

    Hepatocellular carcinomas (HCCs) are liver tumors related to various etiologies, including alcohol intake and infection with hepatitis B (HBV) or C (HCV) virus. Additional risk factors remain to be identified, particularly in patients who develop HCC without cirrhosis. We found clonal integration of adeno-associated virus type 2 (AAV2) in 11 of 193 HCCs. These AAV2 integrations occurred in known cancer driver genes, namely CCNA2 (cyclin A2; four cases), TERT (telomerase reverse transcriptase; one case), CCNE1 (cyclin E1; three cases), TNFSF10 (tumor necrosis factor superfamily member 10; two cases) and KMT2B (lysine-specific methyltransferase 2B; one case), leading to overexpression of the target genes. Tumors with viral integration mainly developed in non-cirrhotic liver (9 of 11 cases) and without known risk factors (6 of 11 cases), suggesting a pathogenic role for AAV2 in these patients. In conclusion, AAV2 is a DNA virus associated with oncogenic insertional mutagenesis in human HCC. PMID:26301494

  3. Particulate matter inhibits DNA repair and enhances mutagenesis.

    PubMed

    Mehta, Manju; Chen, Lung-Chi; Gordon, Terry; Rom, William; Tang, Moon-Shong

    2008-12-01

    Exposure to ambient air pollution has been associated with adverse health effects including lung cancer. A recent epidemiology study has established that each 10 microg/m3 elevation in long-term exposure to average PM2.5 ambient concentration was associated with approximately 8% of lung cancer mortality. The underlying mechanisms of how PM contributes to lung carcinogenesis, however, remain to be elucidated. We have recently found that transition metals such as nickel and chromium and oxidative stress induced lipid peroxidation metabolites such as aldehydes can greatly inhibit nucleotide excision repair (NER) and enhance carcinogen-induced mutations. Because PM is rich in metal and aldehyde content and can induce oxidative stress, we tested the effect of PM on DNA repair capacity in cultured human lung cells using in vitro DNA repair synthesis and host cell reactivation assays. We found that PM greatly inhibits NER for ultraviolet (UV) light and benzo(a)pyrene diol epoxide (BPDE) induced DNA damage in human lung cells. We further demonstrated that PM exposure can significantly increase both spontaneous and UV-induced mutagenesis. These results together suggest that the carcinogenicity of PM may act through its combined effect on suppression of DNA repair and enhancement of DNA replication errors. PMID:18804180

  4. Multiplex Conditional Mutagenesis Using Transgenic Expression of Cas9 and sgRNAs.

    PubMed

    Yin, Linlin; Maddison, Lisette A; Li, Mingyu; Kara, Nergis; LaFave, Matthew C; Varshney, Gaurav K; Burgess, Shawn M; Patton, James G; Chen, Wenbiao

    2015-06-01

    Determining the mechanism of gene function is greatly enhanced using conditional mutagenesis. However, generating engineered conditional alleles is inefficient and has only been widely used in mice. Importantly, multiplex conditional mutagenesis requires extensive breeding. Here we demonstrate a system for one-generation multiplex conditional mutagenesis in zebrafish (Danio rerio) using transgenic expression of both cas9 and multiple single guide RNAs (sgRNAs). We describe five distinct zebrafish U6 promoters for sgRNA expression and demonstrate efficient multiplex biallelic inactivation of tyrosinase and insulin receptor a and b, resulting in defects in pigmentation and glucose homeostasis. Furthermore, we demonstrate temporal and tissue-specific mutagenesis using transgenic expression of Cas9. Heat-shock-inducible expression of cas9 allows temporal control of tyr mutagenesis. Liver-specific expression of cas9 disrupts insulin receptor a and b, causing fasting hypoglycemia and postprandial hyperglycemia. We also show that delivery of sgRNAs targeting ascl1a into the eye leads to impaired damage-induced photoreceptor regeneration. Our findings suggest that CRISPR/Cas9-based conditional mutagenesis in zebrafish is not only feasible but rapid and straightforward. PMID:25855067

  5. Extinction of hepatitis C virus by ribavirin in hepatoma cells involves lethal mutagenesis.

    PubMed

    Ortega-Prieto, Ana M; Sheldon, Julie; Grande-Pérez, Ana; Tejero, Héctor; Gregori, Josep; Quer, Josep; Esteban, Juan I; Domingo, Esteban; Perales, Celia

    2013-01-01

    Lethal mutagenesis, or virus extinction produced by enhanced mutation rates, is under investigation as an antiviral strategy that aims at counteracting the adaptive capacity of viral quasispecies, and avoiding selection of antiviral-escape mutants. To explore lethal mutagenesis of hepatitis C virus (HCV), it is important to establish whether ribavirin, the purine nucleoside analogue used in anti-HCV therapy, acts as a mutagenic agent during virus replication in cell culture. Here we report the effect of ribavirin during serial passages of HCV in human hepatoma Huh-7.5 cells, regarding viral progeny production and complexity of mutant spectra. Ribavirin produced an increase of mutant spectrum complexity and of the transition types associated with ribavirin mutagenesis, resulting in HCV extinction. Ribavirin-mediated depletion of intracellular GTP was not the major contributory factor to mutagenesis since mycophenolic acid evoked a similar decrease in GTP without an increase in mutant spectrum complexity. The intracellular concentration of the other nucleoside-triphosphates was elevated as a result of ribavirin treatment. Mycophenolic acid extinguished HCV without an intervening mutagenic activity. Ribavirin-mediated, but not mycophenolic acid-mediated, extinction of HCV occurred via a decrease of specific infectivity, a feature typical of lethal mutagenesis. We discuss some possibilities to explain disparate results on ribavirin mutagenesis of HCV. PMID:23976977

  6. Genome-Wide Transposon Mutagenesis in Saccharomyces cerevisiae and Candida albicans

    PubMed Central

    Xu, Tao; Bharucha, Nikë; Kumar, Anuj

    2016-01-01

    Transposon mutagenesis is an effective method for generating large sets of random mutations in target DNA, with applicability toward numerous types of genetic screens in prokaryotes, single-celled eukaryotes, and metazoans alike. Relative to methods of random mutagenesis by chemical/UV treatment, transposon insertions can be easily identified in mutants with phenotypes of interest. The construction of transposon insertion mutants is also less labor-intensive on a genome-wide scale than methods for targeted gene replacement, although transposon insertions are not precisely targeted to a specific residue, and thus coverage of the target DNA can be problematic. The collective advantages of transposon mutagenesis have been well demonstrated in studies of the budding yeast Saccharomyces cerevisiae and the related pathogenic yeast Candida albicans, as transposon mutagenesis has been used extensively for phenotypic screens in both yeasts. Consequently, we present here protocols for the generation and utilization of transposon-insertion DNA libraries in S. cerevisiae and C. albicans. Specifically, we present methods for the large-scale introduction of transposon insertion alleles in a desired strain of S. cerevisiae. Methods are also presented for transposon mutagenesis of C. albicans, encompassing both the construction of the plasmid-based transposon-mutagenized DNA library and its introduction into a desired strain of Candida. In total, these methods provide the necessary information to implement transposon mutagenesis in yeast, enabling the construction of large sets of identifiable gene disruption mutations, with particular utility for phenotypic screening in nonstandard genetic backgrounds. PMID:21815095

  7. Alanine screening mutagenesis establishes the critical inactivating damage of irradiated E. coli lactose repressor.

    PubMed

    Goffinont, Stephane; Villette, Sandrine; Spotheim-Maurizot, Melanie

    2012-06-01

    The function of the E. coli lactose operon requires the binding of lactose repressor to operator DNA. We have previously shown that γ rradiation destabilizes the repressor-operator complex because the repressor loses its DNA-binding ability. It was suggested that the observed oxidation of the four tyrosines (Y7, Y12, Y17, Y47) and the concomitant structural changes of the irradiated DNA-binding domains (headpieces) could be responsible for the inactivation. To pinpoint the tyrosine whose oxidation has the strongest effect, four headpieces containing the product of tyrosine oxidation, 3,4-dihydroxyphenylalanine (DOPA), were simulated by molecular dynamics. We have observed that replacing Y47 by DOPA triggers the largest change of structure and stability of the headpiece and have concluded that Y47 oxidation is the greatest contributor to the decrease of repressor binding to DNA. To experimentally verify this conclusion, we applied the alanine screening mutagenesis approach. Tetrameric mutated repressors bearing an alanine instead of each one of the tyrosines were prepared and their binding to operator DNA was checked. Their binding ability is quite similar to that of the wild-type repressor, except for the Y47A mutant whose binding is strongly reduced. Circular dichroism determinations revealed small reductions of the proportion of α helices and of the melting temperature for Y7A, Y12A and Y17A headpieces, but much larger ones were revealed for Y47A headpiece. These results established the critical role of Y47 oxidation in modifying the structure and stability of the headpiece, and in reduction of the binding ability of the whole lactose repressor. PMID:22551504

  8. Perturbation of bacteriochlorophyll molecules in Fenna-Matthews-Olson protein complexes through mutagenesis of cysteine residues.

    PubMed

    Saer, Rafael; Orf, Gregory S; Lu, Xun; Zhang, Hao; Cuneo, Matthew J; Myles, Dean A A; Blankenship, Robert E

    2016-09-01

    The Fenna-Matthews-Olson (FMO) pigment-protein complex in green sulfur bacteria transfers excitation energy from the chlorosome antenna complex to the reaction center. In understanding energy transfer in the FMO protein, the individual contributions of the bacteriochlorophyll pigments to the FMO complex's absorption spectrum could provide detailed information with which molecular and energetic models can be constructed. The absorption properties of the pigments, however, are such that their spectra overlap significantly. To overcome this, we used site-directed mutagenesis to construct a series of mutant FMO complexes in the model green sulfur bacterium Chlorobaculum tepidum (formerly Chlorobium tepidum). Two cysteines at positions 49 and 353 in the C. tepidum FMO complex, which reside near hydrogen bonds between BChls 2 and 3, and their amino acid binding partner serine 73 and tyrosine 15, respectively, were changed to alanine residues. The resulting C49A, C353A, and C49A C353A double mutants were analyzed with a combination of optical absorption and circular dichroism (CD) spectroscopies. Our results revealed changes in the absorption properties of several underlying spectral components in the FMO complex, as well as the redox behavior of the complex in response to the reductant sodium dithionite. A high-resolution X-ray structure of the C49A C353A double mutant reveals that these spectral changes appear to be independent of any major structural rearrangements in the FMO mutants. Our findings provide important tests for theoretical calculations of the C. tepidum FMO absorption spectrum, and additionally highlight a possible role for cysteine residues in the redox activity of the pigment-protein complex. PMID:27114180

  9. Tetragonal Lysozyme Interactions Studied by Site Directed Mutagenesis

    NASA Technical Reports Server (NTRS)

    Crawford, Lisa; Karr, Laurel J.; Nadarajah, Arunan; Pusey, Marc

    1999-01-01

    A number of recent experimental and theoretical studies have indicated that tetragonal lysozyme crystal growth proceeds by the addition of aggregates, formed by reversible self association of the solute molecules in the bulk solution. Periodic bond chain and atomic force microscopy studies have indicated that the probable growth unit is at minimum a 43 tetramer, and most likely an octamer composed of two complete turns about the 43 axis. If these results are correct, then there are intermolecular interactions which are only formed in the solution and others only formed at the joining of the growth unit to the crystal surface. We have set out to study these interactions, and the correctness of this hypothesis, using site directed mutagenesis of specific amino acid residues involved in the different bonds. We had initially expressed wild type lysozyme in S. cervasiae with yields of approximately 5 mg/L, which were eventually raised to approximately 40 mg/L. We are now moving the expression to the Pichia system, with anticipated yields of 300 to (3)500 mg/L, comparable to what can be obtained from egg whites. An additional advantage of using recombinant protein is the greater genetic homogeneity of the material obtained and the absence of any other contaminating egg proteins. The first mutation experiments are TYR 23 (Registered) PHE or ALA and ASN 113 (Registered) ALA or ASP. Both TYR 23 and ASN 113 form part of the postulated dimerization intermolecular binding site which lead to the formation of the 43 helix. Tyrosine also participates in an intermolecular hydrogen bond with ARG 114. The results of these and subsequent experiments will be discussed.

  10. Tetragonal Lysozyme Interactions Studied by Site Directed Mutagenesis

    NASA Technical Reports Server (NTRS)

    Crawford, Lisa; Karr, Laurel; Pusey, Marc

    1998-01-01

    A number of recent experimental and theoretical studies have indicated that tetragonal lysozyme crystal growth proceeds by the addition of aggregates, formed by reversible self association of the solute molecules in the bulk'solution. Periodic bond chain and atomic force microscopy studies have indicated that the probable growth unit is at minimum a 43 tetramer, and most likely an octamer composed of two complete turns about the 4(sub 3) axis. If these results are correct, then there are intermolecular interactions which are only formed in the solution and others only formed at the joining of the growth unit to the crystal surface. We have set out to study these interactions, and the correctness of this hypothesis, using site directed mutagenesis of specific amino acid residues involved in the different bonds. We had initially expressed wild type lysozyme in S. cervasiae with yields of approximately 5 mg/L, which were eventually raised to approximately 40 mg/L. We are now moving the expression to the Pichia system, with anticipated yields of 300 to greater than 500 mg/L, comparable to what can be obtained from egg whites. An additional advantage of using recombinant protein is the greater genetic homogeneity of the material obtained and the absence of any other contaminating egg proteins. The first mutation experiments are TYR 23 yields PHE or ALA and ASN 113 yields ALA or ASP. Both TYR 23 and ASN 113 form part of the postulated dimerization intermolecular binding site which lead to the formation of the 4(sub 3) helix. Tyrosine also participates in an intermolecular hydrogen bond with ARG 114. The results of these and subsequent experiments will be discussed.

  11. The ClosTron: Mutagenesis in Clostridium refined and streamlined.

    PubMed

    Heap, John T; Kuehne, Sarah A; Ehsaan, Muhammad; Cartman, Stephen T; Cooksley, Clare M; Scott, Jamie C; Minton, Nigel P

    2010-01-01

    The recent development of the ClosTron Group II intron directed mutagenesis tool for Clostridium has advanced genetics in this genus, and here we present several significant improvements. We have shown how marker re-cycling can be used to construct strains with multiple mutations, demonstrated using FLP/FRT in Clostridium acetobutylicum; tested the capacity of the system for the delivery of transgenes to the chromosome of Clostridium sporogenes, which proved feasible for 1.0kbp transgenes in addition to a marker; and extended the host range of the system, constructing mutants in Clostridium beijerinckii and, for the first time, in a B1/NAP1/027 'epidemic' strain of Clostridium difficile. Automated intron design bioinformatics are now available free-of-charge at our website http://clostron.com; the out-sourced construction of re-targeted intron plasmids has become cost-effective as well as rapid; and the combination of constitutive intron expression with direct selection for intron insertions has made mutant isolation trivial. These developments mean mutants can now be constructed with very little time and effort for the researcher. Those who prefer to construct plasmids in-house are no longer reliant on a commercial kit, as a mixture of two new plasmids provides unlimited template for intron re-targeting by Splicing by Overlap Extension (SOE) PCR. The new ClosTron plasmids also offer blue-white screening and other options for identification of recombinant plasmids. The improved ClosTron system supersedes the prototype plasmid pMTL007 and the original method, and exploits the potential of Group II introns more fully. PMID:19891996

  12. Structure-based design of combinatorial mutagenesis libraries

    PubMed Central

    Verma, Deeptak; Grigoryan, Gevorg; Bailey-Kellogg, Chris

    2015-01-01

    The development of protein variants with improved properties (thermostability, binding affinity, catalytic activity, etc.) has greatly benefited from the application of high-throughput screens evaluating large, diverse combinatorial libraries. At the same time, since only a very limited portion of sequence space can be experimentally constructed and tested, an attractive possibility is to use computational protein design to focus libraries on a productive portion of the space. We present a general-purpose method, called “Structure-based Optimization of Combinatorial Mutagenesis” (SOCoM), which can optimize arbitrarily large combinatorial mutagenesis libraries directly based on structural energies of their constituents. SOCoM chooses both positions and substitutions, employing a combinatorial optimization framework based on library-averaged energy potentials in order to avoid explicitly modeling every variant in every possible library. In case study applications to green fluorescent protein, β-lactamase, and lipase A, SOCoM optimizes relatively small, focused libraries whose variants achieve energies comparable to or better than previous library design efforts, as well as larger libraries (previously not designable by structure-based methods) whose variants cover greater diversity while still maintaining substantially better energies than would be achieved by representative random library approaches. By allowing the creation of large-scale combinatorial libraries based on structural calculations, SOCoM promises to increase the scope of applicability of computational protein design and improve the hit rate of discovering beneficial variants. While designs presented here focus on variant stability (predicted by total energy), SOCoM can readily incorporate other structure-based assessments, such as the energy gap between alternative conformational or bound states. PMID:25611189

  13. Germinal cell mutagenesis in specially designed maize genotypes.

    PubMed Central

    Plewa, M J; Wagner, E D

    1981-01-01

    We have used three inbreds of Zea mays in our in situ and laboratory studies in environmental mutagenesis. Inbred W22 plants homozygous for wx-C were used in a study to detect the possible mutagenic properties of 32 pesticides or combination of pesticides under modern agricultural conditions. The large numbers of pollen grains analyzed and the ease in detecting mutant pollen grains enabled us to treat the experimental plants with field recommended rates of pesticides. In a current study we are evaluating the possible mutagenicity of Chicago municipal sewage sludge. We are measuring the frequency of mutant pollen grains in inbred M14 at both the wx-C and wx-90 heteroalleles. These plants were exposed to various concentrations of municipal sewage sludge under field conditions. We have inbred Early-Early Synthetic for five generations and tested this inbred with known mutagens. Early-Early Synthetic is a rapidly maturing inbred growing from kernel to anthesis in approximately 4 weeks and attaining a height of approximately 50 cm. Plants of this inbred have been chronically treated with ethylmethanesulfonate (EMS) or maleic hydrazide (MH) under laboratory conditions and forward mutation at the wx locus was measured in the pollen grains. EMS and MH were mutagenic at concentrations of 1 microM and 10 nM, respectively. The concentrations of EMS and MH were calibrated in Early-Early Synthetic to a linear increase in the frequency of forward mutant pollen grains. The construction of a maize monitor for environmental mutagens is currently in progress. This assay will measure forward or reverse mutation at the wx locus in pollen grains, point mutation in somatic cells and will incorporate a cytogenetic endpoint in root-tip cells. Images FIGURE 2. FIGURE 3. FIGURE 5. PMID:6780335

  14. Synthetic approach to stop-codon scanning mutagenesis.

    PubMed

    Nie, Lihua; Lavinder, Jason J; Sarkar, Mohosin; Stephany, Kimberly; Magliery, Thomas J

    2011-04-27

    A general combinatorial mutagenesis strategy using common dimethoxytrityl-protected mononucleotide phosphoramidites and a single orthogonally protected trinucleotide phosphoramidite (Fmoc-TAG; Fmoc = 9-fluorenylmethoxycarbonyl) was developed to scan a gene with the TAG amber stop codon with complete synthetic control. In combination with stop-codon suppressors that insert natural (e.g., alanine) or unnatural (e.g., p-benzoylphenylalanine, Bpa) amino acids, a single DNA library can be used to incorporate different amino acids for diverse purposes. Here, we scanned TAG codons through part of the gene for a model four-helix bundle protein, Rop, which regulates the copy number of ColE1 plasmids. Alanine was incorporated into Rop for mapping its binding site using an in vivo activity screen, and subtle but important differences from in vitro gel-shift studies of Rop function are evident. As a test, Bpa was incorporated using a Phe14 amber mutant isolated from the scanning library. Surprisingly, Phe14Bpa-Rop is weakly active, despite the critical role of Phe14 in Rop activity. Bpa is a photoaffinity label unnatural amino acid that can form covalent bonds with adjacent molecules upon UV irradiation. Irradiation of Phe14Bpa-Rop, which is a dimer in solution like wild-type Rop, results in covalent dimers, trimers, and tetramers. This suggests that Phe14Bpa-Rop weakly associates as a tetramer in solution and highlights the use of Bpa cross-linking as a means of trapping weak and transient interactions. PMID:21452871

  15. Genetic Regulation of Charged Particle Mutagenesis in Human Cells

    NASA Technical Reports Server (NTRS)

    Kronenberg, Amy; Gauny, S.; Cherbonnel-Lasserre, C.; Liu, W.; Wiese, C.

    1999-01-01

    Our studies use a series of syngeneic, and where possible, isogenic human B-lymphoblastoid cell lines to assess the genetic factors that modulate susceptibility apoptosis and their impact on the mutagenic risks of low fluence exposures to 1 GeV Fe ions and 55 MeV protons. These ions are representative of the types of charged particle radiation that are of particular significance for human health in the space radiation environment. The model system employs cell lines derived from the male donor WIL-2. These cells have a single X chromosome and they are hemizygous for one mutation marker, hypoxanthine phosphoribosyltransferase (HPRT). TK6 and WTK1 cells were each derived from descendants of WIL-2 and were each selected as heterozygotes for a second mutation marker, the thymidine kinase (TK) gene located on chromosome 17q. The HPRT and TK loci can detect many different types of mutations, from single basepair substitutions up to large scale loss of heterozygosity (LOH). The single expressing copy of TK in the TK6 and WTKI cell lines is found on the same copy of chromosome 17, and this allele can be identified by a restriction fragment length polymorphism (RFLP) identified when high molecular weight DNA is digested by the SacI restriction endonuclease and hybridized against the cDNA probe for TK. A large series of polymorphic linked markers has been identified that span more than 60 cM of DNA (approx. 60 megabasepairs) and distinguish the copy of chromosome 17 bearing the initially active TK allele from the copy of chromosome 17 bearing the silent TK allele in both TK6 and WTKI cells. TK6 cells express normal p53 protein while WTKI cells express homozygous mutant p53. Expression of mutant p53 can increase susceptibility to x-ray-induced mutations. It's been suggested that the increased mutagenesis in p53 mutant cells might be due to reduced apoptosis.

  16. Radiation mutagenesis from molecular and genetic points of view

    SciTech Connect

    Chen, D.J.C.; Park, M.S.; Okinaka, R.T.; Jaberaboansari, A.

    1993-02-01

    An important biological effect of ionizing radiation on living organisms is mutation induction. Mutation is also a primary event in the etiology of cancer. The chain events, from induction of DNA damage by ionizing radiation to processing of these damages by the cellular repair/replication machinery, that lead to mutation are not well understood. The development of quantitative methods for measuring mutation-induction, such as the HPRT system, in cultured mammalian cells has provided an estimate of the mutagenic effects of x- and {gamma}-rays as wen as of high LET radiation in both rodent and human cells. A major conclusion from these mutagenesis data is that high LET radiation induces mutations more efficiently than g-rays. Molecular analysis of mutations induced by sparsely ionizing radiation have detected major structural alterations at the gene level. Our molecular results based on analysis of human HPRT deficient mutants induced by {gamma}-rays, {alpha}-particles and high energy charged particles indicate that higher LET radiation induce more total and large deletion mutations than {gamma}-rays. Utilizing molecular techniques including polymerase chain reaction (PCR), Single-strand conformation polymorphism (SSCP), denaturing gradient gel electrophoresis (DGGE) and Direct DNA sequencing, mutational spectra induced by ionizing radiation have been compared in different cell systems. Attempts have also been made to determine the mutagenic potential and the nature of mutation induced by low dose rate {gamma}-rays. Defective repair, in the form of either a diminished capability for repair or inaccurate repair, can lead to increased risk of heritable mutations from radiation exposure. Therefore, the effects of DNA repair deficiency on the mutation induction in mammalian cells is reviewed.

  17. Radiation mutagenesis from molecular and genetic points of view

    SciTech Connect

    Chen, D.J.C.; Park, M.S.; Okinaka, R.T.; Jaberaboansari, A.

    1993-01-01

    An important biological effect of ionizing radiation on living organisms is mutation induction. Mutation is also a primary event in the etiology of cancer. The chain events, from induction of DNA damage by ionizing radiation to processing of these damages by the cellular repair/replication machinery, that lead to mutation are not well understood. The development of quantitative methods for measuring mutation-induction, such as the HPRT system, in cultured mammalian cells has provided an estimate of the mutagenic effects of x- and [gamma]-rays as wen as of high LET radiation in both rodent and human cells. A major conclusion from these mutagenesis data is that high LET radiation induces mutations more efficiently than g-rays. Molecular analysis of mutations induced by sparsely ionizing radiation have detected major structural alterations at the gene level. Our molecular results based on analysis of human HPRT deficient mutants induced by [gamma]-rays, [alpha]-particles and high energy charged particles indicate that higher LET radiation induce more total and large deletion mutations than [gamma]-rays. Utilizing molecular techniques including polymerase chain reaction (PCR), Single-strand conformation polymorphism (SSCP), denaturing gradient gel electrophoresis (DGGE) and Direct DNA sequencing, mutational spectra induced by ionizing radiation have been compared in different cell systems. Attempts have also been made to determine the mutagenic potential and the nature of mutation induced by low dose rate [gamma]-rays. Defective repair, in the form of either a diminished capability for repair or inaccurate repair, can lead to increased risk of heritable mutations from radiation exposure. Therefore, the effects of DNA repair deficiency on the mutation induction in mammalian cells is reviewed.

  18. TET2-mediated 5-hydroxymethylcytosine induces genetic instability and mutagenesis.

    PubMed

    Mahfoudhi, Emna; Talhaoui, Ibtissam; Cabagnols, Xenia; Della Valle, Véronique; Secardin, Lise; Rameau, Philippe; Bernard, Olivier A; Ishchenko, Alexander A; Abbes, Salem; Vainchenker, William; Saparbaev, Murat; Plo, Isabelle

    2016-07-01

    The family of Ten-Eleven Translocation (TET) proteins is implicated in the process of active DNA demethylation and thus in epigenetic regulation. TET 1, 2 and 3 proteins are oxygenases that can hydroxylate 5-methylcytosine (5-mC) into 5-hydroxymethylcytosine (5-hmC) and further oxidize 5-hmC into 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC). The base excision repair (BER) pathway removes the resulting 5-fC and 5-caC bases paired with a guanine and replaces them with regular cytosine. The question arises whether active modification of 5-mC residues and their subsequent elimination could affect the genomic DNA stability. Here, we generated two inducible cell lines (Ba/F3-EPOR, and UT7) overexpressing wild-type or catalytically inactive human TET2 proteins. Wild-type TET2 induction resulted in an increased level of 5-hmC and a cell cycle defect in S phase associated with higher level of phosphorylated P53, chromosomal and centrosomal abnormalities. Furthermore, in a thymine-DNA glycosylase (Tdg) deficient context, the TET2-mediated increase of 5-hmC induces mutagenesis characterized by GC>AT transitions in CpG context suggesting a mutagenic potential of 5-hmC metabolites. Altogether, these data suggest that TET2 activity and the levels of 5-hmC and its derivatives should be tightly controlled to avoid genetic and chromosomal instabilities. Moreover, TET2-mediated active demethylation might be a very dangerous process if used to entirely demethylate the genome and might rather be used only at specific loci. PMID:27289557

  19. Biophysical Optimization of a Therapeutic Protein by Nonstandard Mutagenesis

    PubMed Central

    Pandyarajan, Vijay; Phillips, Nelson B.; Cox, Gabriela P.; Yang, Yanwu; Whittaker, Jonathan; Ismail-Beigi, Faramarz; Weiss, Michael A.

    2014-01-01

    Insulin provides a model for the therapeutic application of protein engineering. A paradigm in molecular pharmacology was defined by design of rapid-acting insulin analogs for the prandial control of glycemia. Such analogs, a cornerstone of current diabetes regimens, exhibit accelerated subcutaneous absorption due to more rapid disassembly of oligomeric species relative to wild-type insulin. This strategy is limited by a molecular trade-off between accelerated disassembly and enhanced susceptibility to degradation. Here, we demonstrate that this trade-off may be circumvented by nonstandard mutagenesis. Our studies employed LysB28, ProB29-insulin (“lispro”) as a model prandial analog that is less thermodynamically stable and more susceptible to fibrillation than is wild-type insulin. We have discovered that substitution of an invariant tyrosine adjoining the engineered sites in lispro (TyrB26) by 3-iodo-Tyr (i) augments its thermodynamic stability (ΔΔGu 0.5 ±0.2 kcal/mol), (ii) delays onset of fibrillation (lag time on gentle agitation at 37 °C was prolonged by 4-fold), (iii) enhances affinity for the insulin receptor (1.5 ± 0.1-fold), and (iv) preserves biological activity in a rat model of diabetes mellitus. 1H NMR studies suggest that the bulky iodo-substituent packs within a nonpolar interchain crevice. Remarkably, the 3-iodo-TyrB26 modification stabilizes an oligomeric form of insulin pertinent to pharmaceutical formulation (the R6 zinc hexamer) but preserves rapid disassembly of the oligomeric form pertinent to subcutaneous absorption (T6 hexamer). By exploiting this allosteric switch, 3-iodo-TyrB26-lispro thus illustrates how a nonstandard amino acid substitution can mitigate the unfavorable biophysical properties of an engineered protein while retaining its advantages. PMID:24993826

  20. Mutagenesis of the rapamycin producer Streptomyces hygroscopicus FC904.

    PubMed

    Cheng, Y R; Huang, J; Qiang, H; Lin, W L; Demain, A L

    2001-11-01

    Rapamycin (RPM) is produced by Streptomyces hygroscopicus FC904 isolated from soil in Fuzhou, China. It is a triene macrolide antibiotic with potential application as an immunosuppressant and drug for human gene therapy. In an attempt to improve rapamycin production, mutation and screening of the parent culture have been carried out. Thousands of survivors were obtained after mutagenesis by NTG (3 mg/ml) and UV (30 W, 15 cm, 30 seconds) of spore suspensions. None showed improved production of RPM. We determined the susceptibility to antibiotics of S. hygroscopicus FC904 by two fold dilutions of antibiotics in oatmeal agar plates. It was found that the strain was resistant to penicillin, erythromycin, RPM, tetracycline and chloramphenicol, but susceptible to mitomycin C (MIC, 10 microg/ml) and aminoglycosides such as gentamicin (MIC, 0.1 microg/ml), kanamycin (MIC, 0.1 microg/ml) and streptomycin (MIC, 0.3 microg/ml). Protoplasts of strain FC904 were prepared after finding the best conditions for their formation. They were treated with gentamicin, erythromycin, mitomycin C and NTG. Surprisingly, gentamicin was especially effective for obtaining higher RPM-producing mutants. Mutant C14 was selected by exposing the protoplasts of the parent strain FC904 to 1 microg/ml of gentamicin at 28 degrees C for 2 hours. A higher RPM-producing mutant (C14-1) was obtained from the protoplasts of mutant C14 treated with gentamicin, and its titer was 60% higher than that of the parent strain FC904 by HPLC analysis. Another improved mutant (C14-2) was obtained from the spores of mutant C 14 treated with 1 microg/ml of gentamicin plus 2 mg/ml of NTG at 28 degrees C for 2 hours. Mutant C14-2 had a titer 124% higher than FC904. The possible mechanism for the effect of gentamicin by using protoplasts or spore suspensions will be discussed, i.e. the possibility of gentamicin being a mutagen or a selective agent. PMID:11827040

  1. The effect of adaptive mutagenesis on genetic variation at a linked, neutral locus

    SciTech Connect

    Colby, C.; Williams, S.M.

    1995-07-01

    Based on recent studies in single-celled organisms, it has been argued that a fitness benefit associated with a mutation will increase the probability of that mutation occurring. This increase is independent of mutation rates at other loci and is called adaptive mutagenesis. We modeled the effect of adaptive mutagenesis on populations of haploid organisms with adaptive mutation rates ranging from 0 to 1 x 10{sup -5}. Allele frequencies at the selected locus and a neutral linked locus were tracked. We also observed the amount of linkage disequilibrium during the selective sweep and the final heterozygosity after the sweep. The presence of adaptive mutagenesis increases the number of genetic backgrounds carrying the new fitter allele, making the outcomes more representative of the population before the selection. Therefore, more neutral genetic variation is preserved in simulations with adaptive mutagenesis than in those without it due to hitchhiking. Since adaptive mutagensis is time-dependent, it can generate mutants when other mechanisms of mutation cannot. In addition, adaptive mutagenesis has the potential to confound both phylogeny construction and the detection of natural selection from patterns of nucleotide variation. 27 refs., 4 figs.

  2. Comparison of CRISPR/Cas9 expression constructs for efficient targeted mutagenesis in rice.

    PubMed

    Mikami, Masafumi; Toki, Seiichi; Endo, Masaki

    2015-08-01

    The CRISPR/Cas9 system is an efficient tool used for genome editing in a variety of organisms. Despite several recent reports of successful targeted mutagenesis using the CRISPR/Cas9 system in plants, in each case the target gene of interest, the Cas9 expression system and guide-RNA (gRNA) used, and the tissues used for transformation and subsequent mutagenesis differed, hence the reported frequencies of targeted mutagenesis cannot be compared directly. Here, we evaluated mutation frequency in rice using different Cas9 and/or gRNA expression cassettes under standardized experimental conditions. We introduced Cas9 and gRNA expression cassettes separately or sequentially into rice calli, and assessed the frequency of mutagenesis at the same endogenous targeted sequences. Mutation frequencies differed significantly depending on the Cas9 expression cassette used. In addition, a gRNA driven by the OsU6 promoter was superior to one driven by the OsU3 promoter. Using an all-in-one expression vector harboring the best combined Cas9/gRNA expression cassette resulted in a much improved frequency of targeted mutagenesis in rice calli, and bi-allelic mutant plants were produced in the T0 generation. The approach presented here could be adapted to optimize the construction of Cas9/gRNA cassettes for genome editing in a variety of plants. PMID:26188471

  3. Genome-Wide Synthetic Genetic Screening by Transposon Mutagenesis in Candida albicans

    PubMed Central

    Horton, Brooke N.; Kumar, Anuj

    2016-01-01

    Transposon-based mutagenesis is an effective method for genetic screening on a genome-wide scale, with particular applicability in organisms possessing compact genomes where transforming DNA tends to integrate by homologous recombination. Methods for transposon mutagenesis have been applied with great success in the budding yeast Saccharomyces cerevisiae and in the related pathogenic yeast Candida albicans. In C. albicans, we have implemented transposon mutagenesis to generate heterozygous mutations for the analysis of complex haploinsufficiency, a type of synthetic genetic interaction wherein a pair of non-complementing heterozygous mutations results in a stronger phenotype then either individual mutation in isolation. Genes exhibiting complex haploinsufficiency typically function within a regulatory pathway, in parallel pathways, or in parallel branches within a single pathway. Here, we present protocols to implement transposon mutagenesis for complex haploinsufficiency screening in C. albicans, indicating methods for transposon construction, mutagenesis, phenotypic screening, and identification of insertion sites in strains of interest. In total, the approach is a useful means to implement large-scale synthetic genetic screening in the diploid C. albicans. PMID:25636616

  4. Ds insertion mutagenesis as an efficient tool to produce diverse variations for rice breeding.

    PubMed

    Jiang, Shu-Ye; Bachmann, Doris; La, Honggui; Ma, Zhigang; Venkatesh, Prasanna Nori; Ramamoorthy, Rengasamy; Ramachandran, Srinivasan

    2007-11-01

    The availability of diversified germplasm resources is the most important for developing improved rice varieties with higher seed yield or tolerance to various biotic or abiotic stresses. Here we report an efficient tool to create increased variations in rice by maize Ac/Ds transposon (a gene trap system) insertion mutagenesis. We have generated around 20,000 Ds insertion rice lines of which majority are homozygous for Ds element. We subjected these lines to phenotypic and abiotic stress screens and evaluated these lines with respect to their seed yields and other agronomic traits as well as their tolerance to drought, salinity and cold. Based on this evaluation, we observed that random Ds insertions into rice genome have led to diverse variations including a range of morphological and conditional phenotypes. Such differences in phenotype among these lines were accompanied by differential gene expression revealed by GUS histochemical staining of gene trapped lines. Among the various phenotypes identified, some Ds lines showed significantly higher grain yield compared to wild-type plants under normal growth conditions indicating that rice could be improved in grain yield by disrupting certain endogenous genes. In addition, several 1,000s of Ds lines were subjected to abiotic stresses to identify conditional mutants. Subsequent to these screens, over 800 lines responsive to drought, salinity or cold stress were obtained, suggesting that rice has the genetic potential to survive under abiotic stresses when appropriate endogenous genes were suppressed. The mutant lines that have higher seed yielding potential or display higher tolerance to abiotic stresses may be used for rice breeding by conventional backcrossing combining with molecular marker-assisted selection. In addition, by exploiting the behavior of Ds to leave footprints upon remobilization, we have shown an alternative strategy to develop new rice varieties without foreign DNA sequences in their genome. PMID

  5. In Vivo Transcriptional Profiling of Listeria monocytogenes and Mutagenesis Identify New Virulence Factors Involved in Infection

    PubMed Central

    Camejo, Ana; Buchrieser, Carmen; Couvé, Elisabeth; Carvalho, Filipe; Reis, Olga; Ferreira, Pierre; Sousa, Sandra; Cossart, Pascale; Cabanes, Didier

    2009-01-01

    Listeria monocytogenes is a human intracellular pathogen able to colonize host tissues after ingestion of contaminated food, causing severe invasive infections. In order to gain a better understanding of the nature of host–pathogen interactions, we studied the L. monocytogenes genome expression during mouse infection. In the spleen of infected mice, ≈20% of the Listeria genome is differentially expressed, essentially through gene activation, as compared to exponential growth in rich broth medium. Data presented here show that, during infection, Listeria is in an active multiplication phase, as revealed by the high expression of genes involved in replication, cell division and multiplication. In vivo bacterial growth requires increased expression of genes involved in adaptation of the bacterial metabolism and stress responses, in particular to oxidative stress. Listeria interaction with its host induces cell wall metabolism and surface expression of virulence factors. During infection, L. monocytogenes also activates subversion mechanisms of host defenses, including resistance to cationic peptides, peptidoglycan modifications and release of muramyl peptides. We show that the in vivo differential expression of the Listeria genome is coordinated by a complex regulatory network, with a central role for the PrfA-SigB interplay. In particular, L. monocytogenes up regulates in vivo the two major virulence regulators, PrfA and VirR, and their downstream effectors. Mutagenesis of in vivo induced genes allowed the identification of novel L. monocytogenes virulence factors, including an LPXTG surface protein, suggesting a role for S-layer glycoproteins and for cadmium efflux system in Listeria virulence. PMID:19478867

  6. 5-Azacytidine Can Induce Lethal Mutagenesis in Human Immunodeficiency Virus Type 1▿ †

    PubMed Central

    Dapp, Michael J.; Clouser, Christine L.; Patterson, Steven; Mansky, Louis M.

    2009-01-01

    Ribonucleosides inhibit human immunodeficiency virus type 1 (HIV-1) replication by mechanisms that have not been fully elucidated. Here, we report the antiviral mechanism for the ribonucleoside analog 5-azacytidine (5-AZC). We hypothesized that the anti-HIV-1 activity of 5-AZC was due to an increase in the HIV-1 mutation rate following its incorporation into viral RNA during transcription. However, we demonstrate that 5-AZC's primary antiviral activity can be attributed to its effect on the early phase of HIV-1 replication. Furthermore, the antiviral activity was associated with an increase in the frequency of viral mutants, suggesting that 5-AZC's primary target is reverse transcription. Sequencing analysis showed an enrichment in G-to-C transversion mutations and further supports the idea that reverse transcription is an antiviral target of 5-AZC. These results indicate that 5-AZC is incorporated into viral DNA following reduction to 5-aza-2′-deoxycytidine. Incorporation into the viral DNA leads to an increase in mutant frequency that is consistent with lethal mutagenesis during reverse transcription as the primary antiviral mechanism of 5-AZC. Antiviral activity and increased mutation frequency were also associated with the late phase of HIV-1 replication; however, 5-AZC's effect on the late phase was less robust. These results reveal that the primary antiviral mechanism of 5-AZC can be attributed to its ability to increase the HIV-1 mutation frequency through viral-DNA incorporation during reverse transcription. Our observations indicate that 5-AZC can affect two steps in HIV-1 replication (i.e., transcription and reverse transcription) but that its primary antiviral activity is due to incorporation during reverse transcription. PMID:19726509

  7. Gene-Trap Mutagenesis Identifies Mammalian Genes Contributing to Intoxication by Clostridium perfringens ε-Toxin

    PubMed Central

    Ivie, Susan E.; Fennessey, Christine M.; Sheng, Jinsong; Rubin, Donald H.; McClain, Mark S.

    2011-01-01

    The Clostridium perfringens ε-toxin is an extremely potent toxin associated with lethal toxemias in domesticated ruminants and may be toxic to humans. Intoxication results in fluid accumulation in various tissues, most notably in the brain and kidneys. Previous studies suggest that the toxin is a pore-forming toxin, leading to dysregulated ion homeostasis and ultimately cell death. However, mammalian host factors that likely contribute to ε-toxin-induced cytotoxicity are poorly understood. A library of insertional mutant Madin Darby canine kidney (MDCK) cells, which are highly susceptible to the lethal affects of ε-toxin, was used to select clones of cells resistant to ε-toxin-induced cytotoxicity. The genes mutated in 9 surviving resistant cell clones were identified. We focused additional experiments on one of the identified genes as a means of validating the experimental approach. Gene expression microarray analysis revealed that one of the identified genes, hepatitis A virus cellular receptor 1 (HAVCR1, KIM-1, TIM1), is more abundantly expressed in human kidney cell lines than it is expressed in human cells known to be resistant to ε-toxin. One human kidney cell line, ACHN, was found to be sensitive to the toxin and expresses a larger isoform of the HAVCR1 protein than the HAVCR1 protein expressed by other, toxin-resistant human kidney cell lines. RNA interference studies in MDCK and in ACHN cells confirmed that HAVCR1 contributes to ε-toxin-induced cytotoxicity. Additionally, ε-toxin was shown to bind to HAVCR1 in vitro. The results of this study indicate that HAVCR1 and the other genes identified through the use of gene-trap mutagenesis and RNA interference strategies represent important targets for investigation of the process by which ε-toxin induces cell death and new targets for potential therapeutic intervention. PMID:21412435

  8. A molecular characterization of spontaneous frameshift mutagenesis within the trpA gene of Escherichia coli

    PubMed Central

    Hardin, Aaron; Villalta, Christopher F.; Doan, Michael; Jabri, Mouna; Chockalingham, Valliammal; White, Steven J.; Fowler, Robert G.

    2007-01-01

    Spontaneous frameshift mutations are an important source of genetic variation in all species and cause a large number of genetic disorders in humans. To enhance our understanding of the molecular mechanisms of frameshift mutagenesis, 583 spontaneous Trp+ revertants of two trpA frameshift alleles in Escherichia coli were isolated and DNA sequenced. In order to measure the contribution of methyl-directed mismatch repair to frameshift production, mutational spectra were constructed for both mismatch repair-proficient and repair-defective strains. The molecular origins of practically all of the frameshifts analyzed could be explained by one of six simple models based upon misalignment of the template or nascent DNA strands with or without misincoroporation of primer nucleotides during DNA replication. Most frameshifts occurred within mononucleotide runs as has been shown often in previous studies but the location of the 76 frameshift sites was usually outside of runs. Mismatch repair generally was most effective in preventing the occurrence of frameshifts within runs but there was much variation from site to site. Most frameshift sites outside of runs appear to be refractory to mismatch repair although the small number of occurrences at most of these sites make firm conclusions impossible. There was a dense pattern of reversion sites within the trpA DNA region where reversion events could occur, suggesting that, in general, most DNA sequences are capable of undergoing spontaneous mutational events during replication that can lead to small deletions and insertions. Many of these errors are likely to occur at low frequencies and be tolerated as events too costly to prevent or repair. These studies also revealed an unpredicted flexibility in the primary amino acid sequence of the trpA product, the α subunit of tryptophan synthase. PMID:17084112

  9. Exploring the Enantioselective Mechanism of Halohydrin Dehalogenase from Agrobacterium radiobacter AD1 by Iterative Saturation Mutagenesis

    PubMed Central

    Guo, Chao; Chen, Yanpu; Zheng, Yu; Zhang, Wei; Tao, Yunwen; Feng, Juan

    2015-01-01

    Halohydrin dehalogenase from Agrobacterium radiobacter AD1 (HheC) shows great potential in producing valuable chiral epoxides and β-substituted alcohols. The wild-type (WT) enzyme displays a high R-enantiopreference toward most aromatic substrates, whereas no S-selective HheC has been reported to date. To obtain more enantioselective enzymes, seven noncatalytic active-site residues were subjected to iterative saturation mutagenesis (ISM). After two rounds of screening aspects of both activity and enantioselectivity (E), three outstanding mutants (Thr134Val/Leu142Met, Leu142Phe/Asn176His, and Pro84Val/Phe86Pro/Thr134Ala/Asn176Ala mutants) with divergent enantioselectivity were obtained. The two double mutants displayed approximately 2-fold improvement in R-enantioselectivity toward 2-chloro-1-phenylethanol (2-CPE) without a significant loss of enzyme activity compared with the WT enzyme. Strikingly, the Pro84Val/Phe86Pro/Thr134Ala/Asn176Ala mutant showed an inverted enantioselectivity (from an ER of 65 [WT] to an ES of 101) and approximately 100-fold-enhanced catalytic efficiency toward (S)-2-CPE. Molecular dynamic simulation and docking analysis revealed that the phenyl side chain of (S)-2-CPE bound at a different location than that of its R-counterpart; those mutations generated extra connections for the binding of the favored enantiomer, while the eliminated connections reduced binding of the nonfavored enantiomer, all of which could contribute to the observed inverted enantiopreference. PMID:25681194

  10. Intensive mutagenesis of the nisin hinge leads to the rational design of enhanced derivatives.

    PubMed

    Healy, Brian; Field, Des; O'Connor, Paula M; Hill, Colin; Cotter, Paul D; Ross, R Paul

    2013-01-01

    Nisin A is the most extensively studied lantibiotic and has been used as a preservative by the food industry since 1953. This 34 amino acid peptide contains three dehydrated amino acids and five thioether rings. These rings, resulting from one lanthionine and four methyllanthionine bridges, confer the peptide with its unique structure. Nisin A has two mechanisms of action, with the N-terminal domain of the peptide inhibiting cell wall synthesis through lipid II binding and the C-terminal domain responsible for pore-formation. The focus of this study is the three amino acid 'hinge' region (N 20, M 21 and K 22) which separates these two domains and allows for conformational flexibility. As all lantibiotics are gene encoded, novel variants can be generated through manipulation of the corresponding gene. A number of derivatives in which the hinge region was altered have previously been shown to possess enhanced antimicrobial activity. Here we take this approach further by employing simultaneous, indiscriminate site-saturation mutagenesis of all three hinge residues to create a novel bank of nisin derivative producers. Screening of this bank revealed that producers of peptides with hinge regions consisting of AAK, NAI and SLS displayed enhanced bioactivity against a variety of targets. These and other results suggested a preference for small, chiral amino acids within the hinge region, leading to the design and creation of producers of peptides with hinges consisting of AAA and SAA. These producers, and the corresponding peptides, exhibited enhanced bioactivity against Lactococcus lactis HP, Streptococcus agalactiae ATCC 13813, Mycobacterium smegmatis MC2155 and Staphylococcus aureus RF122 and thus represent the first example of nisin derivatives that possess enhanced activity as a consequence of rational design. PMID:24244524

  11. In silico functional dissection of saturation mutagenesis: Interpreting the relationship between phenotypes and changes in protein stability, interactions and activity

    PubMed Central

    Pires, Douglas E. V.; Chen, Jing; Blundell, Tom L.; Ascher, David B.

    2016-01-01

    Despite interest in associating polymorphisms with clinical or experimental phenotypes, functional interpretation of mutation data has lagged behind generation of data from modern high-throughput techniques and the accurate prediction of the molecular impact of a mutation remains a non-trivial task. We present here an integrated knowledge-driven computational workflow designed to evaluate the effects of experimental and disease missense mutations on protein structure and interactions. We exemplify its application with analyses of saturation mutagenesis of DBR1 and Gal4 and show that the experimental phenotypes for over 80% of the mutations correlate well with predicted effects of mutations on protein stability and RNA binding affinity. We also show that analysis of mutations in VHL using our workflow provides valuable insights into the effects of mutations, and their links to the risk of developing renal carcinoma. Taken together the analyses of the three examples demonstrate that structural bioinformatics tools, when applied in a systematic, integrated way, can rapidly analyse a given system to provide a powerful approach for predicting structural and functional effects of thousands of mutations in order to reveal molecular mechanisms leading to a phenotype. Missense or non-synonymous mutations are nucleotide substitutions that alter the amino acid sequence of a protein. Their effects can range from modifying transcription, translation, processing and splicing, localization, changing stability of the protein, altering its dynamics or interactions with other proteins, nucleic acids and ligands, including small molecules and metal ions. The advent of high-throughput techniques including sequencing and saturation mutagenesis has provided large amounts of phenotypic data linked to mutations. However, one of the hurdles has been understanding and quantifying the effects of a particular mutation, and how they translate into a given phenotype. One approach to overcome

  12. Mutagenesis and phenotyping resources in zebrafish for studying development and human disease.

    PubMed

    Varshney, Gaurav Kumar; Burgess, Shawn Michael

    2014-03-01

    The zebrafish (Danio rerio) is an important model organism for studying development and human disease. The zebrafish has an excellent reference genome and the functions of hundreds of genes have been tested using both forward and reverse genetic approaches. Recent years have seen an increasing number of large-scale mutagenesis projects and the number of mutants or gene knockouts in zebrafish has increased rapidly, including for the first time conditional knockout technologies. In addition, targeted mutagenesis techniques such as zinc finger nucleases, transcription activator-like effector nucleases and clustered regularly interspaced short sequences (CRISPR) or CRISPR-associated (Cas), have all been shown to effectively target zebrafish genes as well as the first reported germline homologous recombination, further expanding the utility and power of zebrafish genetics. Given this explosion of mutagenesis resources, it is now possible to perform systematic, high-throughput phenotype analysis of all zebrafish gene knockouts. PMID:24162064

  13. Targeted Mutagenesis in Rice Using TALENs and the CRISPR/Cas9 System.

    PubMed

    Endo, Masaki; Nishizawa-Yokoi, Ayako; Toki, Seiichi

    2016-01-01

    Sequence-specific nucleases (SSNs), such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspersed short palindromic repeats (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9) system, are powerful tools for understanding gene function and for developing novel traits in plants. In plant species for which transformation and regeneration systems using protoplasts are not yet established, direct delivery to nuclei of SSNs either in the form of RNA or protein is difficult. Thus, Agrobacterium-mediated transformation of SSN expression constructs in cultured cells is a practical means of delivering targeted mutagenesis in some plant species including rice. Because targeted mutagenesis occurs stochastically in transgenic cells and SSN-mediated targeted mutagenesis often leads to no selectable phenotype, identification of highly mutated cell lines is a critical step in obtaining regenerated plants with desired mutations. PMID:27557690

  14. Bromodeoxyuridine mutagenesis in mammalian cells is related to deoxyribonucleotide pool imbalance.

    PubMed Central

    Ashman, C R; Davidson, R L

    1981-01-01

    The relationship between bromodeoxyuridine (BrdUrd) mutagenesis in mammalian cells and the effects of BrdUrd on deoxyribonucleoside triphosphate pools was analyzed. It was found that the exposure of Syrian hamster melanoma cells to mutagenic concentrations of BrdUrd resulted in the formation of a large bromodeoxyuridine triphosphate (BrdUTP) pool, which remained at a high level for several days. In contrast, the size of the deoxycytidine triphosphate (dCTP) pool dropped rapidly after the addition of BrdUrd, reached a minimum at about 6 h, and then expanded gradually to nearly its original level over the next 3 days. The addition of lower concentrations of BrdUrd, which had less of a mutagenic effect, resulted in the formation of a smaller BrdUTP pool and a slightly smaller drop in the dCTP pool. When a high concentration of deoxycytidine was added at the same time as a normally mutagenic concentration of BrdUrd, the drop in the dCTP pool was prevented, as was BrdUrd mutagenesis. In all of these experiments, mutagenesis was related to the ratio of BrdUTP to dCTP in the cells. In addition, it was shown that mutagenesis occurred primarily during the first 24 h of BrdUrd exposure, when the BrdUTP/dCTP ratio was at its highest level. It appears that there is a critical ratio of BrdUTP to dCTP that must be attained for high levels of mutagenesis to occur and that the extent of mutagenesis is related to the ratio of the BrdUrd and dCTP pools. PMID:6965099

  15. Crystal structure and site-directed mutagenesis analyses of haloalkane dehalogenase LinB from Sphingobium sp. strain MI1205.

    PubMed

    Okai, Masahiko; Ohtsuka, Jun; Imai, Lica Fabiana; Mase, Tomoko; Moriuchi, Ryota; Tsuda, Masataka; Nagata, Koji; Nagata, Yuji; Tanokura, Masaru

    2013-06-01

    The enzymes LinB(UT) and LinB(MI) (LinB from Sphingobium japonicum UT26 and Sphingobium sp. MI1205, respectively) catalyze the hydrolytic dechlorination of β-hexachlorocyclohexane (β-HCH) and yield different products, 2,3,4,5,6-pentachlorocyclohexanol (PCHL) and 2,3,5,6-tetrachlorocyclohexane-1,4-diol (TCDL), respectively, despite their 98% identity in amino acid sequence. To reveal the structural basis of their different enzymatic properties, we performed site-directed mutagenesis and X-ray crystallographic studies of LinB(MI) and its seven point mutants. The mutation analysis revealed that the seven amino acid residues uniquely found in LinB(MI) were categorized into three groups based on the efficiency of the first-step (from β-HCH to PCHL) and second-step (from PCHL to TCDL) conversions. Crystal structure analyses of wild-type LinB(MI) and its seven point mutants indicated how each mutated residue contributed to the first- and second-step conversions by LinB(MI). The dynamics simulation analyses of wild-type LinB(MI) and LinB(UT) revealed that the entrance of the substrate access tunnel of LinB(UT) was more flexible than that of LinB(MI), which could lead to the different efficiencies of dehalogenation activity between these dehalogenases. PMID:23564170

  16. Crystal Structure and Site-Directed Mutagenesis Analyses of Haloalkane Dehalogenase LinB from Sphingobium sp. Strain MI1205

    PubMed Central

    Okai, Masahiko; Ohtsuka, Jun; Imai, Lica Fabiana; Mase, Tomoko; Moriuchi, Ryota; Tsuda, Masataka; Nagata, Koji; Nagata, Yuji

    2013-01-01

    The enzymes LinBUT and LinBMI (LinB from Sphingobium japonicum UT26 and Sphingobium sp. MI1205, respectively) catalyze the hydrolytic dechlorination of β-hexachlorocyclohexane (β-HCH) and yield different products, 2,3,4,5,6-pentachlorocyclohexanol (PCHL) and 2,3,5,6-tetrachlorocyclohexane-1,4-diol (TCDL), respectively, despite their 98% identity in amino acid sequence. To reveal the structural basis of their different enzymatic properties, we performed site-directed mutagenesis and X-ray crystallographic studies of LinBMI and its seven point mutants. The mutation analysis revealed that the seven amino acid residues uniquely found in LinBMI were categorized into three groups based on the efficiency of the first-step (from β-HCH to PCHL) and second-step (from PCHL to TCDL) conversions. Crystal structure analyses of wild-type LinBMI and its seven point mutants indicated how each mutated residue contributed to the first- and second-step conversions by LinBMI. The dynamics simulation analyses of wild-type LinBMI and LinBUT revealed that the entrance of the substrate access tunnel of LinBUT was more flexible than that of LinBMI, which could lead to the different efficiencies of dehalogenation activity between these dehalogenases. PMID:23564170

  17. Metabolic effects of CYP2A6 and CYP2A13 on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced gene mutation-A mammalian cell-based mutagenesis approach

    SciTech Connect

    Chiang, Huai-chih; Wang, Chin-Ying; Lee, Hui-Ling; Tsou, Tsui-Chun

    2011-06-01

    Both cytochrome P450 2A6 (CYP2A6) and cytochrome P450 2A13 (CYP2A13) are involved in metabolic activation of tobacco-specific nitrosamines and may play important roles in cigarette smoking-induced lung cancer. Unlike CYP2A6, effects of CYP2A13 on the tobacco-specific nitrosamine-induced mutagenesis in lung cells remain unclear. This study uses a supF mutagenesis assay to examine the relative effects of CYP2A6 and CYP2A13 on metabolic activation of a tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and its resulting mutagenesis in human lung cells. A recombinant adenovirus-mediated CYP2A6/CYP2A13 expression system was established to specifically address the relative effects of these two CYPs. Mutagenesis results revealed that both CYP2A6 and CYP2A13 significantly enhanced the NNK-induced supF mutation and that the mutagenic effect of CYP2A13 was markedly higher than that of CYP2A6. Analysis of NNK metabolism indicated that {>=} 70% of NNK was detoxified to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), either with or without CYP2A6/CYP2A13 expression. Both CYP2A6 and CYP2A13 significantly enhanced the {alpha}-hydroxylation of NNK; and the {alpha}-hydroxylation activity of CYP2A13 was significantly higher than that of CYP2A6. Analysis of the NNK-related DNA adduct formation indicated that, in the presence of CYP2A13, NNK treatments caused marked increases in O{sup 6}-methylguanine (O{sup 6}-MeG). The present results provide the first direct in vitro evidence demonstrating the predominant roles of CYP2A13 in NNK-induced mutagenesis, possibly via metabolic activation of NNK {alpha}-hydroxylation.

  18. Quantitative evaluation of DNA damage and mutation rate by atmospheric and room-temperature plasma (ARTP) and conventional mutagenesis.

    PubMed

    Zhang, Xue; Zhang, Chong; Zhou, Qian-Qian; Zhang, Xiao-Fei; Wang, Li-Yan; Chang, Hai-Bo; Li, He-Ping; Oda, Yoshimitsu; Xing, Xin-Hui

    2015-07-01

    DNA damage is the dominant source of mutation, which is the driving force of evolution. Therefore, it is important to quantitatively analyze the DNA damage caused by different mutagenesis methods, the subsequent mutation rates, and their relationship. Atmospheric and room temperature plasma (ARTP) mutagenesis has been used for the mutation breeding of more than 40 microorganisms. However, ARTP mutagenesis has not been quantitatively compared with conventional mutation methods. In this study, the umu test using a flow-cytometric analysis was developed to quantify the DNA damage in individual viable cells using Salmonella typhimurium NM2009 as the model strain and to determine the mutation rate. The newly developed method was used to evaluate four different mutagenesis systems: a new ARTP tool, ultraviolet radiation, 4-nitroquinoline-1-oxide (4-NQO), and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) mutagenesis. The mutation rate was proportional to the corresponding SOS response induced by DNA damage. ARTP caused greater DNA damage to individual living cells than the other conventional mutagenesis methods, and the mutation rate was also higher. By quantitatively comparing the DNA damage and consequent mutation rate after different types of mutagenesis, we have shown that ARTP is a potentially powerful mutagenesis tool with which to improve the characteristics of microbial cell factories. PMID:26025015

  19. Use of the Photoactic Ability of a Bacterium to Teach the Genetic Principles of Random Mutagenesis & Mutant Screening

    ERIC Educational Resources Information Center

    Din, Neena; Bird, Terry H.; Berleman, James E.

    2007-01-01

    In this article, the authors present a laboratory activity that relies on the use of a very versatile bacterial system to introduce the concept of how mutagenesis can be used for molecular and genetic analysis of living organisms. They have used the techniques of random mutagenesis and selection/screening to obtain strains of the organism "R.…

  20. Adaptive response and enhancement of N-methyl-N-nitro-N-nitrosoguanidine mutagenesis by chloramphenicol in Streptomyces fradiae

    SciTech Connect

    Baltz, R.H.; Stonesifer, J.

    1985-11-01

    Streptomyces fradiae expressed an adaptive response to treatment with small doses of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) that caused a reduction in mutagenesis by treatment with larger doses of MNNG. Treatment of S. fradiae with high levels of MNNG in the presence of chloramphenicol caused enhancement of mutagenesis, independent of the adaptive response.

  1. Random UV-C mutagenesis of Scheffersomyces (formerly Pichia) stipitis NRRL Y-7124 to improve anaerobic growth on lignocellulosic sugars

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Yeast strains for anaerobic conversion of lignocellulosic sugars to ethanol were produced from Scheffersomyces (formerly Pichia) stipitis NRRL Y-7124 using UV-C mutagenesis. Random UV-C mutagenesis potentially produces large numbers of mutations broadly and uniformly over the whole genome to genera...

  2. Comparative mutagenesis of human cells in vivo and in vitro

    SciTech Connect

    Thilly, W.G.

    1990-01-01

    Our goal is to develop the tools of mutational spectrometry in order to discover the cause(s) of genetic change in somatic and germinal cells in humans. Our study of the spectrum of point mutations in human mitochrondrial DNA sequences has revealed that there are multiple point mutation hotspots in each of four separate sequences in the mitochrondrial genome. These spectra were revealed by a combination of high fidelity PCR (modified T{sub 7} polymerase) and denaturing gradient gel electrophoresis which has a limit of detection of about 10{sup {minus}3}. There appear to be identical hotspot mutations in both cultured B cell and fresh human blood T cell samples.

  3. Improvement of Biocatalysts for Industrial and Environmental Purposes by Saturation Mutagenesis

    PubMed Central

    Valetti, Francesca; Gilardi, Gianfranco

    2013-01-01

    Laboratory evolution techniques are becoming increasingly widespread among protein engineers for the development of novel and designed biocatalysts. The palette of different approaches ranges from complete randomized strategies to rational and structure-guided mutagenesis, with a wide variety of costs, impacts, drawbacks and relevance to biotechnology. A technique that convincingly compromises the extremes of fully randomized vs. rational mutagenesis, with a high benefit/cost ratio, is saturation mutagenesis. Here we will present and discuss this approach in its many facets, also tackling the issue of randomization, statistical evaluation of library completeness and throughput efficiency of screening methods. Successful recent applications covering different classes of enzymes will be presented referring to the literature and to research lines pursued in our group. The focus is put on saturation mutagenesis as a tool for designing novel biocatalysts specifically relevant to production of fine chemicals for improving bulk enzymes for industry and engineering technical enzymes involved in treatment of waste, detoxification and production of clean energy from renewable sources. PMID:24970191

  4. Using ultra-sensitive next generation sequencing to dissect DNA damage-induced mutagenesis.

    PubMed

    Wang, Kaile; Ma, Xiaolu; Zhang, Xue; Wu, Dafei; Sun, Chenyi; Sun, Yazhou; Lu, Xuemei; Wu, Chung-I; Guo, Caixia; Ruan, Jue

    2016-01-01

    Next generation sequencing (NGS) technologies have dramatically improved studies in biology and biomedical science. However, no optimal NGS approach is available to conveniently analyze low frequency mutations caused by DNA damage treatments. Here, by developing an exquisite ultra-sensitive NGS (USNGS) platform "EasyMF" and incorporating it with a widely used supF shuttle vector-based mutagenesis system, we can conveniently dissect roles of lesion bypass polymerases in damage-induced mutagenesis. In this improved mutagenesis analysis pipeline, the initial steps are the same as in the supF mutation assay, involving damaging the pSP189 plasmid followed by its transfection into human 293T cells to allow replication to occur. Then "EasyMF" is employed to replace downstream MBM7070 bacterial transformation and other steps for analyzing damage-induced mutation frequencies and spectra. This pipeline was validated by using UV damaged plasmid after its replication in lesion bypass polymerase-deficient 293T cells. The increased throughput and reduced cost of this system will allow us to conveniently screen regulators of translesion DNA synthesis pathway and monitor environmental genotoxic substances, which can ultimately provide insight into the mechanisms of genome stability and mutagenesis. PMID:27122023

  5. Using ultra-sensitive next generation sequencing to dissect DNA damage-induced mutagenesis

    PubMed Central

    Wang, Kaile; Ma, Xiaolu; Zhang, Xue; Wu, Dafei; Sun, Chenyi; Sun, Yazhou; Lu, Xuemei; Wu, Chung-I; Guo, Caixia; Ruan, Jue

    2016-01-01

    Next generation sequencing (NGS) technologies have dramatically improved studies in biology and biomedical science. However, no optimal NGS approach is available to conveniently analyze low frequency mutations caused by DNA damage treatments. Here, by developing an exquisite ultra-sensitive NGS (USNGS) platform “EasyMF” and incorporating it with a widely used supF shuttle vector-based mutagenesis system, we can conveniently dissect roles of lesion bypass polymerases in damage-induced mutagenesis. In this improved mutagenesis analysis pipeline, the initial steps are the same as in the supF mutation assay, involving damaging the pSP189 plasmid followed by its transfection into human 293T cells to allow replication to occur. Then “EasyMF” is employed to replace downstream MBM7070 bacterial transformation and other steps for analyzing damage-induced mutation frequencies and spectra. This pipeline was validated by using UV damaged plasmid after its replication in lesion bypass polymerase-deficient 293T cells. The increased throughput and reduced cost of this system will allow us to conveniently screen regulators of translesion DNA synthesis pathway and monitor environmental genotoxic substances, which can ultimately provide insight into the mechanisms of genome stability and mutagenesis. PMID:27122023

  6. Workshop on ENU Mutagenesis: Planning for Saturation, July 25-28, 2002

    SciTech Connect

    Nadeau, Joseph H

    2002-07-25

    The goal of the conference is to enhance the development of improved technologies and new approaches to the identification of genes underlying chemically-induced mutant phenotypes. The conference brings together ENU mutagenesis experts from the United States and aborad for a small, intensive workshop to consider these issues.

  7. Deletion mutagenesis identifies a haploinsufficient role for gamma-zein in opaque-2 endosperm modification

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Quality Protein Maize (QPM) is a hard kernel variant of the high-lysine mutant, opaque-2. Using gamma irradiation, we created opaque QPM variants to identify opaque-2 modifier genes and to investigate deletion mutagenesis combined with Illumina sequencing as a maize functional genomics tool. A K0326...

  8. The Hermes Transposon of Musca domestica Is an Efficient Tool for the Mutagenesis of Schizosaccharomyces pombe

    PubMed Central

    Evertts, Adam G.; Plymire, Christopher; Craig, Nancy L.; Levin, Henry L.

    2007-01-01

    Currently, no transposon-based method for the mutagenesis of Schizosaccharomyces pombe exists. We have developed such a system based on the introduction of the hermes transposon from the housefly into S. pombe. This system efficiently disrupts open reading frames and allows the insertion sites to be readily identified. PMID:17947404

  9. The hermes transposon of Musca domestica is an efficient tool for the mutagenesis of Schizosaccharomyces pombe.

    PubMed

    Evertts, Adam G; Plymire, Christopher; Craig, Nancy L; Levin, Henry L

    2007-12-01

    Currently, no transposon-based method for the mutagenesis of Schizosaccharomyces pombe exists. We have developed such a system based on the introduction of the hermes transposon from the housefly into S. pombe. This system efficiently disrupts open reading frames and allows the insertion sites to be readily identified. PMID:17947404

  10. KERATINOCYTE CELL-MEDIATED MUTAGENESIS ASSAY: CORRELATION WITH IN VIVO TUMOR STUDIES

    EPA Science Inventory

    A murine keratinocyte cell-mediated mutagenesis assay was characterized and examined as an in vitro model system for studying the biotransformation of promutagens/procarcinogens by mouse skin. The assay used living cultured newborn SENCAR keratinocytes for the metabolic activatio...

  11. Insertion mutagenesis of the yeast Candida famata (Debaryomyces hansenii) by random integration of linear DNA fragments.

    PubMed

    Dmytruk, Kostyantyn V; Voronovsky, Andriy Y; Sibirny, Andriy A

    2006-09-01

    The feasibility of using random insertional mutagenesis to isolate mutants of the flavinogenic yeast Candida famata was explored. Mutagenesis was performed by transformation of the yeast with an integrative plasmid containing the Saccharomyces cerevisiae LEU2 gene as a selective marker. The addition of restriction enzyme together with the plasmid (restriction enzyme-mediated integration, REMI) increased the transformation frequency only slightly. Integration of the linearized plasmid occurred randomly in the C. famata genome. To investigate the potential of insertional mutagenesis, it was used for tagging genes involved in positive regulation of riboflavin synthesis in C. famata. Partial DNA sequencing of tagged genes showed that they were homologous to the S. cerevisiae genes RIB1, MET2, and SEF1. Intact orthologs of these genes isolated from Debaryomyces hansenii restored the wild phenotype of the corresponding mutants, i.e., the ability to overproduce riboflavin under iron limitation. The Staphylococcus aureus ble gene conferring resistance to phleomycin was used successfully in the study as a dominant selection marker for C. famata. The results obtained indicate that insertional mutagenesis is a powerful tool for tagging genes in C. famata. PMID:16770625

  12. Stationary-Phase Mutagenesis in Stressed Bacillus subtilis Cells Operates by Mfd-Dependent Mutagenic Pathways.

    PubMed

    Gómez-Marroquín, Martha; Martin, Holly A; Pepper, Amber; Girard, Mary E; Kidman, Amanda A; Vallin, Carmen; Yasbin, Ronald E; Pedraza-Reyes, Mario; Robleto, Eduardo A

    2016-01-01

    In replication-limited cells of Bacillus subtilis, Mfd is mutagenic at highly transcribed regions, even in the absence of bulky DNA lesions. However, the mechanism leading to increased mutagenesis through Mfd remains currently unknown. Here, we report that Mfd may promote mutagenesis in nutritionally stressed B. subtilis cells by coordinating error-prone repair events mediated by UvrA, MutY and PolI. Using a point-mutated gene conferring leucine auxotrophy as a genetic marker, it was found that the absence of UvrA reduced the Leu⁺ revertants and that a second mutation in mfd reduced mutagenesis further. Moreover, the mfd and polA mutants presented low but similar reversion frequencies compared to the parental strain. These results suggest that Mfd promotes mutagenic events that required the participation of NER pathway and PolI. Remarkably, this Mfd-dependent mutagenic pathway was found to be epistatic onto MutY; however, whereas the MutY-dependent Leu⁺ reversions required Mfd, a direct interaction between these proteins was not apparent. In summary, our results support the concept that Mfd promotes mutagenesis in starved B. subtilis cells by coordinating both known and previously unknown Mfd-associated repair pathways. These mutagenic processes bias the production of genetic diversity towards highly transcribed regions in the genome. PMID:27399782

  13. Stationary-Phase Mutagenesis in Stressed Bacillus subtilis Cells Operates by Mfd-Dependent Mutagenic Pathways

    PubMed Central

    Gómez-Marroquín, Martha; Martin, Holly A.; Pepper, Amber; Girard, Mary E.; Kidman, Amanda A.; Vallin, Carmen; Yasbin, Ronald E.; Pedraza-Reyes, Mario; Robleto, Eduardo A.

    2016-01-01

    In replication-limited cells of Bacillus subtilis, Mfd is mutagenic at highly transcribed regions, even in the absence of bulky DNA lesions. However, the mechanism leading to increased mutagenesis through Mfd remains currently unknown. Here, we report that Mfd may promote mutagenesis in nutritionally stressed B. subtilis cells by coordinating error-prone repair events mediated by UvrA, MutY and PolI. Using a point-mutated gene conferring leucine auxotrophy as a genetic marker, it was found that the absence of UvrA reduced the Leu+ revertants and that a second mutation in mfd reduced mutagenesis further. Moreover, the mfd and polA mutants presented low but similar reversion frequencies compared to the parental strain. These results suggest that Mfd promotes mutagenic events that required the participation of NER pathway and PolI. Remarkably, this Mfd-dependent mutagenic pathway was found to be epistatic onto MutY; however, whereas the MutY-dependent Leu+ reversions required Mfd, a direct interaction between these proteins was not apparent. In summary, our results support the concept that Mfd promotes mutagenesis in starved B. subtilis cells by coordinating both known and previously unknown Mfd-associated repair pathways. These mutagenic processes bias the production of genetic diversity towards highly transcribed regions in the genome. PMID:27399782

  14. Building on the Past, Shaping the Future: The Environmental Mutagenesis and Genomics Society

    EPA Science Inventory

    In late 2012 the members of the Environmental Mutagen Society voted to change its name to the Environmental Mutagenesis and Genomics Society. Here we describe the thought process that led to adoption of the new name, which both respects the rich history of a Society founded in 19...

  15. Development of potent in vivo mutagenesis plasmids with broad mutational spectra.

    PubMed

    Badran, Ahmed H; Liu, David R

    2015-01-01

    Methods to enhance random mutagenesis in cells offer advantages over in vitro mutagenesis, but current in vivo methods suffer from a lack of control, genomic instability, low efficiency and narrow mutational spectra. Using a mechanism-driven approach, we created a potent, inducible, broad-spectrum and vector-based mutagenesis system in E. coli that enhances mutation 322,000-fold over basal levels, surpassing the mutational efficiency and spectra of widely used in vivo and in vitro methods. We demonstrate that this system can be used to evolve antibiotic resistance in wild-type E. coli in <24 h, outperforming chemical mutagens, ultraviolet light and the mutator strain XL1-Red under similar conditions. This system also enables the continuous evolution of T7 RNA polymerase variants capable of initiating transcription using the T3 promoter in <10 h. Our findings enable broad-spectrum mutagenesis of chromosomes, episomes and viruses in vivo, and are applicable to both bacterial and bacteriophage-mediated laboratory evolution platforms. PMID:26443021

  16. Development of potent in vivo mutagenesis plasmids with broad mutational spectra

    PubMed Central

    Badran, Ahmed H.; Liu, David R.

    2015-01-01

    Methods to enhance random mutagenesis in cells offer advantages over in vitro mutagenesis, but current in vivo methods suffer from a lack of control, genomic instability, low efficiency and narrow mutational spectra. Using a mechanism-driven approach, we created a potent, inducible, broad-spectrum and vector-based mutagenesis system in E. coli that enhances mutation 322,000-fold over basal levels, surpassing the mutational efficiency and spectra of widely used in vivo and in vitro methods. We demonstrate that this system can be used to evolve antibiotic resistance in wild-type E. coli in <24 h, outperforming chemical mutagens, ultraviolet light and the mutator strain XL1-Red under similar conditions. This system also enables the continuous evolution of T7 RNA polymerase variants capable of initiating transcription using the T3 promoter in <10 h. Our findings enable broad-spectrum mutagenesis of chromosomes, episomes and viruses in vivo, and are applicable to both bacterial and bacteriophage-mediated laboratory evolution platforms. PMID:26443021

  17. CHEMICAL MUTAGENESIS AND CARCINOGENESIS: INCORPORATION OF MECHANISTIC DATA INTO RISK ASSESSMENT

    EPA Science Inventory

    CHEMICAL MUTAGENESIS AND CARCINOGENESIS: INCORPORATION OF MECHANISTIC DATA INTO RISK ASSESSMENT

    The current understanding of cancer as a genetic disease, requiring a specific set of genomic alterations for a normal cell to form a metastatic tumor, has provided the oppor...

  18. Highly efficient CRISPR/Cas9-mediated targeted mutagenesis of multiple genes in Populus.

    PubMed

    Tingting, Liu; Di, Fan; Lingyu, Ran; Yuanzhong, Jiang; Rui, Liu; Keming, Luo

    2015-10-01

    The typeⅡCRISPR/Cas9 system (Clustered regularly interspaced short palindromic repeats /CRISPR-associated 9) has been widely used in bacteria, yeast, animals and plants as a targeted genome editing technique. In previous work, we have successfully knocked out the endogenous phytoene dehydrogenase (PDS) gene in Populus tomentosa Carr. using this system. To study the effect of target design on the efficiency of CRISPR/Cas9-mediated gene knockout in Populus, we analyzed the efficiency of mutagenesis using different single-guide RNA (sgRNA) that target PDS DNA sequence. We found that mismatches between the sgRNA and the target DNA resulted in decreased efficiency of mutagenesis and even failed mutagenesis. Moreover, complementarity between the 3' end nucleotide of sgRNA and target DNA is especially crucial for efficient mutagenesis. Further sequencing analysis showed that two PDS homologs in Populus, PtPDS1 and PtPDS2, could be knocked out simultaneously using this system with 86.4% and 50% efficiency, respectively. These results indicated the possibility of introducing mutations in two or more endogenous genes efficiently and obtaining multi-mutant strains of Populus using this system. We have indeed generated several knockout mutants of transcription factors and structural genes in Populus, which establishes a foundation for future studies of gene function and genetic improvement of Populus. PMID:26496757

  19. Statistical procedures for the design and analysis of in vitro mutagenesis assays

    SciTech Connect

    Kaldor, J.

    1983-03-01

    In previous statistical treatments of a certain class of mutagenesis assays, stochastic models of mutation and cell growth have not been utilized. In this paper, we review the assumptions under which these models are derived, introduce some further assumptions, and propose ways to estimate and test hypotheses regarding the parameters of the models from assay data. It is shown via simulation and exact calculation that if the models are valid, the proposed statistical procedures provide very accurate Type I error rates for hypothesis tests, and coverage probabilities for confidence intervals. The cases of a linear dose response relationship for mutagenesis, and a comparison of a set of treated cell cultures with a set of control cultures are treated in detail. Approximate power functions for hypothesis tests of interest are then derived, and these are also shown to be satisfactorily close to the true power functions. The approximations are used to develop guidelines for planning aspects of a mutagenesis assay, including the number, spacing and range of dose levels employed. Examples of applications of the procedures are provided, and the paper concludes with a discussion of future statistical work which may be carried out in the area of mutagenesis assays. 38 references, 8 figures, 7 tables.

  20. N-Ethyl-N-Nitrosourea Mutagenesis: Boarding the Mouse Mutant Express

    PubMed Central

    Cordes, Sabine P.

    2005-01-01

    In the mouse, random mutagenesis with N-ethyl-N-nitrosourea (ENU) has been used since the 1970s in forward mutagenesis screens. However, only in the last decade has ENU mutagenesis been harnessed to generate a myriad of new mouse mutations in large-scale genetic screens and focused, smaller efforts. The development of additional genetic tools, such as balancer chromosomes, refinements in genetic mapping strategies, and evolution of specialized assays, has allowed these screens to achieve new levels of sophistication. The impressive productivity of these screens has led to a deluge of mouse mutants that wait to be harnessed. Here the basic large- and small-scale strategies are described, as are the basics of screen design. Finally, and importantly, this review describes the mechanisms by which such mutants may be accessed now and in the future. Thus, this review should serve both as an overview of the power of forward mutagenesis in the mouse and as a resource for those interested in developing their own screens, adding onto existing efforts, or obtaining specific mouse mutants that have already been generated. PMID:16148305

  1. Alleles conferring improved fiber quality from EMS mutagenesis of elite cotton genotypes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The elite gene pool of cotton (Gossypium spp.) has less diversity than those of most other major crops, making identification of novel alleles important to ongoing crop improvement. A total of 3,164 M5 lines resulting from ethyl methanesulfonate mutagenesis of two G. hirsutum breeding lines, TAM 94L...

  2. Natural selection underlies apparent stress-induced mutagenesis in a bacteriophage infection model.

    PubMed

    Yosef, Ido; Edgar, Rotem; Levy, Asaf; Amitai, Gil; Sorek, Rotem; Munitz, Ariel; Qimron, Udi

    2016-01-01

    The emergence of mutations following growth-limiting conditions underlies bacterial drug resistance, viral escape from the immune system and fundamental evolution-driven events. Intriguingly, whether mutations are induced by growth limitation conditions or are randomly generated during growth and then selected by growth limitation conditions remains an open question(1). Here, we show that bacteriophage T7 undergoes apparent stress-induced mutagenesis when selected for improved recognition of its host's receptor. In our unique experimental set-up, the growth limitation condition is physically and temporally separated from mutagenesis: growth limitation occurs while phage DNA is outside the host, and spontaneous mutations occur during phage DNA replication inside the host. We show that the selected beneficial mutations are not pre-existing and that the initial slow phage growth is enabled by the phage particle's low-efficiency DNA injection into the host. Thus, the phage particle allows phage populations to initially extend their host range without mutagenesis by virtue of residual recognition of the host receptor. Mutations appear during non-selective intracellular replication, and the frequency of mutant phages increases by natural selection acting on free phages, which are not capable of mutagenesis. PMID:27572836

  3. IN VITRO MAMMALIAN MUTAGENESIS AS A MODEL FOR GENETIC LESIONS IN HUMAN CANCER

    EPA Science Inventory

    Recently, in vitro mammalian cell assays of mutagenesis have been criticized as being poorly predictive of long-term in vivo rodent assays of carcinogenicity. Yet in vitro as says using mammalian cells might be expected to register types of genetic lesions thought to be important...

  4. Random mutagenesis strategies for construction of large and diverse clone libraries of mutated DNA fragments.

    PubMed

    Chusacultanachai, Sudsanguan; Yuthavong, Yongyuth

    2004-01-01

    The first important step toward a successful preparation of large and diverse DNA libraries with desired complexity is to select a suitable mutagenesis strategy. This chapter describes three different methods for random mutagenesis, the use of XL1-red cells, error-prone polymerase chain reaction (PCR), and degenerate oligonucleotides-Pfu (DOP). These mutagenesis strategies possess different benefits and pitfalls; thus, they are differentially useful for production of DNA libraries with different density and complexity. The use of XL1-red, an engineered Escherichia coli with DNA repair deficiency, is one of the simplest mutagenesis and requires no subcloning step. After plasmid encoding DNA of inter-est is transformed into the cells, the mutations are simply generated during each round of DNA replication. The mutation frequency of this method is reported to be 1 base change per 2000 nucleotides; however, it can be slightly increased by extending the culture period to allow the accumulation of more mutations. This strategy is suitable for generation of random mutations with low frequency in a large target DNA. Error-prone PCR is one of the most widely used random mutagenesis. During DNA amplification, misincorporation of nucleotides can be promoted by altering the nucleotide ratio and the concentration of divalent cations in the reaction. We discovered that, by adjusting template concentration, frequency of mutation could be controlled easily and a library with desired mutation rate could be obtained. Additionally, efficiency of subsequent cloning steps to insert the PCR product into plasmid DNA is also a key factor determining size and complexity of the libraries. DOP mutagenesis is a rapid and effective method for random mutagenesis of small DNA and peptides. This strategy uses two chemically synthesized degenerate oligonucleotides as primers. By controlling the positions and ratios of degenerate nucleotides used during oligonucleotide synthesis, it is possible to

  5. Yeast cytochrome c peroxidase: mutagenesis and expression in Escherichia coli show tryptophan-51 is not the radical site in compound I

    SciTech Connect

    Fishel, L.A.; Villafranca, J.E.; Mauro, J.M.; Kraut, J.

    1987-01-27

    Using oligonucleotide-directed site-specific mutagenesis, they have constructed a system for the mutation and expression of yeast cytochrome c peroxidase (CCP, EC 1.11.1.5) in Escherichia coli and applied it to test the hypothesis that Trp-51 is the locus of the free radical observed in compound I of CCP. The system was created by substituting a CCP gene modified by site-directed mutagenesis, CCP(MI), for the fol gene in a vector previously used for mutagenesis and overexpression of dihydrofolate reductase. E. coli transformed with the resulting plasmid produced the CCP(MI) enzyme in large quantities, more than 15 mg/L of cell culture, of which 10% is holo- and 90% is apo-CCP(MI). The apoenzyme was easily converted to holoenzyme by the addition of bovine hemin. Purified CCP(MI) has the same catalytic activity and spectra as bakers' yeast CCP. A mutation has been made in CCP(MI), Trp-51 to Phe. The Phe-51 mutant protein CCP(MI,F51) is fully active, and the electron paramagnetic resonance (EPR) spectrum, at 89 K, of its oxidized intermediate, compound I, displays a strong sharp resonance at g = 2.004, which is very similar to the signal observed for compound I of both bakers' yeast CCP and CCP(MI). However, UV-visible and EPR spectroscopy revealed that the half-life of CCP(MI,F51) compound I at 23 /sup 0/C is only 1.4% of that observed for the compound I forms of CCP(MI) or bakers' yeast CCP. Thus, Trp-51 is not necessary for the formation of the free radical observed in compound I but appears to exert a significant influence on its stability.

  6. In vivo growth characteristics of leucine and methionine auxotrophic mutants of Mycobacterium bovis BCG generated by transposon mutagenesis.

    PubMed Central

    McAdam, R A; Weisbrod, T R; Martin, J; Scuderi, J D; Brown, A M; Cirillo, J D; Bloom, B R; Jacobs, W R

    1995-01-01

    Insertional mutagenesis in Mycobacterium bovis BCG, a member of the slow-growing M. tuberculosis complex, was accomplished with transposons engineered from the Mycobacterium smegmatis insertion element IS1096. Transposons were created by placing a kanamycin resistance gene in several different positions in IS1096, and the resulting transposons were electroporated into BCG on nonreplicating plasmids. These analyses demonstrated that only one of the two open reading frames was necessary for transposition. A library of insertions was generated. Southern analysis of 23 kanamycin-resistant clones revealed that the transposons had inserted directly, with no evidence of cointegrate formation, into different restriction fragments in each clone. Sequence analysis of nine of the clones revealed junctional direct 8-bp repeats with only a slight similarity in target sites. These results suggest that IS1096-derived transposons transposed into the BCG genome in a relatively random fashion. Three auxotrophs, two for leucine and one for methionine, were isolated from the library of transposon insertions in BCG. They were characterized by sequencing and found to be homologous to the leuD gene of Escherichia coli and a sulfate-binding protein of cyanobacteria, respectively. When inoculated intravenously into C57BL/6 mice, the leucine auxotrophs, in contrast to the parent BCG strain or the methionine auxotroph, showed an inability to grow in vivo and were cleared within 7 weeks from the lungs and spleen. PMID:7868221

  7. Site-directed mutagenesis and high-resolution NMR spectroscopy of the active site of porphobilinogen deaminase

    SciTech Connect

    Scott, A.I.; Roessner, C.A.; Stolowich, N.J.; Karuso, P.; Williams, H.J.; Grant, S.K.; Gonzalez, M.D.; Hoshino, T. )

    1988-10-18

    The active site of porphobilinogen (PBG){sup 1} deaminase from Escherichia coli has been found to contain an unusual dipyrromethane derived from four molecules of 5-aminolevulinic acid (ALA) covalently linked to Cys-242, one of the two cysteine residues conserved in E. coli and human deaminase. By use of a hemA{sup {minus}} strain of E. coli the enzyme was enriched from (5-{sup 13}C)ALA and examined by {sup 1}H-detected multiple quantum coherence spectroscopy, which revealed all of the salient features of a dipyrromethane composed of two PBG units linked heat to tail and terminating in a CH{sub 2}-S bond to a cysteine residue. Site-specific mutagenesis of Cys-99 and Cys-242, respectively, has shown that substitution of Ser for Cys-99 does not affect the enzymatic activity, whereas substitution of Ser for Cys-242 removes essentially all of the catalytic activity as measured by the conversion of the substrate PBG to uro'gen I. The NMR spectrum of the covalent complex of deaminase with the suicide inhibitor 2-bromo-(2,11-{sup 13}C{sub 2})PBG reveals that the aminomethyl terminus of the inhibitor reacts with the enzyme's cofactor at the {alpha}-free pyrrole. NMR spectroscopy of the ES{sub 2} complex confirmed a PBG-derived head-to-tail dipyrromethane attached to the {alpha}-free pyrrole position of the enzyme. A mechanistic rationale for deaminase is presented.

  8. ENU mutagenesis identifies mice modeling Warburg Micro Syndrome with sensory axon degeneration caused by a deletion in Rab18.

    PubMed

    Cheng, Chih-Ya; Wu, Jaw-Ching; Tsai, Jin-Wu; Nian, Fang-Shin; Wu, Pei-Chun; Kao, Lung-Sen; Fann, Ming-Ji; Tsai, Shih-Jen; Liou, Ying-Jay; Tai, Chin-Yin; Hong, Chen-Jee

    2015-05-01

    Mutations in the gene of RAB18, a member of Ras superfamily of small G-proteins, cause Warburg Micro Syndrome (WARBM) which is characterized by defective neurodevelopmental and ophthalmological phenotypes. Despite loss of Rab18 had been reported to induce disruption of the endoplasmic reticulum structure and neuronal cytoskeleton organization, parts of the pathogenic mechanism caused by RAB18 mutation remain unclear. From the N-ethyl-N-nitrosourea (ENU)-induced mutagenesis library, we identified a mouse line whose Rab18 was knocked out. This Rab18(-/-) mouse exhibited stomping gait, smaller testis and eyes, mimicking several features of WARBM. Rab18(-/-) mice were obviously less sensitive to pain and touch than WT mice. Histological examinations on Rab18(-/-) mice revealed progressive axonal degeneration in the optic nerves, dorsal column of the spinal cord and sensory roots of the spinal nerves while the motor roots were spared. All the behavioral and pathological changes that resulted from abnormalities in the sensory axons were prevented by introducing an extra copy of Rab18 transgene in Rab18(-/-) mice. Our results reveal that sensory axonal degeneration is the primary cause of stomping gait and progressive weakness of the hind limbs in Rab18(-/-) mice, and optic nerve degeneration should be the major pathology of progressive optic atrophy in children with WARBM. Our results indicate that the sensory nervous system is more vulnerable to Rab18 deficiency and WARBM is not only a neurodevelopmental but also neurodegenerative disease. PMID:25779931

  9. A Mutant Mouse with a Highly Specific Contextual Fear-Conditioning Deficit Found in an N-Ethyl-N-Nitrosourea (ENU) Mutagenesis Screen

    ERIC Educational Resources Information Center

    Pletcher, Mathew T.; Wiltshire, Tim; Tarantino, Lisa M.; Mayford, Mark; Reijmers, Leon G.; Coats, Jennifer K.

    2006-01-01

    Targeted mutagenesis in mice has shown that genes from a wide variety of gene families are involved in memory formation. The efficient identification of genes involved in learning and memory could be achieved by random mutagenesis combined with high-throughput phenotyping. Here, we provide the first report of a mutagenesis screen that has…

  10. The transcription elongation factor NusA is required for stress-induced mutagenesis in Escherichia coli.

    PubMed

    Cohen, Susan E; Walker, Graham C

    2010-01-12

    Stress-induced mutagenesis describes the accumulation of mutations that occur in nongrowing cells, in contrast to mutagenesis that occurs in actively dividing populations, and has been referred to as stationary-phase or adaptive mutagenesis. The most widely studied system for stress-induced mutagenesis involves monitoring the appearance of Lac(+) revertants of the strain FC40 under starvation conditions in Escherichia coli. The SOS-inducible translesion DNA polymerase DinB plays an important role in this phenomenon. Loss of DinB (DNA pol IV) function results in a severe reduction of Lac(+) revertants. We previously reported that NusA, an essential component of elongating RNA polymerases, interacts with DinB. Here we report our unexpected observation that wild-type NusA function is required for stress-induced mutagenesis. We present evidence that this effect is unlikely to be due to defects in transcription of lac genes but rather is due to an inability to adapt and mutate in response to environmental stress. Furthermore, we extended our analysis to the formation of stress-induced mutants in response to antibiotic treatment, observing the same striking abolition of mutagenesis under entirely different conditions. Our results are the first to implicate NusA as a crucial participant in the phenomenon of stress-induced mutagenesis. PMID:20036541

  11. One-Tube-Only Standardized Site-Directed Mutagenesis: An Alternative Approach to Generate Amino Acid Substitution Collections

    PubMed Central

    Mingo, Janire; Erramuzpe, Asier; Luna, Sandra; Aurtenetxe, Olaia; Amo, Laura; Diez, Ibai; Schepens, Jan T. G.; Hendriks, Wiljan J. A. J.; Cortés, Jesús M.; Pulido, Rafael

    2016-01-01

    Site-directed mutagenesis (SDM) is a powerful tool to create defined collections of protein variants for experimental and clinical purposes, but effectiveness is compromised when a large number of mutations is required. We present here a one-tube-only standardized SDM approach that generates comprehensive collections of amino acid substitution variants, including scanning- and single site-multiple mutations. The approach combines unified mutagenic primer design with the mixing of multiple distinct primer pairs and/or plasmid templates to increase the yield of a single inverse-PCR mutagenesis reaction. Also, a user-friendly program for automatic design of standardized primers for Ala-scanning mutagenesis is made available. Experimental results were compared with a modeling approach together with stochastic simulation data. For single site-multiple mutagenesis purposes and for simultaneous mutagenesis in different plasmid backgrounds, combination of primer sets and/or plasmid templates in a single reaction tube yielded the distinct mutations in a stochastic fashion. For scanning mutagenesis, we found that a combination of overlapping primer sets in a single PCR reaction allowed the yield of different individual mutations, although this yield did not necessarily follow a stochastic trend. Double mutants were generated when the overlap of primer pairs was below 60%. Our results illustrate that one-tube-only SDM effectively reduces the number of reactions required in large-scale mutagenesis strategies, facilitating the generation of comprehensive collections of protein variants suitable for functional analysis. PMID:27548698

  12. One-Tube-Only Standardized Site-Directed Mutagenesis: An Alternative Approach to Generate Amino Acid Substitution Collections.

    PubMed

    Mingo, Janire; Erramuzpe, Asier; Luna, Sandra; Aurtenetxe, Olaia; Amo, Laura; Diez, Ibai; Schepens, Jan T G; Hendriks, Wiljan J A J; Cortés, Jesús M; Pulido, Rafael

    2016-01-01

    Site-directed mutagenesis (SDM) is a powerful tool to create defined collections of protein variants for experimental and clinical purposes, but effectiveness is compromised when a large number of mutations is required. We present here a one-tube-only standardized SDM approach that generates comprehensive collections of amino acid substitution variants, including scanning- and single site-multiple mutations. The approach combines unified mutagenic primer design with the mixing of multiple distinct primer pairs and/or plasmid templates to increase the yield of a single inverse-PCR mutagenesis reaction. Also, a user-friendly program for automatic design of standardized primers for Ala-scanning mutagenesis is made available. Experimental results were compared with a modeling approach together with stochastic simulation data. For single site-multiple mutagenesis purposes and for simultaneous mutagenesis in different plasmid backgrounds, combination of primer sets and/or plasmid templates in a single reaction tube yielded the distinct mutations in a stochastic fashion. For scanning mutagenesis, we found that a combination of overlapping primer sets in a single PCR reaction allowed the yield of different individual mutations, although this yield did not necessarily follow a stochastic trend. Double mutants were generated when the overlap of primer pairs was below 60%. Our results illustrate that one-tube-only SDM effectively reduces the number of reactions required in large-scale mutagenesis strategies, facilitating the generation of comprehensive collections of protein variants suitable for functional analysis. PMID:27548698

  13. EMS mutagenesis in mature seed-derived rice calli as a new method for rapidly obtaining TILLING mutant populations

    PubMed Central

    2014-01-01

    Background TILLING (Targeting Induced Local Lesions IN Genomes) is a reverse genetic method that combines chemical mutagenesis with high-throughput genome-wide screening for point mutation detection in genes of interest. However, this mutation discovery approach faces a particular problem which is how to obtain a mutant population with a sufficiently high mutation density. Furthermore, plant mutagenesis protocols require two successive generations (M1, M2) for mutation fixation to occur before the analysis of the genotype can begin. Results Here, we describe a new TILLING approach for rice based on ethyl methanesulfonate (EMS) mutagenesis of mature seed-derived calli and direct screening of in vitro regenerated plants. A high mutagenesis rate was obtained (i.e. one mutation in every 451 Kb) when plants were screened for two senescence-related genes. Screening was carried out in 2400 individuals from a mutant population of 6912. Seven sense change mutations out of 15 point mutations were identified. Conclusions This new strategy represents a significant advantage in terms of time-savings (i.e. more than eight months), greenhouse space and work during the generation of mutant plant populations. Furthermore, this effective chemical mutagenesis protocol ensures high mutagenesis rates thereby saving in waste removal costs and the total amount of mutagen needed thanks to the mutagenesis volume reduction. PMID:24475756

  14. The myeloperoxidase product hypochlorous acid generates irreversible high-density lipoprotein receptor inhibitors

    PubMed Central

    Binder, Veronika; Ljubojevic, Senka; Haybaeck, Johannes; Holzer, Michael; El-Gamal, Dalia; Schicho, Rudolf; Pieske, Burkert; Heinemann, Akos; Marsche, Gunther

    2014-01-01

    Objective Elevated levels of advanced oxidation protein products (AOPPs) have been described in several chronic inflammatory diseases, like chronic renal insufficiency, rheumatoid arthritis and atherosclerosis. Recent findings revealed that AOPPs are inhibitors of the major high-density lipoprotein (HDL) receptor, scavenger receptor class B, type 1 (SR-BI). Here we investigated what oxidation induced structural alterations convert plasma albumin into an HDL-receptor inhibitor. Approach and Results Exposure of albumin to the physiological oxidant, hypochlorous acid, generated high affinity SR-BI ligands. Protection of albumin lysine-residues prior exposure to hypochlorous acid as well as regeneration of N-chloramines after oxidation of albumin completely prevented binding of oxidized albumin to SR-BI, indicating that modification of albumin lysine-residues is required to generate SR-BI ligands. Of particular interest, N-chloramines within oxidized albumin promoted irreversible binding to SR-BI, resulting in permanent receptor blockade. We observed that the SR-BI inhibitory activity of albumin isolated from chronic kidney disease patients correlated with the content of the myeloperoxidase-specific oxidation product 3-chlorotyrosine and was associated with alterations in the composition of HDL. Conclusion Given that several potential atheroprotective activities of HDL are mediated by SR-BI, the present results raise the possibility that oxidized plasma albumin, through permanent SR-BI blockade, contributes to the pathophysiology of cardiovascular disease. PMID:23493288

  15. Relationship between anion binding and anion permeability revealed by mutagenesis within the cystic fibrosis transmembrane conductance regulator chloride channel pore

    PubMed Central

    Linsdell, Paul

    2001-01-01

    Anion binding within the pores of wild-type and mutant cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channels, expressed in two different mammalian cell lines, was assayed using patch clamp recording. Specifically, experiments measured both the conductance of different anions and the ability of other permeant anions to block Cl− permeation through the pore. Under symmetrical ionic conditions, wild-type CFTR channels showed the conductance sequence Cl− >NO3− >Br−≥formate >F− >SCN−≈ ClO4−. High SCN− conductance was not observed, nor was there an anomalous mole fraction effect of SCN− on conductance under the conditions used. Iodide currents could not be measured under symmetrical ionic conditions, but under bi-ionic conditions I− conductance appeared low. Chloride currents through CFTR channels were blocked by low concentrations (10 mM) of SCN−, I− and ClO4−, implying relatively tight binding of these anions within the pore. Two mutations in CFTR which alter the anion permeability sequence, F337S and T338A, also altered the anion conductance sequence. Furthermore, block by SCN−, I− and ClO4− were weakened in both mutants. Both these effects are consistent with altered anion binding within the pore. The effects of mutations on anion permeability and relative anion conductance suggested that, for most anions, increased permeability was associated with increased conductance. This indicates that the CFTR channel pore does not achieve its anion selectivity by selective anion binding within the mutated region. Instead, it is suggested that entry of anions into the region around F337 and T338 facilitates their passage through the pore. In wild-type CFTR channels, anion entry into this crucial pore region is probably dominated by anion hydration energies. PMID:11179391

  16. Relationship between anion binding and anion permeability revealed by mutagenesis within the cystic fibrosis transmembrane conductance regulator chloride channel pore.

    PubMed

    Linsdell, P

    2001-02-15

    1. Anion binding within the pores of wild-type and mutant cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels, expressed in two different mammalian cell lines, was assayed using patch clamp recording. Specifically, experiments measured both the conductance of different anions and the ability of other permeant anions to block Cl- permeation through the pore. 2. Under symmetrical ionic conditions, wild-type CFTR channels showed the conductance sequence Cl- > NO3- > Br- > or = formate > F- > SCN- congruent to ClO4-. 3. High SCN- conductance was not observed, nor was there an anomalous mole fraction effect of SCN- on conductance under the conditions used. Iodide currents could not be measured under symmetrical ionic conditions, but under bi-ionic conditions I- conductance appeared low. 4. Chloride currents through CFTR channels were blocked by low concentrations (10 mM) of SCN-, I- and ClO4-, implying relatively tight binding of these anions within the pore. 5. Two mutations in CFTR which alter the anion permeability sequence, F337S and T338A, also altered the anion conductance sequence. Furthermore, block by SCN-, I- and ClO4- were weakened in both mutants. Both these effects are consistent with altered anion binding within the pore. 6. The effects of mutations on anion permeability and relative anion conductance suggested that, for most anions, increased permeability was associated with increased conductance. This indicates that the CFTR channel pore does not achieve its anion selectivity by selective anion binding within the mutated region. Instead, it is suggested that entry of anions into the region around F337 and T338 facilitates their passage through the pore. In wild-type CFTR channels, anion entry into this crucial pore region is probably dominated by anion hydration energies. PMID:11179391

  17. Insights into electron leakage in the reaction cycle of cytochrome P450 BM3 revealed by kinetic modeling and mutagenesis.

    PubMed

    Lim, Joseph B; Barker, Kimberly A; Eller, Kristen A; Jiang, Linda; Molina, Veronica; Saifee, Jessica F; Sikes, Hadley D

    2015-11-01

    As a single polypeptide, cytochrome P450 BM3 fuses oxidase and reductase domains and couples each domain's function to perform catalysis with exceptional activity upon binding of substrate for hydroxylation. Mutations introduced into the enzyme to change its substrate specificity often decrease coupling efficiency between the two domains, resulting in unproductive consumption of cofactors and formation of water and/or reactive species. This phenomenon can correlate with leakage, in which P450 BM3 uses electrons from NADPH to reduce oxygen to water and/or reactive species even without bound substrate. The physical basis for leakage is not yet well understood in this particular member of the cytochrome P450 family. To clarify the relationship between leakage and coupling, we used simulations to illustrate how different combinations of kinetic parameters related to substrate-free consumption of NADPH and substrate hydroxylation can lead to either minimal effects on coupling or a dramatic decrease in coupling as a result of leakage. We explored leakage in P450 BM3 by introducing leakage-enhancing mutations and combining these mutations to assess whether doing so increases leakage further. The variants in this study provide evidence that while a transition to high spin may be vital for coupled hydroxylation, it is not required for enhanced leakage; substrate binding and the consequent shift in spin state are not necessary as a redox switch for catalytic oxidation of NADPH. Additionally, the variants in this study suggest a tradeoff between leakage and stability and thus evolvability, as the mutations we investigated were far more deleterious than other mutations that have been used to change substrate specificity. PMID:26311413

  18. Targeted Mutagenesis of the Hypophysiotropic Gnrh3 in Zebrafish (Danio rerio) Reveals No Effects on Reproductive Performance

    PubMed Central

    Spicer, Olivia Smith; Wong, Ten-Tsao; Zmora, Nilli; Zohar, Yonathan

    2016-01-01

    Gnrh is the major neuropeptide regulator of vertebrate reproduction, triggering a cascade of events in the pituitary-gonadal axis that result in reproductive competence. Previous research in mice and humans has demonstrated that Gnrh/GNRH null mutations result in hypogonadotropic hypogonadism and infertility. The goal of this study was to eliminate gnrh3 (the hypophysiotropic Gnrh form) function in zebrafish (Danio rerio) to determine how ontogeny and reproductive performance are affected, as well as factors downstream of Gnrh3 along the reproductive axis. Using the TALEN technology, we developed a gnrh3-/- zebrafish line that harbors a 62 bp deletion in the gnrh3 gene. Our gnrh3-/- zebrafish line represents the first targeted and heritable mutation of a Gnrh isoform in any organism. Using immunohistochemistry, we verified that gnrh3-/- fish do not possess Gnrh3 peptide in any regions of the brain. However, other than changes in mRNA levels of pituitary gonadotropin genes (fshb, lhb, and cga) during early development, which are corrected by adulthood, there were no changes in ontogeny and reproduction in gnrh3-/- fish. The gnrh3-/- zebrafish are fertile, displaying normal gametogenesis and reproductive performance in males and females. Together with our previous results that Gnrh3 cell ablation causes infertility, these results indicate that a compensatory mechanism is being activated, which is probably primed early on upon Gnrh3 neuron differentiation and possibly confined to Gnrh3 neurons. Potential compensation factors and sensitive windows of time for compensation during development and puberty should be explored. PMID:27355207

  19. Lysine-scanning Mutagenesis Reveals an Amendable Face of the Cyclotide Kalata B1 for the Optimization of Nematocidal Activity*

    PubMed Central

    Huang, Yen-Hua; Colgrave, Michelle L.; Clark, Richard J.; Kotze, Andrew C.; Craik, David J.

    2010-01-01

    Cyclotides are a family of macrocyclic peptides that combine the unique features of a head-to-tail cyclic backbone and a cystine knot motif, the combination of which imparts them with extraordinary stability. The prototypic cyclotide kalata B1 is toxic against two economically important gastrointestinal nematode parasites of sheep, Haemonchus contortus and Trichostrongylus colubriformis. A lysine scan was conducted to examine the effect of the incorporation of positive charges into the kalata B1 cyclotide framework. Each of the non-cysteine residues in this 29-amino acid peptide was successively substituted with lysine, and the nematocidal and hemolytic activities of the suite of mutants were determined. Substitution of 11 residues within kalata B1 decreased the nematocidal activity dramatically. On the other hand, six other residues that are clustered on the surface of kalata B1 were tolerant to Lys substitution, and indeed the introduction of positively charged residues into this region increased nematocidal activity. This activity was increased further in double and triple lysine mutants, with a maximal increase (relative to the native kalata B1) of 13-fold obtained with a triple lysine mutant (mutated at positions Thr-20, Asn-29, and Gly-1). Hemolytic activity correlated with the nematocidal activity of all lysine mutants. Our data clearly highlight the residues crucial for nematocidal and hemolytic activity in cyclotides, and demonstrate that the nematocidal activity of cyclotides can be increased by incorporation of basic amino acids. PMID:20103593

  20. Mutagenesis of the novel Hericium erinaceus ribonuclease, RNase He1, reveals critical responsible residues for enzyme stability and activity.

    PubMed

    Kobayashi, Hiroko; Motoyoshi, Naomi; Itagaki, Tadashi; Inokuchi, Norio

    2014-01-01

    Here, we determined the sequence of a cDNA encoding a guanylic acid-specific ribonuclease (RNase He1) from Hericium erinaceus that exhibits high sequence identity (59%) with RNase Po1, an enzyme with anti-cancer activity and which is found in Pleurotus ostreatus. RNase He1 and RNase Po1 have similar structures and heat stabilities; hence, RNase He1 may also have potential as an anti-cancer agent. Therefore, we initiated structure-function studies to further characterize the enzyme. Based on the RNase Po1 structure, RNase He1 is predicted to form 3 disulfide bonds involving Cys7-Cys98, Cys5-Cys83, and Cys47-Cys81 linkages. The Cys5Ala mutant exhibited no RNase activity, whereas the Cys81Ala mutant retained RNase activity, but had reduced heat stability. Therefore, the Cys5-Cys83 bond in RNase He1 is essential for the structure of the RNase active site region. Similarly, the Cys47-Cys81 bond helps maintain the conformational stability of the active site region, and may contribute to the greater heat stability of RNase He1. PMID:25366489

  1. Genome-wide mutagenesis of Xanthomonas axonopodis pv. citri reveals novel genetic determinants and regulation mechanisms of biofilm formation.

    PubMed

    Li, Jinyun; Wang, Nian

    2011-01-01

    Xanthomonas axonopodis pv. citri (Xac) causes citrus canker disease, a major threat to citrus production worldwide. Accumulating evidence suggests that the formation of biofilms on citrus leaves plays an important role in the epiphytic survival of this pathogen prior to the development of canker disease. However, the process of Xac biofilm formation is poorly understood. Here, we report a genome-scale study of Xac biofilm formation in which we identified 92 genes, including 33 novel genes involved in biofilm formation and 7 previously characterized genes, colR, fhaB, fliC, galU, gumD, wxacO, and rbfC, known to be important for Xac biofilm formation. In addition, 52 other genes with defined or putative functions in biofilm formation were identified, even though they had not previously reported been to be associated with biofilm formation. The 92 genes were isolated from 292 biofilm-defective mutants following a screen of a transposon insertion library containing 22,000 Xac strain 306 mutants. Further analyses indicated that 16 of the novel genes are involved in the production of extracellular polysaccharide (EPS) and/or lipopolysaccharide (LPS), 7 genes are involved in signaling and regulatory pathways, and 5 genes have unknown roles in biofilm formation. Furthermore, two novel genes, XAC0482, encoding a haloacid dehalogenase-like phosphatase, and XAC0494 (designated as rbfS), encoding a two-component sensor protein, were confirmed to be biofilm-related genes through complementation assays. Our data demonstrate that the formation of mature biofilm requires EPS, LPS, both flagellum-dependent and flagellum-independent cell motility, secreted proteins and extracellular DNA. Additionally, multiple signaling pathways are involved in Xac biofilm formation. This work is the first report on a genome-wide scale of the genetic processes of biofilm formation in plant pathogenic bacteria. The report provides significant new information about the genetic determinants and regulatory mechanism of biofilm formation. PMID:21750733

  2. Newly identified essential amino acid residues affecting ^8-sphingolipid desaturase activity revealed by site-directed mutagenesis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In order to identify amino acid residues crucial for the enzymatic activity of ^8-sphingolipid desaturases, a sequence comparison was performed among ^8-sphingolipid desaturases and ^6-fatty acid desaturase from various plants. In addition to the known conserved cytb5 (cytochrome b5) HPGG motif and...

  3. Identification of pathogenicity-related genes in the rice pathogen Ustilaginoidea virens through random insertional mutagenesis.

    PubMed

    Yu, Mina; Yu, Junjie; Hu, Jiankun; Huang, Lei; Wang, Yahui; Yin, Xiaole; Nie, Yafeng; Meng, Xiangkun; Wang, Weiduo; Liu, Yongfeng

    2015-03-01

    Agrobacterium tumefaciens-mediated transformation (ATMT) is becoming a popular effective system as an insertional mutagenesis tool in filamentous fungi. To gain more insight into the cellular and molecular mechanisms in the pathogenesis of Ustilaginoidea virens, the causal agent of rice false smut disease, a T-DNA insertion mutant library of U. virens was established using ATMT. We optimized a range of conditions to improve the transformation efficiency. Transformants were most effectively obtained when the optimal co-cultivation time is 72h, with 50μM AS in medium and 100μl A. tumefaciens for co-cultivation, leading to the production of 160-185 hygromycin B resistant transformants per 1×10(5) conidia. Southern blot analysis indicated that 58.14% of transformants had a single T-DNA copy. Among 5600 transformants tested for virulence, 37 mutants with reproducible pathogenic defects were obtained. The flanking sequences of three avirulent tranformants (B20, B1015 and B1465) and two pathogenicity-reduced transformants (B726 and B785) were amplified by high-efficiency thermal asymmetric interlaced PCR. Sequence analyses revealed that single T-DNA insertion in mutant B20 targeted the coding region of a gene encoding a protein highly similar to SUN family protein, and in mutant B726 targeted upstream of a gene with unknown function. The two T-DNA insertion sites in mutant B785 were found in the coding region of a gene encoding C6 transcription factor, but failed in amplified flanking sequence of another T-DNA. Chromosomal rearrangement occurred in the genome of mutant B1016 and B1465 with single T-DNA insertion. Among avirulent mutants, B20 showed altered colony growth and pigmentation. The T-DNA insert in B20 was detected in the coding region of a gene named UvSUN2. Morphophysiological characterization analysis suggested that UvSUN2 might be a virulence factor, and possibly required for proper fungal growth, cell wall construction, and stress responses in U. virens

  4. Efficient gene-driven germ-line point mutagenesis of C57BL/6J mice

    SciTech Connect

    Michaud III, Edward J; Culiat, Cymbeline T; Klebig, Mitch; Barker, Gene; Cain, K T; Carpenter, Debra J S; Easter, Lori L; Foster, Carmen M; Gardner, Alysyn Wallace; Guo, ZY; Houser, Kay J; Hughes, Lori A; Kerley, Marilyn K; Liu, Zhaowei; Olszewski, Robert Edward; Pinn, Irina; Shaw, Ginger D; Shinpock, Sarah G; Wymore, Ann; Rinchik, Eugene M; Johnson, Dabney K

    2005-01-01

    Background: Analysis of an allelic series of point mutations in a gene, generated by N-ethyl-N-nitrosourea (ENU) mutagenesis, is a valuable method for discovering the full scope of its biological function. Here we present an efficient gene-driven approach for identifying ENU-induced point mutations in any gene in C57BL/6J mice. The advantage of such an approach is that it allows one to select any gene of interest in the mouse genome and to go directly from DNA sequence to mutant mice. Results: We produced the Cryopreserved Mutant Mouse Bank (CMMB), which is an archive of DNA, cDNA, tissues, and sperm from 4,000 G1 male offspring of ENU-treated C57BL/6J males mated to untreated C57BL/6J females. Each mouse in the CMMB carries a large number of random heterozygous point mutations throughout the genome. High-throughput Temperature Gradient Capillary Electrophoresis (TGCE) was employed to perform a 32-Mbp sequence-driven screen for mutations in 38 PCR amplicons from 11 genes in DNA and/or cDNA from the CMMB mice. DNA sequence analysis of heteroduplex-forming amplicons identified by TGCE revealed 22 mutations in 10 genes for an overall mutation frequency of 1 in 1.45 Mbp. All 22 mutations are single base pair substitutions, and nine of them (41%) result in nonconservative amino acid substitutions. Intracytoplasmic sperm injection (ICSI) of cryopreserved spermatozoa into B6D2F1 or C57BL/6J ova was used to recover mutant mice for nine of the mutations to date. Conclusions: The inbred C57BL/6J CMMB, together with TGCE mutation screening and ICSI for the recovery of mutant mice, represents a valuable gene-driven approach for the functional annotation of the mammalian genome and for the generation of mouse models of human genetic diseases. The ability of ENU to induce mutations that cause various types of changes in proteins will provide additional insights into the functions of mammalian proteins that may not be detectable by knockout mutations.

  5. [Mutagenesis induced with dioxidin in the Allium-test].

    PubMed

    Shkarupa, V M; Baryliak, I R

    2006-01-01

    The influence of dioxidin in different concentrations (10-100 mg/l) on the cytogenetic parameters of Allium cepa L. has been studied. The mutagenic effect of dioxidin was shown within all the range of the studied concentrations. The curve "dose-effect" has been determined for the concentrations of 10, 20, 30, 40, 50, and 100 mg/l. The peak of the mutagenic effect and significant reduction of the mitotic index were revealed at the concentration of 100 mg/l. It was shown that the mutagenic efficiency of the dioxidin statistically correlated with reduction of the mitotic activities. PMID:17385416

  6. Application of a novel multi-screening signature-tagged mutagenesis assay for identification of Klebsiella pneumoniae genes essential in colonization and infection.

    PubMed

    Struve, Carsten; Forestier, Christiane; Krogfelt, Karen A

    2003-01-01

    Klebsiella pneumoniae is a common cause of urinary tract infections (UTIs) and pneumonia, especially in immunocompromised individuals. Epidemiological studies have revealed that K. pneumoniae infections are frequently preceded by gastrointestinal colonization and the gastrointestinal tract is believed to be the most important reservoir for transmission of the bacteria. To identify genes involved in the ability of K. pneumoniae to colonize the intestine and infect the urinary tract, a novel multi-screening signature-tagged mutagenesis (MS-STM) assay was implemented. In the MS-STM assay, PCR-amplified tags present in the inoculum as well as recovered pools from each infection model are simultaneously subjected to hybridization using each specific tag as a probe. Therefore, screenings of a mutant library in more than one infection model is significantly eased compared to the traditional signature-tagged mutagenesis methodology. From a total of 1,440 K. pneumoniae transposon mutants screened, 13 mutants were identified as attenuated in intestinal colonization as well as the UTI model. In addition, six mutants attenuated only in the UTI model were identified. Transposon insertion sites in attenuated mutants were, among others, in genes encoding well-known K. pneumoniae virulence factors such as lipopolysaccharide and capsule, as well as in genes of unknown function. PMID:12576590

  7. Revealing Rembrandt

    PubMed Central

    Parker, Andrew J.

    2014-01-01

    The power and significance of artwork in shaping human cognition is self-evident. The starting point for our empirical investigations is the view that the task of neuroscience is to integrate itself with other forms of knowledge, rather than to seek to supplant them. In our recent work, we examined a particular aspect of the appreciation of artwork using present-day functional magnetic resonance imaging (fMRI). Our results emphasized the continuity between viewing artwork and other human cognitive activities. We also showed that appreciation of a particular aspect of artwork, namely authenticity, depends upon the co-ordinated activity between the brain regions involved in multiple decision making and those responsible for processing visual information. The findings about brain function probably have no specific consequences for understanding how people respond to the art of Rembrandt in comparison with their response to other artworks. However, the use of images of Rembrandt's portraits, his most intimate and personal works, clearly had a significant impact upon our viewers, even though they have been spatially confined to the interior of an MRI scanner at the time of viewing. Neuroscientific studies of humans viewing artwork have the capacity to reveal the diversity of human cognitive responses that may be induced by external advice or context as people view artwork in a variety of frameworks and settings. PMID:24795552

  8. Materials Data on SrBiAu (SG:194) by Materials Project

    DOE Data Explorer

    Kristin Persson

    2015-03-19

    Computed materials data using density functional theory calculations. These calculations determine the electronic structure of bulk materials by solving approximations to the Schrodinger equation. For more information, see https://materialsproject.org/docs/calculations

  9. Revealing Mercury

    NASA Astrophysics Data System (ADS)

    Prockter, L. M.; Solomon, S. C.; Head, J. W.; Watters, T. R.; Murchie, S. L.; Robinson, M. S.; Chapman, C. R.; McNutt, R. L.

    2009-04-01

    The MErcury Surface, Space ENvironment, GEochemistry, and Ranging (MESSENGER) spacecraft, developed under NASA's Discovery Program, launched in August 2004. En route to insertion into orbit about Mercury in 2011, MESSENGER flies by Mercury three times. The first and second of these encounters were accomplished in January and October of 2008. These flybys viewed portions of Mercury's surface that were not observed by Mariner 10 during its reconnaissance of somewhat less than half of the planet in 1974-1975. All MESSENGER instruments operated during each flyby and returned a wealth of new data. Many of the new observations were focused on the planet's geology, including monochrome imaging at resolutions as high as 100 m/pixel, multispectral imaging in 11 filters at resolutions as high as 500 m/pixel, laser altimetry tracks extending over several thousands of kilometers, and high-resolution spectral measurements of several types of terrain. Here we present an overview of the first inferences on the global geology of Mercury from the MESSENGER observations. Whereas evidence for volcanism was equivocal from Mariner 10 data, the new MESSENGER images and altimetry provide compelling evidence that volcanism was widespread and protracted on Mercury. Color imaging reveals three common spectral units on the surface: a higher-reflectance, relatively red material occurring as a distinct class of smooth plains, typically with distinct embayment relationships interpreted to indicate volcanic emplacement; a lower-reflectance, relatively blue material typically excavated by impact craters and therefore inferred to be more common at depth; and a spectrally intermediate terrain that constitutes much of the uppermost crust. Three more minor spectral units are also seen: fresh crater ejecta, reddish material associated with rimless depressions interpreted to be volcanic centers, and high-reflectance deposits seen in some crater floors. Preliminary measurements of crater size

  10. Transposon mutagenesis as an approach to improved understanding of Borrelia pathogenesis and biology

    PubMed Central

    Lin, Tao; Troy, Erin B.; Hu, Linden T.; Gao, Lihui; Norris, Steven J.

    2014-01-01

    Transposon insertion provides a method for near-random mutation of bacterial genomes, and has been utilized extensively for the study of bacterial pathogenesis and biology. This approach is particularly useful for organisms that are relatively refractory to genetic manipulation, including Lyme disease Borrelia. In this review, progress to date in the application of transposon mutagenesis to the study of Borrelia burgdorferi is reported. An effective Himar1-based transposon vector has been developed and used to acquire a sequence-defined library of nearly 4500 mutants in the infectious, moderately transformable B. burgdorferi B31 derivative 5A18NP1. Analysis of these transposon mutants using signature-tagged mutagenesis (STM) and Tn-seq approaches has begun to yield valuable information regarding the genes important in the pathogenesis and biology of this organism. PMID:24904839

  11. Triplex technology in studies of DNA damage, DNA repair, and mutagenesis

    PubMed Central

    Mukherjee, Anirban; Vasquez, Karen M.

    2012-01-01

    Triplex-forming oligonucleotides (TFOs) can bind to the major groove of homopurine-homopyrimidine stretches of double-stranded DNA in a sequence-specific manner through Hoogsteen hydrogen bonding to form DNA triplexes. TFOs by themselves or conjugated to reactive molecules can be used to direct sequence-specific DNA damage, which in turn results in the induction of several DNA metabolic activities. Triplex technology is highly utilized as a tool to study gene regulation, molecular mechanisms of DNA repair, recombination, and mutagenesis. In addition, TFO targeting of specific genes has been exploited in the development of therapeutic strategies to modulate DNA structure and function. In this review, we discuss advances made in studies of DNA damage, DNA repair, recombination, and mutagenesis by using triplex technology to target specific DNA sequences. PMID:21501652

  12. Triplex technology in studies of DNA damage, DNA repair, and mutagenesis.

    PubMed

    Mukherjee, Anirban; Vasquez, Karen M

    2011-08-01

    Triplex-forming oligonucleotides (TFOs) can bind to the major groove of homopurine-homopyrimidine stretches of double-stranded DNA in a sequence-specific manner through Hoogsteen hydrogen bonding to form DNA triplexes. TFOs by themselves or conjugated to reactive molecules can be used to direct sequence-specific DNA damage, which in turn results in the induction of several DNA metabolic activities. Triplex technology is highly utilized as a tool to study gene regulation, molecular mechanisms of DNA repair, recombination, and mutagenesis. In addition, TFO targeting of specific genes has been exploited in the development of therapeutic strategies to modulate DNA structure and function. In this review, we discuss advances made in studies of DNA damage, DNA repair, recombination, and mutagenesis by using triplex technology to target specific DNA sequences. PMID:21501652

  13. Mutagenesis at the ouabain-resistance locus in human diploid fibroblasts.

    PubMed

    Buchwald, M

    1977-09-01

    The variables affecting the frequency of ouabain-resistant mutant clones have been studied in a strain of foetal lung fibroblasts. Optimum mutant recovery was obtained when cells were selected in 10(-6) M ouabain at a cell density of 2 X 10(4) cells/cm 2 (10(6) cell per 100-mm dish). The spontaneous mutation rate was estimated to be 4 X 10(-8) per cell generation. Treatment with the mutagens ethyl methanesulfonate (EMS), N-methyl-N' -nitro-N-nitrosoguanidine, and UV light increased the frequency of mutant colonies by an order of magnitude. The maximum number of mutants after mutagenesis with EMS occurred after two population doublings of growth in non-selective medium prior to selection and depended on the dose of EMS. Ouabain-resistance is a useful marker for studies of quantitative mutagenesis in human cells. PMID:904650

  14. Altered lipid accumulation in Nannochloropsis salina CCAP849/3 following EMS and UV induced mutagenesis

    PubMed Central

    Beacham, T.A.; Macia, V. Mora; Rooks, P.; White, D.A.; Ali, S.T.

    2015-01-01

    Microalgae have potential as a chemical feed stock in a range of industrial applications. Nannochloropsis salina was subject to EMS mutagenesis and the highest lipid containing cells selected using fluorescence-activated cell sorting. Assessment of growth, lipid content and fatty acid composition identified mutant strains displaying a range of altered traits including changes in the PUFA content and a total FAME increase of up to 156% that of the wild type strain. Combined with a reduction in growth this demonstrated a productivity increase of up to 76%. Following UV mutagenesis, lipid accumulation of the mutant cultures was elevated to more than 3 fold that of the wild type strain, however reduced growth rates resulted in a reduction in overall productivity. Changes observed are indicative of alterations to the regulation of the omega 6 Kennedy pathway. The importance of these variations in physiology for industrial applications such as biofuel production is discussed. PMID:26753128

  15. Software-supported USER cloning strategies for site-directed mutagenesis and DNA assembly.

    PubMed

    Genee, Hans Jasper; Bonde, Mads Tvillinggaard; Bagger, Frederik Otzen; Jespersen, Jakob Berg; Sommer, Morten O A; Wernersson, Rasmus; Olsen, Lars Rønn

    2015-03-20

    USER cloning is a fast and versatile method for engineering of plasmid DNA. We have developed a user friendly Web server tool that automates the design of optimal PCR primers for several distinct USER cloning-based applications. Our Web server, named AMUSER (Automated DNA Modifications with USER cloning), facilitates DNA assembly and introduction of virtually any type of site-directed mutagenesis by designing optimal PCR primers for the desired genetic changes. To demonstrate the utility, we designed primers for a simultaneous two-position site-directed mutagenesis of green fluorescent protein (GFP) to yellow fluorescent protein (YFP), which in a single step reaction resulted in a 94% cloning efficiency. AMUSER also supports degenerate nucleotide primers, single insert combinatorial assembly, and flexible parameters for PCR amplification. AMUSER is freely available online at http://www.cbs.dtu.dk/services/AMUSER/. PMID:24847672

  16. [Application of near-infrared reflectance spectroscopy in grass breeding with space flight mutagenesis].

    PubMed

    Ren, Wei-Bo; Han, Jian-Guo; Zhang, Yun-Wei; Guo, Hui-Qin

    2008-02-01

    Near infrared reflectance spectroscopy is a new fast and efficient analysis method. It has been wildly used in many areas such as evaluation of feedstuff, assessment of soil fertilizer and so on. In the present paper, the principle, technique method and merits of NIRS were introduced. The potential application of NIRS in grass breeding with space flight mutagenesis was discussed in areas such as analysis of grass nutrition, estimate of secondary metabolism compounds, forecast of disease and insects resistance, and evaluation of abiotic stress. The conclusion is that application of NIRS in grass breeding with space mutagenesis is significant in both academic and technical areas because it not only improves the efficiency of mutation selection but helps uncover the mechanism of space mutation breeding. PMID:18479009

  17. Prediction of Enzyme Mutant Activity Using Computational Mutagenesis and Incremental Transduction

    PubMed Central

    Basit, Nada; Wechsler, Harry

    2011-01-01

    Wet laboratory mutagenesis to determine enzyme activity changes is expensive and time consuming. This paper expands on standard one-shot learning by proposing an incremental transductive method (T2bRF) for the prediction of enzyme mutant activity during mutagenesis using Delaunay tessellation and 4-body statistical potentials for representation. Incremental learning is in tune with both eScience and actual experimentation, as it accounts for cumulative annotation effects of enzyme mutant activity over time. The experimental results reported, using cross-validation, show that overall the incremental transductive method proposed, using random forest as base classifier, yields better results compared to one-shot learning methods. T2bRF is shown to yield 90% on T4 and LAC (and 86% on HIV-1). This is significantly better than state-of-the-art competing methods, whose performance yield is at 80% or less using the same datasets. PMID:22007208

  18. Enhancement of thermostability and kinetic efficiency of Aspergillus niger PhyA phytase by site-directed mutagenesis.

    PubMed

    Hesampour, Ardeshir; Siadat, Seyed Ehsan Ranaei; Malboobi, Mohammad Ali; Mohandesi, Nooshin; Arab, Seyed Shahriar; Ghahremanpour, Mohammad Mehdi

    2015-03-01

    Phytase efficiently catalyzes the hydrolysis of phytate to phosphate; it can be utilized as an animal supplement to provide animals their nutrient requirements for phosphate and to mitigate environmental pollution caused by unutilized feed phosphate. Owing to animal feed being commonly pelleted at 70 to 90 °C, phytase with a sufficiently high thermal stability is desirable. Based on the crystal structure of PhyA and bioinformatics analysis at variant heat treatments, 12 single and multiple mutants were introduced by site-directed mutagenesis in order to improve phytase thermostability. Mutated constructs were expressed in Pichia pastoris. The manipulated phytases were purified; their biochemical and kinetic investigation revealed that while the thermostability of six mutants was improved, P9 (T314S Q315R V62N) and P12 (S205N S206A T151A T314S Q315R) showed the highest heat stability (P < 0.05) with 24 and 22.6 % greater retention, respectively, compared with the PhyA of the wild type at 80 °C. The K m value of the improved thermostable P9 and P12 mutant enzymes for sodium phytate were 35 and 20 % lower (P < 0.05) with respect to the wild-type enzyme. In conclusion, it is feasible to simultaneously improve the thermostability and the catalytic efficiency of phytase to be used as an animal feed supplement. PMID:25527139

  19. Mutagenesis and genetic characterisation of amylolytic Aspergillus niger.

    PubMed

    Shafique, Sobiya; Bajwa, Rukhsana; Shafique, Shazia

    2010-07-01

    Aspergillus niger FCBP-198 was genetically modified for its ability to reveal extra cellular alpha-amylase enzyme activity. From 76 efficient mutants isolated after ultraviolet (UV) irradiation, An-UV-5.6 was selected as the most efficient UV mutant, with 76.41 units mL(-1) of alpha-amylase activity compared to wild (34.45 units mL(-1)). In case of ethyl methane sulphonate (EMS), among 242 survivors, 74 were assayed quantitatively and An-Ch-4.7 was found to be the most competent, as it exhibited a three-fold increase in alpha-amylase activity (89.38 units mL(-1)) than the parental strain. Genetic relationships of the mutants of A. niger FCBP-198 were analysed with a randomly amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR). Results obtained from the comparison between genotypes of A. niger FCBP-198 showed differences in the sizes and numbers of amplified fragments per primer for each isolate. The dendrogram showed that genotypes An-Ch-4.7 and An-Ch-4.2 were distinctly classified into one category, while the isolates An-UV-5.6, An-UV-5.1 and A. niger FCBP-198 have the nearest genetic relationship. The five isolates from A. niger FCBP-198 genotypes shared an average of 65% bands. PMID:19764004

  20. A Plasmid-Transposon Hybrid Mutagenesis System Effective in a Broad Range of Enterobacteria

    PubMed Central

    Monson, Rita; Smith, Debra S.; Matilla, Miguel A.; Roberts, Kevin; Richardson, Elizabeth; Drew, Alison; Williamson, Neil; Ramsay, Josh; Welch, Martin; Salmond, George P. C.

    2015-01-01

    Random transposon mutagenesis is a powerful technique used to generate libraries of genetic insertions in many different bacterial strains. Here we develop a system facilitating random transposon mutagenesis in a range of different Gram-negative bacterial strains, including Pectobacterium atrosepticum, Citrobacter rodentium, Serratia sp. ATCC39006, Serratia plymuthica, Dickeya dadantii, and many more. Transposon mutagenesis was optimized in each of these strains and three studies are presented to show the efficacy of this system. Firstly, the important agricultural pathogen D. dadantii was mutagenized. Two mutants that showed reduced protease production and one mutant producing the previously cryptic pigment, indigoidine, were identified and characterized. Secondly, the enterobacterium, Serratia sp. ATCC39006 was mutagenized and mutants incapable of producing gas vesicles, proteinaceous intracellular organelles, were identified. One of these contained a β-galactosidase transcriptional fusion within the gene gvpA1, essential for gas vesicle production. Finally, the system was used to mutate the biosynthetic gene clusters of the antifungal, anti-oomycete and anticancer polyketide, oocydin A, in the plant-associated enterobacterium, Dickeya solani MK10. The mutagenesis system was developed to allow easy identification of transposon insertion sites by sequencing, after facile generation of a replicon encompassing the transposon and adjacent DNA, post-excision. Furthermore, the system can also create transcriptional fusions with either β-galactosidase or β-glucuronidase as reporters, and exploits a variety of drug resistance markers so that multiple selectable fusions can be generated in a single strain. This system of various transposons has wide utility and can be combined in many different ways. PMID:26733980

  1. Shuttle mutagenesis of Neisseria gonorrhoeae: pilin null mutations lower DNA transformation competence.

    PubMed Central

    Seifert, H S; Ajioka, R S; Paruchuri, D; Heffron, F; So, M

    1990-01-01

    The method of shuttle mutagenesis has been extended to Neisseria gonorrhoeae. We have constructed a defective mini-Tn3 derivative that encodes chloramphenicol resistance in both N. gonorrhoeae and Escherichia coli and selected for mutations in the chloramphenicol resistance gene that express higher levels of antibiotic resistance in N. gonorrhoeae. Isogenic N. gonorrhoeae strains that differ only in pilin expression were constructed and used to test the effect of pilin null mutations on DNA transformation competence. PMID:2152910

  2. Single-Plasmid-Based System for Efficient Noncanonical Amino Acid Mutagenesis in Cultured Mammalian Cells.

    PubMed

    Cohen, Sarit; Arbely, Eyal

    2016-06-01

    We describe a new expression system for efficient non-canonical amino acid mutagenesis in cultured mammalian cells by using the pyrrolysine tRNA synthetase/tRNACUA (Pyl) pair. A significant improvement in the incorporation of non-canonical amino acids into proteins was obtained by combining all the required genetic components into a single and compact vector that can be efficiently delivered to different mammalian cell lines by conventional transfection reagents. PMID:27120490

  3. Transposon Mutagenesis of the Extremely Thermophilic Bacterium Thermus thermophilus HB27

    PubMed Central

    Carr, Jennifer F.; Gregory, Steven T.; Dahlberg, Albert E.

    2014-01-01

    Thermus thermophilus is an extremely thermophilic bacterium that grows between 50 °C and 80 °C and is an excellent model organism not only for understanding life at high temperature but also for its biotechnological and industrial applications. Multiple molecular capabilities are available including targeted gene inactivation and the use of shuttle plasmids that replicate in T. thermophilus and Escherichia coli; however the ability to disrupt gene function randomly by transposon insertion has not been developed. Here we report a detailed method of transposon mutagenesis of T. thermophilus HB27 based on the EZ-Tn5 system from Epicentre Biotechnologies. We were able to generate insertion mutations throughout the chromosome by in vitro transposition and transformation with mutagenized genomic DNA. We also report that an additional step, one that fills in single stranded gaps in donor DNA generated by the transposition reaction, was essential for successful mutagenesis. We anticipate that our method of transposon mutagenesis will enable further genetic development of T. thermophilus and may also be valuable for similar endeavors with other under-developed organisms. PMID:24948436

  4. In vitro Inactivation of Latent HSV by Targeted Mutagenesis Using an HSV-specific Homing Endonuclease.

    PubMed

    Aubert, Martine; Boyle, Nicole M; Stone, Daniel; Stensland, Laurence; Huang, Meei-Li; Magaret, Amalia S; Galetto, Roman; Rawlings, David J; Scharenberg, Andrew M; Jerome, Keith R

    2014-01-01

    Following acute infection, herpes simplex virus (HSV) establishes latency in sensory neurons, from which it can reactivate and cause recurrent disease. Available antiviral therapies do not affect latent viral genomes; therefore, they do not prevent reactivation following therapy cessation. One possible curative approach involves the introduction of DNA double strand breaks in latent HSV genomes by rare-cutting endonucleases, leading to mutagenesis of essential viral genes. We tested this approach in an in vitro HSV latency model using the engineered homing endonuclease (HE) HSV1m5, which recognizes a sequence in the HSV-1 gene UL19, encoding the virion protein VP5. Coexpression of the 3'-exonuclease Trex2 with HEs increased HE-mediated mutagenesis frequencies up to sixfold. Following HSV1m5/Trex2 delivery with adeno-associated viral (AAV) vectors, the target site was mutated in latent HSV genomes with no detectable cell toxicity. Importantly, HSV production by latently infected cells after reactivation was decreased after HSV1m5/Trex2 exposure. Exposure to histone deacetylase inhibitors prior to HSV1m5/Trex2 treatment increased mutagenesis frequencies of latent HSV genomes another two- to fivefold, suggesting that chromatin modification may be a useful adjunct to gene-targeting approaches. These results support the continuing development of HEs and other nucleases (ZFNs, TALENs, CRISPRs) for cure of chronic viral infections.Molecular Therapy-Nucleic Acids (2014) 3, e1; doi:10.1038/mtna.2013.75; published online 4 February 2014. PMID:24496438

  5. An SOS-regulated operon involved in damage-inducible mutagenesis in Caulobacter crescentus

    PubMed Central

    Galhardo, Rodrigo S.; Rocha, Raquel P.; Marques, Marilis V.; Menck, Carlos F. M.

    2005-01-01

    DNA polymerases of the Y-family, such as Escherichia coli UmuC and DinB, are specialized enzymes induced by the SOS response, which bypass lesions allowing the continuation of DNA replication. umuDC orthologs are absent in Caulobacter crescentus and other bacteria, raising the question about the existence of SOS mutagenesis in these organisms. Here, we report that the C.crescentus dinB ortholog is not involved in damage-induced mutagenesis. However, an operon composed of two hypothetical genes and dnaE2, encoding a second copy of the catalytic subunit of Pol III, is damage inducible in a recA-dependent manner, and is responsible for most ultraviolet (UV) and mitomycin C-induced mutations in C.crescentus. The results demonstrate that the three genes are required for the error-prone processing of DNA lesions. The two hypothetical genes were named imuA and imuB, after inducible mutagenesis. ImuB is similar to proteins of the Y-family of polymerases, and possibly cooperates with DnaE2 in lesion bypass. The mutations arising as a consequence of the activity of the imuAB dnaE2 operon are rather unusual for UV irradiation, including G:C to C:G transversions. PMID:15886391

  6. Harnessing mutagenic homologous recombination for targeted mutagenesis in vivo by TaGTEAM.

    PubMed

    Finney-Manchester, Shawn P; Maheshri, Narendra

    2013-05-01

    A major hurdle to evolutionary engineering approaches for multigenic phenotypes is the ability to simultaneously modify multiple genes rapidly and selectively. Here, we describe a method for in vivo-targeted mutagenesis in yeast, targeting glycosylases to embedded arrays for mutagenesis (TaGTEAM). By fusing the yeast 3-methyladenine DNA glycosylase MAG1 to a tetR DNA-binding domain, we are able to elevate mutation rates >800 fold in a specific ∼20-kb region of the genome or on a plasmid that contains an array of tetO sites. A wide spectrum of transitions, transversions and single base deletions are observed. We provide evidence that TaGTEAM generated point mutations occur through error-prone homologous recombination (HR) and depend on resectioning and the error-prone polymerase Pol ζ. We show that HR is error-prone in this context because of DNA damage checkpoint activation and base pair lesions and use this knowledge to shift the primary mutagenic outcome of targeted endonuclease breaks from HR-independent rearrangements to HR-dependent point mutations. The ability to switch repair in this way opens up the possibility of using targeted endonucleases in diverse organisms for in vivo-targeted mutagenesis. PMID:23470991

  7. Insertional Mutagenesis by a Hybrid PiggyBac and Sleeping Beauty Transposon in the Rat

    PubMed Central

    Furushima, Kenryo; Jang, Chuan-Wei; Chen, Diane W.; Xiao, Ningna; Overbeek, Paul A.; Behringer, Richard R.

    2012-01-01

    A hybrid piggyBac/Sleeping Beauty transposon-based insertional mutagenesis system that can be mobilized by simple breeding was established in the rat. These transposons were engineered to include gene trap sequences and a tyrosinase (Tyr) pigmentation reporter to rescue the albinism of the genetic background used in the mutagenesis strategy. Single-copy transposon insertions were transposed into the rat genome by co-injection of plasmids carrying the transposon and RNA encoding piggyBac transposase into zygotes. The levels of transgenic Tyr expression were influenced by chromosomal context, leading to transgenic rats with different pigmentation that enabled visual genotyping. Transgenic rats designed to ubiquitously express either piggyBac or Sleeping Beauty transposase were generated by standard zygote injection also on an albino background. Bigenic rats carrying single-copy transposons at known loci and transposase transgenes exhibited coat color mosaicism, indicating somatic transposition. PiggyBac or Sleeping Beauty transposase bigenic rats bred with wild-type albino rats yielded offspring with pigmentation distinct from the initial transposon insertions as a consequence of germline transposition to new loci. The germline transposition frequency for Sleeping Beauty and piggyBac was ∼10% or about one new insertion per litter. Approximately 50% of the insertions occurred in introns. Chimeric transcripts containing endogenous and gene trap sequences were identified in Gabrb1 mutant rats. This mutagenesis system based on simple crosses and visual genotyping can be used to generate a collection of single-gene mutations in the rat. PMID:23023007

  8. CRISPR/Cas9-mediated mutagenesis of the RIN locus that regulates tomato fruit ripening.

    PubMed

    Ito, Yasuhiro; Nishizawa-Yokoi, Ayako; Endo, Masaki; Mikami, Masafumi; Toki, Seiichi

    2015-11-01

    Site-directed mutagenesis using genetic approaches can provide a wealth of resources for crop breeding as well as for biological research. The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated 9 endonuclease (CRISPR/Cas9) system is a novel strategy used to induce mutations in a specific genome region; the system functions in a variety of organisms, including plants. Here, we report application of the CRISPR/Cas9 system to efficient mutagenesis of the tomato genome. In this study, we targeted the tomato RIN gene, which encodes a MADS-box transcription factor regulating fruit ripening. Three regions within the gene were targeted and mutations consisting either of a single base insertion or deletion of more than three bases were found at the Cas9 cleavage sites in T0 regenerated plants. The RIN-protein-defective mutants produced incomplete-ripening fruits in which red color pigmentation was significantly lower than that of wild type, while heterologous mutants expressing the remaining wild-type gene reached full-ripening red color, confirming the important role of RIN in ripening. Several mutations that were generated at three independent target sites were inherited in the T1 progeny, confirming the applicability of this mutagenesis system in tomato. PMID:26408904

  9. Transgenic Chinese hamster V79 cell lines which exhibit variable levels of gpt mutagenesis

    SciTech Connect

    Klein, C.B.; Rossman, T.G. )

    1990-01-01

    The Escherichia coli gpt gene coding for xanthine-guanine phosphoribosyl transferase has been stably transfected into HPRT{sup {minus}} Chinese hamster V79 cells. Several gpt{sup {minus}} cell lines have been established, which retain the sequence(s) even after long-term culture without selection for gpt. While spontaneous mutagenesis to gpt{sup {minus}} occurs rather frequently for most cell lines, it cannot be correlated with either the number of plasmid integration sites or deletion of the plasmid sequence(s). One transgenic cell line (g12), which continuously maintains a low spontaneous mutation frequency was used in comparative mutagenesis studies with wild-type V79 cells (gpt vs. hprt). Alkylating agents such as N-methyl-N{prime}-nitro-N-nitrosoguanidine (MNNG) and {beta}-propiolactone (BPL) are shown to be equally toxic and mutagenic in both g12 and V79 cells. UV and X-rays are also equally toxic to both cell lines. The data presented here suggests that g12 cells may be useful to study mammalian mutagenesis by agents which yield limited response at the hprt locus.

  10. In vitro Inactivation of Latent HSV by Targeted Mutagenesis Using an HSV-specific Homing Endonuclease

    PubMed Central

    Aubert, Martine; Boyle, Nicole M; Stone, Daniel; Stensland, Laurence; Huang, Meei-Li; Magaret, Amalia S; Galetto, Roman; Rawlings, David J; Scharenberg, Andrew M; Jerome, Keith R

    2014-01-01

    Following acute infection, herpes simplex virus (HSV) establishes latency in sensory neurons, from which it can reactivate and cause recurrent disease. Available antiviral therapies do not affect latent viral genomes; therefore, they do not prevent reactivation following therapy cessation. One possible curative approach involves the introduction of DNA double strand breaks in latent HSV genomes by rare-cutting endonucleases, leading to mutagenesis of essential viral genes. We tested this approach in an in vitro HSV latency model using the engineered homing endonuclease (HE) HSV1m5, which recognizes a sequence in the HSV-1 gene UL19, encoding the virion protein VP5. Coexpression of the 3′-exonuclease Trex2 with HEs increased HE-mediated mutagenesis frequencies up to sixfold. Following HSV1m5/Trex2 delivery with adeno-associated viral (AAV) vectors, the target site was mutated in latent HSV genomes with no detectable cell toxicity. Importantly, HSV production by latently infected cells after reactivation was decreased after HSV1m5/Trex2 exposure. Exposure to histone deacetylase inhibitors prior to HSV1m5/Trex2 treatment increased mutagenesis frequencies of latent HSV genomes another two- to fivefold, suggesting that chromatin modification may be a useful adjunct to gene-targeting approaches. These results support the continuing development of HEs and other nucleases (ZFNs, TALENs, CRISPRs) for cure of chronic viral infections. PMID:24496438

  11. Steady-state transposon mutagenesis in inbred maize.

    PubMed

    McCarty, Donald R; Settles, Andrew Mark; Suzuki, Masaharu; Tan, Bao Cai; Latshaw, Susan; Porch, Tim; Robin, Kevin; Baier, John; Avigne, Wayne; Lai, Jinsheng; Messing, Joachim; Koch, Karen E; Hannah, L Curtis

    2005-10-01

    We implement a novel strategy for harnessing the power of high-copy transposons for functional analysis of the maize genome, and report behavioral features of the Mutator system in a uniform inbred background. The unique UniformMu population and database facilitate high-throughput molecular analysis of Mu-tagged mutants and gene knockouts. Key features of the population include: (i) high mutation frequencies (7% independent seed mutations) and moderation of copy number (approximately 57 total Mu elements; 1-2 MuDR copies per plant) were maintained by continuous back-crossing into a phenotypically uniform inbred background; (ii) a bz1-mum9 marker enabled selection of stable lines (loss of MuDR), inhibiting further transpositions in lines selected for molecular analysis; (iii) build-up of mutation load was prevented by screening Mu-active parents to exclude plants carrying pre-existing seed mutations. To create a database of genomic sequences flanking Mu insertions, selected mutant lines were analyzed by sequencing of MuTAIL PCR clone libraries. These sequences were annotated and clustered to facilitate bioinformatic subtraction of ancestral elements and identification of insertions unique to mutant lines. New insertions targeted low-copy, gene-rich sequences, and in silico mapping revealed a random distribution of insertions over the genome. Our results indicate that Mu populations differ markedly in the occurrence of Mu insertion hotspots and the frequency of suppressible mutations. We suggest that controlled MuDR copy number in UniformMu lines is a key determinant of these differences. The public database (http://uniformmu.org; http://endosperm.info) includes pedigree and phenotypic data for over 2000 independent seed mutants selected from a population of 31 548 F2 lines and integrated with analyses of 34 255 MuTAIL sequences. PMID:16167895

  12. Efficient mutagenesis by Cas9 protein-mediated oligonucleotide insertion and large-scale assessment of single-guide RNAs.

    PubMed

    Gagnon, James A; Valen, Eivind; Thyme, Summer B; Huang, Peng; Akhmetova, Laila; Ahkmetova, Laila; Pauli, Andrea; Montague, Tessa G; Zimmerman, Steven; Richter, Constance; Schier, Alexander F

    2014-01-01

    The CRISPR/Cas9 system has been implemented in a variety of model organisms to mediate site-directed mutagenesis. A wide range of mutation rates has been reported, but at a limited number of genomic target sites. To uncover the rules that govern effective Cas9-mediated mutagenesis in zebrafish, we targeted over a hundred genomic loci for mutagenesis using a streamlined and cloning-free method. We generated mutations in 85% of target genes with mutation rates varying across several orders of magnitude, and identified sequence composition rules that influence mutagenesis. We increased rates of mutagenesis by implementing several novel approaches. The activities of poor or unsuccessful single-guide RNAs (sgRNAs) initiating with a 5' adenine were improved by rescuing 5' end homogeneity of the sgRNA. In some cases, direct injection of Cas9 protein/sgRNA complex further increased mutagenic activity. We also observed that low diversity of mutant alleles led to repeated failure to obtain frame-shift mutations. This limitation was overcome by knock-in of a stop codon cassette that ensured coding frame truncation. Our improved methods and detailed protocols make Cas9-mediated mutagenesis an attractive approach for labs of all sizes. PMID:24873830

  13. Mutagenesis by outer space parameters other than cosmic rays

    NASA Astrophysics Data System (ADS)

    Horneck, Gerda; Rabbow, Elke

    We have studied the ability of microorganisms to cope with the complex interplay of the parameters of space in experiments in low Earth orbit and using space simulation facilities on ground. Emphasis was laid on space parameters other than cosmic rays. The studies are directed towards understanding prebiotic chemical evolution and biological evolution processes, and interplanetary transfer of life. Effects of space vacuum: Space experiments have shown that up to 70% of bacterial and fungal spores survived short-term exposure to space vacuum. The chances of survival in space were increased when spores were embedded in chemical protectants such as sugars, or salt crystals, or when they were exposed in multilayer. During the six years lasting LDEF mission up to 80% of bacterial spores survived exposure to space vacuum. A 10-fold increased mutation rate over the spontaneous rate has been observed in spores of Bacillus subtilis after exposure to space vacuum, which is probably based on a unique molecular signature of tandem-double base change at restricted sites in the DNA. In addition, DNA strand breaks have been observed to be induced by vacuum treatment. Effects of extraterrestrial solar UV radiation: Solar UV radiation has been found to be the most deleterious factor of space. The reason for this is the highly energetic UV-C and vacuum UV radiation that is directly absorbed by the DNA and which induces specific photoproducts in the DNA that are highly mutagenic and lethal. The damaging effect of extraterrestrial solar UV radiation was even aggravated, when the spores were simultaneously exposed to both, solar UV radiation and space vacuum. In order to investigate the mutagenic potential of solar UV radiation, DNA of the Escherichia coli plasmid pUC19 was exposed to selected wavebands of UV radiation (from vacuum UV to UV-A) by use of a solar simulator and space simulation facilities. Action spectra revealed that for vacuum UV different kinds of photochemical damage

  14. Identification of the genes affecting the regulation of riboflavin synthesis in the flavinogenic yeast Pichia guilliermondii using insertion mutagenesis

    PubMed Central

    Boretsky, Yuriy R.; Pynyaha, Yuriy V.; Boretsky, Volodymyr Y.; Fedorovych, Dariya V.; Fayura, Lyubov R.; Protchenko, Olha; Philpott, Caroline C.; Sibirny, Andriy A.

    2012-01-01

    Pichia guilliermondii is a representative of a group of so-called flavinogenic yeast species that overproduce riboflavin (vitamin B2) in response to iron limitation. Using insertion mutagenesis, we isolated P. guilliermondii mutants overproducing riboflavin. Analysis of nucleotide sequence of recombination sites revealed that insertion cassettes integrated into the genome disrupting P. guilliermondii genes similar to the VMA1 gene of Ashbya gossypii and Saccharomyces cerevisiae and FES1 and FRA1 genes of S. cerevisiae. The constructed P. guilliermondii Δvma1–17 mutant possessed five- to sevenfold elevated riboflavin production and twofold decreased iron cell content as compared with the parental strain. Pichia guilliermondii Δfra1–45 mutant accumulated 1.8–2.2-fold more iron in the cells and produced five- to sevenfold more riboflavin as compared with the parental strain. Both Δvma1–17 and Δfes1–77 knockout strains could not grow at 37 °C in contrast to the wild-type strain and the Δfra1–45 mutant. Increased riboflavin production by the wild-type strain was observed at 37 °C. Although the Δfes1–77 mutant did not overproduce riboflavin, it showed partial complementation when crossed with previously isolated P. guilliermondii riboflavin-overproducing mutant rib80–22. Complementation analysis revealed that Δvma1–17 and Δfra1–45 mutants are distinct from previously reported riboflavin-producing mutants hit1-1, rib80-22 and rib81-31 of this yeast. PMID:21261808

  15. Identification of the genes affecting the regulation of riboflavin synthesis in the flavinogenic yeast Pichia guilliermondii using insertion mutagenesis.

    PubMed

    Boretsky, Yuriy R; Pynyaha, Yuriy V; Boretsky, Volodymyr Y; Fedorovych, Dariya V; Fayura, Lyubov R; Protchenko, Olha; Philpott, Caroline C; Sibirny, Andriy A

    2011-05-01

    Pichia guilliermondii is a representative of a group of so-called flavinogenic yeast species that overproduce riboflavin (vitamin B(2)) in response to iron limitation. Using insertion mutagenesis, we isolated P. guilliermondii mutants overproducing riboflavin. Analysis of nucleotide sequence of recombination sites revealed that insertion cassettes integrated into the genome disrupting P. guilliermondii genes similar to the VMA1 gene of Ashbya gossypii and Saccharomyces cerevisiae and FES1 and FRA1 genes of S. cerevisiae. The constructed P. guilliermondiiΔvma1-17 mutant possessed five- to sevenfold elevated riboflavin production and twofold decreased iron cell content as compared with the parental strain. Pichia guilliermondiiΔfra1-45 mutant accumulated 1.8-2.2-fold more iron in the cells and produced five- to sevenfold more riboflavin as compared with the parental strain. Both Δvma1-17 and Δfes1-77 knockout strains could not grow at 37 °C in contrast to the wild-type strain and the Δfra1-45 mutant. Increased riboflavin production by the wild-type strain was observed at 37 °C. Although the Δfes1-77 mutant did not overproduce riboflavin, it showed partial complementation when crossed with previously isolated P. guilliermondii riboflavin-overproducing mutant rib80-22. Complementation analysis revealed that Δvma1-17 and Δfra1-45 mutants are distinct from previously reported riboflavin-producing mutants hit1-1, rib80-22 and rib81-31 of this yeast. PMID:21261808

  16. Directed optimization of a newly identified squalene synthase from Mortierella alpine based on sequence truncation and site-directed mutagenesis.

    PubMed

    Huang, Di; Yao, Yongpeng; Zhang, Hang; Mei, Zhu; Wang, Ru; Feng, Lu; Liu, Bin

    2015-10-01

    Terpenoids, a class of isoprenoids usually isolated from plants, are always used as commercial flavor and anticancer drugs. As a key precursor for triterpenes and sterols, biosynthesis of squalene (SQ) can be catalyzed by squalene synthase (SQS) from two farnesyl diphosphate molecules. In this work, the key SQS gene involved in sterols synthesis by Mortierella alpine, an industrial strain often used to produce unsaturated fatty acid such as γ-linolenic acid and arachidonic acid, was identified and characterized. Bioinformatic analysis indicated that MaSQS contained 416 amino acid residues involved in four highly conserved regions. Phylogenetic analysis revealed the closest relationship of MaSQS with Ganoderma lucidum and Aspergillus, which also belonged to the member of the fungus. Subsequently, the recombinant protein was expressed in Escherichia coli BL21(DE3) and detected by SDS-PAGE. To improve the expression and solubility of protein, 17 or 27 amino acids in the C-terminal were deleted. In vitro activity investigation based on gas chromatography-mass spectrometry revealed that both the truncated enzymes could functionally catalyze the reaction from FPP to SQ and the enzymatic activity was optimal at 37 °C, pH 7.2. Moreover, based on the site-directed mutagenesis, the mutant enzyme mMaSQSΔC17 (E186K) displayed a 3.4-fold improvement in catalytic efficiency (k(cat)/K(m)) compared to the control. It was the first report of characterization and modification of SQS from M. alpine, which facilitated the investigation of isoprenoid biosynthesis in the fungus. The engineered mMaSQSΔC17 (E186K) can be a potential candidate of the terpenes and steroids synthesis employed for synthetic biology. PMID:26275528

  17. High Throughput Mutagenesis for Identification of Residues Regulating Human Prostacyclin (hIP) Receptor Expression and Function

    PubMed Central

    Kent, Toby C.; Fawcett, Lindsay; Burchell, Lynn; van Diepen, Michiel T.; Marelli, Anthony; Batalov, Sergey; Miraglia, Loren; Orth, Anthony P.; Renaud, Nicole A.; Charlton, Steven J.; Gosling, Martin; Gaither, L. Alex; Groot-Kormelink, Paul J.

    2014-01-01

    The human prostacyclin receptor (hIP receptor) is a seven-transmembrane G protein-coupled receptor (GPCR) that plays a critical role in vascular smooth muscle relaxation and platelet aggregation. hIP receptor dysfunction has been implicated in numerous cardiovascular abnormalities, including myocardial infarction, hypertension, thrombosis and atherosclerosis. Genomic sequencing has discovered several genetic variations in the PTGIR gene coding for hIP receptor, however, its structure-function relationship has not been sufficiently explored. Here we set out to investigate the applicability of high throughput random mutagenesis to study the structure-function relationship of hIP receptor. While chemical mutagenesis was not suitable to generate a mutagenesis library with sufficient coverage, our data demonstrate error-prone PCR (epPCR) mediated mutagenesis as a valuable method for the unbiased screening of residues regulating hIP receptor function and expression. Here we describe the generation and functional characterization of an epPCR derived mutagenesis library compromising >4000 mutants of the hIP receptor. We introduce next generation sequencing as a useful tool to validate the quality of mutagenesis libraries by providing information about the coverage, mutation rate and mutational bias. We identified 18 mutants of the hIP receptor that were expressed at the cell surface, but demonstrated impaired receptor function. A total of 38 non-synonymous mutations were identified within the coding region of the hIP receptor, mapping to 36 distinct residues, including several mutations previously reported to affect the signaling of the hIP receptor. Thus, our data demonstrates epPCR mediated random mutagenesis as a valuable and practical method to study the structure-function relationship of GPCRs. PMID:24886841

  18. Mutation induced extinction in finite populations: lethal mutagenesis and lethal isolation.

    PubMed

    Wylie, C Scott; Shakhnovich, Eugene I

    2012-01-01

    Reproduction is inherently risky, in part because genomic replication can introduce new mutations that are usually deleterious toward fitness. This risk is especially severe for organisms whose genomes replicate "semi-conservatively," e.g. viruses and bacteria, where no master copy of the genome is preserved. Lethal mutagenesis refers to extinction of populations due to an unbearably high mutation rate (U), and is important both theoretically and clinically, where drugs can extinguish pathogens by increasing their mutation rate. Previous theoretical models of lethal mutagenesis assume infinite population size (N). However, in addition to high U, small N can accelerate extinction by strengthening genetic drift and relaxing selection. Here, we examine how the time until extinction depends jointly on N and U. We first analytically compute the mean time until extinction (τ) in a simplistic model where all mutations are either lethal or neutral. The solution motivates the definition of two distinct regimes: a survival phase and an extinction phase, which differ dramatically in both how τ scales with N and in the coefficient of variation in time until extinction. Next, we perform stochastic population-genetics simulations on a realistic fitness landscape that both (i) features an epistatic distribution of fitness effects that agrees with experimental data on viruses and (ii) is based on the biophysics of protein folding. More specifically, we assume that mutations inflict fitness penalties proportional to the extent that they unfold proteins. We find that decreasing N can cause phase transition-like behavior from survival to extinction, which motivates the concept of "lethal isolation." Furthermore, we find that lethal mutagenesis and lethal isolation interact synergistically, which may have clinical implications for treating infections. Broadly, we conclude that stably folded proteins are only possible in ecological settings that support sufficiently large populations

  19. piggyBac-based insertional mutagenesis in the presence of stably integrated P elements in Drosophila.

    PubMed

    Hacker, Udo; Nystedt, Sverker; Barmchi, Mojgan Padash; Horn, Carsten; Wimmer, Ernst A

    2003-06-24

    P element-mediated mutagenesis has been used to disrupt an estimated 25% of genes essential for Drosophila adult viability. Mutation of all genes in the fly genome, however, poses a problem, because P elements show significant hotspots of integration. In addition, advanced screening scenarios often require the use of P element-based tools like the generation of germ-line mosaics using FLP recombinase-mediated recombination or gene misexpression using the UAS/Gal4 system. These techniques are P element-based and can therefore not be combined with the use of P elements as mutagenic agents. To circumvent these limitations, we have developed an insertional mutagenesis system using non-P element transposons. An enhanced yellow fluorescent protein-marked piggyBac-based mutator element was mobilized by a piggyBac specific transposase source expressed from a Hermes-based jump-starter transposon marked with enhanced cyan fluorescent protein. In a pilot screen, we have generated 798 piggyBac insertions on FRT bearing third chromosomes of which 9% have sustained a putatively piggyBac-related lethal hit. The FRTs present on the target chromosome remained stably integrated during the screen and could subsequently be used to generate germ-line clones associated with maternal and zygotic phenotypes. PCR-based analysis of insertion loci shows that 57% of the insertions are in genes for which no P element insertions have been reported. Our data demonstrate the potential of this technique to facilitate the quest for saturation mutagenesis of the Drosophila genome. The system is Drosophila nonspecific and potentially applicable in a broad spectrum of nonmodel organisms. PMID:12802016

  20. p53 Mutagenesis by benzo[a]pyrene derived radical cations.

    PubMed

    Sen, Sushmita; Bhojnagarwala, Pratik; Francey, Lauren; Lu, Ding; Penning, Trevor M; Field, Jeffrey

    2012-10-15

    Benzo[a]pyrene (B[a]P), a major human carcinogen in combustion products such as cigarette smoke and diesel exhaust, is metabolically activated into DNA-reactive metabolites via three different enzymatic pathways. The pathways are the anti-(+)-benzo[a]pyrene 7,8-diol 9,10-epoxide pathway (P450/epoxide hydrolase catalyzed) (B[a]PDE), the benzo[a]pyrene o-quinone pathway (aldo ketose reductase (AKR) catalyzed) and the B[a]P radical cation pathway (P450 peroxidase catalyzed). We used a yeast p53 mutagenesis system to assess mutagenesis by B[a]P radical cations. Because radical cations are short-lived, they were generated in situ by reacting B[a]P with cumene hydroperoxide (CuOOH) and horse radish peroxidase (HRP) and then monitoring the generation of the more stable downstream products, B[a]P-1,6-dione and B[a]P-3,6-dione. On the basis of B[a]P-1,6 and 3,6-dione formation, approximately 4 μM of radical cation was generated. In the mutagenesis assays, the radical cations produced in situ showed a dose-dependent increase in mutagenicity from 0.25 μM to 10 μM B[a]P with no significant increase seen with further escalation to 50 μM B[a]P. However, mutagenesis was 200-fold less than with the AKR pathway derived B[a]P, 7-8-dione. Mutant p53 plasmids, which yield red colonies, were recovered from the yeast to study the pattern and spectrum of mutations. The mutation pattern observed was G to T (31%) > G to C (29%) > G to A (14%). The frequency of codons mutated by the B[a]P radical cations was essentially random and not enriched at known cancer hotspots. The quinone products of radical cations, B[a]P-1,6-dione and B[a]P-3,6-dione were more mutagenic than the radical cation reactions, but still less mutagenic than AKR derived B[a]P-7,8-dione. We conclude that B[a]P radical cations and their quinone products are weakly mutagenic in this yeast-based system compared to redox cycling PAH o-quinones. PMID:22768918

  1. Isolation of Magnetospirillum magneticum AMB-1 mutants defective in bacterial magnetic particle synthesis by transposon mutagenesis.

    PubMed

    Wahyudi, A T; Takeyama, H; Matsunaga, T

    2001-01-01

    Nonmagnetic mutants of Magnetospirillum magneticum AMB-1 were recovered following mini-Tn5 transposon mutagenesis. Transconjugants with kanamycin resistance were obtained at a frequency of 2.7 x 10(-7) per recipient. Of 3327 transconjugants, 62 were defective for bacterial magnetic particle (BMP) synthesis. The frequency of independent transposition events for nonmagnetic mutants was about 1.4% in transconjugants. Further analysis of DNA sequences flanking transposon by inverted polymerase chain reaction allowed isolation of at least 10 genes or DNA sequences involved in BMP synthesis in M. magneticum AMB-1. PMID:11963843

  2. [Verbena canadensis Britt mutants with altered development of flowers and inflorescences generated by chemical mutagenesis].

    PubMed

    Shirokova, A V

    2008-01-01

    Rose vervain is a little-known ornamental annual plant. The adaptive properties (drought and cold resistance) and long period of flowering make this species promising for growing in flower gardens and containers. Chemical mutagenesis widely used for various plant species was applied to induce character variation in Rose vervain. The properties of development of flowers and inflorescences in lines descending from the M3--M5 mutants generated by the seed exposure to chemical mutagens diethyl sulfate and nitrosomethylurea were considered. PMID:18792639

  3. Transposon mutagenesis identifies genes driving hepatocellular carcinoma in a chronic hepatitis B mouse model

    PubMed Central

    Bard-Chapeau, Emilie A.; Nguyen, Anh-Tuan; Rust, Alistair G.; Sayadi, Ahmed; Lee, Philip; Chua, Belinda Q; New, Lee-Sun; de Jong, Johann; Ward, Jerrold M.; Chin, Christopher KY.; Chew, Valerie; Toh, Han Chong; Abastado, Jean-Pierre; Benoukraf, Touati; Soong, Richie; Bard, Frederic A.; Dupuy, Adam J.; Johnson, Randy L.; Radda, George K.; Chan, Eric CY.; Wessels, Lodewyk FA.; Adams, David J.

    2014-01-01

    The most common risk factor for developing hepatocellular carcinoma (HCC) is chronic infection with hepatitis B virus (HBV). To better understand the evolutionary forces driving HCC we performed a near saturating transposon mutagenesis screen in a mouse HBV model of HCC. This screen identified 21 candidate early stage drivers, and a bewildering number (2860) of candidate later stage drivers, that were enriched for genes mutated, deregulated, or that function in signaling pathways important for human HCC, with a striking 1199 genes linked to cellular metabolic processes. Our study provides a comprehensive overview of the genetic landscape of HCC. PMID:24316982

  4. A facile and efficient transposon mutagenesis method for generation of multi-codon deletions in protein sequences.

    PubMed

    Liu, Shu-Su; Wei, Xuan; Ji, Qun; Xin, Xiu; Jiang, Biao; Liu, Jia

    2016-06-10

    Substitutions, insertions and deletions are all important mutation events in natural and laboratory protein evolution. However, protein engineering using insertions and deletions (indels) is hindered by the lack of a convenient mutagenesis method. Here, we describe a general transposon mutagenesis method that allows for removal of up to five consecutive in-frame codons from a random position of a target protein. This method, referred to as codon deletion mutagenesis (CDM), relies on an engineered Mu transposon that carries asymmetric terminal sequences flanking the MuA transposase recognition sites. CDM requires minimal DNA manipulations, and can generate multi-codon deletions with high efficiency (>90%). As a proof of principle, we constructed five libraries of green fluorescent protein (GFP) containing one to five random codon deletions, respectively. Several variants with multi-codon deletions remained fluorescent, none of which could be easily identified using traditional mutagenesis method. CDM provides a facile and efficient approach to sampling a protein sequence with multi-codon deletions. It will not only facilitate our understanding of the effects of amino acid deletions on protein function but also expedite protein engineering using deletion mutagenesis. PMID:27071724

  5. Mutagenesis and Modeling To Predict Structural and Functional Characteristics of the Staphylococcus aureus MepA Multidrug Efflux Pump

    PubMed Central

    Schindler, Bryan D.; Patel, Diixa; Seo, Susan M.

    2013-01-01

    MepA is a multidrug and toxin extrusion (MATE) family protein and the only MATE protein encoded within the Staphylococcus aureus genome. Structural data for MATE proteins are limited to a single high-resolution example, NorM of Vibrio cholerae. Substitution mutations were created in MepA using gradient plates containing both a substrate and reserpine as an efflux pump inhibitor. Site-directed mutagenesis of plasmid-based mepA was used to reproduce these mutations, as well as unique or low-frequency mutations identified in mepA-overexpressing clinical strains, and to mutagenize conserved acidic residues. The effect of these changes on protein function was quantitated in a norA-disrupted host strain by susceptibility testing with and without inhibitors and by determining the proficiency of ethidium efflux. Up-function substitutions clustered in the carboxy half of MepA, near the cytoplasmic face of the protein. Repeated application of the same gradient plate conditions frequently reproduced identical substitution mutations, suggesting that individual residues are required for interaction with specific substrates. Acidic residues critical to protein function were identified in helices 4 and 5. In silico modeling revealed an outward-facing molecule, with helices 1, 2, 4, 7, 8, and 10 having contact with a central cavity that may represent a substrate translocation pathway. Functionally important residues within this cavity included S81, A161, M291, and A302. These data provide a critical starting point for understanding how MATE multidrug efflux proteins function and will be useful in refining crystallographic data when they are available. PMID:23175649

  6. Gene-scrambling mutagenesis: generation and analysis of insertional mutations in the alginate regulatory region of Pseudomonas aeruginosa.

    PubMed

    Mohr, C D; Deretic, V

    1990-11-01

    A novel method for random mutagenesis of targeted chromosomal regions in Pseudomona aeruginosa was developed. This method can be used with a cloned DNA fragment of indefinite size that contains a putative gene of interest. Cloned DNA is digested to produce small fragments that are then randomly reassembled into long DNA inserts by using cosmid vectors and lambda packaging reaction. This DNA is then transferred into P. aeruginosa and forced into the chromosome via homologous recombination, producing in a single step a random set of insertional mutants along a desired region of the chromosome. Application of this method to extend the analysis of the alginate regulatory region, using a cloned 6.2-kb fragment with the algR gene and the previously uncharacterized flanking regions, produced several insertional mutations. One mutation was obtained in algR, a known transcriptional regulatory of mucoidy in P. aeruginosa. The null mutation of algR was generated in a mucoid derivative of the standard genetic strain PAO responsive to different environmental factors. This mutation was used to demonstrate that the algR gene product was not essential for the regulation of its promoters. Additional insertions were obtained in regions downstream and upstream of algR. A mutation that did not affect mucoidy was generated in a gene located 1 kb upstream of algR. This gene was transcribed in the direction opposite that of algR transcription and encoded a polypeptide of 47 kDa. Partial nucleotide sequence analysis revealed strong homology of its predicted gene product with the human and yeast argininosuccinate lyases. An insertion downstream of algR produced a strain showing reduced induction of mucoidy in response to growth on nitrate as the nitrogen source. PMID:2121708

  7. Gene-scrambling mutagenesis: generation and analysis of insertional mutations in the alginate regulatory region of Pseudomonas aeruginosa.

    PubMed Central

    Mohr, C D; Deretic, V

    1990-01-01

    A novel method for random mutagenesis of targeted chromosomal regions in Pseudomona aeruginosa was developed. This method can be used with a cloned DNA fragment of indefinite size that contains a putative gene of interest. Cloned DNA is digested to produce small fragments that are then randomly reassembled into long DNA inserts by using cosmid vectors and lambda packaging reaction. This DNA is then transferred into P. aeruginosa and forced into the chromosome via homologous recombination, producing in a single step a random set of insertional mutants along a desired region of the chromosome. Application of this method to extend the analysis of the alginate regulatory region, using a cloned 6.2-kb fragment with the algR gene and the previously uncharacterized flanking regions, produced several insertional mutations. One mutation was obtained in algR, a known transcriptional regulatory of mucoidy in P. aeruginosa. The null mutation of algR was generated in a mucoid derivative of the standard genetic strain PAO responsive to different environmental factors. This mutation was used to demonstrate that the algR gene product was not essential for the regulation of its promoters. Additional insertions were obtained in regions downstream and upstream of algR. A mutation that did not affect mucoidy was generated in a gene located 1 kb upstream of algR. This gene was transcribed in the direction opposite that of algR transcription and encoded a polypeptide of 47 kDa. Partial nucleotide sequence analysis revealed strong homology of its predicted gene product with the human and yeast argininosuccinate lyases. An insertion downstream of algR produced a strain showing reduced induction of mucoidy in response to growth on nitrate as the nitrogen source. Images PMID:2121708

  8. Characterization of cyclo-Acetoacetyl-L-Tryptophan Dimethylallyltransferase in Cyclopiazonic Acid Biosynthesis: Substrate Promiscuity and Site Directed Mutagenesis Studies

    PubMed Central

    Liu, Xinyu; Walsh, Christopher T.

    2009-01-01

    The fungal neurotoxin α-cyclopiazonic acid (CPA), a nanomolar inhibitor of Ca2+-ATPase with a unique pentacyclic indole tetramic acid scaffold is assembled by a three enzyme pathway CpaS, CpaD and CpaO in Aspergillus sp. We recently characterized the first pathway-specific enzyme CpaS, a hybrid two module polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) that generates cyclo-acetoacetyl-L-tryptophan (cAATrp). Here we report the characterization of the second pathway-specific enzyme CpaD that regiospecifically dimethylallylates cAATrp to form β-cyclopiazonic acid. By exploring the tryptophan and tetramate moieties of cAATrp, we demonstrate that CpaD discriminates against free Trp but accepts tryptophan-containing thiohydantoins, diketopiperazines and linear peptides as substrates for C4-prenylation and also acts as regiospecific O-dimethylallyltransferase (DMAT) on a tyrosine-derived tetramic acid. Comparative evaluation of CpaDs from A. oryzae RIB40 and A. flavus NRRL3357 indicated the importance of the N-terminal region for its activity. Sequence alignment of CpaD with eleven homologous fungal Trp-DMATs revealed five regions of conservation suggesting the presense of critical motifs that could be diagonostic for discovering additional Trp-DMATs. Subsequent site-directed mutagenesis studies identified five polar/charged residues and five tyrosine residues within these motifs that are critical for CpaD activity. This motif characerization will enable a gene probe-based approach to discover additional biosynthetic Trp-DMATs. PMID:19877600

  9. Proton transfers in a channelrhodopsin-1 studied by Fourier transform infrared (FTIR) difference spectroscopy and site-directed mutagenesis.

    PubMed

    Ogren, John I; Yi, Adrian; Mamaev, Sergey; Li, Hai; Spudich, John L; Rothschild, Kenneth J

    2015-05-15

    Channelrhodopsin-1 from the alga Chlamydomonas augustae (CaChR1) is a low-efficiency light-activated cation channel that exhibits properties useful for optogenetic applications such as a slow light inactivation and a red-shifted visible absorption maximum as compared with the more extensively studied channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2). Previously, both resonance Raman and low-temperature FTIR difference spectroscopy revealed that unlike CrChR2, CaChR1 under our conditions exhibits an almost pure all-trans retinal composition in the unphotolyzed ground state and undergoes an all-trans to 13-cis isomerization during the primary phototransition typical of other microbial rhodopsins such as bacteriorhodopsin (BR). Here, we apply static and rapid-scan FTIR difference spectroscopy along with site-directed mutagenesis to characterize the proton transfer events occurring upon the formation of the long-lived conducting P2 (380) state of CaChR1. Assignment of carboxylic C=O stretch bands indicates that Asp-299 (homolog to Asp-212 in BR) becomes protonated and Asp-169 (homolog to Asp-85 in BR) undergoes a net change in hydrogen bonding relative to the unphotolyzed ground state of CaChR1. These data along with earlier FTIR measurements on the CaChR1 → P1 transition are consistent with a two-step proton relay mechanism that transfers a proton from Glu-169 to Asp-299 during the primary phototransition and from the Schiff base to Glu-169 during P2 (380) formation. The unusual charge neutrality of both Schiff base counterions in the P2 (380) conducting state suggests that these residues may function as part of a cation selective filter in the open channel state of CaChR1 as well as other low-efficiency ChRs. PMID:25802337

  10. Proton Transfers in a Channelrhodopsin-1 Studied by Fourier Transform Infrared (FTIR) Difference Spectroscopy and Site-directed Mutagenesis*

    PubMed Central

    Ogren, John I.; Yi, Adrian; Mamaev, Sergey; Li, Hai; Spudich, John L.; Rothschild, Kenneth J.

    2015-01-01

    Channelrhodopsin-1 from the alga Chlamydomonas augustae (CaChR1) is a low-efficiency light-activated cation channel that exhibits properties useful for optogenetic applications such as a slow light inactivation and a red-shifted visible absorption maximum as compared with the more extensively studied channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2). Previously, both resonance Raman and low-temperature FTIR difference spectroscopy revealed that unlike CrChR2, CaChR1 under our conditions exhibits an almost pure all-trans retinal composition in the unphotolyzed ground state and undergoes an all-trans to 13-cis isomerization during the primary phototransition typical of other microbial rhodopsins such as bacteriorhodopsin (BR). Here, we apply static and rapid-scan FTIR difference spectroscopy along with site-directed mutagenesis to characterize the proton transfer events occurring upon the formation of the long-lived conducting P2380 state of CaChR1. Assignment of carboxylic C=O stretch bands indicates that Asp-299 (homolog to Asp-212 in BR) becomes protonated and Asp-169 (homolog to Asp-85 in BR) undergoes a net change in hydrogen bonding relative to the unphotolyzed ground state of CaChR1. These data along with earlier FTIR measurements on the CaChR1 → P1 transition are consistent with a two-step proton relay mechanism that transfers a proton from Glu-169 to Asp-299 during the primary phototransition and from the Schiff base to Glu-169 during P2380 formation. The unusual charge neutrality of both Schiff base counterions in the P2380 conducting state suggests that these residues may function as part of a cation selective filter in the open channel state of CaChR1 as well as other low-efficiency ChRs. PMID:25802337

  11. Site-directed mutagenesis of tobacco anionic peroxidase: Effect of additional aromatic amino acids on stability and activity.

    PubMed

    Poloznikov, A A; Zakharova, G S; Chubar, T A; Hushpulian, D M; Tishkov, V I; Gazaryan, I G

    2015-08-01

    Tobacco anionic peroxidase (TOP) is known to effectively catalyze luminol oxidation without enhancers, in contrast to horseradish peroxidase (HRP). To pursue structure-activity relationship studies for TOP, two amino acids have been chosen for mutation, namely Thr151, close to the heme plane, and Phe140 at the entrance to the active site pocket. Three mutant forms TOP F140Y, T151W and F140Y/T151W have been expressed in Escherichia coli, and reactivated to yield active enzymes. Single-point mutations introducing additional aromatic amino acid residues at the surface of TOP exhibit a significant effect on the enzyme catalytic activity and stability as judged by the results of steady-state and transient kinetics studies. TOP T151W is up to 4-fold more active towards a number of aromatic substrates including luminol, whereas TOP F140Y is 2-fold more stable against thermal inactivation and 8-fold more stable in the reaction course. These steady-state observations have been rationalized with the help of transient kinetic studies on the enzyme reaction with hydrogen peroxide in a single turnover regime. The stopped-flow data reveal (a) an increased stability of F140Y Compound I towards hydrogen peroxide, and thus, a higher operational stability as compared to the wild-type enzyme, and (b) a lesser leakage of oxidative equivalents from TOP T151W Compound I resulting in the increased catalytic activity. The results obtained show that TOP unique properties can be further improved for practical applications by site-directed mutagenesis. PMID:25957835

  12. Identification and Characterization of Non-Cellulose-Producing Mutants of Gluconacetobacter hansenii Generated by Tn5 Transposon Mutagenesis

    PubMed Central

    Deng, Ying; Nagachar, Nivedita; Xiao, Chaowen; Tien, Ming

    2013-01-01

    The acs operon of Gluconacetobacter is thought to encode AcsA, AcsB, AcsC, and AcsD proteins that constitute the cellulose synthase complex, required for the synthesis and secretion of crystalline cellulose microfibrils. A few other genes have been shown to be involved in this process, but their precise role is unclear. We report here the use of Tn5 transposon insertion mutagenesis to identify and characterize six non-cellulose-producing (Cel−) mutants of Gluconacetobacter hansenii ATCC 23769. The genes disrupted were acsA, acsC, ccpAx (encoding cellulose-complementing protein [the subscript “Ax” indicates genes from organisms formerly classified as Acetobacter xylinum]), dgc1 (encoding guanylate dicyclase), and crp-fnr (encoding a cyclic AMP receptor protein/fumarate nitrate reductase transcriptional regulator). Protein blot analysis revealed that (i) AcsB and AcsC were absent in the acsA mutant, (ii) the levels of AcsB and AcsC were significantly reduced in the ccpAx mutant, and (iii) the level of AcsD was not affected in any of the Cel− mutants. Promoter analysis showed that the acs operon does not include acsD, unlike the organization of the acs operon of several strains of closely related Gluconacetobacter xylinus. Complementation experiments confirmed that the gene disrupted in each Cel− mutant was responsible for the phenotype. Quantitative real-time PCR and protein blotting results suggest that the transcription of bglAx (encoding β-glucosidase and located immediately downstream from acsD) was strongly dependent on Crp/Fnr. A bglAx knockout mutant, generated via homologous recombination, produced only ∼16% of the wild-type cellulose level. Since the crp-fnr mutant did not produce any cellulose, Crp/Fnr may regulate the expression of other gene(s) involved in cellulose biosynthesis. PMID:24013627

  13. Iterative Saturation Mutagenesis of −6 Subsite Residues in Cyclodextrin Glycosyltransferase from Paenibacillus macerans To Improve Maltodextrin Specificity for 2-O-d-Glucopyranosyl-l-Ascorbic Acid Synthesis

    PubMed Central

    Han, Ruizhi; Shin, Hyun-dong; Chen, Rachel R.; Li, Jianghua; Chen, Jian

    2013-01-01

    2-O-d-Glucopyranosyl-l-ascorbic acid (AA-2G), a stable l-ascorbic acid derivative, is usually synthesized by cyclodextrin glycosyltransferase (CGTase), which contains nine substrate-binding subsites (from +2 to −7). In this study, iterative saturation mutagenesis (ISM) was performed on the −6 subsite residues (Y167, G179, G180, and N193) in the CGTase from Paenibacillus macerans to improve its specificity for maltodextrin, which is a cheap and easily soluble glycosyl donor for AA-2G synthesis. Site saturation mutagenesis of four sites—Y167, G179, G180, and N193—was first performed and revealed that four mutants—Y167S, G179R, N193R, and G180R—produced AA-2G yields higher than those of other mutant and wild-type CGTases. ISM was then conducted with the best positive mutant as a template. Under optimal conditions, mutant Y167S/G179K/N193R/G180R produced the highest AA-2G titer of 2.12 g/liter, which was 84% higher than that (1.15 g/liter) produced by the wild-type CGTase. Kinetics analysis of AA-2G synthesis using mutant CGTases confirmed the enhanced maltodextrin specificity and showed that compared to the wild-type CGTase, the mutants had no cyclization activity but high hydrolysis and disproportionation activities. A possible mechanism for the enhanced substrate specificity was also analyzed through structure modeling of the mutant and wild-type CGTases. These results indicated that the −6 subsite played crucial roles in the substrate binding and catalytic reactions of CGTase and that the obtained CGTase mutants, especially Y167S/G179K/N193R/G180R, are promising starting points for further development through protein engineering. PMID:24077706

  14. TALEN mediated targeted mutagenesis of the caffeic acid O-methyltransferase in highly polyploid sugarcane improves cell wall composition for production of bioethanol.

    PubMed

    Jung, Je Hyeong; Altpeter, Fredy

    2016-09-01

    Sugarcane (Saccharum spp. hybrids) is a prime crop for commercial biofuel production. Advanced conversion technology utilizes both, sucrose accumulating in sugarcane stems as well as cell wall bound sugars for commercial ethanol production. Reduction of lignin content significantly improves the conversion of lignocellulosic biomass into ethanol. Conventional mutagenesis is not expected to confer reduction in lignin content in sugarcane due to its high polyploidy (x = 10-13) and functional redundancy among homo(eo)logs. Here we deploy transcription activator-like effector nuclease (TALEN) to induce mutations in a highly conserved region of the caffeic acid O-methyltransferase (COMT) of sugarcane. Capillary electrophoresis (CE) was validated by pyrosequencing as reliable and inexpensive high throughput method for identification and quantitative characterization of TALEN mediated mutations. Targeted COMT mutations were identified by CE in up to 74 % of the lines. In different events 8-99 % of the wild type COMT were converted to mutant COMT as revealed by pyrosequencing. Mutation frequencies among mutant lines were positively correlated to lignin reduction. Events with a mutation frequency of 99 % displayed a 29-32 % reduction of the lignin content compared to non-transgenic controls along with significantly reduced S subunit content and elevated hemicellulose content. CE analysis displayed similar peak patterns between primary COMT mutants and their vegetative progenies suggesting that TALEN mediated mutations were faithfully transmitted to vegetative progenies. This is the first report on genome editing in sugarcane. The findings demonstrate that targeted mutagenesis can improve cell wall characteristics for production of lignocellulosic ethanol in crops with highly complex genomes. PMID:27306903

  15. PAX5 is a tumor suppressor in mouse mutagenesis models of acute lymphoblastic leukemia

    PubMed Central

    Dang, Jinjun; Wei, Lei; de Ridder, Jeroen; Su, Xiaoping; Rust, Alistair G.; Roberts, Kathryn G.; Payne-Turner, Debbie; Cheng, Jinjun; Ma, Jing; Qu, Chunxu; Wu, Gang; Song, Guangchun; Huether, Robert G.; Schulman, Brenda; Janke, Laura; Zhang, Jinghui; Downing, James R.; van der Weyden, Louise; Adams, David J.

    2015-01-01

    Alterations of genes encoding transcriptional regulators of lymphoid development are a hallmark of B-progenitor acute lymphoblastic leukemia (B-ALL) and most commonly involve PAX5, encoding the DNA-binding transcription factor paired-box 5. The majority of PAX5 alterations in ALL are heterozygous, and key PAX5 target genes are expressed in leukemic cells, suggesting that PAX5 may be a haploinsufficient tumor suppressor. To examine the role of PAX5 alterations in leukemogenesis, we performed mutagenesis screens of mice heterozygous for a loss-of-function Pax5 allele. Both chemical and retroviral mutagenesis resulted in a significantly increased penetrance and reduced latency of leukemia, with a shift to B-lymphoid lineage. Genomic profiling identified a high frequency of secondary genomic mutations, deletions, and retroviral insertions targeting B-lymphoid development, including Pax5, and additional genes and pathways mutated in ALL, including tumor suppressors, Ras, and Janus kinase-signal transducer and activator of transcription signaling. These results show that in contrast to simple Pax5 haploinsufficiency, multiple sequential alterations targeting lymphoid development are central to leukemogenesis and contribute to the arrest in lymphoid maturation characteristic of ALL. This cross-species analysis also validates the importance of concomitant alterations of multiple cellular growth, signaling, and tumor suppression pathways in the pathogenesis of B-ALL. PMID:25855603

  16. An inducible tool for random mutagenesis in Aspergillus niger based on the transposon Vader.

    PubMed

    Paun, Linda; Nitsche, Benjamin; Homan, Tim; Ram, Arthur F; Kempken, Frank

    2016-07-01

    The ascomycete Aspergillus niger is widely used in the biotechnology, for instance in producing most of the world's citric acid. It is also known as a major food and feed contaminant. While generation of gene knockouts for functional genomics has become feasible in ku70 mutants, analyzing gene functions or metabolic pathways remains a laborious task. An unbiased transposon-based mutagenesis approach may aid this process of analyzing gene functions by providing mutant libraries in a short time. The Vader transposon is a non-autonomous DNA-transposon, which is activated by the homologous tan1-transposase. However, in the most commonly used lab strain of A. niger (N400 strain and derivatives), we found that the transposase, encoded by the tan1 gene, is mutated and inactive. To establish a Vader transposon-based mutagenesis system in the N400 background, we expressed the functional transposase of A. niger strain CBS 513.88 under the control of an inducible promoter based on the Tet-on system, which is activated in the presence of the antibiotic doxycycline (DOX). Increasing amounts of doxycycline lead to higher Vader excision frequencies, whereas little to none activity of Vader was observed without addition of doxycycline. Hence, this system appears to be suitable for producing stable mutants in the A. niger N400 background. PMID:27003267

  17. Mutagenesis for improvement of activity and thermostability of amylomaltase from Corynebacterium glutamicum.

    PubMed

    Nimpiboon, Pitchanan; Kaulpiboon, Jarunee; Krusong, Kuakarun; Nakamura, Shigeyoshi; Kidokoro, Shun-Ichi; Pongsawasdi, Piamsook

    2016-05-01

    This work aims to improve thermostability of amylomaltase from a mesophilic Corynebacterium glutamicum (CgAM) by random and site-directed mutagenesis. From error prone PCR, a mutated CgAM with higher thermostability at 50°C compared to the wild-type was selected and sequenced. The result showed that the mutant contains a single mutation of A406V. Site-directed mutagenesis was then performed to construct A406V and A406L. Both mutated CgAMs showed higher intermolecular transglucosylation activity with an upward shift in the optimum temperature and a slight increase in the optimum pH for disproportionation and cyclization reactions. Thermostability of both mutated CgAMs at 35-40°C was significantly increased with a higher peak temperature from DSC spectra when compared to the wild-type. A406V had a greater effect on activity and thermostability than A406L. The catalytic efficiency values kcat/Km of A406V- and A406L-CgAMs were 2.9 and 1.4 times higher than that of the wild-type, respectively, mainly due to a significant increase in kcat. LR-CD product analysis demonstrated that A406V gave higher product yield, especially at longer incubation time and higher temperature, in comparison to the wild-type enzyme. PMID:26875536

  18. ProxiMAX randomization: a new technology for non-degenerate saturation mutagenesis of contiguous codons.

    PubMed

    Ashraf, Mohammed; Frigotto, Laura; Smith, Matthew E; Patel, Seema; Hughes, Marcus D; Poole, Andrew J; Hebaishi, Husam R M; Ullman, Christopher G; Hine, Anna V

    2013-10-01

    Back in 2003, we published 'MAX' randomization, a process of non-degenerate saturation mutagenesis using exactly 20 codons (one for each amino acid) or else any required subset of those 20 codons. 'MAX' randomization saturates codons located in isolated positions within a protein, as might be required in enzyme engineering, or else on one face of an α-helix, as in zinc-finger engineering. Since that time, we have been asked for an equivalent process that can saturate multiple contiguous codons in a non-degenerate manner. We have now developed 'ProxiMAX' randomization, which does just that: generating DNA cassettes for saturation mutagenesis without degeneracy or bias. Offering an alternative to trinucleotide phosphoramidite chemistry, ProxiMAX randomization uses nothing more sophisticated than unmodified oligonucleotides and standard molecular biology reagents. Thus it requires no specialized chemistry, reagents or equipment, and simply relies on a process of saturation cycling comprising ligation, amplification and digestion for each cycle. The process can encode both unbiased representation of selected amino acids or else encode them in predefined ratios. Each saturated position can be defined independently of the others. We demonstrate accurate saturation of up to 11 contiguous codons. As such, ProxiMAX randomization is particularly relevant to antibody engineering. PMID:24059507

  19. A mutagenesis and screening strategy to generate optimally thermostabilized membrane proteins for structural studies.

    PubMed

    Magnani, Francesca; Serrano-Vega, Maria J; Shibata, Yoko; Abdul-Hussein, Saba; Lebon, Guillaume; Miller-Gallacher, Jennifer; Singhal, Ankita; Strege, Annette; Thomas, Jennifer A; Tate, Christopher G

    2016-08-01

    The thermostability of an integral membrane protein (MP) in detergent solution is a key parameter that dictates the likelihood of obtaining well-diffracting crystals that are suitable for structure determination. However, many mammalian MPs are too unstable for crystallization. We developed a thermostabilization strategy based on systematic mutagenesis coupled to a radioligand-binding thermostability assay that can be applied to receptors, ion channels and transporters. It takes ∼6-12 months to thermostabilize a G-protein-coupled receptor (GPCR) containing 300 amino acid (aa) residues. The resulting thermostabilized MPs are more easily crystallized and result in high-quality structures. This methodology has facilitated structure-based drug design applied to GPCRs because it is possible to determine multiple structures of the thermostabilized receptors bound to low-affinity ligands. Protocols and advice are given on how to develop thermostability assays for MPs and how to combine mutations to make an optimally stable mutant suitable for structural studies. The steps in the procedure include the generation of ∼300 site-directed mutants by Ala/Leu scanning mutagenesis, the expression of each mutant in mammalian cells by transient transfection and the identification of thermostable mutants using a thermostability assay that is based on binding of an (125)I-labeled radioligand to the unpurified, detergent-solubilized MP. Individual thermostabilizing point mutations are then combined to make an optimally stable MP that is suitable for structural biology and other biophysical studies. PMID:27466713

  20. [Epigenetic mutagenesis as program of age-related protein dysfunction and aging].

    PubMed

    Romanov, G A; Sukhoverov, V S; Vanyushin, B F

    2015-01-01

    DNA methylation plays an important polyfunctional role in ontogenesis of human and mammals. A steep rise in probability of mutational substitution of CpG dinucleotide on TpG dinucleotide in the genome is one of the consequences of DNA methylation. All spectrum (17) of possible DNA and protein mutations caused by CpG-dinucleotide methylation in DNA were characterized, and the three most dangerous mutations (able to result in protein inactivation) were isolated. The computer program that allows one to predict all most probable mutations in the analyzed gene and encoded protein was created. On the example of genes from humans and various mammals, it was demonstrated that the amount of potentially dangerous sites of epigenetic mutagenesis in exons was drastically decreased as a result of genome evolution. But, at the same time, unforced preservation of such sites and their persistence were established, indicating the occurrence of age-related protein dysfunction built into the genome epigenetic program, resulting in apoptosis and aging; this program is based on the set and position of methylated codons in exonic gene regions. It is assumed that the program of epigenetic mutagenesis limits the lifetime of an individual, accelerating the deliverance of the population from long-lived individuals that completed the reproductive period. PMID:26021123

  1. Inducibility of a gene product required for UV and chemical mutagenesis in Escherichia coli.

    PubMed Central

    Bagg, A; Kenyon, C J; Walker, G C

    1981-01-01

    The product of the umuC gene is required for UV and chemical mutagenesis in Escherichia coli. By the use of the Mud(Ap, lac) bacteriophage, we have obtained an operon fusion of the lac structural genes to the promoter/regulatory region of the umuC gene. The strain containing the umuC::Mud(Ap, lac) fusion was identified on the basis of its UV nonmutability. Strains containing this putative null allele of umuC were (i) nonmutable by UV and other agents, (ii) slightly UV sensitive, and (iii) deficient in their ability to carry out Weigle reactivation of UV-irradiation bacteriophage lambda. The UV nonmutability of the strain could be suppressed by a derivative of the mutagenesis-enhancing plasmid pKM101. beta-Galactosidase synthesis in umuC::Mud(Ap, lac) fusion strains was inducible by UV and other DNA-damaging agents. Genetic analysis of the regulation of beta-galactosidase in umuC::Mud(Ap, lac) strains suggests that the lexA protein is the direct repressor of the umuC gene and that a function of the recA protein, probably its protease activity, is required for the removal of the lexA repressor at the time of umuC induction. PMID:7029544

  2. Improving the activity of the subtilisin nattokinase by site-directed mutagenesis and molecular dynamics simulation.

    PubMed

    Weng, Meizhi; Deng, Xiongwei; Bao, Wei; Zhu, Li; Wu, Jieyuan; Cai, Yongjun; Jia, Yan; Zheng, Zhongliang; Zou, Guolin

    2015-09-25

    Nattokinase (NK), a bacterial serine protease from Bacillus subtilis var. natto, is a potential cardiovascular drug exhibiting strong fibrinolytic activity. To broaden its commercial and medical applications, we constructed a single-mutant (I31L) and two double-mutants (M222A/I31L and T220S/I31L) by site-directed mutagenesis. Active enzymes were expressed in Escherichia coli with periplasmic secretion and were purified to homogeneity. The kinetic parameters of enzymes were examined by spectroscopy assay and isothermal titration calorimetry (ITC), and their fibrinolytic activities were determined by fibrin plate method. The substitution of Leu(31) for Ile(31) resulted in about 2-fold enhancement of catalytic efficiency (Kcat/KM) compared with wild-type NK. The specific activities of both double-mutants (M222A/I31L and T220S/I31L) were significantly increased when compared with the single-mutants (M222A and T220S) and the oxidative stability of M222A/I31L mutant was enhanced with respect to wild-type NK. This study demonstrates the feasibility of improving activity of NK by site-directed mutagenesis and shows successful protein engineering cases to improve the activity of NK as a potent therapeutic agent. PMID:26291268

  3. Rational and random mutagenesis of firefly luciferase to identify an efficient emitter of red bioluminescence

    NASA Astrophysics Data System (ADS)

    Branchini, Bruce R.; Southworth, Tara L.; Khattak, Neelum F.; Murtiashaw, Martha H.; Fleet, Sarah E.

    2004-06-01

    Firefly luciferase, which emits yellow-green (557 nm) light, and the corresponding cDNA have been used successfully as a bioluminescence reporter of gene expression. One particularly exciting application is in the area of in vivo bioluminescence imaging. Our interest is in developing improved reagents by identifying Photinus pyralis luciferase mutants that efficiently emit red bioluminescence. In this way, the proven advantages of the P. pyralis protein can be combined with the potential advantages of a red-shifted emitter. Using site-directed mutagenesis techniques, we have identified many mutants emitting red bioluminescence. Unfortunately, these enzymes generally have significantly decreased bioluminescence activity. Interestingly, we discovered a mutation, Ile351Ala, that produced a moderate 16 nm red-shift, while maintaining excellent bioluminescence activity. We then undertook a random mutagenesis approach to identify luciferase mutants that emit further red-shifted bioluminescence with minimal loss of activity. Libraries of mutants were created using an error-prone PCR method and the Ile351Ala luciferase mutant as the template DNA. The libraries were screened by in vivo bacterial assays and the promising mutants were purified to enable accurate determination of bioluminescence emission spectra and total bioluminescence activity. We will report the characterization results, including the identification of the randomly altered amino acids, of several mutants that catalyze bioluminescence with emission maxima of approximately 600 nm.

  4. Ionizing radiation-induced mutagenesis: radiation studies in Neurospora predictive for results in mammalian cells

    NASA Technical Reports Server (NTRS)

    Evans, H. H.; DeMarini, D. M.

    1999-01-01

    Ionizing radiation was the first mutagen discovered and was used to develop the first mutagenicity assay. In the ensuing 70+ years, ionizing radiation became a fundamental tool in understanding mutagenesis and is still a subject of intensive research. Frederick de Serres et al. developed and used the Neurospora crassa ad-3 system initially to explore the mutagenic effects of ionizing radiation. Using this system, de Serres et al. demonstrated the dependence of the frequency and spectra of mutations induced by ionizing radiation on the dose, dose rate, radiation quality, repair capabilities of the cells, and the target gene employed. This work in Neurospora predicted the subsequent observations of the mutagenic effects of ionizing radiation in mammalian cells. Modeled originally on the mouse specific-locus system developed by William L. Russell, the N. crassa ad-3 system developed by de Serres has itself served as a model for interpreting the results in subsequent systems in mammalian cells. This review describes the primary findings on the nature of ionizing radiation-induced mutagenesis in the N. crassa ad-3 system and the parallel observations made years later in mammalian cells.

  5. In vitro mutagenesis and identification of mutants via ISSR in lily (Lilium longiflorum).

    PubMed

    Xi, Mengli; Sun, Lina; Qiu, Shuai; Liu, Juanjuan; Xu, Jin; Shi, Jisen

    2012-06-01

    An efficient in vitro mutagenesis protocol for Lilium longiflorum Thunb. cv. White fox has been established. The effect of 6-BA and NAA on adventitious bud formation from the bulblet-scale thin cell layers was tested. Results showed that the optimal medium for adventitious bud induction is MS basal medium supplemented with 2.0 mg/l 6-BA and 0.1 mg/l NAA. The differentiation frequency and the average number of adventitious buds reached 95.55% and 3.00, respectively. Various doses (0.0, 0.5, 1.0, 1.5, 2.0, and 2.5 Gy) of gamma rays were applied to investigate the effect of radiation on adventitious bud formation from bulblet-scale thin cell layers. The forming capacity of the adventitious buds significantly decreased with the increase of radiation dose. The results suggested that the optimal irradiation dose is 1.0 Gy. Dose of 1.0 Gy treatment resulted in 55.33% survival of irradiated bulblet-scale thin cell layers and 39.27% mutagenesis rate. The genetic variations among the morphological mutants were evaluated by DNA fingerprinting using ISSR molecular marker. The genetic variation frequency reached 36.06% using seven ISSR primers. Out of the 50 mutant lines transferred to the greenhouse, 9 were observed to have significantly different morphological characters than those of the controls. PMID:22228557

  6. Caffeine enhanced measurement of mutagenesis by low levels of [gamma]-irradiation in human lymphocytes

    SciTech Connect

    Puck, T.P.; Johnson, R.; Waldren, C.A. ); Morse, H. )

    1993-09-01

    The well-known action of caffeine in synergizing mutagenesis (including chromosome aberrations) of agents like ionizing radiation by inhibition of cellular repair processes has been incorporated into a rapid procedure for detection of mutagenicity with high sensitivity. Effects of 5-10 rads of [gamma]-irradiation, which approximate the human lifetime dose accumulation from background radiation, can be detected in a two-day procedure using an immortalized human WBC culture. Chromosomally visible lesions are scored on cells incubated for 2 h after irradiation in the presence and absence of 1.0 mg/ml of caffeine. An eightfold amplification of scorable lesions is achieved over the action of radiation alone. This approach provides a closer approximation to absolute mutagenicity unmitigated by repair processes, which can vary in different situations. It is proposed that mutagenesis testing of this kind, using caffiene or other repair-inhibitory agents, be employed to identify mutagens in their effective concentrations to which human populations may be exposed; to detect agents such as caffeine that may synergize mutagenic actions and pose epidemiologic threats; and to discover effective anti-mutagens. Information derived from the use of such procedures may help prevent cancer and newly acquired genetic disease.

  7. Herpesvirus mutagenesis facilitated by infectious bacterial artificial chromosomes (iBACs).

    PubMed

    Robinson, Karl E; Mahony, Timothy J

    2015-01-01

    A critical factor in the study of herpesviruses, their genes and gene functions is the capacity to derive mutants that harbor deletions, truncations, or insertions within the genetic elements of interest. Once constructed the impact of the introduced mutation on the phenotypic properties of the rescued virus can be determined in either in vitro or in vivo systems. However, the construction of such mutants by traditional virological mutagenesis techniques can be a difficult and laborious undertaking. The maintenance of a viral genome as an infectious bacterial artificial chromosome (iBAC), however, endows the capacity to manipulate the viral genome for mutagenesis studies with relative ease. Here, the construction and characterization of two gene deletion mutants of an alphaherpesvirus maintained as iBAC in combination with an inducible homologous recombination system in Escherichia coli is detailed. The methodology is generally applicable to any iBAC and is demonstrated to be a highly efficient and informative approach for mutant virus construction. PMID:25239746

  8. Lentiviral vector-based insertional mutagenesis identifies genes associated with liver cancer

    PubMed Central

    Ranzani, Marco; Cesana, Daniela; Bartholomae, Cynthia C.; Sanvito, Francesca; Pala, Mauro; Benedicenti, Fabrizio; Gallina, Pierangela; Sergi, Lucia Sergi; Merella, Stefania; Bulfone, Alessandro; Doglioni, Claudio; von Kalle, Christof; Kim, Yoon Jun; Schmidt, Manfred; Tonon, Giovanni; Naldini, Luigi; Montini, Eugenio

    2013-01-01

    Transposons and γ-retroviruses have been efficiently used as insertional mutagens in different tissues to identify molecular culprits of cancer. However, these systems are characterized by recurring integrations that accumulate in tumor cells, hampering the identification of early cancer-driving events amongst bystander and progression-related events. We developed an insertional mutagenesis platform based on lentiviral vectors (LVV) by which we could efficiently induce hepatocellular carcinoma (HCC) in 3 different mouse models. By virtue of LVV’s replication-deficient nature and broad genome-wide integration pattern, LVV-based insertional mutagenesis allowed identification of 4 new liver cancer genes from a limited number of integrations. We validated the oncogenic potential of all the identified genes in vivo, with different levels of penetrance. Our newly identified cancer genes are likely to play a role in human disease, since they are upregulated and/or amplified/deleted in human HCCs and can predict clinical outcome of patients. PMID:23314173

  9. Transposon mutagenesis identifies genetic drivers of Braf(V600E) melanoma.

    PubMed

    Mann, Michael B; Black, Michael A; Jones, Devin J; Ward, Jerrold M; Yew, Christopher Chin Kuan; Newberg, Justin Y; Dupuy, Adam J; Rust, Alistair G; Bosenberg, Marcus W; McMahon, Martin; Print, Cristin G; Copeland, Neal G; Jenkins, Nancy A

    2015-05-01

    Although nearly half of human melanomas harbor oncogenic BRAF(V600E) mutations, the genetic events that cooperate with these mutations to drive melanogenesis are still largely unknown. Here we show that Sleeping Beauty (SB) transposon-mediated mutagenesis drives melanoma progression in Braf(V600E) mutant mice and identify 1,232 recurrently mutated candidate cancer genes (CCGs) from 70 SB-driven melanomas. CCGs are enriched in Wnt, PI3K, MAPK and netrin signaling pathway components and are more highly connected to one another than predicted by chance, indicating that SB targets cooperative genetic networks in melanoma. Human orthologs of >500 CCGs are enriched for mutations in human melanoma or showed statistically significant clinical associations between RNA abundance and survival of patients with metastatic melanoma. We also functionally validate CEP350 as a new tumor-suppressor gene in human melanoma. SB mutagenesis has thus helped to catalog the cooperative molecular mechanisms driving BRAF(V600E) melanoma and discover new genes with potential clinical importance in human melanoma. PMID:25848750

  10. Involvement of a joker mutation in a polymerase-independent lethal mutagenesis escape mechanism.

    PubMed

    Agudo, Rubén; de la Higuera, Ignacio; Arias, Armando; Grande-Pérez, Ana; Domingo, Esteban

    2016-07-01

    We previously characterized a foot-and-mouth disease virus (FMDV) with three amino acid replacements in its polymerase (3D) that conferred resistance to the mutagenic nucleoside analogue ribavirin. Here we show that passage of this mutant in the presence of high ribavirin concentrations resulted in selection of viruses with the additional replacement I248T in 2C. This 2C substitution alone (even in the absence of replacements in 3D) increased FMDV fitness mainly in the presence of ribavirin, prevented an incorporation bias in favor of A and U associated with ribavirin mutagenesis, and conferred the ATPase activity of 2C decreased sensitivity to ribavirin-triphosphate. Since in previous studies we described that 2C with I248T was selected under different selective pressures, this replacement qualifies as a joker substitution in FMDV evolution. The results have identified a role of 2C in nucleotide incorporation, and have unveiled a new polymerase-independent mechanism of virus escape to lethal mutagenesis. PMID:27136067

  11. A mutagenesis-derived broad-spectrum disease resistance locus in wheat.

    PubMed

    Campbell, Jackie; Zhang, Hongtao; Giroux, Michael J; Feiz, Leila; Jin, Yue; Wang, Meinan; Chen, Xianming; Huang, Li

    2012-07-01

    Wheat leaf rust, stem rust, stripe rust, and powdery mildew caused by the fungal pathogens Puccinia triticina, P. graminis f. sp. tritici, P. striiformis f. sp. tritici, and Blumeria graminis f. sp. tritici, respectively, are destructive diseases of wheat worldwide. Breeding durable disease resistance cultivars rely largely on continually introgressing new resistance genes, especially the genes with different defense mechanisms, into adapted varieties. Here, we describe a new resistance gene obtained by mutagenesis. The mutant, MNR220 (mutagenesis-derived new resistance), enhances resistance to three rusts and powdery mildew, with the characteristics of delayed disease development at the seedling stage and completed resistance at the adult plant stage. Genetic analysis demonstrated that the resistance in MNR220 is conferred by a single semidominant gene mapped on the short arm of chromosome 2B. Gene expression profiling of several pathogenesis-related genes indicated that MNR220 has an elevated and rapid pathogen-induced response. In addition to its potential use in breeding for resistance to multiple diseases, high-resolution mapping and cloning of the disease resistance locus in MNR220 may lead to a better understanding of the regulation of defense responses in wheat. PMID:22446929

  12. Mutation measurement in mammalian cells. IV: Comparison of gamma-ray and chemical mutagenesis.

    PubMed

    Puck, T T; Johnson, R; Webb, P; Yohrling, G

    1998-01-01

    The interaction of chemical mutagens with mammalian cells is much more complex than that of gamma-irradiation because of the different ways in which chemical agents react with cell and medium components. Nevertheless, the system previously described for analysis of mutagenesis by gamma-radiation appears applicable to chemical mutagenesis. The approach involves measurement of cell survival, use of caffeine to inhibit repair, analysis of mitotic index changes, and quantitation of microscopically visible structural changes in mitotic chromosomes. The behavior of a variety of chemical mutagens and nonmutagens in this system is described and compared with that of gamma-irradiation. The procedure is simple and the results reasonably quantitative though less so than those of gamma-irradiation. The procedure can be used for environmental monitoring, analysis of mutational events, and individual and epidemiological testing. Mutational events should be classified as primary or secondary depending on whether they represent initial genomic insult, or genomic changes resulting from primary mutation followed by structural changes due to metabolic actions. While caffeine has multiple effects on the mammalian genome, when used under the conditions specified here it appears to act principally as an inhibitor of mutation repair, and so affords a measure of the role of repair in the action of different mutagens on cells in the G2 phase of the life cycle. PMID:9776977

  13. A Multifunctional Mutagenesis System for Analysis of Gene Function in Zebrafish

    PubMed Central

    Quach, Helen Ngoc Bao; Tao, Shijie; Vrljicak, Pavle; Joshi, Adita; Ruan, Hua; Sukumaran, Rashmi; Varshney, Gaurav K.; LaFave, Matthew C.; Burgess, Shawn M.; Winkler, Christoph; Emelyanov, Alexander; Parinov, Sergey; Sampath, Karuna

    2015-01-01

    Since the sequencing of the human reference genome, many human disease-related genes have been discovered. However, understanding the functions of all the genes in the genome remains a challenge. The biological activities of these genes are usually investigated in model organisms such as mice and zebrafish. Large-scale mutagenesis screens to generate disruptive mutations are useful for identifying and understanding the activities of genes. Here, we report a multifunctional mutagenesis system in zebrafish using the maize Ds transposon. Integration of the Ds transposable element containing an mCherry reporter for protein trap events and an EGFP reporter for enhancer trap events produced a collection of transgenic lines marking distinct cell and tissue types, and mutagenized genes in the zebrafish genome by trapping and prematurely terminating endogenous protein coding sequences. We obtained 642 zebrafish lines with dynamic reporter gene expression. The characterized fish lines with specific expression patterns will be made available through the European Zebrafish Resource Center (EZRC), and a database of reporter expression is available online (http://fishtrap.warwick.ac.uk/). Our approach complements other efforts using zebrafish to facilitate functional genomic studies in this model of human development and disease. PMID:25840430

  14. Sleeping Beauty transposon insertional mutagenesis based mouse models for cancer gene discovery

    PubMed Central

    Moriarity, Branden S; Largaespada, David A

    2016-01-01

    Large-scale genomic efforts to study human cancer, such as the cancer gene atlas (TCGA), have identified numerous cancer drivers in a wide variety of tumor types. However, there are limitations to this approach, the mutations and expression or copy number changes that are identified are not always clearly functionally relevant, and only annotated genes and genetic elements are thoroughly queried. The use of complimentary, nonbiased, functional approaches to identify drivers of cancer development and progression is ideal to maximize the rate at which cancer discoveries are achieved. One such approach that has been successful is the use of the Sleeping Beauty (SB) transposon-based mutagenesis system in mice. This system uses a conditionally expressed transposase and mutagenic transposon allele to target mutagenesis to somatic cells of a given tissue in mice to cause random mutations leading to tumor development. Analysis of tumors for transposon common insertion sites (CIS) identifies candidate cancer genes specific to that tumor type. While similar screens have been performed in mice with the PiggyBac (PB) transposon and viral approaches, we limit extensive discussion to SB. Here we discuss the basic structure of these screens, screens that have been performed, methods used to identify CIS. PMID:26051241

  15. Mechanisms of mutagenesis: Analysis through the use of alcohol dehydrogenase in Drosophila: Final report

    SciTech Connect

    Sofer, W.H.

    1986-12-01

    Our original objective was to understand the mechanism of mutagenesis of several important mutagens in higher organisms. Our approach was to try to deduce this mechanism by working backwards from its final effects. The strategy that we used in an effort to carry out our studies was to make mutations in the alcohol dehydrogenase gene of Drosophila melanogaster and sequence the modified genes. Most of our work was focused on an array of mutants that we had induced with formaldehyde, a potent mutagen in Drosophila, and with ethyl methane sulfonate. Over the course of the project period we cloned and sequenced the ADH gene from four formalde-induced mutants and from one EMS mutant. We showed that the four formaldehyde-induced mutants contained small deletions within the protein-coding region of their ADH genes ranging in size from between 6 and 34 bp. The one EMS-induced mutant was shown by DNA sequencing to bear an AT to GC sequence change at a tryptophan codon near the c-terminal coding portion of the gene. These results have significantly increased our understanding of the mechanism(s) of mutagenesis in higher organisms. 20 refs., 1 fig.

  16. Mutagenesis by Cytostatic Alkylating Agents in Yeast Strains of Differing Repair Capacities

    PubMed Central

    Ruhland, Axel; Brendel, Martin

    1979-01-01

    Reversion of two nuclear ochre nonsense alleles and cell inactivation induced by mono-, bi-, and tri-functional alkylating agents and by UV has been investigated in stationary-phase haploid cells of yeast strains with differing capacities for DNA repair. The ability to survive alkylation damage is correlated with UV repair capacity, a UV-resistant and UV-mutable strain (RAD REV) being least and a UV-sensitive and UV-nonmutable strain (rad1 rev3) most sensitive. Mutagenicity of alkylating agents is highest in the former and is abolished in the latter strain. Deficiency in excision repair (rad1 rad2) or in the RAD18 function does not lead to enhanced mutability. Mutagenesis by the various agents is characterized by a common pattern of induction of locus-specific revertants and suppressor mutants. Induction kinetics are mostly linear, but UV-induced reversion in the RAD REV strain follows higher-than-linear (probably "quadratic") kinetics. The alkylating agent cyclophosphamide, usually considered inactive without metabolic conversion, reduces colony-forming ability and induces revertants in a manner similar but not identical to the other chemicals tested. These findings are taken to support the concept of mutagenesis by misrepair after alkylation, which albeit sharing common features with the mechanism of UV-induced reversion, can be distinguished therefrom. PMID:387518

  17. Transposon mutagenesis identifies genetic drivers of BrafV600E melanoma

    PubMed Central

    Mann, Michael B; Black, Michael A; Jones, Devin J; Ward, Jerrold M; Yew, Christopher Chin Kuan; Newberg, Justin Y; Dupuy, Adam J; Rust, Alistair G; Bosenberg, Marcus W; McMahon, Martin; Print, Cristin G; Copeland, Neal G; Jenkins, Nancy A

    2016-01-01

    Although nearly half of human melanomas harbor oncogenic BRAFV600E mutations, the genetic events that cooperate with these mutations to drive melanogenesis are still largely unknown. Here we show that Sleeping Beauty (SB) transposon-mediated mutagenesis drives melanoma progression in BrafV600E mutant mice and identify 1,232 recurrently mutated candidate cancer genes (CCGs) from 70 SB-driven melanomas. CCGs are enriched in Wnt, PI3K, MAPK and netrin signaling pathway components and are more highly connected to one another than predicted by chance, indicating that SB targets cooperative genetic networks in melanoma. Human orthologs of >500 CCGs are enriched for mutations in human melanoma or showed statistically significant clinical associations between RNA abundance and survival of patients with metastatic melanoma. We also functionally validate CEP350 as a new tumor-suppressor gene in human melanoma. SB mutagenesis has thus helped to catalog the cooperative molecular mechanisms driving BRAFV600E melanoma and discover new genes with potential clinical importance in human melanoma. PMID:25848750

  18. Random Mutagenesis of the Aspergillus oryzae Genome Results in Fungal Antibacterial Activity.

    PubMed

    Leonard, Cory A; Brown, Stacy D; Hayman, J Russell

    2013-01-01

    Multidrug-resistant bacteria cause severe infections in hospitals and communities. Development of new drugs to combat resistant microorganisms is needed. Natural products of microbial origin are the source of most currently available antibiotics. We hypothesized that random mutagenesis of Aspergillus oryzae would result in secretion of antibacterial compounds. To address this hypothesis, we developed a screen to identify individual A. oryzae mutants that inhibit the growth of Methicillin-resistant Staphylococcus aureus (MRSA) in vitro. To randomly generate A. oryzae mutant strains, spores were treated with ethyl methanesulfonate (EMS). Over 3000 EMS-treated A. oryzae cultures were tested in the screen, and one isolate, CAL220, exhibited altered morphology and antibacterial activity. Culture supernatant from this isolate showed antibacterial activity against Methicillin-sensitive Staphylococcus aureus, MRSA, and Pseudomonas aeruginosa, but not Klebsiella pneumonia or Proteus vulgaris. The results of this study support our hypothesis and suggest that the screen used is sufficient and appropriate to detect secreted antibacterial fungal compounds resulting from mutagenesis of A. oryzae. Because the genome of A. oryzae has been sequenced and systems are available for genetic transformation of this organism, targeted as well as random mutations may be introduced to facilitate the discovery of novel antibacterial compounds using this system. PMID:23983696

  19. Engineering stress tolerance of Escherichia coli by stress-induced mutagenesis (SIM)-based adaptive evolution.

    PubMed

    Zhu, Linjiang; Cai, Zhen; Zhang, Yanping; Li, Yin

    2014-01-01

    Microbial tolerance to toxic products and biomass hydrolysates is a challenge for the production of fuels and chemicals from renewable resources. To improve cellular tolerance to these environmental stresses, a novel adaptive evolutionary strategy based on stress-induced mutagenesis (SIM) was developed using non-dividing cells. The concept of this method was proved using Escherichia coli FC40 as a model strain, which was used to quantitatively evaluate the rate of SIM. By deleting either the mutL or mutS gene to disturb the mismatch repair activity of the host cells, the SIM rate under stressful conditions increased by 92- and 57-fold, respectively. A periodic SIMbased adaptive evolution procedure, which synchronized the mutagenesis and the selection process in a single plate-incubation step, was then developed using the mutL-deleted mutant. E. coli mutants tolerant to high concentrations of butanol (13 g/L), NaCl (95 g/L), and high temperature (50°C) were obtained. These results indicate that stress-induced adaptive evolution in non-dividing cells is an effective approach that can improve microbial tolerance against various stresses and generate robust microbial strains suitable for production of fuels and chemicals. PMID:24106039

  20. Implementation of a large-scale ENU mutagenesis program: towards increasing the mouse mutant resource.

    PubMed

    Nolan, P M; Peters, J; Vizor, L; Strivens, M; Washbourne, R; Hough, T; Wells, C; Glenister, P; Thornton, C; Martin, J; Fisher, E; Rogers, D; Hagan, J; Reavill, C; Gray, I; Wood, J; Spurr, N; Browne, M; Rastan, S; Hunter, J; Brown, S D

    2000-07-01

    Systematic approaches to mouse mutagenesis will be vital for future studies of gene function. We have begun a major ENU mutagenesis program incorporating a large genome-wide screen for dominant mutations. Progeny of ENU-mutagenized mice are screened for visible defects at birth and weaning, and at 5 weeks of age by using a systematic and semi-quantitative screening protocol-SHIRPA. Following this, mice are screened for abnormal locomotor activity and for deficits in prepulse inhibition of the acoustic startle response. Moreover, in the primary screen, blood is collected from mice and subjected to a comprehensive clinical biochemical analysis. Subsequently, secondary and tertiary screens of increasing complexity can be used on animals demonstrating deficits in the primary screen. Frozen sperm is archived from all the male mice passing through the screen. In addition, tail tips are stored for DNA. Overall, the program will provide an extensive new resource of mutant and phenotype data to the mouse and human genetics communities at large. The challenge now is to employ the expanding mouse mutant resource to improve the mutant map of the mouse. An improved mutant map of the mouse will be an important asset in exploiting the growing gene map of the mouse and assisting with the identification of genes underlying novel mutations-with consequent benefits for the analysis of gene function and the identification of novel pathways. PMID:10886012

  1. Improving isopropanol tolerance and production of Clostridium beijerinckii DSM 6423 by random mutagenesis and genome shuffling.

    PubMed

    Gérando, H Máté de; Fayolle-Guichard, F; Rudant, L; Millah, S K; Monot, F; Ferreira, Nicolas Lopes; López-Contreras, A M

    2016-06-01

    Random mutagenesis and genome shuffling was applied to improve solvent tolerance and isopropanol/butanol/ethanol (IBE) production in the strictly anaerobic bacteria Clostridium beijerinckii DSM 6423. Following chemical mutagenesis with N-methyl-N-nitro-N-nitrosoguanidine (NTG), screening of putatively improved strains was done by submitting the mutants to toxic levels of inhibitory chemicals or by screening for their tolerance to isopropanol (>35 g/L). Suicide substrates, such as ethyl or methyl bromobutyrate or alcohol dehydrogenase inhibitors like allyl alcohol, were tested and, finally, 36 mutants were isolated. The fermentation profiles of these NTG mutant strains were characterized, and the best performing mutants were used for consecutive rounds of genome shuffling. Screening of strains with further enhancement in isopropanol tolerance at each recursive shuffling step was then used to spot additionally improved strains. Three highly tolerant strains were finally isolated and able to withstand up to 50 g/L isopropanol on plates. Even if increased tolerance to the desired end product was not always accompanied by higher production capabilities, some shuffled strains showed increased solvent titers compared to the parental strains and the original C. beijerinckii DSM 6423. This study confirms the efficiency of genome shuffling to generate improved strains toward a desired phenotype such as alcohol tolerance. This tool also offers the possibility of obtaining improved strains of Clostridium species for which targeted genetic engineering approaches have not been described yet. PMID:26852409

  2. Fragile DNA Motifs Trigger Mutagenesis at Distant Chromosomal Loci in Saccharomyces cerevisiae

    PubMed Central

    Saini, Natalie; Zhang, Yu; Nishida, Yuri; Sheng, Ziwei; Choudhury, Shilpa; Mieczkowski, Piotr; Lobachev, Kirill S.

    2013-01-01

    DNA sequences capable of adopting non-canonical secondary structures have been associated with gross-chromosomal rearrangements in humans and model organisms. Previously, we have shown that long inverted repeats that form hairpin and cruciform structures and triplex-forming GAA/TTC repeats induce the formation of double-strand breaks which trigger genome instability in yeast. In this study, we demonstrate that breakage at both inverted repeats and GAA/TTC repeats is augmented by defects in DNA replication. Increased fragility is associated with increased mutation levels in the reporter genes located as far as 8 kb from both sides of the repeats. The increase in mutations was dependent on the presence of inverted or GAA/TTC repeats and activity of the translesion polymerase Polζ. Mutagenesis induced by inverted repeats also required Sae2 which opens hairpin-capped breaks and initiates end resection. The amount of breakage at the repeats is an important determinant of mutations as a perfect palindromic sequence with inherently increased fragility was also found to elevate mutation rates even in replication-proficient strains. We hypothesize that the underlying mechanism for mutagenesis induced by fragile motifs involves the formation of long single-stranded regions in the broken chromosome, invasion of the undamaged sister chromatid for repair, and faulty DNA synthesis employing Polζ. These data demonstrate that repeat-mediated breaks pose a dual threat to eukaryotic genome integrity by inducing chromosomal aberrations as well as mutations in flanking genes. PMID:23785298

  3. Expression-independent gene trap vectors for random and targeted mutagenesis in embryonic stem cells

    PubMed Central

    Tsakiridis, Anestis; Tzouanacou, Elena; Rahman, Afifah; Colby, Douglas; Axton, Richard; Chambers, Ian; Wilson, Valerie; Forrester, Lesley; Brickman, Joshua M.

    2009-01-01

    Promoterless gene trap vectors have been widely used for high-efficiency gene targeting and random mutagenesis in embryonic stem (ES) cells. Unfortunately, such vectors are only effective for genes expressed in ES cells and this has prompted the development of expression-independent vectors. These polyadenylation (poly A) trap vectors employ a splice donor to capture an endogenous gene's polyadenylation sequence and provide transcript stability. However, the spectrum of mutations generated by these vectors appears largely restricted to the last intron of target loci due to nonsense-mediated mRNA decay (NMD) making them unsuitable for gene targeting applications. Here, we present novel poly A trap vectors that overcome the effect of NMD and also employ RNA instability sequences to improve splicing efficiency. The set of random insertions generated with these vectors show a significantly reduced insertional bias and the vectors can be targeted directly to a 5′ intron. We also show that this relative positional independence is linked to the human β-actin promoter and is most likely a result of its transcriptional activity in ES cells. Taken together our data indicate that these vectors are an effective tool for insertional mutagenesis that can be used for either gene trapping or gene targeting. PMID:19692586

  4. piggyBac transposon somatic mutagenesis with an activated reporter and tracker (PB-SMART) for genetic screens in mice.

    PubMed

    Landrette, Sean F; Cornett, Jonathan C; Ni, Thomas K; Bosenberg, Marcus W; Xu, Tian

    2011-01-01

    Somatic forward genetic screens have the power to interrogate thousands of genes in a single animal. Retroviral and transposon mutagenesis systems in mice have been designed and deployed in somatic tissues for surveying hematopoietic and solid tumor formation. In the context of cancer, the ability to visually mark mutant cells would present tremendous advantages for identifying tumor formation, monitoring tumor growth over time, and tracking tumor infiltrations and metastases into wild-type tissues. Furthermore, locating mutant clones is a prerequisite for screening and analyzing most other somatic phenotypes. For this purpose, we developed a system using the piggyBac (PB) transposon for somatic mutagenesis with an activated reporter and tracker, called PB-SMART. The PB-SMART mouse genetic screening system can simultaneously induce somatic mutations and mark mutated cells using bioluminescence or fluorescence. The marking of mutant cells enable analyses that are not possible with current somatic mutagenesis systems, such as tracking cell proliferation and tumor growth, detecting tumor cell infiltrations, and reporting tissue mutagenesis levels by a simple ex vivo visual readout. We demonstrate that PB-SMART is highly mutagenic, capable of tumor induction with low copy transposons, which facilitates the mapping and identification of causative insertions. We further integrated a conditional transposase with the PB-SMART system, permitting tissue-specific mutagenesis with a single cross to any available Cre line. Targeting the germline, the system could also be used to conduct F1 screens. With these features, PB-SMART provides an integrated platform for individual investigators to harness the power of somatic mutagenesis and phenotypic screens to decipher the genetic basis of mammalian biology and disease. PMID:22039523

  5. Use of a simian virus 40-based shuttle vector to analyze enhanced mutagenesis in mitomycin C-treated monkey cells

    SciTech Connect

    Roilides, E.; Munson, P.J.; Levine, A.S.; Dixon, K.

    1988-09-01

    When monkey cells were treated with mitomycin C 24 h before transfection with UV-irradiated pZ189 (a simian virus 40-based shuttle vector), there was a twofold increase in the frequency of mutations in the supF gene of the vector. These results suggest the existence of an enhancible mutagenesis pathway in mammalian cells. However, DNA sequence analysis of the SupF- mutants suggested no dramatic changes in the mechanisms of mutagenesis due to mitomycin C treatment of the cells.

  6. Rational development of an attenuated recombinant cyprinid herpesvirus 3 vaccine using prokaryotic mutagenesis and in vivo bioluminescent imaging

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cyprinid herpesvirus 3 (CyHV-3) is causing severe economic losses worldwide in the carp industry, and a safe and efficacious attenuated vaccine compatible with mass vaccination is needed. We produced single deleted recombinants using prokaryotic mutagenesis. When producing a recombinant lacking open...

  7. [Measurement of mutagenesis to study the effects of chemical agents]. Final report, August 1, 1993--July 31, 1994

    SciTech Connect

    Puck, T.T.

    1994-12-31

    This is the final report of a study conducted at the Eleanor Roosevelt Institute for Cancer Research, Inc. This study looked at mutagenesis as a measurement of the effects of chemical agents. Topics discussed in this report include: development of a new theory for the role of lipids and lipoproteins in the interactions of macromolecules; the action of caffeine in synergizing mutagenesis of agents like ionizing radiation by inhibition of cellular repair processes which was incorporated into a rapid procedure for detection of mutagenicity with high sensitivity; quantitative theoretical analysis of the mutagenesis process in cells exposed to physical and chemical mutagenic agents; theoretical analysis was developed leading to the conclusion that the visible chromosomal lesions described will also include a significant proportion of point mutations; application of this methodology for meaningful measurement of mutagenesis to study the effects of chemical agents was begun; and investigation of the cell cytoskeleton`s effect of genome exposure operating in the course of the differentiation process.

  8. Incorporation of a lambda phage recombination system and EGFP detection to simplify mutagenesis of Herpes simplex virus bacterial artificial chromosomes

    PubMed Central

    Schmeisser, Falko; Weir, Jerry P

    2007-01-01

    Background Targeted mutagenesis of the herpesvirus genomes has been facilitated by the use of bacterial artificial chromosome (BAC) technology. Such modified genomes have potential uses in understanding viral pathogenesis, gene identification and characterization, and the development of new viral vectors and vaccines. We have previously described the construction of a herpes simplex virus 2 (HSV-2) BAC and the use of an allele replacement strategy to construct HSV-2 recombinants. While the BAC mutagenesis procedure is a powerful method to generate HSV-2 recombinants, particularly in the absence of selective marker in eukaryotic culture, the mutagenesis procedure is still difficult and cumbersome. Results Here we describe the incorporation of a phage lambda recombination system into an allele replacement vector. This strategy enables any DNA fragment containing the phage attL recombination sites to be efficiently inserted into the attR sites of the allele replacement vector using phage lambda clonase. We also describe how the incorporation of EGFP into the allele replacement vector can facilitate the selection of the desired cross-over recombinant BACs when the allele replacement reaction is a viral gene deletion. Finally, we incorporate the lambda phage recombination sites directly into an HSV-2 BAC vector for direct recombination of gene cassettes using the phage lambda clonase-driven recombination reaction. Conclusion Together, these improvements to the techniques of HSV BAC mutagenesis will facilitate the construction of recombinant herpes simplex viruses and viral vectors. PMID:17501993

  9. RAT BLADDER CELL-MEDIATED MUTAGENESIS OF CHINESE HAMSTER V79 CELLS AND METABOLISM OF BENZO(A)PYRENE

    EPA Science Inventory

    Primary rat bladder epithelial cells were coculivated with Chinese hamster V79 cells in the presence of carcinogens, and the induction of 6-thioguanine resistance in the V79 cells was used as a marker of cell-mediated mutagenesis. The carcinogens dimethylnitrosamine, 7, 12-dimeth...

  10. Agrobacterium tumefaciens-mediated transformation: An efficient tool for insertional mutagenesis and targeted gene disruption in Harpophora oryzae.

    PubMed

    Liu, Ning; Chen, Guo-Qing; Ning, Guo-Ao; Shi, Huan-Bin; Zhang, Chu-Long; Lu, Jian-Ping; Mao, Li-Juan; Feng, Xiao-Xiao; Liu, Xiao-Hong; Su, Zhen-Zhu; Lin, Fu-Cheng

    2016-01-01

    The endophytic filamentous fungus Harpophora oryzae is a beneficial endosymbiont isolated from the wild rice. H. oryzae could not only effectively improve growth rate and biomass yield of rice crops, but also induce systemic resistance against the rice blast fungus, Magnaporthe oryzae. In this study, Agrobacterium tumefaciens-mediated transformation (ATMT) was employed and optimized to modify the H. oryzae genes by either random DNA fragment integration or targeted gene replacement. Our results showed that co-cultivation of H. oryzae conidia with A. tumefaciens in the presence of acetosyringone for 48 h at 22 °C could lead to a relatively highest frequency of transformation, and 200 μM acetosyringone (AS) pre-cultivation of A. tumefaciens is also suggested. ATMT-mediated knockout mutagenesis was accomplished with the gene-deletion cassettes using a yeast homologous recombination method with a yeast-Escherichia-Agrobacterium shuttle vector pKOHo. Using the ATMT-mediated knockout mutagenesis, we successfully deleted three genes of H. oryzae (HoATG5, HoATG7, and HoATG8), and then got the null mutants ΔHoatg5, ΔHoatg7, and ΔHoatg8. These results suggest that ATMT is an efficient tool for gene modification including randomly insertional mutagenesis and gene deletion mutagenesis in H. oryzae. PMID:26686612

  11. Random T-DNA mutagenesis identifies a Cu-Zn-superoxide dismutase gene as a virulence factor of Sclerotinia sclerotiorum

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Ascomycetous fungus Sclerotinia sclerotiorum is a devastating pathogen capable of infecting more than 400 plant species including many economically important crops. In order to gain a better mechanistic understanding of its non-specific host-pathogen interactions, random mutagenesis through Agro...

  12. Inducible mutagenesis in TEPC 2372, a mouse plasmacytoma cell line that harbors the transgenic shuttle vector lambdaLIZ.

    PubMed

    Felix, K; Kovalchuk, A L; Park, S S; Coleman, A E; Ramsay, E S; Qian, M; Kelliher, K A; Jones, G M; Ried, T; Bornkamm, G W; Janz, S

    2001-01-25

    The plasmacytoma cell line, TEPC 2372, was derived from a malignant plasma cell tumor that developed in the peritoneal cavity of a BALB/c mouse that harbored the transgenic shuttle vector for the assessment of mutagenesis in vivo, lambdaLIZ. TEPC 2372 was found to display the typical features of a BALB/c plasmacytoma. It consisted of pleomorphic plasma cells that secreted a monoclonal immunoglobulin (IgG2b/lambda), was initially dependent on the presence of IL-6 to grow in cell culture, contained a hyperdiploid chromosome complement with a tendency to undergo tetraploidization, and harbored a constitutively active c-myc gene by virtue of a T(6;15) chromosomal translocation. TEPC 2372 was further characterized by the ability to respond to in vitro exposure with 4-NQO (4-nitroquinoline-1-oxide), an oxidative model mutagen, with a vigorous dose-dependent increase in mutagenesis that peaked at a 7.85-fold elevation of mutant rates in lambdaLIZ when compared to background mutant rates in untreated controls. Cotreatment with 4-NQO and BSO (buthionine sulfoximine), a glutathione-depleting compound that causes endogenous oxidative stress, resulted in a 9.03-fold increase in the mutant frequency in lambdaLIZ. These results demonstrated that TEPC 2372, the malignant plasma cell counterpart of the lambdaLIZ-based in vivo mutagenesis assay, may be useful as an in vitro reference point for the further elucidation of oxidative mutagenesis in lymphoid tissues. PMID:11166031

  13. From Green to Blue: Site-Directed Mutagenesis of the Green Fluorescent Protein to Teach Protein Structure-Function Relationships

    ERIC Educational Resources Information Center

    Giron, Maria D.; Salto, Rafael

    2011-01-01

    Structure-function relationship studies in proteins are essential in modern Cell Biology. Laboratory exercises that allow students to familiarize themselves with basic mutagenesis techniques are essential in all Genetic Engineering courses to teach the relevance of protein structure. We have implemented a laboratory course based on the…

  14. Mutagenesis and heterologous expression in yeast of a plant Delta6-fatty acid desaturase.

    PubMed

    Sayanova, O; Beaudoin, F; Libisch, B; Castel, A; Shewry, P R; Napier, J A

    2001-07-01

    Membrane-bound microsomal fatty acid desaturases are known to have three conserved histidine boxes, comprising a total of up to eight histidine residues. Recently, a number of deviations from this consensus have been reported, with the substitution of a glutamine for the first histidine residue of the third histidine box being present in the so called 'front end' desaturases. These enzymes are also characterized by the presence of a cytochrome b5 domain at the protein N-terminus. Site-directed mutagenesis has been used to probe the functional importance of a number of amino acid residues which comprise the third histidine box of a 'front end' desaturase, the borage Delta6-fatty acid desaturase. This showed that the variant glutamine in the third histidine box is essential for enzyme activity and that histidine is not able to substitute for this residue. PMID:11457919

  15. KP-1212/1461, a nucleoside designed for the treatment of HIV by viral mutagenesis.

    PubMed

    Harris, Kevin S; Brabant, William; Styrchak, Sheila; Gall, Alexander; Daifuku, Richard

    2005-07-01

    We report the activities of a novel nucleoside analog against HIV. This nucleoside (KP-1212) is not a chain terminator but exerts its antiviral effects via mutagenesis of the viral genome. Serial passaging of HIV in the presence of KP-1212 causes an increase in the mutation rate of the virus leading to viral ablation. HIV strains resistant to KP-1212 have not yet been isolated. Quite to the contrary, virus treated with KP-1212 exhibited an increased sensitivity not only to KP-1212 but also to another nucleoside reverse transcriptase inhibitor (NRTI), zidovudine. HIV strains resistant to other NRTIs (e.g. zidovudine, lamivudine, stavudine, abacavir, etc.) exhibited no cross-resistance towards KP-1212. Multiple assays confirmed that KP-1212 has a favorable (low) genotoxicity profile when compared to some approved antiviral nucleosides. In addition, KP-1212 is not toxic to mitochondria nor does it exhibit any inhibitory effects on mitochondrial DNA synthesis. PMID:15890415

  16. Spectrum of Bmp5 mutations from germline mutagenesis experiments in mice

    SciTech Connect

    Marker, P.C.; Kwonjune Seung; Bland, A.E.

    1997-02-01

    Over 40 years of mutagenesis experiments using the mouse specific-locus test have produced a large number of induced germline mutations at seven loci, among them the short ear locus. We have previously shown that the short ear locus encodes bone morphogenetic protein 5 (BMP5), a member of a large family of secreted signaling molecules that play key roles in axis formation, tissue differentiation, mesenchymal-epithelial interactions, and skeletal development. Here we examine 24 chemical- and radiation-induced mutations at the short ear locus. Sequence changes in the Bmp5 open reading frame confirm the importance of cysteine residues in the function of TGF{beta} superfamily members. The spectrum of N-ethyl-N-nitrosourea-induced mutations also provides new information about the basepair, sequence context, and strand specificity of germline mutations in mammals. 52 refs., 3 figs., 2 tabs.

  17. Crystal structure and site-directed mutagenesis of a nitroalkane oxidase from Streptomyces ansochromogenes.

    PubMed

    Li, Yanhua; Gao, Zengqiang; Hou, Haifeng; Li, Lei; Zhang, Jihui; Yang, Haihua; Dong, Yuhui; Tan, Huarong

    2011-02-18

    Nitroalkane oxidase (NAO) catalyzes neutral nitroalkanes to their corresponding aldehydes or ketones, hydrogen peroxide and nitrite. The crystal structure of NAO from Streptomyces ansochromogenes was determined; it consists of two domains, a TIM barrel domain bound to FMN and C-terminal domain with a novel folding pattern. Site-directed mutagenesis of His179, which is spatially adjacent to FMN, resulted in the loss of enzyme activity, demonstrating that this amino acid residue is important for catalysis. The crystal structure of mutant H179D-nitroethane was also analyzed. Interestingly, Sa-NAO shows the typical function as nitroalkane oxidase but its structure is similar to that of 2-nitropropane dioxygenase. Overall, these results suggest that Sa-NAO is a novel nitroalkane oxidase with TIM barrel structure. PMID:21147069

  18. Tryptophan Lyase (NosL): Mechanistic Insights from Substrate Analogues and Mutagenesis.

    PubMed

    Bhandari, Dhananjay M; Xu, Hui; Nicolet, Yvain; Fontecilla-Camps, Juan C; Begley, Tadhg P

    2015-08-11

    NosL is a member of a family of radical S-adenosylmethionine enzymes that catalyze the cleavage of the Cα-Cβ bond of aromatic amino acids. In this paper, we describe a set of experiments with substrate analogues and mutants for probing the early steps of the NosL mechanism. We provide biochemical evidence in support of the structural studies showing that the 5'-deoxyadenosyl radical abstracts a hydrogen atom from the amino group of tryptophan. We demonstrate that d-tryptophan is a substrate for NosL but shows relaxed regio control of the first β-scission reaction. Mutagenesis studies confirm that Arg323 is important for controlling the regiochemistry of the β-scission reaction and that Ser340 binds the substrate by hydrogen bonding to the indolic N1 atom. PMID:26204056

  19. Cloning of human epidermal growth factor as a bacterial secretory protein, its properties and mutagenesis

    SciTech Connect

    Engler, D.A.; Matsunami, R.K.; Campion, S.R.; Foote, R.S.; Mural, R.J.; Larimer, F.W.; Stevens, A.; Niyogi, S.K.

    1987-05-01

    A chimeric gene, containing the DNA coding for the human epidermal growth factor (EGF) and that for the signal peptide of E. coli alkaline phosphatase, was constructed by the annealing and subsequent ligation of appropriate DNA oligonucleotides synthesized in an automated DNA synthesizer. The gene was then cloned into a bacterial plasmid under the transcriptional control of the E. coli trp-lac (tac) promoter, and then transformed into E. coli. Following induction with isopropylthiogalactoside, the secretion of EGF into the E. coli periplasmic space and some into the growth medium was confirmed by its specific binding to the EGF receptor and stimulation of the EGF receptor tyrosine kinase activity. The size and physicochemical properties of the purified protein mimicked those of authentic human EGF. Studies of structure/function relationships by specific alterations of targeted amino acid residues in the EGF molecule have been initiated by utilizing site-directed mutagenesis.

  20. Site-directed mutagenesis studies of acetylglutamate synthase delineate the site for the arginine inhibitor.

    PubMed

    Sancho-Vaello, Enea; Fernández-Murga, M Leonor; Rubio, Vicente

    2008-04-01

    N-acetyl-L-glutamate synthase (NAGS), the first enzyme of bacterial/plant arginine biosynthesis and an essential activator of the urea cycle in animals, is, respectively, arginine-inhibited and activated. Site-directed mutagenesis of recombinant Pseudomonas aeruginosa NAGS (PaNAGS) delineates the arginine site in the PaNAGS acetylglutamate kinase-like domain, and, by extension, in human NAGS. Key residues for glutamate binding are identified in the acetyltransferase domain. However, the acetylglutamate kinase-like domain may modulate glutamate binding, since one mutation affecting this domain increases the K(m) for glutamate. The effects on PaNAGS of two mutations found in human NAGS deficiency support the similarity of bacterial and human NAGSs despite their low sequence identity. PMID:18319063

  1. X-Ray Structure and Mutagenesis Studies of the N-Isopropylammelide Isopropylaminohydrolase, AtzC

    PubMed Central

    Newman, Janet; Briggs, Lyndall J.; Scott, Colin; Peat, Thomas S.

    2015-01-01

    The N-isopropylammelide isopropylaminohydrolase from Pseudomonas sp. strain ADP, AtzC, provides the third hydrolytic step in the mineralization of s-triazine herbicides, such as atrazine. We obtained the X-ray crystal structure of AtzC at 1.84 Å with a weak inhibitor bound in the active site and then used a combination of in silico docking and site-directed mutagenesis to understand the interactions between AtzC and its substrate, isopropylammelide. The substitution of an active site histidine residue (His249) for an alanine abolished the enzyme’s catalytic activity. We propose a plausible catalytic mechanism, consistent with the biochemical and crystallographic data obtained that is similar to that found in carbonic anhydrase and other members of subtype III of the amidohydrolase family PMID:26390431

  2. Mutagenesis as a Tool in Plant Genetics, Functional Genomics, and Breeding

    PubMed Central

    Sikora, Per; Chawade, Aakash; Larsson, Mikael; Olsson, Johanna; Olsson, Olof

    2011-01-01

    Plant mutagenesis is rapidly coming of age in the aftermath of recent developments in high-resolution molecular and biochemical techniques. By combining the high variation of mutagenised populations with novel screening methods, traits that are almost impossible to identify by conventional breeding are now being developed and characterised at the molecular level. This paper provides a comprehensive overview of the various techniques and workflows available to researchers today in the field of molecular breeding, and how these tools complement the ones already used in traditional breeding. Both genetic (Targeting Induced Local Lesions in Genomes; TILLING) and phenotypic screens are evaluated. Finally, different ways of bridging the gap between genotype and phenotype are discussed. PMID:22315587

  3. Mouse Models of Cancer: Sleeping Beauty Transposons for Insertional Mutagenesis Screens and Reverse Genetic Studies

    PubMed Central

    Tschida, Barbara R.; Largaespada, David A.; Keng, Vincent W.

    2014-01-01

    The genetic complexity and heterogeneity of cancer has posed a problem in designing rationally targeted therapies effective in a large proportion of human cancer. Genomic characterization of many cancer types has provided a staggering amount of data that needs to be interpreted to further our understanding of this disease. Forward genetic screening in mice using Sleeping Beauty (SB) based insertional mutagenesis is an effective method for candidate cancer gene discovery that can aid in distinguishing driver from passenger mutations in human cancer. This system has been adapted for unbiased screens to identify drivers of multiple cancer types. These screens have already identified hundreds of candidate cancer-promoting mutations. These can be used to develop new mouse models for further study, which may prove useful for therapeutic testing. SB technology may also hold the key for rapid generation of reverse genetic mouse models of cancer, and has already been used to model glioblastoma and liver cancer. PMID:24468652

  4. Molecular basis of transcriptional fidelity and DNA lesion-induced transcriptional mutagenesis

    PubMed Central

    Xu, Liang; Da, Lintai; Plouffe, Steven W.; Chong, Jenny; Kool, Eric; Wang, Dong

    2014-01-01

    Maintaining high transcriptional fidelity is essential for life. Some DNA lesions lead to significant changes in transcriptional fidelity. In this review, we will summarize recent progress towards understanding the molecular basis of RNA polymerase II (Pol II) transcriptional fidelity and DNA lesion-induced transcriptional mutagenesis. In particular, we will focus on the three key checkpoint steps of controlling Pol II transcriptional fidelity: insertion (specific nucleotide selection and incorporation), extension (differentiation of RNA transcript extension of a matched over mismatched 3'-RNA terminus), and proofreading (preferential removal of misincorporated nucleotides from the 3'-RNA end). We will also discuss some novel insights into the molecular basis and chemical perspectives of controlling Pol II transcriptional fidelity through structural, computational, and chemical biology approaches. PMID:24767259

  5. A Transposon-Based Tool for Transformation and Mutagenesis in Trypanosomatid Protozoa

    PubMed Central

    Damasceno, Jeziel D.; Beverley, Stephen M.; Tosi, Luiz R.O.

    2014-01-01

    The ability of transposable elements to mobilize across genomes and affect the expression of genes makes them exceptional tools for genetic manipulation methodologies. Several transposon-based systems have been modified and incorporated into shuttle mutagenesis approaches in a variety of organisms. We have found that the Mos1 element, a DNA transposon from Drosophila mauritiana, is suitable and readily adaptable to a variety of strategies to the study of trypanosomatid parasitic protozoa. Trypanosomatids are the causative agents of a wide range of neglected diseases in underdeveloped regions of the globe. In this chapter we describe the basic elements and the available protocols for the in vitro use of Mos1 derivatives in the protozoan parasite Leishmania. PMID:25388118

  6. Genetic modification through oligonucleotide-mediated mutagenesis. A GMO regulatory challenge?

    PubMed

    Breyer, Didier; Herman, Philippe; Brandenburger, Annick; Gheysen, Godelieve; Remaut, Erik; Soumillion, Patrice; Van Doorsselaere, Jan; Custers, René; Pauwels, Katia; Sneyers, Myriam; Reheul, Dirk

    2009-01-01

    In the European Union, the definition of a GMO is technology-based. This means that a novel organism will be regulated under the GMO regulatory framework only if it has been developed with the use of defined techniques. This approach is now challenged with the emergence of new techniques. In this paper, we describe regulatory and safety issues associated with the use of oligonucleotide-mediated mutagenesis to develop novel organisms. We present scientific arguments for not having organisms developed through this technique fall within the scope of the EU regulation on GMOs. We conclude that any political decision on this issue should be taken on the basis of a broad reflection at EU level, while avoiding discrepancies at international level. PMID:19833073

  7. Isolation of temperature-sensitive Abelson virus mutants by site-directed mutagenesis.

    PubMed Central

    Engelman, A; Rosenberg, N

    1987-01-01

    Mutants of Abelson virus encoding temperature-sensitive protein-tyrosine kinase (EC 2.7.1.112) were created by site-directed mutagenesis using sequence information from temperature-sensitive mutants of the related v-src oncogene. Expression of these two independent mutations in Escherichia coli resulted in reduced phosphorylation of the mutant proteins at high temperature. Viruses containing one of the mutations induced conditional transformation of both NIH 3T3 and lymphoid cells when expressed in the context of a truncated transforming protein. These results underscore the functional homology between protein-tyrosine kinases and suggest that transfer of mutations within a related gene family may provide a rapid method to create mutants. Images PMID:2825174

  8. Random Transposon Mutagenesis for Cell-Envelope Resistant to Phage Infection.

    PubMed

    Reyes-Cortés, Ruth; Arguijo-Hernández, Emma S; Carballo-Ontiveros, Marco A; Martínez-Peñafiel, Eva; Kameyama, Luis

    2016-01-01

    In order to identify host components involved in the infective process of bacteriophages, we developed a wide-range strategy to obtain cell envelope mutants, using Escherichia coli W3110 and its specific phage mEp213. The strategy consisted in four steps: (1) random mutagenesis using transposon miniTn10Km(r); (2) selection of phage-resistant mutants by replica-plating; (3) electroporation of the phage-resistant mutants with mEp213 genome, followed by selection of those allowing phage development; and (4) sequencing of the transposon-disrupted genes. This strategy allowed us to distinguish the host factors related to phage development or multiplication within the cell, from those involved in phage infection at the level of the cell envelope. PMID:27311665

  9. Excavating the Genome: Large Scale Mutagenesis Screening for the Discovery of New Mouse Models

    PubMed Central

    Sundberg, John P.; Dadras, Soheil S.; Silva, Kathleen A.; Kennedy, Victoria E.; Murray, Stephen A.; Denegre, James; Schofield, Paul N.; King, Lloyd E.; Wiles, Michael; Pratt, C. Herbert

    2016-01-01

    Technology now exists for rapid screening of mutated laboratory mice to identify phenotypes associated with specific genetic mutations. Large repositories exist for spontaneous mutants and those induced by chemical mutagenesis, many of which have never been studied or comprehensively evaluated. To supplement these resources, a variety of techniques have been consolidated in an international effort to create mutations in all known protein coding genes in the mouse. With targeted embryonic stem cell lines now available for almost all protein coding genes and more recently CRISPR/Cas9 technology, large-scale efforts are underway to create novel mutant mouse strains and to characterize their phenotypes. However, accurate diagnosis of skin, hair, and nail diseases still relies on careful gross and histological analysis. While not automated to the level of the physiological phenotyping, histopathology provides the most direct and accurate diagnosis and correlation with human diseases. As a result of these efforts, many new mouse dermatological disease models are being developed. PMID:26551941

  10. Evaluating Risks of Insertional Mutagenesis by DNA Transposons in Gene Therapy

    PubMed Central

    Hackett, Perry B.; Largaespada, David A.; Switzer, Kirsten C.; Cooper, Laurence J.N.

    2013-01-01

    Investigational therapy can be successfully undertaken using viral- and non-viral-mediated ex vivo gene transfer. Indeed, recent clinical trials have established the potential for genetically modified T cells to improve and restore health. Recently the Sleeping Beauty (SB) transposon/transposase system has been applied in clinical trials to stably insert a chimeric antigen receptor (CAR) to redirect T-cell specificity. We discuss the context in which the SB system can be harnessed for gene therapy and describe the human application of SB-modified CAR+ T cells. We have focused on theoretical issues relating to insertional mutagenesis in the context of human genomes that are naturally subjected to remobilization of transposons and the experimental evidence over the last decade of employing SB transposons for defining genes that induce cancer. These findings are put into the context of the use of SB transposons in the treatment of human disease. PMID:23313630

  11. Rapid fine conformational epitope mapping using comprehensive mutagenesis and deep sequencing.

    PubMed

    Kowalsky, Caitlin A; Faber, Matthew S; Nath, Aritro; Dann, Hailey E; Kelly, Vince W; Liu, Li; Shanker, Purva; Wagner, Ellen K; Maynard, Jennifer A; Chan, Christina; Whitehead, Timothy A

    2015-10-30

    Knowledge of the fine location of neutralizing and non-neutralizing epitopes on human pathogens affords a better understanding of the structural basis of antibody efficacy, which will expedite rational design of vaccines, prophylactics, and therapeutics. However, full utilization of the wealth of information from single cell techniques and antibody repertoire sequencing awaits the development of a high throughput, inexpensive method to map the conformational epitopes for antibody-antigen interactions. Here we show such an approach that combines comprehensive mutagenesis, cell surface display, and DNA deep sequencing. We develop analytical equations to identify epitope positions and show the method effectiveness by mapping the fine epitope for different antibodies targeting TNF, pertussis toxin, and the cancer target TROP2. In all three cases, the experimentally determined conformational epitope was consistent with previous experimental datasets, confirming the reliability of the experimental pipeline. Once the comprehensive library is generated, fine conformational epitope maps can be prepared at a rate of four per day. PMID:26296891

  12. Site-Directed Mutagenesis to Improve Sensitivity of a Synthetic Two-Component Signaling System.

    PubMed

    Olshefsky, Audrey; Shehata, Laila; Kuldell, Natalie

    2016-01-01

    Two-component signaling (2CS) systems enable bacterial cells to respond to changes in their local environment, often using a membrane-bound sensor protein and a cytoplasmic responder protein to regulate gene expression. Previous work has shown that Escherichia coli's natural EnvZ/OmpR 2CS could be modified to construct a light-sensing bacterial photography system. The resulting bacterial photographs, or "coliroids," rely on a phosphotransfer reaction between Cph8, a synthetic version of EnvZ that senses red light, and OmpR. Gene expression changes can be visualized through upregulation of a LacZ reporter gene by phosphorylated OmpR. Unfortunately, basal LacZ expression leads to a detectable reporter signal even when cells are grown in the light, diminishing the contrast of the coliroids. We performed site-directed mutagenesis near the phosphotransfer site of Cph8 to isolate mutants with potentially improved image contrast. Five mutants were examined, but only one of the mutants, T541S, increased the ratio of dark/light gene expression, as measured by β-galactosidase activity. The ratio changed from 2.57 fold in the starting strain to 5.59 in the T541S mutant. The ratio decreased in the four other mutant strains we examined. The phenotype observed in the T541S mutant strain may arise because the serine sidechain is chemically similar but physically smaller than the threonine sidechain. This may minimally change the protein's local structure, but may be less sterically constrained when compared to threonine, resulting in a higher probability of a phosphotransfer event. Our initial success pairing synthetic biology and site-directed mutagenesis to optimize the bacterial photography system's performance encourages us to imagine further improvements to the performance of this and other synthetic systems, especially those based on 2CS signaling. PMID:26799494

  13. Protective effect of basil (Ocimum basilicum L.) against oxidative DNA damage and mutagenesis.

    PubMed

    Berić, Tanja; Nikolić, Biljana; Stanojević, Jasna; Vuković-Gacić, Branka; Knezević-Vukcević, Jelena

    2008-02-01

    Mutagenic and antimutagenic properties of essential oil (EO) of basil and its major constituent Linalool, reported to possess antioxidative properties, were examined in microbial tests. In Salmonella/microsome and Escherichia. coli WP2 reversion assays both derivatives (0.25-2.0 microl/plate) showed no mutagenic effect. Salmonella. typhimurium TA98, TA100 and TA102 strains displayed similar sensitivity to both basil derivatives as non-permeable E. coli WP2 strains IC185 and IC202 oxyR. Moreover, the toxicity of basil derivatives to WP2 strains did not depend on OxyR function. The reduction of t-BOOH-induced mutagenesis by EO and Linalool (30-60%) was obtained in repair proficient strains of the E. coli K12 assay (Nikolić, B., Stanojević, J., Mitić, D., Vuković-Gacić, B., Knezević-Vukcević, J., Simić, D., 2004. Comparative study of the antimutagenic potential of vitamin E in different E. coli strains. Mutat. Res. 564, 31-38), as well as in E. coli WP2 IC202 strain. EO and Linalool reduced spontaneous mutagenesis in mismatch repair deficient E. coli K12 strains (27-44%). In all tests, antimutagenic effect of basil derivatives was comparable with that obtained with model antioxidant vitamin E. Linalool and vitamin E induced DNA strand breaks in Comet assay on S. cerevisiae 3A cells, but at non-genotoxic concentrations (0.075 and 0.025 microg/ml, respectively) they reduced the number of H(2)O(2)-induced comets (45-70% Linalool and 80-93% vitamin E). Obtained results indicate that antigenotoxic potential of basil derivatives could be attributed to their antioxidative properties. PMID:17980946

  14. Gene Deletion by Fluorescence-Reported Allelic Exchange Mutagenesis in Chlamydia trachomatis

    PubMed Central

    Mueller, Konrad E.; Wolf, Katerina

    2016-01-01

    ABSTRACT Although progress in Chlamydia genetics has been rapid, genomic modification has previously been limited to point mutations and group II intron insertions which truncate protein products. The bacterium has thus far been intractable to gene deletion or more-complex genomic integrations such as allelic exchange. Herein, we present a novel suicide vector dependent on inducible expression of a chlamydial gene that renders Chlamydia trachomatis fully genetically tractable and permits rapid reverse genetics by fluorescence-reported allelic exchange mutagenesis (FRAEM). We describe the first available system of targeting chlamydial genes for deletion or allelic exchange as well as curing plasmids from C. trachomatis serovar L2. Furthermore, this approach permits the monitoring of mutagenesis by fluorescence microscopy without disturbing bacterial growth, a significant asset when manipulating obligate intracellular organisms. As proof of principle, trpA was successfully deleted and replaced with a sequence encoding both green fluorescent protein (GFP) and β-lactamase. The trpA-deficient strain was unable to grow in indole-containing medium, and this phenotype was reversed by complementation with trpA expressed in trans. To assess reproducibility at alternate sites, FRAEM was repeated for genes encoding type III secretion effectors CTL0063, CTL0064, and CTL0065. In all four cases, stable mutants were recovered one passage after the observation of transformants, and allelic exchange was limited to the specific target gene, as confirmed by whole-genome sequencing. Deleted sequences were not detected by quantitative real-time PCR (qPCR) from isogenic mutant populations. We demonstrate that utilization of the chlamydial suicide vector with FRAEM renders C. trachomatis highly amenable to versatile and efficient genetic manipulation. PMID:26787828

  15. Trapping Cardiac Recessive Mutants via Expression-based Insertional Mutagenesis Screening

    PubMed Central

    Ding, Yonghe; Liu, Weibin; Deng, Yun; Jomok, Beninio; Yang, Jingchun; Huang, Wei; Clark, Karl J.; Zhong, Tao P.; Lin, Xueying; Ekker, Stephen C.; Xu, Xiaolei

    2013-01-01

    Rationale Mutagenesis screening is a powerful genetic tool for probing biological mechanisms underlying vertebrate development and human diseases. However, the increased colony management efforts in vertebrates impose a significant challenge for identifying genes affecting a particular organ such as the heart, especially those exhibiting adult phenotypes upon depletion. Objective We aim to develop a facile approach that streamlines colony management efforts via enriching cardiac mutants, which enables us to screen for adult phenotypes. Methods and Results The transparency of the zebrafish embryos enabled us to score 67 stable transgenic lines generated from an insertional mutagenesis screen using a transposon-based protein trapping vector. Fifteen lines with cardiac monomeric red fluorescent protein (mRFP) reporter expression were identified. We defined the molecular nature for 10 lines and bred them to homozygosity, which led to the identification of one embryonic lethal, one larval lethal, and one adult recessive mutant exhibiting cardiac hypertrophy at one year of age. Further characterization of these mutants uncovered an essential function of methionine adenosyltransferase II, alpha a (mat2aa) in cardiogenesis, an essential function of mitochondrial ribosomal protein S18B (mrps18b) in cardiac mitochondrial homeostasis, as well as a function of DnaJ (Hsp40) homolog, subfamily B, member 6b (dnajb6b) in adult cardiac hypertrophy. Conclusions We demonstrate that transposon-based gene trapping is an efficient approach for identifying both embryonic and adult recessive mutants with cardiac expression. The generation of a Zebrafish Insertional Cardiac (ZIC) mutant collection shall facilitate the annotation of a vertebrate cardiac genome, as well as enable heart-based adult screens. PMID:23283723

  16. 5-Azacytidine Enhances the Mutagenesis of HIV-1 by Reduction to 5-Aza-2'-Deoxycytidine.

    PubMed

    Rawson, Jonathan M O; Daly, Michele B; Xie, Jiashu; Clouser, Christine L; Landman, Sean R; Reilly, Cavan S; Bonnac, Laurent; Kim, Baek; Patterson, Steven E; Mansky, Louis M

    2016-04-01

    5-Azacytidine (5-aza-C) is a ribonucleoside analog that induces the lethal mutagenesis of human immunodeficiency virus type 1 (HIV-1) by causing predominantly G-to-C transversions during reverse transcription. 5-Aza-C could potentially act primarily as a ribonucleotide (5-aza-CTP) or as a deoxyribonucleotide (5-aza-2'-deoxycytidine triphosphate [5-aza-dCTP]) during reverse transcription. In order to determine the primary form of 5-aza-C that is active against HIV-1, Illumina sequencing was performed using proviral DNA from cells treated with 5-aza-C or 5-aza-dC. 5-Aza-C and 5-aza-dC were found to induce highly similar patterns of mutation in HIV-1 in terms of the types of mutations observed, the magnitudes of effects, and the distributions of mutations at individual sequence positions. Further, 5-aza-dCTP was detected by liquid chromatography-tandem mass spectrometry in cells treated with 5-aza-C, demonstrating that 5-aza-C was a substrate for ribonucleotide reductase. Notably, levels of 5-aza-dCTP were similar in cells treated with equivalent effective concentrations of 5-aza-C or 5-aza-dC. Lastly, HIV-1 reverse transcriptase was found to incorporate 5-aza-CTPin vitroat least 10,000-fold less efficiently than 5-aza-dCTP. Taken together, these data support the model that 5-aza-C enhances the mutagenesis of HIV-1 primarily after reduction to 5-aza-dC, which can then be incorporated during reverse transcription and lead to G-to-C hypermutation. These findings may have important implications for the design of new ribonucleoside analogs directed against retroviruses. PMID:26833151

  17. Site-Directed Mutagenesis to Improve Sensitivity of a Synthetic Two-Component Signaling System

    PubMed Central

    Kuldell, Natalie

    2016-01-01

    Two-component signaling (2CS) systems enable bacterial cells to respond to changes in their local environment, often using a membrane-bound sensor protein and a cytoplasmic responder protein to regulate gene expression. Previous work has shown that Escherichia coli’s natural EnvZ/OmpR 2CS could be modified to construct a light-sensing bacterial photography system. The resulting bacterial photographs, or “coliroids,” rely on a phosphotransfer reaction between Cph8, a synthetic version of EnvZ that senses red light, and OmpR. Gene expression changes can be visualized through upregulation of a LacZ reporter gene by phosphorylated OmpR. Unfortunately, basal LacZ expression leads to a detectable reporter signal even when cells are grown in the light, diminishing the contrast of the coliroids. We performed site-directed mutagenesis near the phosphotransfer site of Cph8 to isolate mutants with potentially improved image contrast. Five mutants were examined, but only one of the mutants, T541S, increased the ratio of dark/light gene expression, as measured by β-galactosidase activity. The ratio changed from 2.57 fold in the starting strain to 5.59 in the T541S mutant. The ratio decreased in the four other mutant strains we examined. The phenotype observed in the T541S mutant strain may arise because the serine sidechain is chemically similar but physically smaller than the threonine sidechain. This may minimally change the protein’s local structure, but may be less sterically constrained when compared to threonine, resulting in a higher probability of a phosphotransfer event. Our initial success pairing synthetic biology and site-directed mutagenesis to optimize the bacterial photography system’s performance encourages us to imagine further improvements to the performance of this and other synthetic systems, especially those based on 2CS signaling. PMID:26799494

  18. Sleeping Beauty transposon mutagenesis identifies genes that cooperate with mutant Smad4 in gastric cancer development.

    PubMed

    Takeda, Haruna; Rust, Alistair G; Ward, Jerrold M; Yew, Christopher Chin Kuan; Jenkins, Nancy A; Copeland, Neal G

    2016-04-01

    Mutations in SMAD4 predispose to the development of gastrointestinal cancer, which is the third leading cause of cancer-related deaths. To identify genes driving gastric cancer (GC) development, we performed a Sleeping Beauty (SB) transposon mutagenesis screen in the stomach of Smad4(+/-) mutant mice. This screen identified 59 candidate GC trunk drivers and a much larger number of candidate GC progression genes. Strikingly, 22 SB-identified trunk drivers are known or candidate cancer genes, whereas four SB-identified trunk drivers, including PTEN, SMAD4, RNF43, and NF1, are known human GC trunk drivers. Similar to human GC, pathway analyses identified WNT, TGF-β, and PI3K-PTEN signaling, ubiquitin-mediated proteolysis, adherens junctions, and RNA degradation in addition to genes involved in chromatin modification and organization as highly deregulated pathways in GC. Comparative oncogenomic filtering of the complete list of SB-identified genes showed that they are highly enriched for genes mutated in human GC and identified many candidate human GC genes. Finally, by comparing our complete list of SB-identified genes against the list of mutated genes identified in five large-scale human GC sequencing studies, we identified LDL receptor-related protein 1B (LRP1B) as a previously unidentified human candidate GC tumor suppressor gene. In LRP1B, 129 mutations were found in 462 human GC samples sequenced, and LRP1B is one of the top 10 most deleted genes identified in a panel of 3,312 human cancers. SB mutagenesis has, thus, helped to catalog the cooperative molecular mechanisms driving SMAD4-induced GC growth and discover genes with potential clinical importance in human GC. PMID:27006499

  19. Development of Therapeutic Chimeric Uricase by Exon Replacement/Restoration and Site-Directed Mutagenesis.

    PubMed

    Xie, Guangrong; Yang, Weizhen; Chen, Jing; Li, Miaomiao; Jiang, Nan; Zhao, Baixue; Chen, Si; Wang, Min; Chen, Jianhua

    2016-01-01

    The activity of urate oxidase was lost during hominoid evolution, resulting in high susceptibility to hyperuricemia and gout in humans. In order to develop a more "human-like" uricase for therapeutic use, exon replacement/restoration and site-directed mutagenesis were performed to obtain porcine-human uricase with higher homology to deduced human uricase (dHU) and increased uricolytic activity. In an exon replacement study, substitution of exon 6 in wild porcine uricase (wPU) gene with corresponding exon in dhu totally abolished its activity. Substitutions of exon 5, 3, and 1-2 led to 85%, 60%, and 45% loss of activity, respectively. However, replacement of exon 4 and 7-8 did not significantly change the enzyme activity. When exon 5, 6, and 3 in dhu were replaced by their counterparts in wpu, the resulting chimera H1-2P₃H₄P5-6H7-8 was active, but only about 28% of wPU. Multiple sequence alignment and homology modeling predicted that mutations of E24D and E83G in H1-2P₃H₄P5-6H7-8 were favorable for further increase of its activity. After site-directed mutagenesis, H1-2P₃H₄P5-6H7-8 (E24D & E83G) with increased homology (91.45%) with dHU and higher activity and catalytic efficiency than the FDA-approved porcine-baboon chimera (PBC) was obtained. It showed optimum activity at pH 8.5 and 35 °C and was stable in a pH range of 6.5-11.0 and temperature range of 20-40 °C. PMID:27213357

  20. Enhancing the efficiency and regioselectivity of P450 oxidation catalysts by unnatural amino acid mutagenesis.

    PubMed

    Kolev, Joshua N; Zaengle, Jacqueline M; Ravikumar, Rajesh; Fasan, Rudi

    2014-05-01

    The development of effective strategies for modulating the reactivity and selectivity of cytochrome P450 enzymes represents a key step toward expediting the use of these biocatalysts for synthetic applications. We have investigated the potential of unnatural amino acid mutagenesis to aid efforts in this direction. Four unnatural amino acids with diverse aromatic side chains were incorporated at 11 active-site positions of a substrate-promiscuous CYP102A1 variant. The resulting "uP450s" were then tested for their catalytic activity and regioselectivity in the oxidation of two representative substrates: a small-molecule drug and a natural product. Large shifts in regioselectivity resulted from these single mutations, and in particular, for para-acetyl-Phe substitutions at positions close to the heme cofactor. Screening this mini library of uP450s enabled us to identify P450 catalysts for the selective hydroxylation of four aliphatic positions in the target substrates, including a C(sp(3))-H site not oxidized by the parent enzyme. Furthermore, we discovered a general activity-enhancing effect of active-site substitutions involving the unnatural amino acid para-amino-Phe, which resulted in P450 catalysts capable of supporting the highest total turnover number reported to date on a complex molecule (34,650). The functional changes induced by the unnatural amino acids could not be reproduced by any of the 20 natural amino acids. This study thus demonstrates that unnatural amino acid mutagenesis constitutes a promising new strategy for improving the catalytic activity and regioselectivity of P450 oxidation catalysts. PMID:24692265

  1. Enhancing the Efficiency and Regioselectivity of P450 Oxidation Catalysts via Unnatural Amino Acid Mutagenesis

    PubMed Central

    Kolev, Joshua N.; Zaengle, Jacqueline M.; Ravikumar, Rajesh

    2014-01-01

    The development of effective strategies for modulating the reactivity and selectivity of cytochrome P450 enzymes represents a key step toward expediting the use of these biocatalysts for synthetic applications. In this work, we investigated the potential of unnatural amino acid mutagenesis to aid efforts in this direction. To this end, four unnatural amino acids comprising a diverse set of aromatic side-chain groups were incorporated into eleven active site positions of a substrate-promiscuous CYP102A1 variant. The resulting ‘uP450s’ were then tested for their catalytic activity and regioselectivity in the oxidation of two representative substrates consisting of a small-molecule drug and a natural product. Large shifts in regioselectivity were obtained as a result of these single mutations and, in particular, via para-acetyl-Phe substitutions at positions in close proximity to the heme cofactor. Notably, screening of this mini library of uP450s enabled the rapid identification of P450 catalysts for the selective hydroxylation of four aliphatic positions in the target substrates, including a C(sp3)—H site not oxidized by the parent enzyme. Furthermore, our studies led to the discovery of a general activity-enhancing effect of active site substitutions involving the unnatural amino acid para-amino-Phe, resulting in P450 catalysts capable of supporting the highest total turnover number reported to date on a complex molecule (34,650 turnovers). The functional changes induced by the unnatural amino acids could not be recapitulated by any of the twenty natural amino acids. This study thus demonstrates that unnatural amino acid mutagenesis constitutes a promising, new strategy for improving the catalytic activity and regioselectivity of P450 oxidation catalysts. PMID:24692265

  2. An underlying mechanism for the increased mutagenesis of lagging-strand genes in Bacillus subtilis

    PubMed Central

    Million-Weaver, Samuel; Samadpour, Ariana N.; Moreno-Habel, Daniela A.; Nugent, Patrick; Brittnacher, Mitchell J.; Weiss, Eli; Hayden, Hillary S.; Miller, Samuel I.; Liachko, Ivan; Merrikh, Houra

    2015-01-01

    We previously reported that lagging-strand genes accumulate mutations faster than those encoded on the leading strand in Bacillus subtilis. Although we proposed that orientation-specific encounters between replication and transcription underlie this phenomenon, the mechanism leading to the increased mutagenesis of lagging-strand genes remained unknown. Here, we report that the transcription-dependent and orientation-specific differences in mutation rates of genes require the B. subtilis Y-family polymerase, PolY1 (yqjH). We find that without PolY1, association of the replicative helicase, DnaC, and the recombination protein, RecA, with lagging-strand genes increases in a transcription-dependent manner. These data suggest that PolY1 promotes efficient replisome progression through lagging-strand genes, thereby reducing potentially detrimental breaks and single-stranded DNA at these loci. Y-family polymerases can alleviate potential obstacles to replisome progression by facilitating DNA lesion bypass, extension of D-loops, or excision repair. We find that the nucleotide excision repair (NER) proteins UvrA, UvrB, and UvrC, but not RecA, are required for transcription-dependent asymmetry in mutation rates of genes in the two orientations. Furthermore, we find that the transcription-coupling repair factor Mfd functions in the same pathway as PolY1 and is also required for increased mutagenesis of lagging-strand genes. Experimental and SNP analyses of B. subtilis genomes show mutational footprints consistent with these findings. We propose that the interplay between replication and transcription increases lesion susceptibility of, specifically, lagging-strand genes, activating an Mfd-dependent error-prone NER mechanism. We propose that this process, at least partially, underlies the accelerated evolution of lagging-strand genes. PMID:25713353

  3. Development of Therapeutic Chimeric Uricase by Exon Replacement/Restoration and Site-Directed Mutagenesis

    PubMed Central

    Xie, Guangrong; Yang, Weizhen; Chen, Jing; Li, Miaomiao; Jiang, Nan; Zhao, Baixue; Chen, Si; Wang, Min; Chen, Jianhua

    2016-01-01

    The activity of urate oxidase was lost during hominoid evolution, resulting in high susceptibility to hyperuricemia and gout in humans. In order to develop a more “human-like” uricase for therapeutic use, exon replacement/restoration and site-directed mutagenesis were performed to obtain porcine–human uricase with higher homology to deduced human uricase (dHU) and increased uricolytic activity. In an exon replacement study, substitution of exon 6 in wild porcine uricase (wPU) gene with corresponding exon in dhu totally abolished its activity. Substitutions of exon 5, 3, and 1–2 led to 85%, 60%, and 45% loss of activity, respectively. However, replacement of exon 4 and 7–8 did not significantly change the enzyme activity. When exon 5, 6, and 3 in dhu were replaced by their counterparts in wpu, the resulting chimera H1-2P3H4P5-6H7-8 was active, but only about 28% of wPU. Multiple sequence alignment and homology modeling predicted that mutations of E24D and E83G in H1-2P3H4P5-6H7-8 were favorable for further increase of its activity. After site-directed mutagenesis, H1-2P3H4P5-6H7-8 (E24D & E83G) with increased homology (91.45%) with dHU and higher activity and catalytic efficiency than the FDA-approved porcine–baboon chimera (PBC) was obtained. It showed optimum activity at pH 8.5 and 35 °C and was stable in a pH range of 6.5–11.0 and temperature range of 20–40 °C. PMID:27213357

  4. Targeted mutagenesis in soybean using the CRISPR-Cas9 system.

    PubMed

    Sun, Xianjun; Hu, Zheng; Chen, Rui; Jiang, Qiyang; Song, Guohua; Zhang, Hui; Xi, Yajun

    2015-01-01

    Genome editing is a valuable technique for gene function analysis and crop improvement. Over the past two years, the CRISPR-Cas9 system has emerged as a powerful tool for precisely targeted gene editing. In this study, we predicted 11 U6 genes in soybean (Glycine max L.). We then constructed two vectors (pCas9-GmU6-sgRNA and pCas9-AtU6-sgRNA) using the soybean U6-10 and Arabidopsis U6-26 promoters, respectively, to produce synthetic guide RNAs (sgRNAs) for targeted gene mutagenesis. Three genes, Glyma06g14180, Glyma08g02290 and Glyma12g37050, were selected as targets. Mutations of these three genes were detected in soybean protoplasts. The vectors were then transformed into soybean hairy roots by Agrobacterium rhizogenes infection, resulting in efficient target gene editing. Mutation efficiencies ranged from 3.2-9.7% using the pCas9-AtU6-sgRNA vector and 14.7-20.2% with the pCas9-GmU6-sgRNA vector. Biallelic mutations in Glyma06g14180 and Glyma08g02290 were detected in transgenic hairy roots. Off-target activities associated with Glyma06g14180 and Glyma12g37050 were also detected. Off-target activity would improve mutation efficiency for the construction of a saturated gene mutation library in soybean. Targeted mutagenesis using the CRISPR-Cas9 system should advance soybean functional genomic research, especially that of genes involved in the roots and nodules. PMID:26022141

  5. BAX and tumor suppressor TRP53 are important in regulating mutagenesis in spermatogenic cells in mice.

    PubMed

    Xu, Guogang; Vogel, Kristine S; McMahan, C Alex; Herbert, Damon C; Walter, Christi A

    2010-12-01

    During the first wave of spermatogenesis, and in response to ionizing radiation, elevated mutant frequencies are reduced to a low level by unidentified mechanisms. Apoptosis is occurring in the same time frame that the mutant frequency declines. We examined the role of apoptosis in regulating mutant frequency during spermatogenesis. Apoptosis and mutant frequencies were determined in spermatogenic cells obtained from Bax-null or Trp53-null mice. The results showed that spermatogenic lineage apoptosis was markedly decreased in Bax-null mice and was accompanied by a significantly increased spontaneous mutant frequency in seminiferous tubule cells compared to that of wild-type mice. Apoptosis profiles in the seminiferous tubules for Trp53-null were similar to control mice. Spontaneous mutant frequencies in pachytene spermatocytes and in round spermatids from Trp53-null mice were not significantly different from those of wild-type mice. However, epididymal spermatozoa from Trp53-null mice displayed a greater spontaneous mutant frequency compared to that from wild-type mice. A greater proportion of spontaneous transversions and a greater proportion of insertions/deletions 15 days after ionizing radiation were observed in Trp53-null mice compared to wild-type mice. Base excision repair activity in mixed germ cell nuclear extracts prepared from Trp53-null mice was significantly lower than that for wild-type controls. These data indicate that BAX-mediated apoptosis plays a significant role in regulating spontaneous mutagenesis in seminiferous tubule cells obtained from neonatal mice, whereas tumor suppressor TRP53 plays a significant role in regulating spontaneous mutagenesis between postmeiotic round spermatid and epididymal spermatozoon stages of spermiogenesis. PMID:20739667

  6. Evaluation of conformational epitopes on thyroid peroxidase by antipeptide antibody binding and mutagenesis

    PubMed Central

    GORA, M; GARDAS, A; WIKTOROWICZ, W; HOBBY, P; WATSON, P F; WEETMAN, A P; SUTTON, B J; BANGA, J P

    2004-01-01

    Autoantibodies to thyroid peroxidase (TPO) recognize predominantly conformational epitopes, which are restricted to two distinct determinants, termed immunodominant domain region (IDR) A and B. These dominant determinants reside in the region with structural homology to myeloperoxidase (MPO)-like domain and may extend into the adjacent complement control protein (CCP) domain. We have explored the location of these determinants on the MPO-like domain of the structural model of TPO, by identifying exposed hydrophilic loops that are potential candidates for the autoantigenic sites, generating rabbit antipeptide antisera, and competing with well characterized murine monoclonal antibodies (mabs) specific for these two IDRs. We recently defined the location of IDR-B, and here report our findings on the location of IDR-A and its relationship to IDR-B, defined with a new panel of 15 antipeptide antisera. Moreover, in combination with single amino acid replacements by in vitro mutagenesis, we have defined the limits of the IDR-B region on the TPO model. The combination of antisera to peptides P12 (aa 549–563), P14 (aa 599–617) and P18 (aa 210–225) inhibited the binding of the mab specific for IDR-A (mab 2) by 75. The same combination inhibited the binding of autoantibodies to native TPO from 67 to 94% (mean 81·5%) at autoantibody levels of 5 IU. Fabs prepared from the antipeptide IgG and pooled in this combination were also effective in competition assays, thus defining the epitopes more precisely. IDR-A was found to lie immediately adjacent to IDR-B and thus the two immunodominant epitopes form an extended patch on the surface of TPO. Finally, by single amino acid mutagenesis, we show that IDR-B extends to residue N642, thus further localizing the boundary of this autoantigenic region on the structural model. PMID:15030525

  7. A computer program for the estimation of protein and nucleic acid sequence diversity in random point mutagenesis libraries

    PubMed Central

    Volles, Michael J.; Lansbury, Peter T.

    2005-01-01

    A computer program for the generation and analysis of in silico random point mutagenesis libraries is described. The program operates by mutagenizing an input nucleic acid sequence according to mutation parameters specified by the user for each sequence position and type of point mutation. The program can mimic almost any type of random mutagenesis library, including those produced via error-prone PCR (ep-PCR), mutator Escherichia coli strains, chemical mutagenesis, and doped or random oligonucleotide synthesis. The program analyzes the generated nucleic acid sequences and/or the associated protein library to produce several estimates of library diversity (number of unique sequences, point mutations, and single point mutants) and the rate of saturation of these diversities during experimental screening or selection of clones. This information allows one to select the optimal screen size for a given mutagenesis library, necessary to efficiently obtain a certain coverage of the sequence-space. The program also reports the abundance of each specific protein mutation at each sequence position, which is useful as a measure of the level and type of mutation bias in the library. Alternatively, one can use the program to evaluate the relative merits of preexisting libraries, or to examine various hypothetical mutation schemes to determine the optimal method for creating a library that serves the screen/selection of interest. Simulated libraries of at least 109 sequences are accessible by the numerical algorithm with currently available personal computers; an analytical algorithm is also available which can rapidly calculate a subset of the numerical statistics in libraries of arbitrarily large size. A multi-type double-strand stochastic model of ep-PCR is developed in an appendix to demonstrate the applicability of the algorithm to amplifying mutagenesis procedures. Estimators of DNA polymerase mutation-type-specific error rates are derived using the model. Analyses of an

  8. Role of copper and ceruloplasmin in oxidative mutagenesis induced by the gluthathione-{gamma}-glutamyl transpeptidase system and by other thiols

    SciTech Connect

    Stark, A.A.; Glass, G.A.

    1997-10-01

    Glutathione is activated to a mutagen by {gamma}-glutamyl transpeptidase. Other thiols, such as cysteine, penicillamine, cysteine ethylester, and cysteinylglycine, are direct mutagens in the Ames Salmonella mutagenicity test. Thiol mutagenesis is oxidative in nature and involves H{sub 2}O{sub 2} and possibly hydroxyl radicals. Transition metals are crucial for thiol autoxidation. The role of copper and ceruloplasmin (CP) in thiol-dependent mutagenesis was studied in Salmonella typhimurium strain TA 102. Cu and CP at low concentrations enhanced thiol-dependent mutagenesis in the presence, but not in the absence, and added Fe. The degree of enhancement depended on the type of thiol. At high Cu or CP concentrations, thiol mutagenesis was inhibited. Cu also decreased the mutagenicity of H{sub 2}O{sub 2}. Cu- and CP-enhanced mutagenesis were inhibited by radical scavengers, catalase, and peroxidase but not by superoxide dismutase. The effects of Cu and CP on thiol-dependent mutagenesis were similar to their effects on thiol-driven lipid peroxidation. The results indicate that the role of Cu and CP in the enhancement of thiol mutagenesis is the facilitation of the transfer of electrons from a thiol to iron, rather than in catalysis of the Fenton reaction. 34 refs., 7 figs., 2 tabs.

  9. Insight into the mechanism of phosphoenolpyruvate mutase catalysis derived from site-directed mutagenesis studies of active site residues.

    PubMed

    Jia, Y; Lu, Z; Huang, K; Herzberg, O; Dunaway-Mariano, D

    1999-10-26

    PEP mutase catalyzes the conversion of phosphoenolpyruvate (PEP) to phosphonopyruvate in biosynthetic pathways leading to phosphonate secondary metabolites. A recent X-ray structure [Huang, K., Li, Z., Jia, Y., Dunaway-Mariano, D., and Herzberg, O. (1999) Structure (in press)] of the Mytilus edulis enzyme complexed with the Mg(II) cofactor and oxalate inhibitor reveals an alpha/beta-barrel backbone-fold housing an active site in which Mg(II) is bound by the two carboxylate groups of the oxalate ligand and the side chain of D85 and, via bridging water molecules, by the side chains of D58, D85, D87, and E114. The oxalate ligand, in turn, interacts with the side chains of R159, W44, and S46 and the backbone amide NHs of G47 and L48. Modeling studies identified two feasible PEP binding modes: model A in which PEP replaces oxalate with its carboxylate group interacting with R159 and its phosphoryl group positioned close to D58 and Mg(II) shifting slightly from its original position in the crystal structure, and model B in which PEP replaces oxalate with its phosphoryl group interacting with R159 and Mg(II) retaining its original position. Site-directed mutagenesis studies of the key mutase active site residues (R159, D58, D85, D87, and E114) were carried out in order to evaluate the catalytic roles predicted by the two models. The observed retention of low catalytic activity in the mutants R159A, D85A, D87A, and E114A, coupled with the absence of detectable catalytic activity in D58A, was interpreted as evidence for model A in which D58 functions in nucleophilic catalysis (phosphoryl transfer), R159 functions in PEP carboxylate group binding, and the carboxylates of D85, D87 and E114 function in Mg(II) binding. These results also provide evidence against model B in which R159 serves to mediate the phosphoryl transfer. A catalytic motif, which could serve both the phosphoryl transfer and the C-C cleavage enzymes of the PEP mutase superfamily, is proposed. PMID:10571990

  10. Space mutagenesis of genetically engineered bacteria expressing recombinant human interferon α1b and screening of higher yielding strains.

    PubMed

    Wang, Junfeng; Liu, Changting; Liu, Jinyi; Fang, Xiangqun; Xu, Chen; Guo, Yinghua; Chang, De; Su, Longxiang

    2014-03-01

    The aim of this study was to investigate the space mutagenesis of genetically engineered bacteria expressing recombinant human interferon α1b. The genetically engineered bacteria expressing the recombinant interferon α1b were sent into outer space on the Chinese Shenzhou VIII spacecraft. After the 17 day space flight, mutant strains that highly expressed the target gene were identified. After a series of screening of spaceflight-treated bacteria and the quantitative comparison of the mutant strains and original strain, we found five strains that showed a significantly higher production of target proteins, compared with the original strain. Our results support the notion that the outer space environment has unique effects on the mutation breeding of microorganisms, including genetically engineered strains. Mutant strains that highly express the target protein could be obtained through spaceflight-induced mutagenesis. PMID:24096450

  11. Tailor-Made Protein Tyrosine Phosphatases: In Vitro Site-Directed Mutagenesis of PTEN and PTPRZ-B.

    PubMed

    Luna, Sandra; Mingo, Janire; Aurtenetxe, Olaia; Blanco, Lorena; Amo, Laura; Schepens, Jan; Hendriks, Wiljan J; Pulido, Rafael

    2016-01-01

    In vitro site-directed mutagenesis (SDM) of protein tyrosine phosphatases (PTPs) is a commonly used approach to experimentally analyze PTP functions at the molecular and cellular level and to establish functional correlations with PTP alterations found in human disease. Here, using the tumor-suppressor PTEN and the receptor-type PTPRZ-B (short isoform from PTPRZ1 gene) phosphatases as examples, we provide a brief insight into the utility of specific mutations in the experimental analysis of PTP functions. We describe a standardized, rapid, and simple method of mutagenesis to perform single and multiple amino acid substitutions, as well as deletions of short nucleotide sequences, based on one-step inverse PCR and DpnI restriction enzyme treatment. This method of SDM is generally applicable to any other protein of interest. PMID:27514801

  12. Site-specific incorporation of probes into RNA polymerase by unnatural-amino-acid mutagenesis and Staudinger-Bertozzi ligation

    PubMed Central

    Chakraborty, Anirban; Mazumder, Abhishek; Lin, Miaoxin; Hasemeyer, Adam; Xu, Qumiao; Wang, Dongye; Ebright, Yon W.; Ebright, Richard H.

    2015-01-01

    Summary A three-step procedure comprising (i) unnatural-amino-acid mutagenesis with 4-azido-phenylalanine, (ii) Staudinger-Bertozzi ligation with a probe-phosphine derivative, and (iii) in vitro reconstitution of RNA polymerase (RNAP) enables the efficient site-specific incorporation of a fluorescent probe, a spin label, a crosslinking agent, a cleaving agent, an affinity tag, or any other biochemical or biophysical probe, at any site of interest in RNAP. Straightforward extensions of the procedure enable the efficient site-specific incorporation of two or more different probes in two or more different subunits of RNAP. We present protocols for synthesis of probe-phosphine derivatives, preparation of RNAP subunits and the transcription initiation factor σ, unnatural amino acid mutagenesis of RNAP subunits and σ, Staudinger ligation with unnatural-amino-acid-containing RNAP subunits and σ, quantitation of labelling efficiency and labelling specificity, and reconstitution of RNAP. PMID:25665560

  13. Transcriptional mutagenesis by 8-oxodG in α-synuclein aggregation and the pathogenesis of Parkinson's disease

    PubMed Central

    Basu, Sambuddha; Je, Goun; Kim, Yoon-Seong

    2015-01-01

    Parkinson's disease (PD) is an age-related progressive neurodegenerative disease associated with selective loss of dopaminergic neurons. The characteristic hallmark of the disease is intracytoplasmic proteinacious inclusion bodies called Lewy bodies, primarily consisting of a presynaptic protein α-synuclein. Oxidative stress-mediated damage to macromolecules have been shown to occur frequently in PD. Oxidative damage to DNA in the form of oxidized guanine (8-oxodG) accumulates in both the mitochondrial and nuclear DNA of dopaminergic neurons of the substantia nigra in PD. 8-oxodG-mediated transcriptional mutagenesis has been shown to have the potential to alter phenotype of cells through production of mutant pool of proteins. This review comprehensively summarizes the role of oxidative stress-mediated damage incurred during neurodegeneration, and highlights the scope of transcriptional mutagenesis event in leading to α-synuclein aggregation as seen in PD. PMID:26315598

  14. Optimizing glycosyltransferase specificity via ‘hot spot’ saturation mutagenesis presents a new catalyst for novobiocin glycorandomization

    PubMed Central

    Williams, Gavin J.; Goff, Randal D.; Zhang, Changsheng; Thorson, Jon S.

    2010-01-01

    Summary A comprehensive two phase ‘hot spot’ saturation mutagenesis strategy to rapidly evolve glycosyltransferase specificity for non-natural acceptors is described. Specifically, the application of a high throughput screen (based upon the fluorescent acceptor umbelliferone) was used to identify key amino acid ‘hot spots’ that contribute to GT proficiency and/or promiscuity. Saturation mutagenesis of the corresponding hot spots facilitated the utilization of a lower throughput screen to provide OleD prodigy capable of efficiently glycosylating the non-natural acceptor novobiocic acid with an array of unique sugars. Incredibly, even in the absence of a high-throughput screen for novobiocic acid glycosylation, this approach rapidly led to improvements in the desired catalytic activity of several hundred-fold. PMID:18420146

  15. Construction and characterization of Pseudomonas aeruginosa protein F-deficient mutants after in vitro and in vivo insertion mutagenesis of the cloned gene.

    PubMed Central

    Woodruff, W A; Hancock, R E

    1988-01-01

    Mutants with insertion mutations in the Pseudomonas aeruginosa protein F (oprF) gene were created in vivo by Tn1 mutagenesis of the cloned gene in Escherichia coli and in vitro by insertion of the streptomycin resistance-encoding omega fragment into the cloned gene, followed by transfer of the mutated protein F gene back to P. aeruginosa. Homologous recombination into the P. aeruginosa chromosome was driven by a bacteriophage F116L transduction method in the oprF::Tn1 mutants or Tn5-instability in the oprF::omega mutants. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western immunoblotting demonstrated that the resultant oprF insertion mutants had lost protein F, whereas restriction digestion and Southern blotting experiments proved that the mutants contained a single chromosomal oprF gene with either Tn1 or omega inserted into it. It has been proposed that protein F has a role in antibiotic uptake in P. aeruginosa. Measurement of antibiotic resistance levels showed small to marginal increases in resistance, compared with that of the parent P. aeruginosa strain, to a variety of beta-lactam antibiotics. Protein F-deficient mutants had altered barrier properties as revealed by a three- to fivefold increase in the uptake of the hydrophobic fluorescent probe 1-N-phenylnaphthylamine. Images PMID:2836364

  16. Identification of the O antigen polymerase (rfc) gene in Escherichia coli O4 by insertional mutagenesis using a nonpolar chloramphenicol resistance cassette.

    PubMed Central

    Lukomski, S; Hull, R A; Hull, S I

    1996-01-01

    Computer analysis of the O4 polysaccharide gene cluster of Escherichia coli revealed the presence of two open reading frames (ORFs) encoding strongly hydrophobic polypeptides. O antigen polymerase, which is encoded by the rfc gene, is a potential membrane protein and therefore should be hydrophobic. To identify the rfc gene, these two ORFs were subjected to insertional mutagenesis. A chloramphenicol resistance cassette was designed which, when properly inserted, does not cause a polar effect in downstream genes. Each of two ORFs, cloned into a plasmid vector, was inactivated with this cassette. Two types of mutants bearing chromosomal insertions of the cassettes in each ORF were constructed by homologous recombination. These mutants were characterized by PCR, Southern blotting, and transverse-alternating-field electrophoresis. Only one class of mutants exhibited the expected O polymerase-deficient phenotype; they produced O4-specific, semirough lipopolysaccharide. Therefore, this ORF was identified as the rfc gene. The chromosomal rfc mutation was complemented in trans by the rfc gene expressed from a plasmid vector. PMID:8550424

  17. Molecular cloning, nucleotide sequence, and marker exchange mutagenesis of the exo-poly-alpha-D-galacturonosidase-encoding pehX gene of Erwinia chrysanthemi EC16.

    PubMed Central

    He, S Y; Collmer, A

    1990-01-01

    The pehX gene encoding extracellular exo-poly-alpha-D-galacturonosidase (exoPG; EC 3.2.1.82) was isolated from a genomic library of the pectate lyase-deficient Erwinia chrysanthemi mutant UM1005 (a Nalr Kanr delta pelABCE derivative of EC16) by immunoscreening 2,800 Escherichia coli HB101 transformants with an antibody against exoPG protein. The cloned pehX gene was expressed highly from its own promoter in E. coli, and most of the enzyme was localized in the periplasm. The nucleotide sequence of pehX revealed the presence of an amino-terminal signal peptide and an open reading frame encoding a preprotein of 64,608 daltons. The cloned pehX gene was insertionally inactivated with TnphoA and used to mutate the chromosomal pehX gene of E. chrysanthemi AC4150 (Nalr) and CUCPB5006 (Nalr Kans delta pelABCE) by marker exchange mutagenesis. Analysis of the resulting mutants, CUCPB5008 (Pel+ Peh-) and CUCPB5009 (Pel- Peh-), indicated that exoPG can contribute significantly to bacterial utilization of polygalacturonate and the induction of pectate lyase in the presence of extracellular pectic polymers. CUCPB5009 retained a slight ability to pit polygalacturonate semisolid agar and macerated chrysanthemum pith tissues when large numbers of bacteria were inoculated. Images PMID:2168372

  18. Identification of active site lysyl residues of phenylalanine dehydrogenase by chemical modification with methyl acetyl phosphate combined with site-directed mutagenesis.

    PubMed

    Kataoka, K; Tanizawa, K; Fukui, T; Ueno, H; Yoshimura, T; Esaki, N; Soda, K

    1994-12-01

    A monoanionic acetylation reagent, methyl acetyl phosphate, was used to acetylate lysyl residues of the recombinant thermostable phenylalanine dehydrogenase from Thermoactinomyces intermedius. The enzyme was irreversibly inactivated with the reagent in a time- and dose-dependent manner. Simultaneous addition of substrate and coenzyme markedly protected the enzyme from inactivation. Acetylated lysyl residues presumably occurring at the active site were determined by differential modification; the enzyme was first modified with a cold reagent in the presence of both substrate and coenzyme and, after removal of the added substances by gel filtration, was then labeled with a radioactive reagent. At least 7 lysyl residues per enzyme subunit were radiolabeled by this method. To further specify the lysyl residue(s) whose modification results in inactivation of the enzyme, 5 lysyl residues highly conserved in various amino acid dehydrogenase sequences were replaced with Ala by site-directed mutagenesis. Although all of the single mutant enzymes were inactivated with the reagent as effectively as the wild-type enzyme, a double mutant enzyme in which both Lys-69 and Lys-81 were replaced with Ala was found to be inactivated very slowly. These results suggest that the reagent can acetylate both of these lysyl residues and inactivate the enzyme. Kinetic analyses of the single Lys-69 and Lys-81 mutant enzymes revealed that they are involved in substrate binding and catalysis, respectively, like the corresponding residues in the homologous leucine dehydrogenase. PMID:7706231

  19. Increasing frequencies of site-specific mutagenesis and gene targeting in Arabidopsis by manipulating DNA repair pathways.

    PubMed

    Qi, Yiping; Zhang, Yong; Zhang, Feng; Baller, Joshua A; Cleland, Spencer C; Ryu, Yungil; Starker, Colby G; Voytas, Daniel F

    2013-03-01

    Improved methods for engineering sequence-specific nucleases, including zinc finger nucleases (ZFNs) and TAL effector nucleases (TALENs), have made it possible to precisely modify plant genomes. However, the success of genome modification is largely dependent on the intrinsic activity of the engineered nucleases. In this study, we sought to enhance ZFN-mediated targeted mutagenesis and gene targeting (GT) in Arabidopsis by manipulating DNA repair pathways. Using a ZFN that creates a double-strand break (DSB) at the endogenous ADH1 locus, we analyzed repair outcomes in the absence of DNA repair proteins such as KU70 and LIG4 (both involved in classic nonhomologous end-joining, NHEJ) and SMC6B (involved in sister-chromatid-based homologous recombination, HR). We achieved a fivefold to 16-fold enhancement in HR-based GT in a ku70 mutant and a threefold to fourfold enhancement in GT in the lig4 mutant. Although the NHEJ mutagenesis frequency was not significantly changed in ku70 or lig4, DNA repair was shifted to microhomology-dependent alternative NHEJ. As a result, mutations in both ku70 and lig4 were predominantly large deletions, which facilitates easy screening for mutations by PCR. Interestingly, NHEJ mutagenesis and GT at the ADH1 locus were enhanced by sixfold to eightfold and threefold to fourfold, respectively, in a smc6b mutant. The increase in NHEJ-mediated mutagenesis by loss of SMC6B was further confirmed using ZFNs that target two other Arabidopsis genes, namely, TT4 and MPK8. Considering that components of DNA repair pathways are highly conserved across species, mutations in DNA repair genes likely provide a universal strategy for harnessing repair pathways to achieve desired targeted genome modifications. PMID:23282329

  20. Analysis of repair and mutagenesis of chromium-induced DNA damage in yeast, mammalian cells, and transgenic mice.

    PubMed Central

    Cheng, L; Liu, S; Dixon, K

    1998-01-01

    Chromium (Cr) is a widespread environmental contaminant and a known human carcinogen. We have used shuttle vector systems in yeast, mammalian cells, and transgenic mice to characterize the mutational specificity and premutational DNA damage induced by Cr(VI) and its reduction intermediates in order to elucidate the mechanism by which Cr induces mutations. In the yeast system, treatment of vector-containing cells with Cr(VI) results in a dose-dependent increase in mutations in the SUP4-o target gene of the vector; mutagenesis is enhanced in an apn-1 yeast mutant, deficient in the capacity to repair oxidative-type DNA damage. In vector-containing mammalian cells, treatment with Cr(VI) also results in a dose-dependent increase in mutations in the vector target gene supF. The Cr-induced mutations in supF occurred mostly at G:C base pairs and were widely distributed across the gene, a pattern similar to those observed with ionizing radiation or hydrogen peroxide. These results support the hypothesis that Cr(VI)-induced oxidative-type DNA damage is responsible for Cr mutagenesis in the cell. Recently these studies were extended into the Big Blue transgenic mouse system in which Cr-induced mutagenesis was observed in the lung, the target organ for Cr carcinogenesis in humans. Analysis of the spectrum of these mutations will test whether Cr mutagenesis occurs by similar mechanisms in the intact animal as in cell culture systems and yeast. Images Figure 2 Figure 3 PMID:9703488

  1. Development of an Efficient In Vivo System (Pjunc-TpaseIS1223) for Random Transposon Mutagenesis of Lactobacillus casei

    PubMed Central

    Licandro-Seraut, Hélène; Brinster, Sophie; van de Guchte, Maarten; Scornec, Hélène; Maguin, Emmanuelle; Sansonetti, Philippe

    2012-01-01

    The random transposon mutagenesis system Pjunc-TpaseIS1223 is composed of plasmids pVI129, expressing IS1223 transposase, and pVI110, a suicide transposon plasmid carrying the Pjunc sequence, the substrate of the IS1223 transposase. This system is particularly efficient in Lactobacillus casei, as more than 10,000 stable, random mutants were routinely obtained via electroporation. PMID:22610425

  2. Application of an in vivo mutagenesis system to assess aminothiol effects on neutron-induced genotoxic damage in mouse spleenocytes

    SciTech Connect

    Basic, I. . Dept. of Animal Physiology); Grdina, D.J.; Lyons, T. )

    1989-01-01

    A cloning technique has been developed to quantitate and study {ital in vivo} somatic mutations at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in human lymphocytes. In this paper we describe a modification of this assay to quantify HGPRT mutations in mouse spleenocytes. In particular, we have investigated the effects of the aminothiol on mutagenesis induced by single doses of whole body exposures to fission-spectrum neutrons from the JANUS reactor at Argonne National Laboratory. 7 refs., 3 tabs.

  3. Site-directed mutagenesis identifies a tyrosine radical involved in the photosynthesized oxygen-evolving system

    SciTech Connect

    Debus, R.J.; Barry, B.A.; Babcock, G.T.; McIntosh, L.

    1988-01-01

    Photosynthetic oxygen evolution takes place in the thylakoid protein complex known as photosystem II. The reaction center core of this photosystem, where photochemistry occurs, is a heterodimer of homologous polypeptides called D1 and D2. Besides chlorophyll and quinone, photosystem II contains other organic cofactors, including two known as Z and D. Z transfers electrons from the site of water oxidation to the oxidized reaction center primary donor, P/sub 680//sup +/, while D /center dot//sup +/ gives rise to the dark-stable EPR spectrum known as signal II. D/center dot//sup +/ has recently been shown to be a tyrosine radical. Z is probably a second tyrosine located in a similar environment. Indirect evidence indicates that Z and D are associated with the D1 and E2 polypeptides, respectively. To identify the specific tyrosine residue corresponding to D, the authors have changed Tyr-160 of the D2 polypeptide to phenylalanine by site-directed mutagenesis of a psbD gene in the cyanobacterium Synechocystis 6803. The resulting mutant grows photosynthetically, but it lacks the EPR signal of D/center dot//sup +/. The authors conclude that D is Tyr-160 of the D2 polypeptide. They suggest that the C/sub 2/ symmetry in photosystem II extends beyond P/sub 680/ to its immediate electron donor and conclude that Z is Try-161 of the D1 polypeptide.

  4. Enhanced maltose production through mutagenesis of acceptor binding subsite +2 in Bacillus stearothermophilus maltogenic amylase.

    PubMed

    Sun, Yecheng; Duan, Xuguo; Wang, Lei; Wu, Jing

    2016-01-10

    Maltogenic amylases are used to decrease the maltotriose content of high maltose syrups. However, due to the interplay between the hydrolysis and transglycosylation activities of maltogenic amylases, the maltotriose contents of these syrups are still greater than that necessary for pure maltose preparation. In this study, the maltogenic amylase from Bacillus stearothermophilus was engineered to decrease its transglycosylation activity with the expectation that this would enhance maltose production. Site-directed mutagenesis was used to generate Trp 177 variants W177F, W177Y, W177L, W177N, and W177S. The transglycosylation activities of the mutant enzymes decreased as the hydrophilicity of the residue at position 177 increased. The mutant enzymes exhibited notable enhancements in maltose production, with a minimum of maltotriose contents of 0.2%, compared with 3.2% for the wild-type enzyme. Detailed characterization of the mutant enzymes suggests that the best of them, W177S, will deliver performance superior to that of the wild-type under industrial conditions. PMID:26597712

  5. [Enhancing glutamate decarboxylase activity by site-directed mutagenesis: an insight from Ramachandran plot].

    PubMed

    Ke, Piyu; Huang, Jun; Hu, Sheng; Zhao, Weirui; Lü, Changjiang; Yu, Kai; Lei, Yinlin; Wang, Jinbo; Mei, Lehe

    2016-01-01

    Glutamate decarboxylase (GAD) can catalyze the decarboxylation of glutamate into γ-aminobutyrate (GABA) and is the only enzyme of GABA biosynthesis. Improving GAD activity and thermostability will be helpful for the highly efficient biosynthesis of GABA. According to the Ramachandran plot information of GAD 1407 three-dimensional structure from Lactobacillus brevis CGMCC No. 1306, we identified the unstable site K413 as the mutation target, constructed the mutant GAD by site-directed mutagenesis and measured the thermostability and activity of the wide type and mutant GAD. Mutant K413A led to a remarkably slower inactivation rate, and its half-life at 50 °C reached 105 min which was 2.1-fold higher than the wild type GAD1407. Moreover, mutant K413I exhibited 1.6-fold higher activity in comparison with the wide type GAD1407, although it had little improvement in thermostability of GAD. Ramachandran plot can be considered as a potential approach to increase GAD thermostability and activity. PMID:27443004

  6. 2-(N-acetoxy-N-acetylamino)fluorene mutagenesis in mammalian cells: sequence-specific hot spot.

    PubMed Central

    Gentil, A; Margot, A; Sarasin, A

    1986-01-01

    Mutations induced by 2-(N-acetoxy-N-acetylamino)fluorene were studied using temperature-sensitive simian virus 40 (SV40) mutants as probe in monkey kidney cells. In vitro treatment of the SV40 virions with 2-(N-acetoxy-N-acetylamino)fluorene increased mutagenesis and decreased survival in the viral progeny. A lethal hit of approximately 85 acetylaminofluorene adducts per SV40 genome was calculated. UV irradiation of cells prior to infection did not modify the results. Molecular analysis of independent SV40 revertants showed that 2-(N-acetoxy-N-acetylamino)fluorene induces base substitutions that are located not opposite putative acetylaminofluorene adducts but next to them. Moreover, a hot spot of mutation restoring a true wild-type genotype was observed in 10 of the 16 revertants analyzed. This hot spot, not targeted opposite a major DNA lesion, was not observed using UV light as damaging agent in the same genetic assay. Two models involving the stabilization, by acetylaminofluorene adducts, of the secondary structure of a specific quasipalindromic SV40 sequence are proposed to explain this sequence-specific hot spot. PMID:3025845

  7. The significance of disulfide bonding in biological activity of HB-EGF, a mutagenesis approach

    SciTech Connect

    Hoskins, J.T.; Zhou, Z.; Harding, P.A.

    2008-10-31

    A site-directed mutagenesis approach was taken to disrupt each of 3 disulfide bonds within human HB-EGF by substituting serine for both cysteine residues that contribute to disulfide bonding. Each HB-EGF disulfide analogue (HB-EGF-Cys/Ser{sub 108/121}, HB-EGF-Cys/Ser{sub 116/132}, and HB-EGF-Cys/Ser{sub 134/143}) was cloned under the regulation of the mouse metallothionein (MT) promoter and stably expressed in mouse fibroblasts. HB-EGF immunoreactive proteins with M{sub r} of 6.5, 21 and 24 kDa were observed from lysates of HB-EGF and each HB-EGF disulfide analogue. HB-EGF immunohistochemical analyses of each HB-EGF stable cell line demonstrated ubiquitous protein expression except HB-EGF-Cys/Ser{sub 108/121} and HB-EGF-Cys/Ser{sub 116/132} stable cell lines which exhibited accumulated expression immediately outside the nucleus. rHB-EGF, HB-EGF, and HB-EGF{sub 134/143} proteins competed with {sup 125}I-EGF in an A431 competitive binding assay, whereas HB-EGF-Cys/Ser{sub 108/121} and HB-EGF-Cys/Ser{sub 116/132} failed to compete. Each HB-EGF disulfide analogue lacked the ability to stimulate tyrosine phosphorylation of the 170 kDa EGFR. These results suggest that HB-EGF-Cys/Ser{sub 134/143} antagonizes EGFRs.

  8. Mutagenesis of mNeptune Red-Shifts Emission Spectrum to 681-685 nm

    PubMed Central

    Li, ZhaoYang; Zhang, ZhiPing; Bi, LiJun; Cui, ZongQiang; Deng, JiaoYu; Wang, DianBing; Zhang, Xian-En

    2016-01-01

    GFP-like fluorescent proteins with diverse emission wavelengths have been developed through mutagenesis, offering many possible choices in cellular and tissue imaging, such as multi-targets imaging, deep tissue imaging that require longer emission wavelength. Here, we utilized a combined approach of random mutation and structure-based rational design to develop new NIR fluorescent proteins on the basis of a far-red fluorescent protein, mNeptune (Ex/Em: 600/650 nm). We created a number of new monomeric NIR fluorescent proteins with the emission range of 681–685 nm, which exhibit the largest Stocks shifts (77–80 nm) compared to other fluorescent proteins. Among them, mNeptune681 and mNeptune684 exhibit more than 30 nm redshift in emission relative to mNeptune, owing to the major role of the extensive hydrogen-bond network around the chromophore and contributions of individual mutations to the observed redshift. Furthermore, the two variants still maintain monomeric state in solution, which is a trait crucial for their use as protein tags. In conclusion, our results suggest that there is untapped potential for developing fluorescent proteins with desired properties. PMID:27119418

  9. Mutagenesis of mNeptune Red-Shifts Emission Spectrum to 681-685 nm.

    PubMed

    Li, ZhaoYang; Zhang, ZhiPing; Bi, LiJun; Cui, ZongQiang; Deng, JiaoYu; Wang, DianBing; Zhang, Xian-En

    2016-01-01

    GFP-like fluorescent proteins with diverse emission wavelengths have been developed through mutagenesis, offering many possible choices in cellular and tissue imaging, such as multi-targets imaging, deep tissue imaging that require longer emission wavelength. Here, we utilized a combined approach of random mutation and structure-based rational design to develop new NIR fluorescent proteins on the basis of a far-red fluorescent protein, mNeptune (Ex/Em: 600/650 nm). We created a number of new monomeric NIR fluorescent proteins with the emission range of 681-685 nm, which exhibit the largest Stocks shifts (77-80 nm) compared to other fluorescent proteins. Among them, mNeptune681 and mNeptune684 exhibit more than 30 nm redshift in emission relative to mNeptune, owing to the major role of the extensive hydrogen-bond network around the chromophore and contributions of individual mutations to the observed redshift. Furthermore, the two variants still maintain monomeric state in solution, which is a trait crucial for their use as protein tags. In conclusion, our results suggest that there is untapped potential for developing fluorescent proteins with desired properties. PMID:27119418

  10. Multiplexed DNA repair assays for multiple lesions and multiple doses via transcription inhibition and transcriptional mutagenesis.

    PubMed

    Nagel, Zachary D; Margulies, Carrie M; Chaim, Isaac A; McRee, Siobhan K; Mazzucato, Patrizia; Ahmad, Anwaar; Abo, Ryan P; Butty, Vincent L; Forget, Anthony L; Samson, Leona D

    2014-05-01

    The capacity to repair different types of DNA damage varies among individuals, making them more or less susceptible to the detrimental health consequences of damage exposures. Current methods for measuring DNA repair capacity (DRC) are relatively labor intensive, often indirect, and usually limited to a single repair pathway. Here, we describe a fluorescence-based multiplex flow-cytometric host cell reactivation assay (FM-HCR) that measures the ability of human cells to repair plasmid reporters, each bearing a different type of DNA damage or different doses of the same type of DNA damage. FM-HCR simultaneously measures repair capacity in any four of the following pathways: nucleotide excision repair, mismatch repair, base excision repair, nonhomologous end joining, homologous recombination, and methylguanine methyltransferase. We show that FM-HCR can measure interindividual DRC differences in a panel of 24 cell lines derived from genetically diverse, apparently healthy individuals, and we show that FM-HCR may be used to identify inhibitors or enhancers of DRC. We further develop a next-generation sequencing-based HCR assay (HCR-Seq) that detects rare transcriptional mutagenesis events due to lesion bypass by RNA polymerase, providing an added dimension to DRC measurements. FM-HCR and HCR-Seq provide powerful tools for exploring relationships among global DRC, disease susceptibility, and optimal treatment. PMID:24757057

  11. Multiplexed DNA repair assays for multiple lesions and multiple doses via transcription inhibition and transcriptional mutagenesis

    PubMed Central

    Nagel, Zachary D.; Margulies, Carrie M.; Chaim, Isaac A.; McRee, Siobhan K.; Mazzucato, Patrizia; Ahmad, Anwaar; Abo, Ryan P.; Butty, Vincent L.; Forget, Anthony L.; Samson, Leona D.

    2014-01-01

    The capacity to repair different types of DNA damage varies among individuals, making them more or less susceptible to the detrimental health consequences of damage exposures. Current methods for measuring DNA repair capacity (DRC) are relatively labor intensive, often indirect, and usually limited to a single repair pathway. Here, we describe a fluorescence-based multiplex flow-cytometric host cell reactivation assay (FM-HCR) that measures the ability of human cells to repair plasmid reporters, each bearing a different type of DNA damage or different doses of the same type of DNA damage. FM-HCR simultaneously measures repair capacity in any four of the following pathways: nucleotide excision repair, mismatch repair, base excision repair, nonhomologous end joining, homologous recombination, and methylguanine methyltransferase. We show that FM-HCR can measure interindividual DRC differences in a panel of 24 cell lines derived from genetically diverse, apparently healthy individuals, and we show that FM-HCR may be used to identify inhibitors or enhancers of DRC. We further develop a next-generation sequencing-based HCR assay (HCR-Seq) that detects rare transcriptional mutagenesis events due to lesion bypass by RNA polymerase, providing an added dimension to DRC measurements. FM-HCR and HCR-Seq provide powerful tools for exploring relationships among global DRC, disease susceptibility, and optimal treatment. PMID:24757057

  12. Transposon mutagenesis of Campylobacter jejuni identifies a bipartite energy taxis system required for motility.

    PubMed

    Hendrixson, D R; Akerley, B J; DiRita, V J

    2001-04-01

    Campylobacter jejuni constitutes the leading cause of bacterial gastroenteritis in the United States and a major cause of diarrhoea worldwide. Little is known about virulence mechanisms in this organism because of the scarcity of suitable genetic tools. We have developed an efficient system of in vitro transposon mutagenesis using a mariner-based transposon and purified mariner transposase. Through in vitro transposition of C. jejuni chromosomal DNA followed by natural transformation of the transposed DNA, large random transposon mutant libraries consisting of approximately 16 000 individual mutants were generated. The first genetic screen of C. jejuni using a transposon-generated mutant library identified 28 mutants defective for flagellar motility, one of the few known virulence determinants of this pathogen. We developed a second genetic system, which allows for the construction of defined chromosomal deletions in C. jejuni, and demonstrated the requirement of sigma28 and sigma54 for motility. In addition, we show that sigma28 is involved in the transcription of flaA and that sigma54 is required for transcription of three other flagellar genes, flaB and flgDE. We also identified two previously uncharacterized genes required for motility encoding proteins that we call CetA and CetB, which mediate energy taxis responses. Through our analysis of the Cet proteins, we propose a unique mechanism for sensing energy levels and mediating energy taxis in C. jejuni. PMID:11298288

  13. Mutagenesis of beryllium-specific TCRs suggests an unusual binding topology for antigen recognition.

    PubMed

    Bowerman, Natalie A; Falta, Michael T; Mack, Douglas G; Kappler, John W; Fontenot, Andrew P

    2011-10-01

    Unconventional Ags, such as metals, stimulate T cells in a very specific manner. To delineate the binding landscape for metal-specific T cell recognition, alanine screens were performed on a set of Be-specific TCRs derived from the lung of a chronic beryllium disease patient. These TCRs are HLA-DP2-restricted and express nearly identical TCR Vβ5.1 chains coupled with different TCR α-chains. Site-specific mutagenesis of all amino acids comprising the CDRs of the TCRA and TCRB genes showed a dominant role for Vβ5.1 residues in Be recognition, with little contribution from the TCR α-chain. Solvent-exposed residues along the α-helices of the HLA-DP2 α- and β-chains were also mutated to alanine. Two β-chain residues, located near the proposed Be binding site of HLA-DP2, played a dominant role in T cell recognition with no contribution from the HLA-DP2 α-chain. These findings suggest that Be-specific T cells recognize Ag using an unconventional binding topology, with the majority of interactions contributed by TCR Vβ5.1 residues and the HLA-DP2 β1-chain. Thus, unusual docking topologies are not exclusively used by autoreactive T cells, but also for the recognition of unconventional metal Ags, such as Be. PMID:21873524

  14. Structure-function study of MalF protein by random mutagenesis.

    PubMed

    Tapia, M I; Mourez, M; Hofnung, M; Dassa, E

    1999-04-01

    MalF is one of the two integral inner membrane proteins of the maltose-maltodextrin transport system. To identify functional regions in this protein, we characterized a collection of malF mutants obtained by random mutagenesis. We analyzed their growth on maltose and maltodextrins, the steady-state levels and subcellular localization of the mutant proteins, and the subcellular localization of MalK. Only 2 of the 21 MalF mutant proteins allowed growth on maltose and maltodextrins. Most mutations resulting in immunodetectable proteins mapped to hydrophilic domains, indicating that insertions affecting transmembrane segments gave rise to unstable or lethal proteins. All MalF mutant proteins, even those C-terminally truncated or with large N-terminal deletions, were inserted into the cytoplasmic membrane. Having identified mutations leading to reduced steady-state level, to partial mislocation, and/or to misfolding, we were able to assign to some regions of MalF a role in the assembly of the MalFGK2 complex and/or in the transport mechanism. PMID:10094708

  15. Functional characterization of the Sindbis virus E2 glycoprotein by transposon linker-insertion mutagenesis

    SciTech Connect

    Navaratnarajah, Chanakha K.; Kuhn, Richard J. . E-mail: kuhnr@purdue.edu

    2007-06-20

    The glycoprotein envelope of alphaviruses consists of two proteins, E1 and E2. E1 is responsible for fusion and E2 is responsible for receptor binding. An atomic structure is available for E1, but one for E2 has not been reported. In this study, transposon linker-insertion mutagenesis was used to probe the function of different domains of E2. A library of mutants, containing 19 amino acid insertions in the E2 glycoprotein sequence of the prototype alphavirus, Sindbis virus (SINV), was generated. Fifty-seven independent E2 insertions were characterized, of which more than half (67%) gave rise to viable virus. The wild-type-like mutants identify regions that accommodate insertions without perturbing virus production and can be used to insert targeting moieties to direct SINV to specific receptors. The defective and lethal mutants give insight into regions of E2 important for protein stability, transport to the cell membrane, E1-E2 contacts, and receptor binding.

  16. Spectrum of T-DNA integrations for insertional mutagenesis of Histoplasma capsulatum

    PubMed Central

    Kemski, Megan M.; Stevens, Bryan; Rappleye, Chad A.

    2012-01-01

    Agrobacterium-mediated transformation is being increasingly used for insertional mutagenesis of fungi. To better evaluate its effectiveness as a mutagen for the fungal pathogen Histoplasma capsulatum, we analyzed a collection of randomly selected T-DNA insertion mutants. Testing of different T-DNA element vectors engineered for transformation of fungi showed that pBHt2 provides the highest transformation efficiency and the lowest rate of vector backbone carryover. Sixty-eight individual T-DNA integrations were characterized by recovery of T-DNA ends and flanking genomic sequences. The right border end of the T-DNA is largely preserved whereas the left border end is frequently truncated. Analysis of T-DNA insertion sites confirms the lack of any integration hotspots in the Histoplasma genome. Relative to genes, T-DNA integrations show significant bias towards promoter regions at the expense of coding sequences. With consideration for potential promoter interruption and the demonstrated efficacy of intronic insertions, 61% of mapped T-DNA insertions should impair gene expression or function. Mapping of T-DNA flanking sequences demonstrates 67% of T-DNA integrations are integrations at a single chromosomal site and 31% of T-DNA integrations are associated with large-scale chromosomal rearrangements. This characterization of T-DNA insertions in mutants selected without regard to phenotype supports application of Agrobacterium-mediated transformation as an insertional mutagen for genome-based screens and functional discovery of genes in Histoplasma. PMID:23332832

  17. Rapid cloning of disease-resistance genes in plants using mutagenesis and sequence capture.

    PubMed

    Steuernagel, Burkhard; Periyannan, Sambasivam K; Hernández-Pinzón, Inmaculada; Witek, Kamil; Rouse, Matthew N; Yu, Guotai; Hatta, Asyraf; Ayliffe, Mick; Bariana, Harbans; Jones, Jonathan D G; Lagudah, Evans S; Wulff, Brande B H

    2016-06-01

    Wild relatives of domesticated crop species harbor multiple, diverse, disease resistance (R) genes that could be used to engineer sustainable disease control. However, breeding R genes into crop lines often requires long breeding timelines of 5-15 years to break linkage between R genes and deleterious alleles (linkage drag). Further, when R genes are bred one at a time into crop lines, the protection that they confer is often overcome within a few seasons by pathogen evolution. If several cloned R genes were available, it would be possible to pyramid R genes in a crop, which might provide more durable resistance. We describe a three-step method (MutRenSeq)-that combines chemical mutagenesis with exome capture and sequencing for rapid R gene cloning. We applied MutRenSeq to clone stem rust resistance genes Sr22 and Sr45 from hexaploid bread wheat. MutRenSeq can be applied to other commercially relevant crops and their relatives, including, for example, pea, bean, barley, oat, rye, rice and maize. PMID:27111722

  18. Insertional mutagenesis identifies multiple networks of cooperating genes driving intestinal tumorigenesis.

    PubMed

    March, H Nikki; Rust, Alistair G; Wright, Nicholas A; ten Hoeve, Jelle; de Ridder, Jeroen; Eldridge, Matthew; van der Weyden, Louise; Berns, Anton; Gadiot, Jules; Uren, Anthony; Kemp, Richard; Arends, Mark J; Wessels, Lodewyk F A; Winton, Douglas J; Adams, David J

    2011-12-01

    The evolution of colorectal cancer suggests the involvement of many genes. To identify new drivers of intestinal cancer, we performed insertional mutagenesis using the Sleeping Beauty transposon system in mice carrying germline or somatic Apc mutations. By analyzing common insertion sites (CISs) isolated from 446 tumors, we identified many hundreds of candidate cancer drivers. Comparison to human data sets suggested that 234 CIS-targeted genes are also dysregulated in human colorectal cancers. In addition, we found 183 CIS-containing genes that are candidate Wnt targets and showed that 20 CISs-containing genes are newly discovered modifiers of canonical Wnt signaling. We also identified mutations associated with a subset of tumors containing an expanded number of Paneth cells, a hallmark of deregulated Wnt signaling, and genes associated with more severe dysplasia included those encoding members of the FGF signaling cascade. Some 70 genes had co-occurrence of CIS pairs, clustering into 38 sub-networks that may regulate tumor development. PMID:22057237

  19. Identification of Legionella pneumophila Genes Important for Infection of Amoebas by Signature-Tagged Mutagenesis

    PubMed Central

    Polesky, Andrea H.; Ross, Julianna T. D.; Falkow, Stanley; Tompkins, Lucy S.

    2001-01-01

    Legionella pneumophila is a facultative intracellular gram-negative rod that causes pneumonia in humans. Free-living amoebas are thought to serve as a reservoir for Legionella infections. Signature-tagged mutagenesis was employed to identify Legionella pneumophila genes necessary for survival in the amoeba Acanthamoeba castellanii. Six mutant strains were defective in assays of invasion and intracellular growth. Four mutants also exhibited invasion and replication defects in Hartmannella vermiformis, an amoeba linked to hospital outbreaks of Legionella pneumonia. The six mutants also were tested in macrophages derived from peripheral blood mononuclear cells. Two mutants had intracellular replication defects, and two different strains entered cells less efficiently. Two transposon insertions were in known L. pneumophila genes, lspK and aroB. The other four were in novel genes. One gene has similarity to a cytochrome c-type biogenesis protein of Pseudomonas fluorescens. Another has similarity to a transcriptional activator regulating flagellar biosynthesis in Vibrio cholera. The third is similar to traA of Rhizobium sp. strain NGR234, which is involved in conjugal transfer of DNA. The fourth has no homology. By using survival in amoeba as a selection, we have isolated mutant strains with a range of phenotypes; and we have potentially identified new L. pneumophila virulence genes. PMID:11159993

  20. Rates and mechanisms of bacterial mutagenesis from maximum-depth sequencing.

    PubMed

    Jee, Justin; Rasouly, Aviram; Shamovsky, Ilya; Akivis, Yonatan; Steinman, Susan R; Mishra, Bud; Nudler, Evgeny

    2016-06-30

    In 1943, Luria and Delbrück used a phage-resistance assay to establish spontaneous mutation as a driving force of microbial diversity. Mutation rates are still studied using such assays, but these can only be used to examine the small minority of mutations conferring survival in a particular condition. Newer approaches, such as long-term evolution followed by whole-genome sequencing, may be skewed by mutational ‘hot’ or ‘cold’ spots. Both approaches are affected by numerous caveats. Here we devise a method, maximum-depth sequencing (MDS), to detect extremely rare variants in a population of cells through error-corrected, high-throughput sequencing. We directly measure locus-specific mutation rates in Escherichia coli and show that they vary across the genome by at least an order of magnitude. Our data suggest that certain types of nucleotide misincorporation occur 10(4)-fold more frequently than the basal rate of mutations, but are repaired in vivo. Our data also suggest specific mechanisms of antibiotic-induced mutagenesis, including downregulation of mismatch repair via oxidative stress, transcription–replication conflicts, and, in the case of fluoroquinolones, direct damage to DNA. PMID:27338792

  1. Structure-Function Study of MalF Protein by Random Mutagenesis

    PubMed Central

    Tapia, María Isabel; Mourez, Michaël; Hofnung, Maurice; Dassa, Elie

    1999-01-01

    MalF is one of the two integral inner membrane proteins of the maltose-maltodextrin transport system. To identify functional regions in this protein, we characterized a collection of malF mutants obtained by random mutagenesis. We analyzed their growth on maltose and maltodextrins, the steady-state levels and subcellular localization of the mutant proteins, and the subcellular localization of MalK. Only 2 of the 21 MalF mutant proteins allowed growth on maltose and maltodextrins. Most mutations resulting in immunodetectable proteins mapped to hydrophilic domains, indicating that insertions affecting transmembrane segments gave rise to unstable or lethal proteins. All MalF mutant proteins, even those C-terminally truncated or with large N-terminal deletions, were inserted into the cytoplasmic membrane. Having identified mutations leading to reduced steady-state level, to partial mislocation, and/or to misfolding, we were able to assign to some regions of MalF a role in the assembly of the MalFGK2 complex and/or in the transport mechanism. PMID:10094708

  2. Mechanisms of mutagenesis in human cells exposed to 55 MeV protons

    NASA Technical Reports Server (NTRS)

    Gauny, S.; Wiese, C.; Kronenberg, A.

    2001-01-01

    Protons represent the major type of charged particle radiation in spaceflight environments. The purpose of this study was to assess mutations arising in human lymphoid cells exposed to protons. Mutations were quantitated at the thymidine kinase (TK1) locus in cell lines derived from the same donor: TK6 cells (wt TP53) and WTK1 cells (mutant TP53). WTK1 cells were much more susceptible to mutagenesis following proton exposure than TK6 cells. Intragenic deletions were observed among early-arising TK1 mutants in TK6 cells, but not in WTK1 cells where all of the mutants arose by LOH. Deletion was the predominant mode of LOH in TK6 cells, while allelic recombination was the major mode of LOH in WTK1 cells. Deletions were of variable lengths, from <1 cM to 64 cM, while mutations that arose by allelic recombination often extended to the telomere. In summary, proton exposures elicited many types of mutations at an autosomal locus in human cells. Most involved large scale loss of genetic information, either through deletion or by recombination.

  3. Mutagenesis and functional analysis of the pore-forming toxin HALT-1 from Hydra magnipapillata.

    PubMed

    Liew, Yvonne Jing Mei; Soh, Wai Tuck; Jiemy, William Febry; Hwang, Jung Shan

    2015-02-01

    Actinoporins are small 18.5 kDa pore-forming toxins. A family of six actinoporin genes has been identified in the genome of Hydra magnipapillata, and HALT-1 (Hydra actinoporin-like toxin-1) has been shown to have haemolytic activity. In this study, we have used site-directed mutagenesis to investigate the role of amino acids in the pore-forming N-terminal region and the conserved aromatic cluster required for cell membrane binding. A total of 10 mutants of HALT-1 were constructed and tested for their haemolytic and cytolytic activity on human erythrocytes and HeLa cells, respectively. Insertion of 1-4 negatively charged residues in the N-terminal region of HALT-1 strongly reduced haemolytic and cytolytic activity, suggesting that the length or charge of the N-terminal region is critical for pore-forming activity. Moreover, substitution of amino acids in the conserved aromatic cluster reduced haemolytic and cytolytic activity by more than 80%, suggesting that these aromatic amino acids are important for attachment to the lipid membrane as shown for other actinoporins. The results suggest that HALT-1 and other actinoporins share similar mechanisms of pore formation and that it is critical for HALT-1 to maintain an amphipathic helix at the N-terminus and an aromatic amino acid-rich segment at the site of membrane binding. PMID:25654788

  4. Mutagenesis and Functional Analysis of the Pore-Forming Toxin HALT-1 from Hydra magnipapillata

    PubMed Central

    Liew, Yvonne Jing Mei; Soh, Wai Tuck; Jiemy, William Febry; Hwang, Jung Shan

    2015-01-01

    Actinoporins are small 18.5 kDa pore-forming toxins. A family of six actinoporin genes has been identified in the genome of Hydra magnipapillata, and HALT-1 (Hydra actinoporin-like toxin-1) has been shown to have haemolytic activity. In this study, we have used site-directed mutagenesis to investigate the role of amino acids in the pore-forming N-terminal region and the conserved aromatic cluster required for cell membrane binding. A total of 10 mutants of HALT-1 were constructed and tested for their haemolytic and cytolytic activity on human erythrocytes and HeLa cells, respectively. Insertion of 1–4 negatively charged residues in the N-terminal region of HALT-1 strongly reduced haemolytic and cytolytic activity, suggesting that the length or charge of the N-terminal region is critical for pore-forming activity. Moreover, substitution of amino acids in the conserved aromatic cluster reduced haemolytic and cytolytic activity by more than 80%, suggesting that these aromatic amino acids are important for attachment to the lipid membrane as shown for other actinoporins. The results suggest that HALT-1 and other actinoporins share similar mechanisms of pore formation and that it is critical for HALT-1 to maintain an amphipathic helix at the N-terminus and an aromatic amino acid-rich segment at the site of membrane binding. PMID:25654788

  5. Thermosensitivity of a barosensitive Saccharomyces cerevisiae mutant obtained by UV mutagenesis

    NASA Astrophysics Data System (ADS)

    Shigematsu, Toru; Nomura, Kazuki; Nasuhara, Yusuke; Ikarashi, Kenta; Nagai, Gen; Hirayama, Masao; Hayashi, Mayumi; Ueno, Shigeaki; Fujii, Tomoyuki

    2010-12-01

    Using UV mutagenesis, a high pressure (HP)-sensitive (barosensitive) mutant of Saccharomyces cerevisiae was obtained. The mutant strain a924E1 showed a significant loss of viability at HP levels of 175 to 250 MPa at 20 °C compared with the parent strain. This strain also showed a significant loss of viability following heat treatment at 50-58 °C at 0.1 MPa. These results showed that the mutation caused a significant thermosensitivity as well as barosensitivity. The activation volume and activation energy values for the inactivation of strain a924E1 were equivalent to those of the parent strain. This suggested that the mechanism for the HP and thermal inactivation reaction of strain a924E1 was basically the same as that of the parent strain. Strain a924E1 showed no deficiency in growth and fermentation ability as well as auxotrophic property. Although the identification of the genetic sites of mutation introduced is underway, these phenotypes are favorable for the application of HP treatment and heat-assisted HP treatment on fermentation control.

  6. Generation of orthogonally selective bacterial riboswitches by targeted mutagenesis and in vivo screening.

    PubMed

    Vincent, Helen A; Robinson, Christopher J; Wu, Ming-Cheng; Dixon, Neil; Micklefield, Jason

    2014-01-01

    Riboswitches are naturally occurring RNA-based genetic switches that control gene expression in response to the binding of small-molecule ligands, typically through modulation of transcription or translation. Their simple mechanism of action and the expanding diversity of riboswitch classes make them attractive targets for the development of novel gene expression tools. The essential first step in realizing this potential is to generate artificial riboswitches that respond to nonnatural, synthetic ligands, thereby avoiding disruption of normal cellular function. Here we describe a strategy for engineering orthogonally selective riboswitches based on natural switches. The approach begins with saturation mutagenesis of the ligand-binding pocket of a naturally occurring riboswitch to generate a library of riboswitch mutants. These mutants are then screened in vivo against a synthetic compound library to identify functional riboswitch-ligand combinations. Promising riboswitch-ligand pairs are then further characterized both in vivo and in vitro. Using this method, a series of artificial riboswitches can be generated that are versatile synthetic biology tools for use in protein production, gene functional analysis, metabolic engineering, and other biotechnological applications. PMID:24549615

  7. [Application of XRF to the analysis of Scutellaria baicalensis with space mutagenesis].

    PubMed

    Wang, Zhi-Zhou; Jing, Xi-Li; Guo, Xi-Hua; Zhu, Yan-Ying

    2011-04-01

    X-ray fluorescence spectrum (XRF) analytic method was applied to the determination of the contents and varieties of mineral elements in the space flight mutagenesis breeding scutellaria baicalensis Georgi, and compared with the ground group which were planted and collected together under the same conditions, in an attempt to search for the influence of space environment upon mutation on scutellaria baicalensis Georgi. The result indicates that the varieties of the main mineral elements in the two samples are samely basically, therefore, the two samples have same absorption and enrichment capability of specific element, but the contents of elements Ca, Na, Zn and S increase by 0.3, 0.3, 0.04 and 0.7 times, respectively, in the space group. This modern testing method played an important role in the study. It is recommended that the testing method should be used in the selection of space bred herbal seeds for its advantages, such as quick, simple, highly sensitive, and of wide measure range. To conclude, New breeds can be selected by space breeding, it is significant for the selection and growing of space herbal medicine seeds, and it also has broad application in space breeding of medical plants. PMID:21714276

  8. Role of electrostatic potential in the in silico prediction of molecular bioactivation and mutagenesis.

    PubMed

    Ford, Kevin A

    2013-04-01

    Electrostatic potential (ESP) is a useful physicochemical property of a molecule that provides insights into inter- and intramolecular associations, as well as prediction of likely sites of electrophilic and nucleophilic metabolic attack. Knowledge of sites of metabolic attack is of paramount importance in DMPK research since drugs frequently fail in clinical trials due to the formation of bioactivated metabolites which are often difficult to measure experimentally due to their reactive nature and relatively short half-lives. Computational chemistry methods have proven invaluable in recent years as a means to predict and study bioactivated metabolites without the need for chemical syntheses, or testing on experimental animals. Additional molecular properties (heat of formation, heat of solvation and E(LUMO) - E(HOMO)) are discussed in this paper as complementary indicators of the behavior of metabolites in vivo. Five diverse examples are presented (acetaminophen, aniline/phenylamines, imidacloprid, nefazodone and vinyl chloride) which illustrate the utility of this multidimensional approach in predicting bioactivation, and in each case the predicted data agreed with experimental data described in the scientific literature. A further example of the usefulness of calculating ESP, in combination with the molecular properties mentioned above, is provided by an examination of the use of these parameters in providing an explanation for the sites of nucleophilic attack of the nucleic acid cytosine. Exploration of sites of nucleophilic attack of nucleic acids is important as adducts of DNA have the potential to result in mutagenesis. PMID:23323940

  9. Site-directed mutagenesis of a conserved Asn450 residue of Bacillus licheniformis γ-glutamyltranspeptidase.

    PubMed

    Lin, Min-Guan; Chi, Meng-Chun; Chen, Yu-Yi; Wang, Tzu-Fan; Lo, Hui-Fen; Lin, Long-Liu

    2016-10-01

    Bacillus licheniformis γ-glutamyltranspeptidase (BlGGT) belongs to N-terminal nucleophile hydrolase superfamily in which all inclusive members are synthetized as single-chain precursors, and then self-processed to form mature enzymes. Here we investigated the role of a conserved Asn450 residue in BlGGT through site-directed mutagenesis and molecular characterization of four relevant variants. Substitution of Asn450 by arginine resulted in a significant reduction in the catalytic activity of BlGGT. Conversely, N450A and N450D displayed an enhanced activity. The catalytic efficiency of BlGGT was calculated to be 16.04mM(-1)s(-1), but this value was either decreased to 8.93mM(-1)s(-1) in N450K or increased to more than 123.65mM(-1)s(-1) in N450A and N450D. In addition, the ratio of transpeptidation to hydrolysis was increased from 3.5 to more than 7.6 by the mutations. Structural analyses showed that fluorescence, circular dichroism spectra and thermal denaturation profiles of mutant proteins were essentially consistent with those of BlGGT. However, guanidine hydrochloride (GdnHCl)-induced transition was significantly reduced in comparison with the wild-type enzyme. Molecular modeling suggests that residue Asn450 of BlGGT is important to create suitable environments for both autoprocessing and catalytic reactions. PMID:27246377

  10. piggyBac transposon-based insertional mutagenesis in mouse haploid embryonic stem cells.

    PubMed

    Pettitt, Stephen J; Tan, E-Pien; Yusa, Kosuke

    2015-01-01

    Forward genetic screening is a powerful non-hypothesis-driven approach to unveil the molecular mechanisms and pathways underlying phenotypes of interest. In this approach, a genome-wide mutant library is first generated and then screened for a phenotype of interest. Subsequently, genes responsible for the phenotype are identified. There have been a number of successful screens in yeasts, Caenorhabditis elegans and Drosophila. These model organisms all allow loss-of-function mutants to be generated easily on a genome-wide scale: yeasts have a haploid stage in their reproductive cycles and the latter two organisms have short generation times, allowing mutations to be systematically bred to homozygosity. However, in mammals, the diploid genome and long generation time have always hampered rapid and efficient production of homozygous mutant cells and animals. The recent discovery of several haploid mammalian cell lines promises to revolutionize recessive genetic screens in mammalian cells. In this protocol, we describe an overview of insertional mutagenesis, focusing on DNA transposons, and provide a method for an efficient generation of genome-wide mutant libraries using mouse haploid embryonic stem cells. PMID:25408399

  11. Stem cell gene therapy: the risks of insertional mutagenesis and approaches to minimize genotoxicity

    PubMed Central

    Wu, Chuanfeng

    2012-01-01

    Virus-based vectors are widely used in hematopoietic stem cell (HSC) gene therapy, and have the ability to integrate permanently into genomic DNA, thus driving long-term expression of corrective genes in all hematopoietic lineages. To date, HSC gene therapy has been successfully employed in the clinic for improving clinical outcomes in small numbers of patients with X-linked severe combined immunodeficiency (SCID-X1), adenosine deaminase deficiency (ADA-SCID), adrenoleukodystrophy (ALD), thalassemia, chronic granulomatous disease (CGD), and Wiskott-Aldrich syndrome (WAS). However, adverse events were observed during some of these HSC gene therapy clinical trials, linked to insertional activation of proto-oncogenes by integrated proviral vectors leading to clonal expansion and eventual development of leukemia. Numerous studies have been performed to understand the molecular basis of vector-mediated genotoxicity, with the aim of developing safer vectors and lower-risk gene therapy protocols. This review will summarize current information on the mechanisms of insertional mutagenesis in hematopoietic stem and progenitor cells due to integrating gene transfer vectors, discuss the available assays for predicting genotoxicity and mapping vector integration sites, and introduce newly-developed approaches for minimizing genotoxicity as a way to further move HSC gene therapy forward into broader clinical application. PMID:22198747

  12. Yellow fluorescent protein phiYFPv (Phialidium): structure and structure-based mutagenesis

    SciTech Connect

    Pletneva, Nadya V.; Pletnev, Vladimir Z. Souslova, Ekaterina; Chudakov, Dmitry M.; Lukyanov, Sergey; Martynov, Vladimir I.; Arhipova, Svetlena; Artemyev, Igor; Wlodawer, Alexander; Dauter, Zbigniew; Pletnev, Sergei

    2013-06-01

    The yellow fluorescent protein phiYFPv with improved folding has been developed from the spectrally identical wild-type phiYFP found in the marine jellyfish Phialidium. The yellow fluorescent protein phiYFPv (λ{sub em}{sup max} ≃ 537 nm) with improved folding has been developed from the spectrally identical wild-type phiYFP found in the marine jellyfish Phialidium. The latter fluorescent protein is one of only two known cases of naturally occurring proteins that exhibit emission spectra in the yellow–orange range (535–555 nm). Here, the crystal structure of phiYFPv has been determined at 2.05 Å resolution. The ‘yellow’ chromophore formed from the sequence triad Thr65-Tyr66-Gly67 adopts the bicyclic structure typical of fluorophores emitting in the green spectral range. It was demonstrated that perfect antiparallel π-stacking of chromophore Tyr66 and the proximal Tyr203, as well as Val205, facing the chromophore phenolic ring are chiefly responsible for the observed yellow emission of phiYFPv at 537 nm. Structure-based site-directed mutagenesis has been used to identify the key functional residues in the chromophore environment. The obtained results have been utilized to improve the properties of phiYFPv and its homologous monomeric biomarker tagYFP.

  13. A mutagenesis-free approach to assignment of (19)F NMR resonances in biosynthetically labeled proteins.

    PubMed

    Kitevski-LeBlanc, Julianne L; Al-Abdul-Wahid, M Sameer; Prosser, R Scott

    2009-02-18

    Solution NMR studies of protein structure and dynamics using fluorinated amino acid probes are a valuable addition to the repertoire of existing (13)C, (15)N, and (1)H experiments. Despite the numerous advantages of the (19)F nucleus in NMR, protein studies are complicated by the dependence of resonance assignments on site-directed mutagenesis methods which are laborious and often problematic. Here we report an NMR-based route to the assignment of fluorine resonances in (13)C,(15)N-3-fluoro-l-tyrosine labeled calmodulin. The assignment begins with the correlation of the fluorine nucleus to the delta proton in the novel (13)C,(15)N-enriched probe which is achieved using a CT-HCCF-COSY experiment. Connection to the backbone is made through two additional solution NMR experiments, namely the (H(beta))C(beta)(C(gamma)C(delta))H(delta) and HNCACB. Assignments are completed using either previously published backbone chemical shift data or obtained experimentally provided uniform (13)C,(15)N labeling procedures are employed during protein expression. Additional benefits of the (13)C,(15)N-3-fluoro-l-tyrosine probe include the reduction of spectral overlap through ((13)C(19)F) CT-HSQCs, as well as the ability to monitor side chain dynamics using (19)F T(1), T(2), and the (13)C-(19)F NOE. PMID:19173647

  14. US132 Cyclodextrin Glucanotransferase Engineering by Random Mutagenesis for an Anti-Staling Purpose.

    PubMed

    Jemli, Sonia; Jaoua, Mouna; Bejar, Samir

    2016-09-01

    The use of the cyclodextrin glucanotransferase (CGTase) of the US132 strain, which is an effective anti-staling agent, has been hampered by its high cyclization activity. Since that random mutagenesis using error-prone PCR is nowadays a method of choice for enzymes engineering, we have optimized this method by adjusting manganese concentration in order to obtain a high percentage of active CGTase mutants. Therefore, the amplification of the gene encoding the US132 CGTase was performed using a MnCl2 concentration ranging between 0 and 0.5 mM. The finding showed that a manganese concentration of 0.04 mM allowed for 90 % of active mutants. A simple method to rapidly screen the obtained mutants was also developed. After the examination of a small library (of less than 1000 clones), the active mutant named MJ13 was selected for a significant decrease in the cyclization activity, thereby showing a remarkable change in the enzyme specificity towards starch dextrinizing. Sequence analysis showed that MJ13 is a triple mutant with two mutations in the catalytic domain (K47E and S382P) and one substitution in the starch binding domain (N655S). PMID:27271016

  15. Insertional Mutagenesis Identifies a STAT3/Arid1b/β-catenin Pathway Driving Neurofibroma Initiation.

    PubMed

    Wu, Jianqiang; Keng, Vincent W; Patmore, Deanna M; Kendall, Jed J; Patel, Ami V; Jousma, Edwin; Jessen, Walter J; Choi, Kwangmin; Tschida, Barbara R; Silverstein, Kevin A T; Fan, Danhua; Schwartz, Eric B; Fuchs, James R; Zou, Yuanshu; Kim, Mi-Ok; Dombi, Eva; Levy, David E; Huang, Gang; Cancelas, Jose A; Stemmer-Rachamimov, Anat O; Spinner, Robert J; Largaespada, David A; Ratner, Nancy

    2016-03-01

    To identify genes and signaling pathways that initiate Neurofibromatosis type 1 (NF1) neurofibromas, we used unbiased insertional mutagenesis screening, mouse models, and molecular analyses. We mapped an Nf1-Stat3-Arid1b/β-catenin pathway that becomes active in the context of Nf1 loss. Genetic deletion of Stat3 in Schwann cell progenitors (SCPs) and Schwann cells (SCs) prevents neurofibroma formation, decreasing SCP self-renewal and β-catenin activity. β-catenin expression rescues effects of Stat3 loss in SCPs. Importantly, P-STAT3 and β-catenin expression correlate in human neurofibromas. Mechanistically, P-Stat3 represses Gsk3β and the SWI/SNF gene Arid1b to increase β-catenin. Knockdown of Arid1b or Gsk3β in Stat3(fl/fl);Nf1(fl/fl);DhhCre SCPs rescues neurofibroma formation after in vivo transplantation. Stat3 represses Arid1b through histone modification in a Brg1-dependent manner, indicating that epigenetic modification plays a role in early tumorigenesis. Our data map a neural tumorigenesis pathway and support testing JAK/STAT and Wnt/β-catenin pathway inhibitors in neurofibroma therapeutic trials. PMID:26904939

  16. Facilitating Structure-Function Studies of CFTR Modulator Sites with Efficiencies in Mutagenesis and Functional Screening.

    PubMed

    Molinski, Steven V; Ahmadi, Saumel; Hung, Maurita; Bear, Christine E

    2015-12-01

    There are nearly 2000 mutations in the CFTR gene associated with cystic fibrosis disease, and to date, the only approved drug, Kalydeco, has been effective in rescuing the functional expression of a small subset of these mutant proteins with defects in channel activation. However, there is currently an urgent need to assess other mutations for possible rescue by Kalydeco, and further, definition of the binding site of such modulators on CFTR would enhance our understanding of the mechanism of action of such therapeutics. Here, we describe a simple and rapid one-step PCR-based site-directed mutagenesis method to generate mutations in the CFTR gene. This method was used to generate CFTR mutants bearing deletions (p.Gln2_Trp846del, p.Ser700_Asp835del, p.Ile1234_Arg1239del) and truncation with polyhistidine tag insertion (p.Glu1172-3Gly-6-His*), which either recapitulate a disease phenotype or render tools for modulator binding site identification, with subsequent evaluation of drug responses using a high-throughput (384-well) membrane potential-sensitive fluorescence assay of CFTR channel activity within a 1 wk time frame. This proof-of-concept study shows that these methods enable rapid and quantitative comparison of multiple CFTR mutants to emerging drugs, facilitating future large-scale efforts to stratify mutants according to their "theratype" or most promising targeted therapy. PMID:26385858

  17. Residue proximity information and protein model discrimination using saturation-suppressor mutagenesis

    PubMed Central

    Sahoo, Anusmita; Khare, Shruti; Devanarayanan, Sivasankar; Jain, Pankaj C.; Varadarajan, Raghavan

    2015-01-01

    Identification of residue-residue contacts from primary sequence can be used to guide protein structure prediction. Using Escherichia coli CcdB as the test case, we describe an experimental method termed saturation-suppressor mutagenesis to acquire residue contact information. In this methodology, for each of five inactive CcdB mutants, exhaustive screens for suppressors were performed. Proximal suppressors were accurately discriminated from distal suppressors based on their phenotypes when present as single mutants. Experimentally identified putative proximal pairs formed spatial constraints to recover >98% of native-like models of CcdB from a decoy dataset. Suppressor methodology was also applied to the integral membrane protein, diacylglycerol kinase A where the structures determined by X-ray crystallography and NMR were significantly different. Suppressor as well as sequence co-variation data clearly point to the X-ray structure being the functional one adopted in vivo. The methodology is applicable to any macromolecular system for which a convenient phenotypic assay exists. DOI: http://dx.doi.org/10.7554/eLife.09532.001 PMID:26716404

  18. Excavating the Genome: Large-Scale Mutagenesis Screening for the Discovery of New Mouse Models.

    PubMed

    Sundberg, John P; Dadras, Soheil S; Silva, Kathleen A; Kennedy, Victoria E; Murray, Stephen A; Denegre, James M; Schofield, Paul N; King, Lloyd E; Wiles, Michael V; Pratt, C Herbert

    2015-11-01

    Technology now exists for rapid screening of mutated laboratory mice to identify phenotypes associated with specific genetic mutations. Large repositories exist for spontaneous mutants and those induced by chemical mutagenesis, many of which have never been fully studied or comprehensively evaluated. To supplement these resources, a variety of techniques have been consolidated in an international effort to create mutations in all known protein coding genes in the mouse. With targeted embryonic stem cell lines now available for almost all protein coding genes and more recently CRISPR/Cas9 technology, large-scale efforts are underway to create further novel mutant mouse strains and to characterize their phenotypes. However, accurate diagnosis of skin, hair, and nail diseases still relies on careful gross and histological analysis, and while not automated to the level of the physiological phenotyping, histopathology still provides the most direct and accurate diagnosis and correlation with human diseases. As a result of these efforts, many new mouse dermatological disease models are being characterized and developed. PMID:26551941

  19. Efficiency and Inheritance of Targeted Mutagenesis in Maize Using CRISPR-Cas9.

    PubMed

    Zhu, Jinjie; Song, Ning; Sun, Silong; Yang, Weilong; Zhao, Haiming; Song, Weibin; Lai, Jinsheng

    2016-01-20

    CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins) is an adaptive immune system in bacteria and archaea to defend against invasion from foreign DNA fragments. Recently, it has been developed as a powerful targeted genome editing tool for a wide variety of species. However, its application in maize has only been tested with transiently expressed somatic cells or with a limited number of stable transgenic T0 plants. The exact efficiency and specificity of the CRISPR/Cas system in the highly complex maize genome has not been documented yet. Here we report an extensive study of the well-studied type II CRISPR-Cas9 system for targeted genome editing in maize, with the codon-optimized Cas9 protein and the short non-coding guide RNA generated through a functional maize U6 snRNA promoter. Targeted gene mutagenesis was detected for 90 loci by maize protoplast assay, with an average cleavage efficiency of 10.67%. Stable knockout transformants for maize phytoene synthase gene (PSY1) were obtained. Mutations occurred in germ cells can be stably inherited to the next generation. Moreover, no off-target effect was detected at the computationally predicted putative off-target loci. No significant difference between the transcriptomes of the Cas9 expressed and non-expressed lines was detected. Our results confirmed that the CRISPR-Cas9 could be successfully applied as a robust targeted genome editing system in maize. PMID:26842991

  20. Retrovirus-induced insertional mutagenesis: mechanism of collagen mutation in Mov13 mice.

    PubMed Central

    Barker, D D; Wu, H; Hartung, S; Breindl, M; Jaenisch, R

    1991-01-01

    The Mov13 mouse strain carries a mutation in the alpha 1(I) procollagen gene which is due to the insertion of a Moloney murine leukemia provirus into the first intron. This insertion results in the de novo methylation of the provirus and flanking DNA, the alteration of chromatin structure, and the transcriptional inactivity of the collagen promoter. To address the mechanism of mutagenesis, we reintroduced a cloned and therefore demethylated version of the Mov13 mutant allele into mouse fibroblasts. The transfected gene was not transcribed, indicating that the transcriptional defect was not due to the hypermethylation. Rather, this result strongly suggests that the mutation is due to the displacement or disruption of cis-acting regulatory DNA sequences within the first intron. We also constructed a Mov13 variant allele containing a single long terminal repeat instead of the whole provirus. This construct also failed to express mRNA, indicating that the Mov13 mutation does not revert by provirus excision as has been observed for other retrovirus-induced mutations. Images PMID:1922037